IFN-τ Displays Anti-Inflammatory Effects on Staphylococcus aureus … · 2017. 3. 22. ·...
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Research Article IFN- Displays Anti-Inflammatory Effects on Staphylococcus aureus Endometritis via Inhibiting the Activation of the NF-B and MAPK Pathways in Mice Zhenbiao Zhang, Yingfang Guo, Yuzhu Liu, Chengye Li, Mengyao Guo, and Ganzhen Deng Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China Correspondence should be addressed to Ganzhen Deng; [email protected] Received 21 June 2016; Revised 22 August 2016; Accepted 12 January 2017; Published 26 February 2017 Academic Editor: Andr´ e Talvani Copyright © 2017 Zhenbiao Zhang et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e aim of the present study was to determine the anti-inflammatory effect of IFN- on endometritis using a mouse model of S. aureus-induced endometritis and to elucidate the mechanism of action underlying these effects. In the present study, the effect of IFN- on S. aureus growth was monitored by turbidimeter at 600 nm. IFN- did not affect S. aureus growth. e histopathological changes indicated that IFN- had a protective effect on uterus tissues with S. aureus infection. e ELISA and qPCR results showed the production of the proinflammatory cytokines TNF-, IL-1, and IL-6 was decreased with IFN- treatment. In contrast, the level of the anti-inflammatory cytokine IL-10 was increased. We further studied the signaling pathway associated with these observations, and the qPCR results showed that the expression of TLR2 was repressed by IFN-. Furthermore, the western blotting results showed the phosphorylation of IB, NF-B p65, and MAPKs (p38, JNK, and ERK) was inhibited by IFN- treatment. e results suggested that IFN- may be a potential drug for the treatment of uterine infection due to S. aureus or other infectious inflammatory diseases. 1. Introduction Endometritis, which is a type of inflammation, can be caused by bacterial, viral, fungal, mycoplasma, and other infections or a combination of the above [1–3]. e clinical symptoms of endometritis include abnormal vaginal discharge and the dilation of the uterus and cervix. e diagnosis of subclinical cases requires the use of specific ultrasound examination, cytology, or biopsy [4]. e cause of endometritis is very complicated, and most studies have shown that an infection by pathogens is the major cause of endometritis [5]. Staphy- lococcus aureus (S. aureus) is the most common pathogenic bacteria [6] and has been reported to induce both clinical and subclinical endometritis. Additionally, it can promote the production of proinflammatory cytokines by keratinocytes, triggering inflammatory disease [7]. Interferon- (IFN-) was originally identified as a preg- nancy-associated protein in ruminants. It is produced by the trophoblast and is important for preventing the degradation of the corpus luteum. Additionally, it is also an early sign of female animal pregnancy [8, 9]. However, compared with the other known IFNs, IFN- also has high antiviral and antiproliferative activity but is not cytotoxic [10]. In contrast, the expression of related Type I IFN genes occurs in response to virus and other pathogens in a variety of inflammatory syndromes [11]. IFN- has a wide range of cross-species activity and functions in various cells, including lymphocytes, macrophages, and epithelial cells [12]. e specific function of IFN- is relatively unknown, but it has been shown to have antiviral activity [13]. IFN- has excellent antiviral activity, which is at least equivalent to that of IFN- in the same species [14, 15]. IFN- has important implications in regulating cytokine networks, such as that involved in inflam- mation and angiogenesis, in the uterus during pregnancy [16]. Previous studies have shown that IFN- prevents the secretion of proinflammatory cytokines, thereby inhibiting Candida albicans-induced inflammation [17]. It is known that gram-negative and gram-positive bacte- ria are the most common microbial cause of inflammatory Hindawi Publishing Corporation BioMed Research International Volume 2017, Article ID 2350482, 12 pages http://dx.doi.org/10.1155/2017/2350482
Transcript of IFN-τ Displays Anti-Inflammatory Effects on Staphylococcus aureus … · 2017. 3. 22. ·...
Research ArticleIFN-120591 Displays Anti-Inflammatory Effects onStaphylococcus aureus Endometritis via Inhibiting theActivation of the NF-120581B and MAPK Pathways in Mice
Zhenbiao Zhang Yingfang Guo Yuzhu Liu Chengye Li Mengyao Guo and Ganzhen Deng
Department of Clinical VeterinaryMedicine College of VeterinaryMedicine HuazhongAgricultural UniversityWuhan 430070 China
Correspondence should be addressed to Ganzhen Deng gzdeng1963sohucom
Received 21 June 2016 Revised 22 August 2016 Accepted 12 January 2017 Published 26 February 2017
Academic Editor Andre Talvani
Copyright copy 2017 Zhenbiao Zhang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
The aim of the present study was to determine the anti-inflammatory effect of IFN-120591 on endometritis using a mouse model of Saureus-induced endometritis and to elucidate the mechanism of action underlying these effects In the present study the effect ofIFN-120591 on S aureus growth was monitored by turbidimeter at 600 nm IFN-120591 did not affect S aureus growthThe histopathologicalchanges indicated that IFN-120591 had a protective effect on uterus tissues with S aureus infectionThe ELISA and qPCR results showedthe production of the proinflammatory cytokines TNF-120572 IL-1120573 and IL-6 was decreased with IFN-120591 treatment In contrast the levelof the anti-inflammatory cytokine IL-10was increasedWe further studied the signaling pathway associatedwith these observationsand the qPCR results showed that the expression of TLR2was repressed by IFN-120591 Furthermore the western blotting results showedthe phosphorylation of I120581B NF-120581B p65 andMAPKs (p38 JNK and ERK) was inhibited by IFN-120591 treatmentThe results suggestedthat IFN-120591may be a potential drug for the treatment of uterine infection due to S aureus or other infectious inflammatory diseases
1 Introduction
Endometritis which is a type of inflammation can be causedby bacterial viral fungal mycoplasma and other infectionsor a combination of the above [1ndash3] The clinical symptomsof endometritis include abnormal vaginal discharge and thedilation of the uterus and cervix The diagnosis of subclinicalcases requires the use of specific ultrasound examinationcytology or biopsy [4] The cause of endometritis is verycomplicated and most studies have shown that an infectionby pathogens is the major cause of endometritis [5] Staphy-lococcus aureus (S aureus) is the most common pathogenicbacteria [6] and has been reported to induce both clinicaland subclinical endometritis Additionally it can promote theproduction of proinflammatory cytokines by keratinocytestriggering inflammatory disease [7]
Interferon-120591 (IFN-120591) was originally identified as a preg-nancy-associated protein in ruminants It is produced by thetrophoblast and is important for preventing the degradationof the corpus luteum Additionally it is also an early sign
of female animal pregnancy [8 9] However compared withthe other known IFNs IFN-120591 also has high antiviral andantiproliferative activity but is not cytotoxic [10] In contrastthe expression of related Type I IFN genes occurs in responseto virus and other pathogens in a variety of inflammatorysyndromes [11] IFN-120591 has a wide range of cross-speciesactivity and functions in various cells including lymphocytesmacrophages and epithelial cells [12] The specific functionof IFN-120591 is relatively unknown but it has been shown tohave antiviral activity [13] IFN-120591 has excellent antiviralactivity which is at least equivalent to that of IFN-120572 in thesame species [14 15] IFN-120591 has important implications inregulating cytokine networks such as that involved in inflam-mation and angiogenesis in the uterus during pregnancy[16] Previous studies have shown that IFN-120591 prevents thesecretion of proinflammatory cytokines thereby inhibitingCandida albicans-induced inflammation [17]
It is known that gram-negative and gram-positive bacte-ria are the most common microbial cause of inflammatory
Hindawi Publishing CorporationBioMed Research InternationalVolume 2017 Article ID 2350482 12 pageshttpdxdoiorg10115520172350482
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diseases [18] S aureus is a well-known gram-positive bacte-ria which can lead to inflammation from both community-acquired and nosocomial infections [19 20] S aureus is alsothe main pathogenic microbe that causes endometritis andenhances the expression of proinflammatory cytokines [21]It has been shown IFN-120591 has immunomodulatory properties[22] A prominent feature of IFN-120591 is its low cytotoxicitycompared with other Type I IFN which has been observed incell culture as well as in vivo [23] In various clinical studiesIFN-120591 has been shown to have anti-inflammatory propertiesand low cytotoxicity As such this study sought to verify theanti-inflammatory effect of IFN-120591 inmice Additionallymorestudies on the functional role of IFN-120591 in mediating anti-inflammatory effects are needed
2 Materials and Methods
21 S aureus Growth S aureus strain ATCC 25923 wascultured at 37∘C in Mueller-Hinton II cation adjusted broth(MH BD Biosciences Sparks MD USA) 180 rpm withIFN-120591 (Recombinant Ovine Interferon-tau IFNT-29O Cre-ative Bioarray New York USA) treatment (0 50 100 and200 ngmL) The absorbance was measured once every 1 h at600 nm until the bacteria reached the stationary phase
22 Animals and Experiment Groups Female BALBc mice(6ndash8 weeks 18ndash20 g) were purchased from the Center ofExperimental Animals of Wuhan Institute All experimentalprocedures were conducted with the approval of the Insti-tutional Animal Care and were approved by the HuazhongAgricultural University Animal Care and Use CommitteeThe mice were housed in microisolator cages and receivedfood and water ad libitum Mice were given an adaptationperiod of 4ndash6 days prior to experimentation
The sixty mice were randomly divided into six groups (a)the control group (CG) in which the mice did not receiveany treatment (b) the S aureus-infected group (S aureus) inwhich the mice were infected with S aureus via an injectionof 100 120583L S aureus into each uterine horn (a total volumeof 200120583L) through a microsyringe followed by a stimulus of24 h (c) IFN-120591 groups inwhich themicewith S aureus endo-metritis were treated with different concentrations of IFN-120591(2 4 8mgkg) and (d) the dexamethasone group (DEX) inwhich the mice with S aureus endometritis were treatmentwith DEX (5mgkg 5mgmL) Finally all the mice wereeuthanized with sodium pentobarbital and sacrificed Theuterine tissues were quickly collected and were kept frozenat minus80∘C until they were used for subsequent experiments
23 Histological Analysis The harvested uterine tissues werefixed in 10 formaldehyde solution and embedded in paraffinwax Sections (4 120583m) were deparaffinized with xylene andrehydrated through graded alcohols for staining Then sec-tions were stained with hematoxylin and eosin (HampE) andlast examined with a microscope (Olympus Japan)
24 Immunohistochemical Analysis Six serial sections ofeach uterus area were collected and mounted on polylysine
coated slides A 4120583m section was prepared from the paraffin-embedded block and dehydrated and decreasing concentra-tions of ethanol and then washed for 5min in running waterFollowing washing sections were treated with 90 formicacid for 5mins and blocked for endogenous peroxidase activ-ity in 5H
2O2 A Tris-HCl (005M pH 766) was introduced
at room temperature for 20min and with a 1 200 dilution(vv) of the MPO antibody (Abcam) was added to the tissuesand incubated at 4∘C overnight As for the negative controlthe primary antibody was replaced with PBS The secondaryantibodies (Tianjin Sungene Biotech Co Ltd China) wereadded as appropriate and 331015840-diaminobenzidine stainingwas visualized using the hematoxylin stain Two pathologistsscored the slides respectively For MPO staining assessmentmounting with glycerin and observing used a microscopewith a camera system (Olympus DP-71 MN USA)
25 HEK293 Cell Culture and Transfection HEK293 cellswere purchased from American Type Culture Collection(ATCC Manassas VA USA) and were cultured in DMEMcontaining 10 FBS with 5 CO
2at 37∘CThe culture media
were changed once every 24 hThe cells were always added 1 hprior to S aureus treatment and were subsequently incubatedin the presence or absence of various concentrations of IFN-120591 HEK293 cells were transfected with pEGFP-N1-mTLR2plasmids using FuGENE HD transfection reagent accordingto the manufacturerrsquos instructions (Roche Applied ScienceIndianapolis IN USA)
26 ELISA Analysis The uterus samples were weighed andhomogenized with phosphate-buffered saline (wv 1 9) onice and then centrifuged at 12000 rpm for 15min at 4∘C Thesupernatant was collected The cytokine TNF-120572 IL-1120573 IL-6 and IL-10 assays were evaluated with the correspondingELISA kits (Biolegend USA) according to the instructionsof the manufacturer Results are expressed as means plusmn SD ofthree independent experiments
27 Quantitative Real-Time Polymerase Chain Reaction TotalRNA from endometrial samples were isolated using TRI-zol reagent (Invitrogen Corporation) according to man-ufacturerrsquos instructions The concentration and purity ofthe total RNA were determined spectrophotometrically at260280 nm The RNA was reverse transcribed into cDNAusing a Revert Aid First Strand cDNA Synthesis Kit(Thermo)Theprimers (TNF-120572 IL-1120573 IL-6 IL-10 andTLR2)used for qPCR are listed in Table 1 (Designed by softwarePrimer 50) The qPCR were executed by a 7500 Fast Real-Time PCR System (Applied Biosystems) with the SYBR greenPlus reagent kit (Roche Applied Science Mannheim Ger-many) in a 20120583L system Each sample set three repetitionsResults were expressed as 2minusΔΔCt comparative method ΔΔCt= (target gene Ct of experimental group minus reference gene Ctof experimental group) minus (target gene Ct of control group minusreference gene Ct of control group) GAPDH was used as thereference gene
28 Western Blot Analysis The protein of collected tissuesamples were extracted using PMSF (10 120583L) and RIPA (1mL)
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Table 1 Sequences of primers used for qPCR
Gene GenBankaccession number
Sequence (51015840-31015840)sense and antisense Product size (bp)
TNF-120572 NM 0136933 Sense 51015840-CTTCTCATTCCTGCTTGTG-31015840 198Antisense 51015840-ACTTGGTGGTTTGCTACG-31015840
IL-1120573 NM 0083613464 Sense 51015840-AGGCTCCGAGATGAACAA-31015840 464Antisense 51015840-AAGGCATTAGAAACAGTCC-31015840
IL-6 NM 0311681 Sense 51015840-TTCTTGGGACTGATGCTG-31015840 380Antisense 51015840-CTGGCTTTGTCTTTCTTGTT-31015840
IL-10 NM 0105482 Sense 51015840-AACATACTGCTAACCGACTC-31015840 286Antisense 51015840-TGGCCTTGTAGACACCTT-31015840
TLR2 NM 0119053 Sense 51015840-TTTGCTCCTGCGAACTCC-31015840 267Antisense 51015840-GCCACGCCCACATCATTC-31015840
GAPDH NM 0012897261 Sense 51015840-TGTTTCCTCGTCCCGTAG-31015840 108Antisense 51015840-CAATCTCCACTTTGCCACT-31015840
function 30minThen protein concentrationwas determinedusing the BCA protein assay kit (Thermo USA) The crudeprotein per sample was subjected to 10 SDS-PAGE usingTris-HCl Precast Gels and subsequently transferred ontoPVDF membranes The membrane was blocked with Trisbuffer saline containing 005 Tween-20 (TBS-T) supple-mented with 5 skim milk at room temperature for 15 h ona rotary shaker following by TBS-T washed The membraneswere washed with a specific primary antibody (Cell SignalingTechnology Inc Danvers MA) diluted in TBS-T containingskim milk (VV = 1 1000) at 4∘C overnight Subsequentlythe membranes were washed with TBS-T The membranewas washed and treated with secondary antibody (TianjinSungene Biotech Co Ltd Tianjin China) diluted in TBS-T(VV = 1 5000) at room temperature for 2 h In the last stepmembranes developed with the ECL Plus Western BlottingDetection System (Amersham Life Science UK) 120573-Actin(Tianjin China) was used as a control
29 Statistical Analysis Statistical analyses of the data wereperformed using the SPSS software (ver 17 for WindowsSPSS Inc Chicago IL USA) All values are expressed asthe means plusmn SD The data were assessed using the Tukey-Kramer method for multiple comparisons Significance wasdetermined by a 1-way ANOVA using a significance level of119901 lt 005
3 Results
31 Effect of IFN-120591 on S aureus Growth The effect of IFN-120591 (50ndash200 ngmL) on S aureus growth was monitored byturbidimeter at 600 nm for 12 hThe results showed that IFN-120591 had no effect on S aureus growth Specifically when com-pared to the untreated controls S aureus grown in the pres-ence of IFN-120591 (50ndash200 ngmL) were not affected (Figure 1)
32 Histopathological Changes Tissues were collected 24hours after the injection of S aureus and were stained withhematoxylin and eosin (HampE) There were no pathologicalchanges in the uterine tissue of the CG (Figure 2(a)) The
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uterine morphology of the mice inoculated with S aureusdisplayed inflammatory cell infiltration endometrial conges-tion severe destruction of the myometrium cells and thedisappearance of the uterus acinar structure (Figure 2(b))However IFN-120591 significantly reduced the inflammation stim-ulated by S aureus and the uterine tissue structure of themice treated with IFN-120591 was almost complete with onlyminor endometrial pathology and an insignificant amount ofinflammatory cell infiltration (Figures 2(c) 2(d) and 2(e))
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(a) (b) (c)
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Figure 2 Histopathological changes (HE times200) HampE staining of the harvested uterine tissues (a) Control group (CG) in which the micewere not treated (b) S aureus-infected group (S aureus) in which the mice were infected with S aureus (c) Dexamethasone group (DEX)in which the mice inoculated with S aureus and developed endometritis were treated with DEX (d)ndash(f) IFN-120591 groups in which the miceinoculated with S aureus and developed endometritis were treated with different concentrations of IFN-120591 (2 4 8mgkg)
Representative pictures are shown to demonstrate the effectsdose-dependent effects of IFN-120591The degree of inflammatoryinjury was also very slight in the DEX group as shown inFigure 2(f)
33 Effects of IFN-120591 onMPO Activity in Uterus Tissues MPOactivity has been used as a quantitative index of inflammationand can illustrate the level of neutrophil infiltrationThe effectof IFN-120591 on MPO activity was examined in mice with Saureus-induced endometritis Immunohistochemical stain-ing showed that MPO specific immunolabelling was scarcelyfound in the neutrophil andmononuclear cells of lamina pro-pria of mucosa in normal uterus in CG (Figure 3(a)) whereasthis expression was found elevated in cells of surface epithe-lium and in cells of the inflammatory infiltrate in S aureus-infected group (Figure 3(b)) The MPO activity was veryslight in DEX group as shown in Figure 3(c) In contrastmice treated with different doses of IFN-120591 uterus showed alower level of MPO activity the immunoreactivity of whichshowed the suppressive effects of IFN-120591 increased in a dose-dependent manner in uterus samples of mice (Figures 3(d)3(e) and 3(f)) The activity of MPO from different micegroups was also measured by MPO assay kits The resultsillustrates that the activity of MPO was low in CG and dra-matically increased in S aureus group (Figure 3(g)) while theS aureus-infected mice treated with different concentrationsof IFN-120591 (2 4 8mgkg) significantly inhibited the activity ofMPO (compare with S aureus group) The results indicatedthat reduction of neutrophil infiltration in uterine tissuescould be one mechanism underlying the protective effect ofIFN-120591
34 Effects of IFN-120591 on Inflammatory Cytokines To deter-mine the effect of IFN-120591 on the S aureus endometritis thelevels of TNF-120572 IL-1120573 IL-6 and IL-10 were measured byqPCR and ELISA The results are shown in Figure 4 Theexpressions of TNF-120572 IL-1120573 and IL-6 were increased signif-icantly in the mice inoculated with S aureus compared tothe CG The increases in TNF-120572 IL-1120573 and IL-6 expressionwhich were induced by S aureus were significantly inhibitedby IFN-120591 Compared with themice inoculated with S aureusthe mRNA and protein expression of TNF-120572 IL-1120573 and IL-6 was significantly decreased following IFN-120591 treatment Theeffects of IFN-120591 increased in a dose-dependent mannerWithincreasing concentrations of IFN-120591 more significant effectswere observed However the effect on IL-10 was differentThe mRNA and protein expression of IL-10 was increasedin the mice inoculated with S aureus compared to the CGCompared with the mice inoculated with S aureus theexpression of IL-10 further increased upon IFN-120591 treatmentWith increasing concentrations of IFN-120591 more significanteffects on IL-10 were observed
35 Effects of IFN-120591 on TLR2 Expression TLR2 signalingplays an important role in the innate inflammatory responseThe secretion of TLR2 by S aureus-infected can induce theactivation of the NF-120581B and MAPK pathways and subse-quently result in the release of proinflammatory cytokinesCompared to the CG TLR2 mRNA levels were significantlyincreased in the S aureus groupThe results showed that IFN-120591 had a suppressive effect on the expression of TLR2 mRNAwhich was originally upregulated by S aureus in the uterinetissues (Figure 5(a))
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Figure 3 Effects of IFN-120591 onMPO activity in uterus tissues (HE times200) Immunohistochemical illustratesMPO activity in sections of uterinetissues (a) Control group (CG) the mice were without any treatment (b) S aureus-infected group (S aureus) the mice were infected withS aureus (c) Dexamethasone group (DEX) the mice of S aureus endometritis were treatment with DEX (d)ndash(f) IFN-120591 groups the miceof S aureus endometritis were treatment with different concentrations of IFN-120591 (2 4 8mgkg) (g) Effects of different doses of IFN-120591 (2 48mgkg) and dexamethasone (5mgkg) on myeloperoxidase activity in mice with S aureus-infected uterine tissues Data are expressed asthe means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
To further confirm that IFN-120591 inhibited the inflammatoryresponse via TLR2 the effect of IFN-120591 on the productionof IL-8 in S aureus-stimulated HEK293-mTLR2 cells wasassessedThe results showed that IFN-120591 inhibited the expres-sion of TLR2 and IL-8 (Figure 5(b)) in S aureus-stimulatedHEK293-mTLR2 cells With increasing doses of IFN-120591 theeffect became increasingly significant
36 IFN-120591 Affected the Activation of the NF-120581B PathwayNF-120581B is an important signaling molecule in the devel-opment of inflammatory diseases Once activated NF-120581Binduces the production of proinflammatory cytokines In thepresent study we examined NF-120581B activation by westernblot analysis As shown in Figure 6 the phosphorylation ofthe I120581B120572 and p65 proteins was activated in mice inoculatedwith S aureus but not in CG The phosphorylation levelin mice inoculated with S aureus was significantly higher
than that in the IFN-120591 treatment groups These observationsindicate that IFN-120591 can suppress the activation of NF-120581B through the inhibition of the phosphorylation of I120581B120572and p65 Compared to the mice inoculated with S aureusthe level of phosphorylation was significantly reduced withincreasing IFN-120591 concentrations in the treatment groupsTheresults showed that IFN-120591 can inhibit NF-120581B I120581B120572 and p65phosphorylation in a dose-dependent manner
37 IFN-120591 Inhibits the Activation of the MAPK PathwayMAPK is an important signaling molecule in the develop-ment of inflammatory diseases The activation of p38 JNKand ERK can induce MyD88-dependent signaling pathwayswhich in turn induce MAPK activation We found that Saureus-infected uterine tissues showed increased phospho-rylation of three MAPKs In the mice inoculated with Saureus the phosphorylation levels of p38 ERK and JNK
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Figure 4 Cytokine concentrations (a) TNF-120572 IL-1120573 IL-6 and IL-10 mRNA levels in the uterine tissue (b) TNF-120572 IL-1120573 IL-6 and IL-10protein levels in the uterine tissueThe data represent the contents of 1mL supernatant of uterine homogenate and are presented as the meansplusmn SD (119899 = 10) The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that were not given anydrug treatment 2 4 and 8 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasone treatmentgroup lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
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Figure 5 Effects of IFN-120591 on TLR2 (a) The TLR2 mRNA levels in the uterine tissues (b) The TLR2 and IL-8 protein levels in HEK293-mTLR2 cells qPCR was performed to assess the mRNA levels of TLR2 Western blotting was performed to detect the protein levels of TLR2120573-Actin was used as a control The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that werenot given any drug treatment 8 4 and 2 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasonetreatment group lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
were significantly increased compared with CG (Figure 7)Additionally the phosphorylation levels of the three MAPKsin themice inoculatedwith S aureuswere significantly higherthan in the mice treated with IFN-120591 These data indicate thatIFN-120591 can suppress the phosphorylation of p38 JNK andERK Compared to the mice inoculated with S aureus in thetreatment groups the level of phosphorylation was reducedsignificantly with increasing concentrations of IFN-120591 Theseresults showed that IFN-120591 can inhibit MAPK p38 JNK andERK phosphorylation in a dose-dependent manner
4 Discussion
IFN-120591 a novel Type I IFN is secreted by the trophoblastectoderm of ruminants in the early stages of pregnancy[22] IFN-120591 regulates the expression of numerous genes inthe endometrium and has been shown to play an impor-tant biological role in the implantation stage of pregnancy[24] Endometritis is a local inflammatory condition whichdelays uterine degeneration and has a significant economicimpact on dairy production [25 26] However infection withmicrobes such as S aureus can cause the destruction of theuterine horn structure subsequently resulting in the loss inthe ability to conceive This has been found to be due toinflammation and necrosis as determined by studies utilizingmorphological and image measurement tools [27ndash29] In thepresent study based on histopathological observations wefound that IFN-120591 has anti-inflammatory functions as well
as a significant protective effect on uterine tissues followinginfection with S aureus
A key component of the host immune response toinfection is the upregulation of cytokine production [30] Tofurther study the effect of IFN-120591 on S aureus endometritisthe levels of TNF-120572 IL-1120573 IL-6 and IL-10 production wereevaluatedThe results showed that the levels of TNF-120572 IL-1120573and IL-6 which are induced by S aureus were significantlyinhibited by IFN-120591 treatment in a dose-dependent mannerDuring the inflammatory response TNF-120572 is induced byregulated genes acting as effective inducers and subsequentlyregulates the expression of cytokines chemokines and celladhesion molecules [31 32] In vivo TNF-120572 can activatethe intracellular I120581B120572 and JNK signaling pathways throughTLRs thus contributing to the production of proinflamma-tory cytokines [33] The proinflammatory cytokine IL-1120573 isa key regulator of acute inflammatory processes in the cen-tral nervous system [34 35] Pathogen-associated molecularpatterns of S aureus can activate inflammatory pathwaysand when bound to innate monocytesmacrophages theyresult in a significant increase in the secretion of TNF-120572 andIL-6 [36 37] IL-6 is a pleiotropic cytokine that regulatesmultiple biological processes including the developmentof inflammation immune responses and the acute phasereaction [38] IL-10 has been shown to be beneficial inimproving systemic immunity by having anti-inflammatoryeffects and promoting the repair of immune dysfunction afterthe invasion of different pathogens [39] Our experimental
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tio
(b)
Figure 6 Effect of IFN-120591 on NF-120581B activation (a) The NF-120581B and I-120581B protein levels in uterine tissues (b) The ratio of phosphorylationproteins levels as well as 120573-actin levels Western blot was performed to detect the levels of total and phosphorylated NF-120581B and I-120581B proteinsin the control group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups(2 4 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
results showed that the expression of IL-10 mRNA andprotein was increased in mice inoculated with S aureusand the expression of IL-10 was further increased with IFN-120591 treatment With increasing concentrations of IFN-120591 theeffect on IL-10 was more significant IFN-120591 can promotethe secretion of inflammatory cytokines including IL-10thereby inhibiting the subsequent secretion of TNF-120572 IL-1120573and IL-6 [40] TLRs are transmembrane pattern recognitionreceptors which are part of the innate immune systemand are the key element in identifying viral and bacterialcomponents [37 41] TLR2 senses the inflammatory reactionscaused by S aureusmost rapidly and sensitively and presentspathogen-derived antigens to various types of cells [42]Our results showed TLR2 increased in mice infected withS aureus and its secretion was significantly reduced afterIFN-120591 treatment TLR2 plays a key role in the responseto both innate and adaptive immunity as it can identifyexogenous pathogen-associated molecular patterns (PAMPs)
and induce proinflammatory signaling pathways [43] In thisstudy IFN-120591 inhibited the secretion of TLR2 in a dose-dependent manner
NF-120581B and MAPKs are two important inflammation-associated pathways and they canmodulate the developmentof inflammation by mediating downstream signaling net-works [44] To further investigate the mechanism by whichIFN-120591 affects inflammation we analyzed the NF-120581B andMAPK pathways The NF-120581B signaling pathway is involvedin the regulation of genes that mediate inflammation as wellas various cytokines and is associatedwith numerous chronicinflammatory diseases [45] Normally theNF-120581Bp65 subunitis bound to I120581B in the cytosol When stimulated by variouspathogens NF-120581B p65 is released due to the phosphorylationof I120581B leading to its activation and nuclear translocationwhich then triggers inflammation [46] Western blottinganalysis showed the phosphorylation levels of I120581B120572 and p65in mice infected with S aureus were significantly higher than
BioMed Research International 9
CG S aureusDEX 248
p38
p-p38
120573-Actin
ERK
p-ERK
JNK
p-JNK
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
p-JN
K120573
-act
in ra
tio
0
02
04
06
08
1
p-p3
8120573
-act
in ra
tio
0
02
04
06
08
1
12
0
02
04
06
08
1
12
14
16
18
p-ER
K120573
-act
in ra
tio
(b)
Figure 7 Effect of IFN-120591 on MAPK activation (a) The protein levels of MAPKs in uterine tissues (b) The ratio of phosphorylation proteinsas well as 120573-actin levels Western blotting was performed to measure the levels of total and phosphorylated p38 ERK and JNK protein in theControl group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups (24 and 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
10 BioMed Research International
that in the IFN-120591 groups These results indicated that IFN-120591 can inhibit the phosphorylation of I120581B120572 and p65 therebyblocking NF-120581B activation In addition to NF-120581B MAPKsare another key regulator of signal transduction pathwaysthat can modulate the levels of inflammatory mediators [47]Specifically p38 JNK and ERK are the major effectors of theMAPK pathwayThe phosphorylation of these three proteinscan cause the translocation of Activator Protein-1 (AP-1) tothe nucleus thereby promoting the inflammatory response[48 49] In the current study we found an increase in thephosphorylation levels of p38 JNK and ERK in the miceinoculated with S aureus However in the groups treatedwith IFN-120591 the phosphorylation of these proteins was signif-icantly inhibited Recent studies have indicated that the inhi-bition of phosphorylation of MAPKs with specific inhibitorscould significantly attenuate inflammation by decreasingthe number of neutrophils Similar effects were observedfollowing the inhibition of proinflammatory cytokines andchemokines [50] In our study we demonstrated that IFN-120591 significantly inhibited the phosphorylation of proteinsinvolved in MAPK pathway activation Taken together ourresults showed that blocking the NF-120581B andMAPK signalingpathways and inhibiting the secretion of proinflammatorycytokines are the main mechanisms by which IFN-120591 abatesendometritis
5 Conclusion
Our study showed that S aureus increases the expression ofTNF-120572 IL-1120573 and IL-6 thereby activating TLR2 signalingpathways including NF-120581B and MAPK The experimentspresented herein show that IFN-120591 reduces the secretion ofproinflammatory cytokines such as TNF-120572 IL-1120573 and IL-6 while promoting the production of the anti-inflammatorycytokine IL-10 This reduction inhibits inflammation andenhances the repair of uterine tissues In addition IFN-120591 inhibited the expression of TLR2 thereby blocking theactivation of the NF-120581B and MAPK signaling pathwaysand abating endometritis Additional studies are requiredto validate the efficacy of IFN-120591 as a treatment for uterineinfection due to S aureus or other infectious pathogens
Competing Interests
The authors have no conflict of interest to declare
Acknowledgments
This work was supported by a grant from the Funda-mental Research Funds for the Central Universities (no2662014BQ024) the National Natural Science Foundationof China (nos 31272631 31472254 and 31502130) andUndergraduate Special Science and Technology Innovationof Huazhong Agricultural University (no 2015BC009)
References
[1] B Polat M Cengiz O Cannazik et al ldquoEndometrial echotex-ture variables in postpartum cows with subclinical endometri-tisrdquo Animal Reproduction Science vol 155 pp 50ndash55 2015
[2] J Roberson D Moll and G Saunders ldquoChronic Staphylococcusaureus endometritis in a virgin giltrdquo Veterinary Record vol 161no 24 pp 821ndash822 2007
[3] M Guo G Wang T Lv et al ldquoEndometrial inflammation andabnormal expression of extracellular matrix proteins inducedbyMycoplasma bovis in dairy cowsrdquoTheriogenology vol 81 no5 pp 669ndash674 2014
[4] I M Sheldon J Cronin L Goetze G Donofrio and H-J Schuberth ldquoDefining postpartum uterine disease and themechanisms of infection and immunity in the female reproduc-tive tract in cattlerdquo Biology of Reproduction vol 81 no 6 pp1025ndash1032 2009
[5] P W Farin L Ball J D Olson et al ldquoEffect of Actinomyces pyo-genes and gram-negative anaerobic bacteria on the developmentof bovine pyometrardquoTheriogenology vol 31 no 5 pp 979ndash9891989
[6] M Guo Y Cao TWang et al ldquoBaicalin inhibits Staphylococcusaureus-induced apoptosis by regulating TLR2 andTLR2-relatedapoptotic factors in the mouse mammary glandsrdquo EuropeanJournal of Pharmacology vol 723 no 1 pp 481ndash488 2014
[7] A K Syed T J Reed K L Clark B R Boles and J MKahlenberg ldquoStaphlyococcus aureus phenol-soluble modulinsstimulate the release of proinflammatory cytokines from ker-atinocytes and are required for induction of skin inflammationrdquoInfection and Immunity vol 83 no 9 pp 3428ndash3437 2015
[8] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[9] T W Chon and S Bixler ldquoInterferon-tau current applicationsand potential in antiviral therapyrdquo Journal of Interferon ampCytokine Research vol 30 no 7 pp 477ndash485 2010
[10] C H Pontzer T L Ott F W Bazer and H M JohnsonldquoStructurefunction studies with interferon tau evidence formultiple active sitesrdquo Journal of Interferon Research vol 14 no3 pp 133ndash141 1994
[11] R M Roberts ldquoInterferon-tau a Type 1 interferon involved inmaternal recognition of pregnancyrdquo Cytokine amp Growth FactorReviews vol 18 no 5-6 pp 403ndash408 2007
[12] A P Alexenko AD Ealy andRM Roberts ldquoThe cross-speciesantiviral activities of different IFN-tau subtypes on bovinemurine and human cells contradictory evidence for therapeu-tic potentialrdquo Journal of Interferon amp Cytokine Research vol 19no 12 pp 1335ndash1341 1999
[13] J A Neira D Tainturier R M LrsquoHaridon and J Martal ldquoCom-parative IFN-tau secretion after hatching by bovine blastocystsderived ex vivo and completely produced in vitrordquoReproductionin Domestic Animals vol 42 no 1 pp 68ndash75 2007
[14] A D Ealy J A Green A P Alexenko D H Keisler and R MRoberts ldquoDifferent ovine interferon-tau genes are not expressedidentically and their protein products display different activi-tiesrdquo Biology of Reproduction vol 58 no 2 pp 566ndash573 1998
[15] K A Naivar S K Ward K J Austin D W Moore and T RHansen ldquoSecretion of bovine uterine proteins in response toType I interferonsrdquo Biology of Reproduction vol 52 no 4 pp848ndash854 1995
[16] M G Teixeira K J Austin D J Perry et al ldquoBovine granulo-cyte chemotactic protein-2 is secreted by the endometrium inresponse to interferon-tau (IFN-tau)rdquo Endocrine vol 6 no 1pp 31ndash37 1997
BioMed Research International 11
[17] G Guarda M Braun F Staehli et al ldquoType I interferon inhibitsinterleukin-1 production and inflammasome activationrdquo Immu-nity vol 34 no 2 pp 213ndash223 2011
[18] T J Foster J A Geoghegan V K Ganesh and M HookldquoAdhesion invasion and evasion the many functions of thesurface proteins of Staphylococcus aureusrdquo Nature ReviewsMicrobiology vol 12 no 1 pp 49ndash62 2014
[19] B A Diep S R Gill R F Chang et al ldquoComplete genomesequence of USA300 an epidemic clone of community-acquiredmeticillin-resistant Staphylococcus aureusrdquoTheLancetvol 367 no 9512 pp 731ndash739 2006
[20] D Wang N Xu Z Zhang et al ldquoSophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstreampathways on LPS-induced mastitis in the mammary gland ofmicerdquo International Immunopharmacology vol 35 pp 111ndash1182016
[21] J-L Zhao Y-X Ding H-X Zhao et al ldquoPresence of super-antigen genes and antimicrobial resistance in Staphylococcusisolates obtained from the uteri of dairy cows with clinicalendometritisrdquo Veterinary Record vol 175 no 14 article 3522014
[22] D K Tennakoon R Smith M D Stewart T E Spencer MNayak and C J Welsh ldquoOvine IFN-tau modulates the expres-sion of MHC antigens on murine cerebrovascular endothelialcells and inhibits replication of Theilerrsquos virusrdquo Journal ofInterferonampCytokine Research vol 21 no 10 pp 785ndash792 2001
[23] J M Soos P S Subramaniam A C Hobeika J Schiffen-bauer and H M Johnson ldquoThe IFN pregnancy recognitionhormone IFN-tau blocks both development and superantigenreactivation of experimental allergic encephalomyelitis withoutassociated toxicityrdquo The Journal of Immunology vol 155 no 5pp 2747ndash2753 1995
[24] G Song T E Spencer and FW Bazer ldquoCathepsins in the ovineuterus regulation by pregnancy progesterone and interferontaurdquo Endocrinology vol 146 no 11 pp 4825ndash4833 2005
[25] R Grio C Giobbe A Cellula et al ldquoInflammation of theuterine corpus endometritisrdquoMinervaGinecologica vol 42 no4 pp 99ndash102 1990
[26] I M Sheldon and H Dobson ldquoPostpartum uterine health incattlerdquo Animal Reproduction Science vol 82-83 pp 295ndash3062004
[27] S A Kurganov ldquoUterine eosinophils and infertility in theratrdquo Rossiiskii Fiziologicheskii Zhurnal Imeni IM Sechen-ovaRossiiskaia Akademiia Nauk vol 96 no 2 pp 138ndash1462010
[28] K Shirasuna F Usui T Karasawa et al ldquoNanosilica-inducedplacental inflammation and pregnancy complications differentroles of the inflammasome components NLRP3 and ASCrdquoNanotoxicology vol 9 no 5 pp 554ndash567 2015
[29] Y Liu C QiuW Li WMu C Li andM Guo ldquoSelenium playsa protective role in Staphylococcus aureus-induced endometritisin the uterine tissue of ratsrdquo Biological Trace Element Researchvol 173 no 2 pp 345ndash353 2016
[30] A A M Lima L G Spınola G Baccan et al ldquoEvaluation ofcorticosterone and IL-1120573 IL-6 IL-10 and TNF-120572 expressionafter 670-nm laser photobiomodulation in ratsrdquo Lasers inMedical Science vol 29 no 2 pp 709ndash715 2014
[31] C-J Liang S-H Wang Y-H Chen et al ldquoViscolin reducesVCAM-1 expression in TNF-120572-treated endothelial cells viathe JNKNF-120581B and ROS pathwayrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1337ndash1346 2011
[32] H-K Tsou H-T Chen C-H Chang W-Y Yang and C-HTang ldquoApoptosis signal-regulating kinase 1 is mediated in TNF-120572-induced CCL2 expression in human synovial fibroblastsrdquoJournal of Cellular Biochemistry vol 113 no 11 pp 3509ndash35192012
[33] H Zhang J Kovacs-Nolan T Kodera Y Eto and Y Mine ldquo120574-Glutamyl cysteine and 120574-glutamyl valine inhibit TNF-120572 signal-ing in intestinal epithelial cells and reduce inflammation in amouse model of colitis via allosteric activation of the calcium-sensing receptorrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1852 no 5 pp 792ndash804 2015
[34] M L Diamond J A Boles A C Ritter et al ldquoIn response tocomments on IL-1120573 associations with posttraumatic epilepsydevelopment a genetics and biomarker cohort studyrdquo Epilepsiavol 55 no 8 pp 1313ndash1314 2014
[35] A Vezzani J French T Bartfai and T Z Baram ldquoThe role ofinflammation in epilepsyrdquo Nature Reviews Neurology vol 7 no1 pp 31ndash40 2011
[36] B Fournier and D J Philpott ldquoRecognition of Staphylococcusaureus by the innate immune systemrdquo Clinical MicrobiologyReviews vol 18 no 3 pp 521ndash540 2005
[37] J E Wang P F Joslashrgensen M Almlof et al ldquoPeptidoglycanand lipoteichoic acid from Staphylococcus aureus induce tumornecrosis factor alpha interleukin 6 (IL-6) and IL-10 productionin both T cells and monocytes in a human whole blood modelrdquoInfection and Immunity vol 68 no 7 pp 3965ndash3970 2000
[38] D Zhu D-Y Yang Y-Y Guo et al ldquoIntracameral interleukin1120573 6 8 10 12p tumor necrosis factor 120572 and vascular endothelialgrowth factor and axial length in patients with cataractrdquo PLoSONE vol 10 no 2 Article ID e0117777 2015
[39] P Lichte R Pfeifer P Kobbe et al ldquoInhalative IL-10 treatmentafter bilateral femoral fractures affect pulmonary inflammationin micerdquo Annals of Anatomy vol 200 pp 73ndash78 2015
[40] K Hara K Shirasuna F Usui et al ldquoInterferon-tau attenuatesuptake of nanoparticles and secretion of interleukin-1120573 inmacrophagesrdquoPLoSONE vol 9 no 12 Article ID e113974 2014
[41] A Mansson Kvarnhammar L Tengroth M Adner and L-OCardell ldquoInnate immune receptors in human airway smoothmuscle cells activation by TLR12 TLR3 TLR4 TLR7 andNOD1 agonistsrdquo PLoSONE vol 8 no 7 Article ID e68701 2013
[42] B Fournier ldquoThe function of TLR2 during staphylococcaldiseasesrdquo Frontiers in Cellular and Infection Microbiology vol2 article 167 2013
[43] L-H Ding D Liu M Xu et al ldquoTLR2ndashMyD88ndashNF-120581Bpathway is involved in tubulointerstitial inflammation causedby proteinuriardquoThe International Journal of Biochemistry amp CellBiology vol 69 pp 114ndash120 2015
[44] Z Zhang X Gao Y Cao et al ldquoSelenium deficiency facilitatesinflammation through the regulation of TLR4 and TLR4-related signaling pathways in the mice uterusrdquo Inflammationvol 38 no 3 pp 1347ndash1356 2015
[45] C Xu G Shen C Chen C Gelinas and A-N T KongldquoSuppression of NF-120581B and NF-120581B-regulated gene expressionby sulforaphane and PEITC through I120581B120572 IKK pathway inhuman prostate cancer PC-3 cellsrdquo Oncogene vol 24 no 28pp 4486ndash4495 2005
[46] W Zhang R Zhang T Wang et al ldquoSelenium inhibits LPS-induced pro-inflammatory gene expression by modulatingMAPK and NF-120581B signaling pathways in mouse mammaryepithelial cells in primary culturerdquo Inflammation vol 37 no 2pp 478ndash485 2014
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
2 BioMed Research International
diseases [18] S aureus is a well-known gram-positive bacte-ria which can lead to inflammation from both community-acquired and nosocomial infections [19 20] S aureus is alsothe main pathogenic microbe that causes endometritis andenhances the expression of proinflammatory cytokines [21]It has been shown IFN-120591 has immunomodulatory properties[22] A prominent feature of IFN-120591 is its low cytotoxicitycompared with other Type I IFN which has been observed incell culture as well as in vivo [23] In various clinical studiesIFN-120591 has been shown to have anti-inflammatory propertiesand low cytotoxicity As such this study sought to verify theanti-inflammatory effect of IFN-120591 inmice Additionallymorestudies on the functional role of IFN-120591 in mediating anti-inflammatory effects are needed
2 Materials and Methods
21 S aureus Growth S aureus strain ATCC 25923 wascultured at 37∘C in Mueller-Hinton II cation adjusted broth(MH BD Biosciences Sparks MD USA) 180 rpm withIFN-120591 (Recombinant Ovine Interferon-tau IFNT-29O Cre-ative Bioarray New York USA) treatment (0 50 100 and200 ngmL) The absorbance was measured once every 1 h at600 nm until the bacteria reached the stationary phase
22 Animals and Experiment Groups Female BALBc mice(6ndash8 weeks 18ndash20 g) were purchased from the Center ofExperimental Animals of Wuhan Institute All experimentalprocedures were conducted with the approval of the Insti-tutional Animal Care and were approved by the HuazhongAgricultural University Animal Care and Use CommitteeThe mice were housed in microisolator cages and receivedfood and water ad libitum Mice were given an adaptationperiod of 4ndash6 days prior to experimentation
The sixty mice were randomly divided into six groups (a)the control group (CG) in which the mice did not receiveany treatment (b) the S aureus-infected group (S aureus) inwhich the mice were infected with S aureus via an injectionof 100 120583L S aureus into each uterine horn (a total volumeof 200120583L) through a microsyringe followed by a stimulus of24 h (c) IFN-120591 groups inwhich themicewith S aureus endo-metritis were treated with different concentrations of IFN-120591(2 4 8mgkg) and (d) the dexamethasone group (DEX) inwhich the mice with S aureus endometritis were treatmentwith DEX (5mgkg 5mgmL) Finally all the mice wereeuthanized with sodium pentobarbital and sacrificed Theuterine tissues were quickly collected and were kept frozenat minus80∘C until they were used for subsequent experiments
23 Histological Analysis The harvested uterine tissues werefixed in 10 formaldehyde solution and embedded in paraffinwax Sections (4 120583m) were deparaffinized with xylene andrehydrated through graded alcohols for staining Then sec-tions were stained with hematoxylin and eosin (HampE) andlast examined with a microscope (Olympus Japan)
24 Immunohistochemical Analysis Six serial sections ofeach uterus area were collected and mounted on polylysine
coated slides A 4120583m section was prepared from the paraffin-embedded block and dehydrated and decreasing concentra-tions of ethanol and then washed for 5min in running waterFollowing washing sections were treated with 90 formicacid for 5mins and blocked for endogenous peroxidase activ-ity in 5H
2O2 A Tris-HCl (005M pH 766) was introduced
at room temperature for 20min and with a 1 200 dilution(vv) of the MPO antibody (Abcam) was added to the tissuesand incubated at 4∘C overnight As for the negative controlthe primary antibody was replaced with PBS The secondaryantibodies (Tianjin Sungene Biotech Co Ltd China) wereadded as appropriate and 331015840-diaminobenzidine stainingwas visualized using the hematoxylin stain Two pathologistsscored the slides respectively For MPO staining assessmentmounting with glycerin and observing used a microscopewith a camera system (Olympus DP-71 MN USA)
25 HEK293 Cell Culture and Transfection HEK293 cellswere purchased from American Type Culture Collection(ATCC Manassas VA USA) and were cultured in DMEMcontaining 10 FBS with 5 CO
2at 37∘CThe culture media
were changed once every 24 hThe cells were always added 1 hprior to S aureus treatment and were subsequently incubatedin the presence or absence of various concentrations of IFN-120591 HEK293 cells were transfected with pEGFP-N1-mTLR2plasmids using FuGENE HD transfection reagent accordingto the manufacturerrsquos instructions (Roche Applied ScienceIndianapolis IN USA)
26 ELISA Analysis The uterus samples were weighed andhomogenized with phosphate-buffered saline (wv 1 9) onice and then centrifuged at 12000 rpm for 15min at 4∘C Thesupernatant was collected The cytokine TNF-120572 IL-1120573 IL-6 and IL-10 assays were evaluated with the correspondingELISA kits (Biolegend USA) according to the instructionsof the manufacturer Results are expressed as means plusmn SD ofthree independent experiments
27 Quantitative Real-Time Polymerase Chain Reaction TotalRNA from endometrial samples were isolated using TRI-zol reagent (Invitrogen Corporation) according to man-ufacturerrsquos instructions The concentration and purity ofthe total RNA were determined spectrophotometrically at260280 nm The RNA was reverse transcribed into cDNAusing a Revert Aid First Strand cDNA Synthesis Kit(Thermo)Theprimers (TNF-120572 IL-1120573 IL-6 IL-10 andTLR2)used for qPCR are listed in Table 1 (Designed by softwarePrimer 50) The qPCR were executed by a 7500 Fast Real-Time PCR System (Applied Biosystems) with the SYBR greenPlus reagent kit (Roche Applied Science Mannheim Ger-many) in a 20120583L system Each sample set three repetitionsResults were expressed as 2minusΔΔCt comparative method ΔΔCt= (target gene Ct of experimental group minus reference gene Ctof experimental group) minus (target gene Ct of control group minusreference gene Ct of control group) GAPDH was used as thereference gene
28 Western Blot Analysis The protein of collected tissuesamples were extracted using PMSF (10 120583L) and RIPA (1mL)
BioMed Research International 3
Table 1 Sequences of primers used for qPCR
Gene GenBankaccession number
Sequence (51015840-31015840)sense and antisense Product size (bp)
TNF-120572 NM 0136933 Sense 51015840-CTTCTCATTCCTGCTTGTG-31015840 198Antisense 51015840-ACTTGGTGGTTTGCTACG-31015840
IL-1120573 NM 0083613464 Sense 51015840-AGGCTCCGAGATGAACAA-31015840 464Antisense 51015840-AAGGCATTAGAAACAGTCC-31015840
IL-6 NM 0311681 Sense 51015840-TTCTTGGGACTGATGCTG-31015840 380Antisense 51015840-CTGGCTTTGTCTTTCTTGTT-31015840
IL-10 NM 0105482 Sense 51015840-AACATACTGCTAACCGACTC-31015840 286Antisense 51015840-TGGCCTTGTAGACACCTT-31015840
TLR2 NM 0119053 Sense 51015840-TTTGCTCCTGCGAACTCC-31015840 267Antisense 51015840-GCCACGCCCACATCATTC-31015840
GAPDH NM 0012897261 Sense 51015840-TGTTTCCTCGTCCCGTAG-31015840 108Antisense 51015840-CAATCTCCACTTTGCCACT-31015840
function 30minThen protein concentrationwas determinedusing the BCA protein assay kit (Thermo USA) The crudeprotein per sample was subjected to 10 SDS-PAGE usingTris-HCl Precast Gels and subsequently transferred ontoPVDF membranes The membrane was blocked with Trisbuffer saline containing 005 Tween-20 (TBS-T) supple-mented with 5 skim milk at room temperature for 15 h ona rotary shaker following by TBS-T washed The membraneswere washed with a specific primary antibody (Cell SignalingTechnology Inc Danvers MA) diluted in TBS-T containingskim milk (VV = 1 1000) at 4∘C overnight Subsequentlythe membranes were washed with TBS-T The membranewas washed and treated with secondary antibody (TianjinSungene Biotech Co Ltd Tianjin China) diluted in TBS-T(VV = 1 5000) at room temperature for 2 h In the last stepmembranes developed with the ECL Plus Western BlottingDetection System (Amersham Life Science UK) 120573-Actin(Tianjin China) was used as a control
29 Statistical Analysis Statistical analyses of the data wereperformed using the SPSS software (ver 17 for WindowsSPSS Inc Chicago IL USA) All values are expressed asthe means plusmn SD The data were assessed using the Tukey-Kramer method for multiple comparisons Significance wasdetermined by a 1-way ANOVA using a significance level of119901 lt 005
3 Results
31 Effect of IFN-120591 on S aureus Growth The effect of IFN-120591 (50ndash200 ngmL) on S aureus growth was monitored byturbidimeter at 600 nm for 12 hThe results showed that IFN-120591 had no effect on S aureus growth Specifically when com-pared to the untreated controls S aureus grown in the pres-ence of IFN-120591 (50ndash200 ngmL) were not affected (Figure 1)
32 Histopathological Changes Tissues were collected 24hours after the injection of S aureus and were stained withhematoxylin and eosin (HampE) There were no pathologicalchanges in the uterine tissue of the CG (Figure 2(a)) The
Effec
t of I
FN-120591
onS
aur
eusg
row
th (C
FUm
L)
107
109
1011
1013
1015
1017
1019
1021
1023
109876 11 124321 50Time (h)
IFN-120591 (50ngmL)IFN-120591 (0ngmL) IFN-120591 (100ngmL)
IFN-120591 (200ngmL)
Figure 1 Effect of IFN-120591 on S aureus growth S aureus (18times 107 CFUmL) was cultured in MH broth supplemented withdifferent concentrations of IFN-120591 (50ndash200 ngmL) in each of theexperiments S aureus growth was monitored turbidimetrically(600 nm) after 12 hThe data represents the mean of triplicates plusmn SDfrom three independent experiments
uterine morphology of the mice inoculated with S aureusdisplayed inflammatory cell infiltration endometrial conges-tion severe destruction of the myometrium cells and thedisappearance of the uterus acinar structure (Figure 2(b))However IFN-120591 significantly reduced the inflammation stim-ulated by S aureus and the uterine tissue structure of themice treated with IFN-120591 was almost complete with onlyminor endometrial pathology and an insignificant amount ofinflammatory cell infiltration (Figures 2(c) 2(d) and 2(e))
4 BioMed Research International
(a) (b) (c)
(d) (e) (f)
Figure 2 Histopathological changes (HE times200) HampE staining of the harvested uterine tissues (a) Control group (CG) in which the micewere not treated (b) S aureus-infected group (S aureus) in which the mice were infected with S aureus (c) Dexamethasone group (DEX)in which the mice inoculated with S aureus and developed endometritis were treated with DEX (d)ndash(f) IFN-120591 groups in which the miceinoculated with S aureus and developed endometritis were treated with different concentrations of IFN-120591 (2 4 8mgkg)
Representative pictures are shown to demonstrate the effectsdose-dependent effects of IFN-120591The degree of inflammatoryinjury was also very slight in the DEX group as shown inFigure 2(f)
33 Effects of IFN-120591 onMPO Activity in Uterus Tissues MPOactivity has been used as a quantitative index of inflammationand can illustrate the level of neutrophil infiltrationThe effectof IFN-120591 on MPO activity was examined in mice with Saureus-induced endometritis Immunohistochemical stain-ing showed that MPO specific immunolabelling was scarcelyfound in the neutrophil andmononuclear cells of lamina pro-pria of mucosa in normal uterus in CG (Figure 3(a)) whereasthis expression was found elevated in cells of surface epithe-lium and in cells of the inflammatory infiltrate in S aureus-infected group (Figure 3(b)) The MPO activity was veryslight in DEX group as shown in Figure 3(c) In contrastmice treated with different doses of IFN-120591 uterus showed alower level of MPO activity the immunoreactivity of whichshowed the suppressive effects of IFN-120591 increased in a dose-dependent manner in uterus samples of mice (Figures 3(d)3(e) and 3(f)) The activity of MPO from different micegroups was also measured by MPO assay kits The resultsillustrates that the activity of MPO was low in CG and dra-matically increased in S aureus group (Figure 3(g)) while theS aureus-infected mice treated with different concentrationsof IFN-120591 (2 4 8mgkg) significantly inhibited the activity ofMPO (compare with S aureus group) The results indicatedthat reduction of neutrophil infiltration in uterine tissuescould be one mechanism underlying the protective effect ofIFN-120591
34 Effects of IFN-120591 on Inflammatory Cytokines To deter-mine the effect of IFN-120591 on the S aureus endometritis thelevels of TNF-120572 IL-1120573 IL-6 and IL-10 were measured byqPCR and ELISA The results are shown in Figure 4 Theexpressions of TNF-120572 IL-1120573 and IL-6 were increased signif-icantly in the mice inoculated with S aureus compared tothe CG The increases in TNF-120572 IL-1120573 and IL-6 expressionwhich were induced by S aureus were significantly inhibitedby IFN-120591 Compared with themice inoculated with S aureusthe mRNA and protein expression of TNF-120572 IL-1120573 and IL-6 was significantly decreased following IFN-120591 treatment Theeffects of IFN-120591 increased in a dose-dependent mannerWithincreasing concentrations of IFN-120591 more significant effectswere observed However the effect on IL-10 was differentThe mRNA and protein expression of IL-10 was increasedin the mice inoculated with S aureus compared to the CGCompared with the mice inoculated with S aureus theexpression of IL-10 further increased upon IFN-120591 treatmentWith increasing concentrations of IFN-120591 more significanteffects on IL-10 were observed
35 Effects of IFN-120591 on TLR2 Expression TLR2 signalingplays an important role in the innate inflammatory responseThe secretion of TLR2 by S aureus-infected can induce theactivation of the NF-120581B and MAPK pathways and subse-quently result in the release of proinflammatory cytokinesCompared to the CG TLR2 mRNA levels were significantlyincreased in the S aureus groupThe results showed that IFN-120591 had a suppressive effect on the expression of TLR2 mRNAwhich was originally upregulated by S aureus in the uterinetissues (Figure 5(a))
BioMed Research International 5
CG S aureus DEX 2 4 8
lowast
lowast
lowast
lowast lowast
(a) (b) (c)
(d) (e) (f)
(g)
02468
10121416
MPO
activ
ity (U
mg)
Figure 3 Effects of IFN-120591 onMPO activity in uterus tissues (HE times200) Immunohistochemical illustratesMPO activity in sections of uterinetissues (a) Control group (CG) the mice were without any treatment (b) S aureus-infected group (S aureus) the mice were infected withS aureus (c) Dexamethasone group (DEX) the mice of S aureus endometritis were treatment with DEX (d)ndash(f) IFN-120591 groups the miceof S aureus endometritis were treatment with different concentrations of IFN-120591 (2 4 8mgkg) (g) Effects of different doses of IFN-120591 (2 48mgkg) and dexamethasone (5mgkg) on myeloperoxidase activity in mice with S aureus-infected uterine tissues Data are expressed asthe means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
To further confirm that IFN-120591 inhibited the inflammatoryresponse via TLR2 the effect of IFN-120591 on the productionof IL-8 in S aureus-stimulated HEK293-mTLR2 cells wasassessedThe results showed that IFN-120591 inhibited the expres-sion of TLR2 and IL-8 (Figure 5(b)) in S aureus-stimulatedHEK293-mTLR2 cells With increasing doses of IFN-120591 theeffect became increasingly significant
36 IFN-120591 Affected the Activation of the NF-120581B PathwayNF-120581B is an important signaling molecule in the devel-opment of inflammatory diseases Once activated NF-120581Binduces the production of proinflammatory cytokines In thepresent study we examined NF-120581B activation by westernblot analysis As shown in Figure 6 the phosphorylation ofthe I120581B120572 and p65 proteins was activated in mice inoculatedwith S aureus but not in CG The phosphorylation levelin mice inoculated with S aureus was significantly higher
than that in the IFN-120591 treatment groups These observationsindicate that IFN-120591 can suppress the activation of NF-120581B through the inhibition of the phosphorylation of I120581B120572and p65 Compared to the mice inoculated with S aureusthe level of phosphorylation was significantly reduced withincreasing IFN-120591 concentrations in the treatment groupsTheresults showed that IFN-120591 can inhibit NF-120581B I120581B120572 and p65phosphorylation in a dose-dependent manner
37 IFN-120591 Inhibits the Activation of the MAPK PathwayMAPK is an important signaling molecule in the develop-ment of inflammatory diseases The activation of p38 JNKand ERK can induce MyD88-dependent signaling pathwayswhich in turn induce MAPK activation We found that Saureus-infected uterine tissues showed increased phospho-rylation of three MAPKs In the mice inoculated with Saureus the phosphorylation levels of p38 ERK and JNK
6 BioMed Research International
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
IL-1120573
mRN
A le
vel
02468
1012141618202224
0
2
4
6
8
10
12
14
16
TNF-
120572m
RNA
leve
l
0123456789
10
IL-6
mRN
A le
vel
0
2
4
6
8
10
12
14
16
IL-10
mRN
A le
vel
lowast
lowastlowast
lowast
lowast
lowastlowast
lowast lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
0
500
1000
1500
2000
2500
3000
3500
4000
IL-10
(pg
mL)
0
500
1000
1500
2000
2500
IL-6
(pg
mL)
0500
1000150020002500300035004000450050005500
TNF-
120572(p
gm
L)
0500
100015002000250030003500400045005000550060006500700075008000
IL-1120573
(pg
mL) lowast
lowast
lowastlowast
lowast lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowastlowast
lowast
(b)
Figure 4 Cytokine concentrations (a) TNF-120572 IL-1120573 IL-6 and IL-10 mRNA levels in the uterine tissue (b) TNF-120572 IL-1120573 IL-6 and IL-10protein levels in the uterine tissueThe data represent the contents of 1mL supernatant of uterine homogenate and are presented as the meansplusmn SD (119899 = 10) The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that were not given anydrug treatment 2 4 and 8 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasone treatmentgroup lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
BioMed Research International 7
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
0
1
2
3
4
5
6
7
8
9
TLR2
mRN
A le
vel
(a)CG S aureus248
CG S aureus248
lowast
lowast
lowast
lowast
TLR2
120573-Actin
0
500
1000
1500
2000
2500
3000
3500
4000
IL-8
(pg
mL)
(b)
Figure 5 Effects of IFN-120591 on TLR2 (a) The TLR2 mRNA levels in the uterine tissues (b) The TLR2 and IL-8 protein levels in HEK293-mTLR2 cells qPCR was performed to assess the mRNA levels of TLR2 Western blotting was performed to detect the protein levels of TLR2120573-Actin was used as a control The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that werenot given any drug treatment 8 4 and 2 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasonetreatment group lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
were significantly increased compared with CG (Figure 7)Additionally the phosphorylation levels of the three MAPKsin themice inoculatedwith S aureuswere significantly higherthan in the mice treated with IFN-120591 These data indicate thatIFN-120591 can suppress the phosphorylation of p38 JNK andERK Compared to the mice inoculated with S aureus in thetreatment groups the level of phosphorylation was reducedsignificantly with increasing concentrations of IFN-120591 Theseresults showed that IFN-120591 can inhibit MAPK p38 JNK andERK phosphorylation in a dose-dependent manner
4 Discussion
IFN-120591 a novel Type I IFN is secreted by the trophoblastectoderm of ruminants in the early stages of pregnancy[22] IFN-120591 regulates the expression of numerous genes inthe endometrium and has been shown to play an impor-tant biological role in the implantation stage of pregnancy[24] Endometritis is a local inflammatory condition whichdelays uterine degeneration and has a significant economicimpact on dairy production [25 26] However infection withmicrobes such as S aureus can cause the destruction of theuterine horn structure subsequently resulting in the loss inthe ability to conceive This has been found to be due toinflammation and necrosis as determined by studies utilizingmorphological and image measurement tools [27ndash29] In thepresent study based on histopathological observations wefound that IFN-120591 has anti-inflammatory functions as well
as a significant protective effect on uterine tissues followinginfection with S aureus
A key component of the host immune response toinfection is the upregulation of cytokine production [30] Tofurther study the effect of IFN-120591 on S aureus endometritisthe levels of TNF-120572 IL-1120573 IL-6 and IL-10 production wereevaluatedThe results showed that the levels of TNF-120572 IL-1120573and IL-6 which are induced by S aureus were significantlyinhibited by IFN-120591 treatment in a dose-dependent mannerDuring the inflammatory response TNF-120572 is induced byregulated genes acting as effective inducers and subsequentlyregulates the expression of cytokines chemokines and celladhesion molecules [31 32] In vivo TNF-120572 can activatethe intracellular I120581B120572 and JNK signaling pathways throughTLRs thus contributing to the production of proinflamma-tory cytokines [33] The proinflammatory cytokine IL-1120573 isa key regulator of acute inflammatory processes in the cen-tral nervous system [34 35] Pathogen-associated molecularpatterns of S aureus can activate inflammatory pathwaysand when bound to innate monocytesmacrophages theyresult in a significant increase in the secretion of TNF-120572 andIL-6 [36 37] IL-6 is a pleiotropic cytokine that regulatesmultiple biological processes including the developmentof inflammation immune responses and the acute phasereaction [38] IL-10 has been shown to be beneficial inimproving systemic immunity by having anti-inflammatoryeffects and promoting the repair of immune dysfunction afterthe invasion of different pathogens [39] Our experimental
8 BioMed Research International
CG S aureusDEX 248
I120581B
p-I120581B
p65
p-p65
120573-Actin
(a)CG S aureusDEX 248
CG S aureusDEX 248
lowastlowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
pNF-
120581B
120573-a
ctin
ratio
0
02
04
06
08
1
12
14
16
18
2
22
24
0
02
04
06
08
1
12
14
p-I120581
B120573
-act
in ra
tio
(b)
Figure 6 Effect of IFN-120591 on NF-120581B activation (a) The NF-120581B and I-120581B protein levels in uterine tissues (b) The ratio of phosphorylationproteins levels as well as 120573-actin levels Western blot was performed to detect the levels of total and phosphorylated NF-120581B and I-120581B proteinsin the control group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups(2 4 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
results showed that the expression of IL-10 mRNA andprotein was increased in mice inoculated with S aureusand the expression of IL-10 was further increased with IFN-120591 treatment With increasing concentrations of IFN-120591 theeffect on IL-10 was more significant IFN-120591 can promotethe secretion of inflammatory cytokines including IL-10thereby inhibiting the subsequent secretion of TNF-120572 IL-1120573and IL-6 [40] TLRs are transmembrane pattern recognitionreceptors which are part of the innate immune systemand are the key element in identifying viral and bacterialcomponents [37 41] TLR2 senses the inflammatory reactionscaused by S aureusmost rapidly and sensitively and presentspathogen-derived antigens to various types of cells [42]Our results showed TLR2 increased in mice infected withS aureus and its secretion was significantly reduced afterIFN-120591 treatment TLR2 plays a key role in the responseto both innate and adaptive immunity as it can identifyexogenous pathogen-associated molecular patterns (PAMPs)
and induce proinflammatory signaling pathways [43] In thisstudy IFN-120591 inhibited the secretion of TLR2 in a dose-dependent manner
NF-120581B and MAPKs are two important inflammation-associated pathways and they canmodulate the developmentof inflammation by mediating downstream signaling net-works [44] To further investigate the mechanism by whichIFN-120591 affects inflammation we analyzed the NF-120581B andMAPK pathways The NF-120581B signaling pathway is involvedin the regulation of genes that mediate inflammation as wellas various cytokines and is associatedwith numerous chronicinflammatory diseases [45] Normally theNF-120581Bp65 subunitis bound to I120581B in the cytosol When stimulated by variouspathogens NF-120581B p65 is released due to the phosphorylationof I120581B leading to its activation and nuclear translocationwhich then triggers inflammation [46] Western blottinganalysis showed the phosphorylation levels of I120581B120572 and p65in mice infected with S aureus were significantly higher than
BioMed Research International 9
CG S aureusDEX 248
p38
p-p38
120573-Actin
ERK
p-ERK
JNK
p-JNK
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
p-JN
K120573
-act
in ra
tio
0
02
04
06
08
1
p-p3
8120573
-act
in ra
tio
0
02
04
06
08
1
12
0
02
04
06
08
1
12
14
16
18
p-ER
K120573
-act
in ra
tio
(b)
Figure 7 Effect of IFN-120591 on MAPK activation (a) The protein levels of MAPKs in uterine tissues (b) The ratio of phosphorylation proteinsas well as 120573-actin levels Western blotting was performed to measure the levels of total and phosphorylated p38 ERK and JNK protein in theControl group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups (24 and 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
10 BioMed Research International
that in the IFN-120591 groups These results indicated that IFN-120591 can inhibit the phosphorylation of I120581B120572 and p65 therebyblocking NF-120581B activation In addition to NF-120581B MAPKsare another key regulator of signal transduction pathwaysthat can modulate the levels of inflammatory mediators [47]Specifically p38 JNK and ERK are the major effectors of theMAPK pathwayThe phosphorylation of these three proteinscan cause the translocation of Activator Protein-1 (AP-1) tothe nucleus thereby promoting the inflammatory response[48 49] In the current study we found an increase in thephosphorylation levels of p38 JNK and ERK in the miceinoculated with S aureus However in the groups treatedwith IFN-120591 the phosphorylation of these proteins was signif-icantly inhibited Recent studies have indicated that the inhi-bition of phosphorylation of MAPKs with specific inhibitorscould significantly attenuate inflammation by decreasingthe number of neutrophils Similar effects were observedfollowing the inhibition of proinflammatory cytokines andchemokines [50] In our study we demonstrated that IFN-120591 significantly inhibited the phosphorylation of proteinsinvolved in MAPK pathway activation Taken together ourresults showed that blocking the NF-120581B andMAPK signalingpathways and inhibiting the secretion of proinflammatorycytokines are the main mechanisms by which IFN-120591 abatesendometritis
5 Conclusion
Our study showed that S aureus increases the expression ofTNF-120572 IL-1120573 and IL-6 thereby activating TLR2 signalingpathways including NF-120581B and MAPK The experimentspresented herein show that IFN-120591 reduces the secretion ofproinflammatory cytokines such as TNF-120572 IL-1120573 and IL-6 while promoting the production of the anti-inflammatorycytokine IL-10 This reduction inhibits inflammation andenhances the repair of uterine tissues In addition IFN-120591 inhibited the expression of TLR2 thereby blocking theactivation of the NF-120581B and MAPK signaling pathwaysand abating endometritis Additional studies are requiredto validate the efficacy of IFN-120591 as a treatment for uterineinfection due to S aureus or other infectious pathogens
Competing Interests
The authors have no conflict of interest to declare
Acknowledgments
This work was supported by a grant from the Funda-mental Research Funds for the Central Universities (no2662014BQ024) the National Natural Science Foundationof China (nos 31272631 31472254 and 31502130) andUndergraduate Special Science and Technology Innovationof Huazhong Agricultural University (no 2015BC009)
References
[1] B Polat M Cengiz O Cannazik et al ldquoEndometrial echotex-ture variables in postpartum cows with subclinical endometri-tisrdquo Animal Reproduction Science vol 155 pp 50ndash55 2015
[2] J Roberson D Moll and G Saunders ldquoChronic Staphylococcusaureus endometritis in a virgin giltrdquo Veterinary Record vol 161no 24 pp 821ndash822 2007
[3] M Guo G Wang T Lv et al ldquoEndometrial inflammation andabnormal expression of extracellular matrix proteins inducedbyMycoplasma bovis in dairy cowsrdquoTheriogenology vol 81 no5 pp 669ndash674 2014
[4] I M Sheldon J Cronin L Goetze G Donofrio and H-J Schuberth ldquoDefining postpartum uterine disease and themechanisms of infection and immunity in the female reproduc-tive tract in cattlerdquo Biology of Reproduction vol 81 no 6 pp1025ndash1032 2009
[5] P W Farin L Ball J D Olson et al ldquoEffect of Actinomyces pyo-genes and gram-negative anaerobic bacteria on the developmentof bovine pyometrardquoTheriogenology vol 31 no 5 pp 979ndash9891989
[6] M Guo Y Cao TWang et al ldquoBaicalin inhibits Staphylococcusaureus-induced apoptosis by regulating TLR2 andTLR2-relatedapoptotic factors in the mouse mammary glandsrdquo EuropeanJournal of Pharmacology vol 723 no 1 pp 481ndash488 2014
[7] A K Syed T J Reed K L Clark B R Boles and J MKahlenberg ldquoStaphlyococcus aureus phenol-soluble modulinsstimulate the release of proinflammatory cytokines from ker-atinocytes and are required for induction of skin inflammationrdquoInfection and Immunity vol 83 no 9 pp 3428ndash3437 2015
[8] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[9] T W Chon and S Bixler ldquoInterferon-tau current applicationsand potential in antiviral therapyrdquo Journal of Interferon ampCytokine Research vol 30 no 7 pp 477ndash485 2010
[10] C H Pontzer T L Ott F W Bazer and H M JohnsonldquoStructurefunction studies with interferon tau evidence formultiple active sitesrdquo Journal of Interferon Research vol 14 no3 pp 133ndash141 1994
[11] R M Roberts ldquoInterferon-tau a Type 1 interferon involved inmaternal recognition of pregnancyrdquo Cytokine amp Growth FactorReviews vol 18 no 5-6 pp 403ndash408 2007
[12] A P Alexenko AD Ealy andRM Roberts ldquoThe cross-speciesantiviral activities of different IFN-tau subtypes on bovinemurine and human cells contradictory evidence for therapeu-tic potentialrdquo Journal of Interferon amp Cytokine Research vol 19no 12 pp 1335ndash1341 1999
[13] J A Neira D Tainturier R M LrsquoHaridon and J Martal ldquoCom-parative IFN-tau secretion after hatching by bovine blastocystsderived ex vivo and completely produced in vitrordquoReproductionin Domestic Animals vol 42 no 1 pp 68ndash75 2007
[14] A D Ealy J A Green A P Alexenko D H Keisler and R MRoberts ldquoDifferent ovine interferon-tau genes are not expressedidentically and their protein products display different activi-tiesrdquo Biology of Reproduction vol 58 no 2 pp 566ndash573 1998
[15] K A Naivar S K Ward K J Austin D W Moore and T RHansen ldquoSecretion of bovine uterine proteins in response toType I interferonsrdquo Biology of Reproduction vol 52 no 4 pp848ndash854 1995
[16] M G Teixeira K J Austin D J Perry et al ldquoBovine granulo-cyte chemotactic protein-2 is secreted by the endometrium inresponse to interferon-tau (IFN-tau)rdquo Endocrine vol 6 no 1pp 31ndash37 1997
BioMed Research International 11
[17] G Guarda M Braun F Staehli et al ldquoType I interferon inhibitsinterleukin-1 production and inflammasome activationrdquo Immu-nity vol 34 no 2 pp 213ndash223 2011
[18] T J Foster J A Geoghegan V K Ganesh and M HookldquoAdhesion invasion and evasion the many functions of thesurface proteins of Staphylococcus aureusrdquo Nature ReviewsMicrobiology vol 12 no 1 pp 49ndash62 2014
[19] B A Diep S R Gill R F Chang et al ldquoComplete genomesequence of USA300 an epidemic clone of community-acquiredmeticillin-resistant Staphylococcus aureusrdquoTheLancetvol 367 no 9512 pp 731ndash739 2006
[20] D Wang N Xu Z Zhang et al ldquoSophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstreampathways on LPS-induced mastitis in the mammary gland ofmicerdquo International Immunopharmacology vol 35 pp 111ndash1182016
[21] J-L Zhao Y-X Ding H-X Zhao et al ldquoPresence of super-antigen genes and antimicrobial resistance in Staphylococcusisolates obtained from the uteri of dairy cows with clinicalendometritisrdquo Veterinary Record vol 175 no 14 article 3522014
[22] D K Tennakoon R Smith M D Stewart T E Spencer MNayak and C J Welsh ldquoOvine IFN-tau modulates the expres-sion of MHC antigens on murine cerebrovascular endothelialcells and inhibits replication of Theilerrsquos virusrdquo Journal ofInterferonampCytokine Research vol 21 no 10 pp 785ndash792 2001
[23] J M Soos P S Subramaniam A C Hobeika J Schiffen-bauer and H M Johnson ldquoThe IFN pregnancy recognitionhormone IFN-tau blocks both development and superantigenreactivation of experimental allergic encephalomyelitis withoutassociated toxicityrdquo The Journal of Immunology vol 155 no 5pp 2747ndash2753 1995
[24] G Song T E Spencer and FW Bazer ldquoCathepsins in the ovineuterus regulation by pregnancy progesterone and interferontaurdquo Endocrinology vol 146 no 11 pp 4825ndash4833 2005
[25] R Grio C Giobbe A Cellula et al ldquoInflammation of theuterine corpus endometritisrdquoMinervaGinecologica vol 42 no4 pp 99ndash102 1990
[26] I M Sheldon and H Dobson ldquoPostpartum uterine health incattlerdquo Animal Reproduction Science vol 82-83 pp 295ndash3062004
[27] S A Kurganov ldquoUterine eosinophils and infertility in theratrdquo Rossiiskii Fiziologicheskii Zhurnal Imeni IM Sechen-ovaRossiiskaia Akademiia Nauk vol 96 no 2 pp 138ndash1462010
[28] K Shirasuna F Usui T Karasawa et al ldquoNanosilica-inducedplacental inflammation and pregnancy complications differentroles of the inflammasome components NLRP3 and ASCrdquoNanotoxicology vol 9 no 5 pp 554ndash567 2015
[29] Y Liu C QiuW Li WMu C Li andM Guo ldquoSelenium playsa protective role in Staphylococcus aureus-induced endometritisin the uterine tissue of ratsrdquo Biological Trace Element Researchvol 173 no 2 pp 345ndash353 2016
[30] A A M Lima L G Spınola G Baccan et al ldquoEvaluation ofcorticosterone and IL-1120573 IL-6 IL-10 and TNF-120572 expressionafter 670-nm laser photobiomodulation in ratsrdquo Lasers inMedical Science vol 29 no 2 pp 709ndash715 2014
[31] C-J Liang S-H Wang Y-H Chen et al ldquoViscolin reducesVCAM-1 expression in TNF-120572-treated endothelial cells viathe JNKNF-120581B and ROS pathwayrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1337ndash1346 2011
[32] H-K Tsou H-T Chen C-H Chang W-Y Yang and C-HTang ldquoApoptosis signal-regulating kinase 1 is mediated in TNF-120572-induced CCL2 expression in human synovial fibroblastsrdquoJournal of Cellular Biochemistry vol 113 no 11 pp 3509ndash35192012
[33] H Zhang J Kovacs-Nolan T Kodera Y Eto and Y Mine ldquo120574-Glutamyl cysteine and 120574-glutamyl valine inhibit TNF-120572 signal-ing in intestinal epithelial cells and reduce inflammation in amouse model of colitis via allosteric activation of the calcium-sensing receptorrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1852 no 5 pp 792ndash804 2015
[34] M L Diamond J A Boles A C Ritter et al ldquoIn response tocomments on IL-1120573 associations with posttraumatic epilepsydevelopment a genetics and biomarker cohort studyrdquo Epilepsiavol 55 no 8 pp 1313ndash1314 2014
[35] A Vezzani J French T Bartfai and T Z Baram ldquoThe role ofinflammation in epilepsyrdquo Nature Reviews Neurology vol 7 no1 pp 31ndash40 2011
[36] B Fournier and D J Philpott ldquoRecognition of Staphylococcusaureus by the innate immune systemrdquo Clinical MicrobiologyReviews vol 18 no 3 pp 521ndash540 2005
[37] J E Wang P F Joslashrgensen M Almlof et al ldquoPeptidoglycanand lipoteichoic acid from Staphylococcus aureus induce tumornecrosis factor alpha interleukin 6 (IL-6) and IL-10 productionin both T cells and monocytes in a human whole blood modelrdquoInfection and Immunity vol 68 no 7 pp 3965ndash3970 2000
[38] D Zhu D-Y Yang Y-Y Guo et al ldquoIntracameral interleukin1120573 6 8 10 12p tumor necrosis factor 120572 and vascular endothelialgrowth factor and axial length in patients with cataractrdquo PLoSONE vol 10 no 2 Article ID e0117777 2015
[39] P Lichte R Pfeifer P Kobbe et al ldquoInhalative IL-10 treatmentafter bilateral femoral fractures affect pulmonary inflammationin micerdquo Annals of Anatomy vol 200 pp 73ndash78 2015
[40] K Hara K Shirasuna F Usui et al ldquoInterferon-tau attenuatesuptake of nanoparticles and secretion of interleukin-1120573 inmacrophagesrdquoPLoSONE vol 9 no 12 Article ID e113974 2014
[41] A Mansson Kvarnhammar L Tengroth M Adner and L-OCardell ldquoInnate immune receptors in human airway smoothmuscle cells activation by TLR12 TLR3 TLR4 TLR7 andNOD1 agonistsrdquo PLoSONE vol 8 no 7 Article ID e68701 2013
[42] B Fournier ldquoThe function of TLR2 during staphylococcaldiseasesrdquo Frontiers in Cellular and Infection Microbiology vol2 article 167 2013
[43] L-H Ding D Liu M Xu et al ldquoTLR2ndashMyD88ndashNF-120581Bpathway is involved in tubulointerstitial inflammation causedby proteinuriardquoThe International Journal of Biochemistry amp CellBiology vol 69 pp 114ndash120 2015
[44] Z Zhang X Gao Y Cao et al ldquoSelenium deficiency facilitatesinflammation through the regulation of TLR4 and TLR4-related signaling pathways in the mice uterusrdquo Inflammationvol 38 no 3 pp 1347ndash1356 2015
[45] C Xu G Shen C Chen C Gelinas and A-N T KongldquoSuppression of NF-120581B and NF-120581B-regulated gene expressionby sulforaphane and PEITC through I120581B120572 IKK pathway inhuman prostate cancer PC-3 cellsrdquo Oncogene vol 24 no 28pp 4486ndash4495 2005
[46] W Zhang R Zhang T Wang et al ldquoSelenium inhibits LPS-induced pro-inflammatory gene expression by modulatingMAPK and NF-120581B signaling pathways in mouse mammaryepithelial cells in primary culturerdquo Inflammation vol 37 no 2pp 478ndash485 2014
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
BioMed Research International 3
Table 1 Sequences of primers used for qPCR
Gene GenBankaccession number
Sequence (51015840-31015840)sense and antisense Product size (bp)
TNF-120572 NM 0136933 Sense 51015840-CTTCTCATTCCTGCTTGTG-31015840 198Antisense 51015840-ACTTGGTGGTTTGCTACG-31015840
IL-1120573 NM 0083613464 Sense 51015840-AGGCTCCGAGATGAACAA-31015840 464Antisense 51015840-AAGGCATTAGAAACAGTCC-31015840
IL-6 NM 0311681 Sense 51015840-TTCTTGGGACTGATGCTG-31015840 380Antisense 51015840-CTGGCTTTGTCTTTCTTGTT-31015840
IL-10 NM 0105482 Sense 51015840-AACATACTGCTAACCGACTC-31015840 286Antisense 51015840-TGGCCTTGTAGACACCTT-31015840
TLR2 NM 0119053 Sense 51015840-TTTGCTCCTGCGAACTCC-31015840 267Antisense 51015840-GCCACGCCCACATCATTC-31015840
GAPDH NM 0012897261 Sense 51015840-TGTTTCCTCGTCCCGTAG-31015840 108Antisense 51015840-CAATCTCCACTTTGCCACT-31015840
function 30minThen protein concentrationwas determinedusing the BCA protein assay kit (Thermo USA) The crudeprotein per sample was subjected to 10 SDS-PAGE usingTris-HCl Precast Gels and subsequently transferred ontoPVDF membranes The membrane was blocked with Trisbuffer saline containing 005 Tween-20 (TBS-T) supple-mented with 5 skim milk at room temperature for 15 h ona rotary shaker following by TBS-T washed The membraneswere washed with a specific primary antibody (Cell SignalingTechnology Inc Danvers MA) diluted in TBS-T containingskim milk (VV = 1 1000) at 4∘C overnight Subsequentlythe membranes were washed with TBS-T The membranewas washed and treated with secondary antibody (TianjinSungene Biotech Co Ltd Tianjin China) diluted in TBS-T(VV = 1 5000) at room temperature for 2 h In the last stepmembranes developed with the ECL Plus Western BlottingDetection System (Amersham Life Science UK) 120573-Actin(Tianjin China) was used as a control
29 Statistical Analysis Statistical analyses of the data wereperformed using the SPSS software (ver 17 for WindowsSPSS Inc Chicago IL USA) All values are expressed asthe means plusmn SD The data were assessed using the Tukey-Kramer method for multiple comparisons Significance wasdetermined by a 1-way ANOVA using a significance level of119901 lt 005
3 Results
31 Effect of IFN-120591 on S aureus Growth The effect of IFN-120591 (50ndash200 ngmL) on S aureus growth was monitored byturbidimeter at 600 nm for 12 hThe results showed that IFN-120591 had no effect on S aureus growth Specifically when com-pared to the untreated controls S aureus grown in the pres-ence of IFN-120591 (50ndash200 ngmL) were not affected (Figure 1)
32 Histopathological Changes Tissues were collected 24hours after the injection of S aureus and were stained withhematoxylin and eosin (HampE) There were no pathologicalchanges in the uterine tissue of the CG (Figure 2(a)) The
Effec
t of I
FN-120591
onS
aur
eusg
row
th (C
FUm
L)
107
109
1011
1013
1015
1017
1019
1021
1023
109876 11 124321 50Time (h)
IFN-120591 (50ngmL)IFN-120591 (0ngmL) IFN-120591 (100ngmL)
IFN-120591 (200ngmL)
Figure 1 Effect of IFN-120591 on S aureus growth S aureus (18times 107 CFUmL) was cultured in MH broth supplemented withdifferent concentrations of IFN-120591 (50ndash200 ngmL) in each of theexperiments S aureus growth was monitored turbidimetrically(600 nm) after 12 hThe data represents the mean of triplicates plusmn SDfrom three independent experiments
uterine morphology of the mice inoculated with S aureusdisplayed inflammatory cell infiltration endometrial conges-tion severe destruction of the myometrium cells and thedisappearance of the uterus acinar structure (Figure 2(b))However IFN-120591 significantly reduced the inflammation stim-ulated by S aureus and the uterine tissue structure of themice treated with IFN-120591 was almost complete with onlyminor endometrial pathology and an insignificant amount ofinflammatory cell infiltration (Figures 2(c) 2(d) and 2(e))
4 BioMed Research International
(a) (b) (c)
(d) (e) (f)
Figure 2 Histopathological changes (HE times200) HampE staining of the harvested uterine tissues (a) Control group (CG) in which the micewere not treated (b) S aureus-infected group (S aureus) in which the mice were infected with S aureus (c) Dexamethasone group (DEX)in which the mice inoculated with S aureus and developed endometritis were treated with DEX (d)ndash(f) IFN-120591 groups in which the miceinoculated with S aureus and developed endometritis were treated with different concentrations of IFN-120591 (2 4 8mgkg)
Representative pictures are shown to demonstrate the effectsdose-dependent effects of IFN-120591The degree of inflammatoryinjury was also very slight in the DEX group as shown inFigure 2(f)
33 Effects of IFN-120591 onMPO Activity in Uterus Tissues MPOactivity has been used as a quantitative index of inflammationand can illustrate the level of neutrophil infiltrationThe effectof IFN-120591 on MPO activity was examined in mice with Saureus-induced endometritis Immunohistochemical stain-ing showed that MPO specific immunolabelling was scarcelyfound in the neutrophil andmononuclear cells of lamina pro-pria of mucosa in normal uterus in CG (Figure 3(a)) whereasthis expression was found elevated in cells of surface epithe-lium and in cells of the inflammatory infiltrate in S aureus-infected group (Figure 3(b)) The MPO activity was veryslight in DEX group as shown in Figure 3(c) In contrastmice treated with different doses of IFN-120591 uterus showed alower level of MPO activity the immunoreactivity of whichshowed the suppressive effects of IFN-120591 increased in a dose-dependent manner in uterus samples of mice (Figures 3(d)3(e) and 3(f)) The activity of MPO from different micegroups was also measured by MPO assay kits The resultsillustrates that the activity of MPO was low in CG and dra-matically increased in S aureus group (Figure 3(g)) while theS aureus-infected mice treated with different concentrationsof IFN-120591 (2 4 8mgkg) significantly inhibited the activity ofMPO (compare with S aureus group) The results indicatedthat reduction of neutrophil infiltration in uterine tissuescould be one mechanism underlying the protective effect ofIFN-120591
34 Effects of IFN-120591 on Inflammatory Cytokines To deter-mine the effect of IFN-120591 on the S aureus endometritis thelevels of TNF-120572 IL-1120573 IL-6 and IL-10 were measured byqPCR and ELISA The results are shown in Figure 4 Theexpressions of TNF-120572 IL-1120573 and IL-6 were increased signif-icantly in the mice inoculated with S aureus compared tothe CG The increases in TNF-120572 IL-1120573 and IL-6 expressionwhich were induced by S aureus were significantly inhibitedby IFN-120591 Compared with themice inoculated with S aureusthe mRNA and protein expression of TNF-120572 IL-1120573 and IL-6 was significantly decreased following IFN-120591 treatment Theeffects of IFN-120591 increased in a dose-dependent mannerWithincreasing concentrations of IFN-120591 more significant effectswere observed However the effect on IL-10 was differentThe mRNA and protein expression of IL-10 was increasedin the mice inoculated with S aureus compared to the CGCompared with the mice inoculated with S aureus theexpression of IL-10 further increased upon IFN-120591 treatmentWith increasing concentrations of IFN-120591 more significanteffects on IL-10 were observed
35 Effects of IFN-120591 on TLR2 Expression TLR2 signalingplays an important role in the innate inflammatory responseThe secretion of TLR2 by S aureus-infected can induce theactivation of the NF-120581B and MAPK pathways and subse-quently result in the release of proinflammatory cytokinesCompared to the CG TLR2 mRNA levels were significantlyincreased in the S aureus groupThe results showed that IFN-120591 had a suppressive effect on the expression of TLR2 mRNAwhich was originally upregulated by S aureus in the uterinetissues (Figure 5(a))
BioMed Research International 5
CG S aureus DEX 2 4 8
lowast
lowast
lowast
lowast lowast
(a) (b) (c)
(d) (e) (f)
(g)
02468
10121416
MPO
activ
ity (U
mg)
Figure 3 Effects of IFN-120591 onMPO activity in uterus tissues (HE times200) Immunohistochemical illustratesMPO activity in sections of uterinetissues (a) Control group (CG) the mice were without any treatment (b) S aureus-infected group (S aureus) the mice were infected withS aureus (c) Dexamethasone group (DEX) the mice of S aureus endometritis were treatment with DEX (d)ndash(f) IFN-120591 groups the miceof S aureus endometritis were treatment with different concentrations of IFN-120591 (2 4 8mgkg) (g) Effects of different doses of IFN-120591 (2 48mgkg) and dexamethasone (5mgkg) on myeloperoxidase activity in mice with S aureus-infected uterine tissues Data are expressed asthe means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
To further confirm that IFN-120591 inhibited the inflammatoryresponse via TLR2 the effect of IFN-120591 on the productionof IL-8 in S aureus-stimulated HEK293-mTLR2 cells wasassessedThe results showed that IFN-120591 inhibited the expres-sion of TLR2 and IL-8 (Figure 5(b)) in S aureus-stimulatedHEK293-mTLR2 cells With increasing doses of IFN-120591 theeffect became increasingly significant
36 IFN-120591 Affected the Activation of the NF-120581B PathwayNF-120581B is an important signaling molecule in the devel-opment of inflammatory diseases Once activated NF-120581Binduces the production of proinflammatory cytokines In thepresent study we examined NF-120581B activation by westernblot analysis As shown in Figure 6 the phosphorylation ofthe I120581B120572 and p65 proteins was activated in mice inoculatedwith S aureus but not in CG The phosphorylation levelin mice inoculated with S aureus was significantly higher
than that in the IFN-120591 treatment groups These observationsindicate that IFN-120591 can suppress the activation of NF-120581B through the inhibition of the phosphorylation of I120581B120572and p65 Compared to the mice inoculated with S aureusthe level of phosphorylation was significantly reduced withincreasing IFN-120591 concentrations in the treatment groupsTheresults showed that IFN-120591 can inhibit NF-120581B I120581B120572 and p65phosphorylation in a dose-dependent manner
37 IFN-120591 Inhibits the Activation of the MAPK PathwayMAPK is an important signaling molecule in the develop-ment of inflammatory diseases The activation of p38 JNKand ERK can induce MyD88-dependent signaling pathwayswhich in turn induce MAPK activation We found that Saureus-infected uterine tissues showed increased phospho-rylation of three MAPKs In the mice inoculated with Saureus the phosphorylation levels of p38 ERK and JNK
6 BioMed Research International
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
IL-1120573
mRN
A le
vel
02468
1012141618202224
0
2
4
6
8
10
12
14
16
TNF-
120572m
RNA
leve
l
0123456789
10
IL-6
mRN
A le
vel
0
2
4
6
8
10
12
14
16
IL-10
mRN
A le
vel
lowast
lowastlowast
lowast
lowast
lowastlowast
lowast lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
0
500
1000
1500
2000
2500
3000
3500
4000
IL-10
(pg
mL)
0
500
1000
1500
2000
2500
IL-6
(pg
mL)
0500
1000150020002500300035004000450050005500
TNF-
120572(p
gm
L)
0500
100015002000250030003500400045005000550060006500700075008000
IL-1120573
(pg
mL) lowast
lowast
lowastlowast
lowast lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowastlowast
lowast
(b)
Figure 4 Cytokine concentrations (a) TNF-120572 IL-1120573 IL-6 and IL-10 mRNA levels in the uterine tissue (b) TNF-120572 IL-1120573 IL-6 and IL-10protein levels in the uterine tissueThe data represent the contents of 1mL supernatant of uterine homogenate and are presented as the meansplusmn SD (119899 = 10) The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that were not given anydrug treatment 2 4 and 8 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasone treatmentgroup lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
BioMed Research International 7
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
0
1
2
3
4
5
6
7
8
9
TLR2
mRN
A le
vel
(a)CG S aureus248
CG S aureus248
lowast
lowast
lowast
lowast
TLR2
120573-Actin
0
500
1000
1500
2000
2500
3000
3500
4000
IL-8
(pg
mL)
(b)
Figure 5 Effects of IFN-120591 on TLR2 (a) The TLR2 mRNA levels in the uterine tissues (b) The TLR2 and IL-8 protein levels in HEK293-mTLR2 cells qPCR was performed to assess the mRNA levels of TLR2 Western blotting was performed to detect the protein levels of TLR2120573-Actin was used as a control The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that werenot given any drug treatment 8 4 and 2 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasonetreatment group lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
were significantly increased compared with CG (Figure 7)Additionally the phosphorylation levels of the three MAPKsin themice inoculatedwith S aureuswere significantly higherthan in the mice treated with IFN-120591 These data indicate thatIFN-120591 can suppress the phosphorylation of p38 JNK andERK Compared to the mice inoculated with S aureus in thetreatment groups the level of phosphorylation was reducedsignificantly with increasing concentrations of IFN-120591 Theseresults showed that IFN-120591 can inhibit MAPK p38 JNK andERK phosphorylation in a dose-dependent manner
4 Discussion
IFN-120591 a novel Type I IFN is secreted by the trophoblastectoderm of ruminants in the early stages of pregnancy[22] IFN-120591 regulates the expression of numerous genes inthe endometrium and has been shown to play an impor-tant biological role in the implantation stage of pregnancy[24] Endometritis is a local inflammatory condition whichdelays uterine degeneration and has a significant economicimpact on dairy production [25 26] However infection withmicrobes such as S aureus can cause the destruction of theuterine horn structure subsequently resulting in the loss inthe ability to conceive This has been found to be due toinflammation and necrosis as determined by studies utilizingmorphological and image measurement tools [27ndash29] In thepresent study based on histopathological observations wefound that IFN-120591 has anti-inflammatory functions as well
as a significant protective effect on uterine tissues followinginfection with S aureus
A key component of the host immune response toinfection is the upregulation of cytokine production [30] Tofurther study the effect of IFN-120591 on S aureus endometritisthe levels of TNF-120572 IL-1120573 IL-6 and IL-10 production wereevaluatedThe results showed that the levels of TNF-120572 IL-1120573and IL-6 which are induced by S aureus were significantlyinhibited by IFN-120591 treatment in a dose-dependent mannerDuring the inflammatory response TNF-120572 is induced byregulated genes acting as effective inducers and subsequentlyregulates the expression of cytokines chemokines and celladhesion molecules [31 32] In vivo TNF-120572 can activatethe intracellular I120581B120572 and JNK signaling pathways throughTLRs thus contributing to the production of proinflamma-tory cytokines [33] The proinflammatory cytokine IL-1120573 isa key regulator of acute inflammatory processes in the cen-tral nervous system [34 35] Pathogen-associated molecularpatterns of S aureus can activate inflammatory pathwaysand when bound to innate monocytesmacrophages theyresult in a significant increase in the secretion of TNF-120572 andIL-6 [36 37] IL-6 is a pleiotropic cytokine that regulatesmultiple biological processes including the developmentof inflammation immune responses and the acute phasereaction [38] IL-10 has been shown to be beneficial inimproving systemic immunity by having anti-inflammatoryeffects and promoting the repair of immune dysfunction afterthe invasion of different pathogens [39] Our experimental
8 BioMed Research International
CG S aureusDEX 248
I120581B
p-I120581B
p65
p-p65
120573-Actin
(a)CG S aureusDEX 248
CG S aureusDEX 248
lowastlowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
pNF-
120581B
120573-a
ctin
ratio
0
02
04
06
08
1
12
14
16
18
2
22
24
0
02
04
06
08
1
12
14
p-I120581
B120573
-act
in ra
tio
(b)
Figure 6 Effect of IFN-120591 on NF-120581B activation (a) The NF-120581B and I-120581B protein levels in uterine tissues (b) The ratio of phosphorylationproteins levels as well as 120573-actin levels Western blot was performed to detect the levels of total and phosphorylated NF-120581B and I-120581B proteinsin the control group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups(2 4 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
results showed that the expression of IL-10 mRNA andprotein was increased in mice inoculated with S aureusand the expression of IL-10 was further increased with IFN-120591 treatment With increasing concentrations of IFN-120591 theeffect on IL-10 was more significant IFN-120591 can promotethe secretion of inflammatory cytokines including IL-10thereby inhibiting the subsequent secretion of TNF-120572 IL-1120573and IL-6 [40] TLRs are transmembrane pattern recognitionreceptors which are part of the innate immune systemand are the key element in identifying viral and bacterialcomponents [37 41] TLR2 senses the inflammatory reactionscaused by S aureusmost rapidly and sensitively and presentspathogen-derived antigens to various types of cells [42]Our results showed TLR2 increased in mice infected withS aureus and its secretion was significantly reduced afterIFN-120591 treatment TLR2 plays a key role in the responseto both innate and adaptive immunity as it can identifyexogenous pathogen-associated molecular patterns (PAMPs)
and induce proinflammatory signaling pathways [43] In thisstudy IFN-120591 inhibited the secretion of TLR2 in a dose-dependent manner
NF-120581B and MAPKs are two important inflammation-associated pathways and they canmodulate the developmentof inflammation by mediating downstream signaling net-works [44] To further investigate the mechanism by whichIFN-120591 affects inflammation we analyzed the NF-120581B andMAPK pathways The NF-120581B signaling pathway is involvedin the regulation of genes that mediate inflammation as wellas various cytokines and is associatedwith numerous chronicinflammatory diseases [45] Normally theNF-120581Bp65 subunitis bound to I120581B in the cytosol When stimulated by variouspathogens NF-120581B p65 is released due to the phosphorylationof I120581B leading to its activation and nuclear translocationwhich then triggers inflammation [46] Western blottinganalysis showed the phosphorylation levels of I120581B120572 and p65in mice infected with S aureus were significantly higher than
BioMed Research International 9
CG S aureusDEX 248
p38
p-p38
120573-Actin
ERK
p-ERK
JNK
p-JNK
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
p-JN
K120573
-act
in ra
tio
0
02
04
06
08
1
p-p3
8120573
-act
in ra
tio
0
02
04
06
08
1
12
0
02
04
06
08
1
12
14
16
18
p-ER
K120573
-act
in ra
tio
(b)
Figure 7 Effect of IFN-120591 on MAPK activation (a) The protein levels of MAPKs in uterine tissues (b) The ratio of phosphorylation proteinsas well as 120573-actin levels Western blotting was performed to measure the levels of total and phosphorylated p38 ERK and JNK protein in theControl group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups (24 and 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
10 BioMed Research International
that in the IFN-120591 groups These results indicated that IFN-120591 can inhibit the phosphorylation of I120581B120572 and p65 therebyblocking NF-120581B activation In addition to NF-120581B MAPKsare another key regulator of signal transduction pathwaysthat can modulate the levels of inflammatory mediators [47]Specifically p38 JNK and ERK are the major effectors of theMAPK pathwayThe phosphorylation of these three proteinscan cause the translocation of Activator Protein-1 (AP-1) tothe nucleus thereby promoting the inflammatory response[48 49] In the current study we found an increase in thephosphorylation levels of p38 JNK and ERK in the miceinoculated with S aureus However in the groups treatedwith IFN-120591 the phosphorylation of these proteins was signif-icantly inhibited Recent studies have indicated that the inhi-bition of phosphorylation of MAPKs with specific inhibitorscould significantly attenuate inflammation by decreasingthe number of neutrophils Similar effects were observedfollowing the inhibition of proinflammatory cytokines andchemokines [50] In our study we demonstrated that IFN-120591 significantly inhibited the phosphorylation of proteinsinvolved in MAPK pathway activation Taken together ourresults showed that blocking the NF-120581B andMAPK signalingpathways and inhibiting the secretion of proinflammatorycytokines are the main mechanisms by which IFN-120591 abatesendometritis
5 Conclusion
Our study showed that S aureus increases the expression ofTNF-120572 IL-1120573 and IL-6 thereby activating TLR2 signalingpathways including NF-120581B and MAPK The experimentspresented herein show that IFN-120591 reduces the secretion ofproinflammatory cytokines such as TNF-120572 IL-1120573 and IL-6 while promoting the production of the anti-inflammatorycytokine IL-10 This reduction inhibits inflammation andenhances the repair of uterine tissues In addition IFN-120591 inhibited the expression of TLR2 thereby blocking theactivation of the NF-120581B and MAPK signaling pathwaysand abating endometritis Additional studies are requiredto validate the efficacy of IFN-120591 as a treatment for uterineinfection due to S aureus or other infectious pathogens
Competing Interests
The authors have no conflict of interest to declare
Acknowledgments
This work was supported by a grant from the Funda-mental Research Funds for the Central Universities (no2662014BQ024) the National Natural Science Foundationof China (nos 31272631 31472254 and 31502130) andUndergraduate Special Science and Technology Innovationof Huazhong Agricultural University (no 2015BC009)
References
[1] B Polat M Cengiz O Cannazik et al ldquoEndometrial echotex-ture variables in postpartum cows with subclinical endometri-tisrdquo Animal Reproduction Science vol 155 pp 50ndash55 2015
[2] J Roberson D Moll and G Saunders ldquoChronic Staphylococcusaureus endometritis in a virgin giltrdquo Veterinary Record vol 161no 24 pp 821ndash822 2007
[3] M Guo G Wang T Lv et al ldquoEndometrial inflammation andabnormal expression of extracellular matrix proteins inducedbyMycoplasma bovis in dairy cowsrdquoTheriogenology vol 81 no5 pp 669ndash674 2014
[4] I M Sheldon J Cronin L Goetze G Donofrio and H-J Schuberth ldquoDefining postpartum uterine disease and themechanisms of infection and immunity in the female reproduc-tive tract in cattlerdquo Biology of Reproduction vol 81 no 6 pp1025ndash1032 2009
[5] P W Farin L Ball J D Olson et al ldquoEffect of Actinomyces pyo-genes and gram-negative anaerobic bacteria on the developmentof bovine pyometrardquoTheriogenology vol 31 no 5 pp 979ndash9891989
[6] M Guo Y Cao TWang et al ldquoBaicalin inhibits Staphylococcusaureus-induced apoptosis by regulating TLR2 andTLR2-relatedapoptotic factors in the mouse mammary glandsrdquo EuropeanJournal of Pharmacology vol 723 no 1 pp 481ndash488 2014
[7] A K Syed T J Reed K L Clark B R Boles and J MKahlenberg ldquoStaphlyococcus aureus phenol-soluble modulinsstimulate the release of proinflammatory cytokines from ker-atinocytes and are required for induction of skin inflammationrdquoInfection and Immunity vol 83 no 9 pp 3428ndash3437 2015
[8] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[9] T W Chon and S Bixler ldquoInterferon-tau current applicationsand potential in antiviral therapyrdquo Journal of Interferon ampCytokine Research vol 30 no 7 pp 477ndash485 2010
[10] C H Pontzer T L Ott F W Bazer and H M JohnsonldquoStructurefunction studies with interferon tau evidence formultiple active sitesrdquo Journal of Interferon Research vol 14 no3 pp 133ndash141 1994
[11] R M Roberts ldquoInterferon-tau a Type 1 interferon involved inmaternal recognition of pregnancyrdquo Cytokine amp Growth FactorReviews vol 18 no 5-6 pp 403ndash408 2007
[12] A P Alexenko AD Ealy andRM Roberts ldquoThe cross-speciesantiviral activities of different IFN-tau subtypes on bovinemurine and human cells contradictory evidence for therapeu-tic potentialrdquo Journal of Interferon amp Cytokine Research vol 19no 12 pp 1335ndash1341 1999
[13] J A Neira D Tainturier R M LrsquoHaridon and J Martal ldquoCom-parative IFN-tau secretion after hatching by bovine blastocystsderived ex vivo and completely produced in vitrordquoReproductionin Domestic Animals vol 42 no 1 pp 68ndash75 2007
[14] A D Ealy J A Green A P Alexenko D H Keisler and R MRoberts ldquoDifferent ovine interferon-tau genes are not expressedidentically and their protein products display different activi-tiesrdquo Biology of Reproduction vol 58 no 2 pp 566ndash573 1998
[15] K A Naivar S K Ward K J Austin D W Moore and T RHansen ldquoSecretion of bovine uterine proteins in response toType I interferonsrdquo Biology of Reproduction vol 52 no 4 pp848ndash854 1995
[16] M G Teixeira K J Austin D J Perry et al ldquoBovine granulo-cyte chemotactic protein-2 is secreted by the endometrium inresponse to interferon-tau (IFN-tau)rdquo Endocrine vol 6 no 1pp 31ndash37 1997
BioMed Research International 11
[17] G Guarda M Braun F Staehli et al ldquoType I interferon inhibitsinterleukin-1 production and inflammasome activationrdquo Immu-nity vol 34 no 2 pp 213ndash223 2011
[18] T J Foster J A Geoghegan V K Ganesh and M HookldquoAdhesion invasion and evasion the many functions of thesurface proteins of Staphylococcus aureusrdquo Nature ReviewsMicrobiology vol 12 no 1 pp 49ndash62 2014
[19] B A Diep S R Gill R F Chang et al ldquoComplete genomesequence of USA300 an epidemic clone of community-acquiredmeticillin-resistant Staphylococcus aureusrdquoTheLancetvol 367 no 9512 pp 731ndash739 2006
[20] D Wang N Xu Z Zhang et al ldquoSophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstreampathways on LPS-induced mastitis in the mammary gland ofmicerdquo International Immunopharmacology vol 35 pp 111ndash1182016
[21] J-L Zhao Y-X Ding H-X Zhao et al ldquoPresence of super-antigen genes and antimicrobial resistance in Staphylococcusisolates obtained from the uteri of dairy cows with clinicalendometritisrdquo Veterinary Record vol 175 no 14 article 3522014
[22] D K Tennakoon R Smith M D Stewart T E Spencer MNayak and C J Welsh ldquoOvine IFN-tau modulates the expres-sion of MHC antigens on murine cerebrovascular endothelialcells and inhibits replication of Theilerrsquos virusrdquo Journal ofInterferonampCytokine Research vol 21 no 10 pp 785ndash792 2001
[23] J M Soos P S Subramaniam A C Hobeika J Schiffen-bauer and H M Johnson ldquoThe IFN pregnancy recognitionhormone IFN-tau blocks both development and superantigenreactivation of experimental allergic encephalomyelitis withoutassociated toxicityrdquo The Journal of Immunology vol 155 no 5pp 2747ndash2753 1995
[24] G Song T E Spencer and FW Bazer ldquoCathepsins in the ovineuterus regulation by pregnancy progesterone and interferontaurdquo Endocrinology vol 146 no 11 pp 4825ndash4833 2005
[25] R Grio C Giobbe A Cellula et al ldquoInflammation of theuterine corpus endometritisrdquoMinervaGinecologica vol 42 no4 pp 99ndash102 1990
[26] I M Sheldon and H Dobson ldquoPostpartum uterine health incattlerdquo Animal Reproduction Science vol 82-83 pp 295ndash3062004
[27] S A Kurganov ldquoUterine eosinophils and infertility in theratrdquo Rossiiskii Fiziologicheskii Zhurnal Imeni IM Sechen-ovaRossiiskaia Akademiia Nauk vol 96 no 2 pp 138ndash1462010
[28] K Shirasuna F Usui T Karasawa et al ldquoNanosilica-inducedplacental inflammation and pregnancy complications differentroles of the inflammasome components NLRP3 and ASCrdquoNanotoxicology vol 9 no 5 pp 554ndash567 2015
[29] Y Liu C QiuW Li WMu C Li andM Guo ldquoSelenium playsa protective role in Staphylococcus aureus-induced endometritisin the uterine tissue of ratsrdquo Biological Trace Element Researchvol 173 no 2 pp 345ndash353 2016
[30] A A M Lima L G Spınola G Baccan et al ldquoEvaluation ofcorticosterone and IL-1120573 IL-6 IL-10 and TNF-120572 expressionafter 670-nm laser photobiomodulation in ratsrdquo Lasers inMedical Science vol 29 no 2 pp 709ndash715 2014
[31] C-J Liang S-H Wang Y-H Chen et al ldquoViscolin reducesVCAM-1 expression in TNF-120572-treated endothelial cells viathe JNKNF-120581B and ROS pathwayrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1337ndash1346 2011
[32] H-K Tsou H-T Chen C-H Chang W-Y Yang and C-HTang ldquoApoptosis signal-regulating kinase 1 is mediated in TNF-120572-induced CCL2 expression in human synovial fibroblastsrdquoJournal of Cellular Biochemistry vol 113 no 11 pp 3509ndash35192012
[33] H Zhang J Kovacs-Nolan T Kodera Y Eto and Y Mine ldquo120574-Glutamyl cysteine and 120574-glutamyl valine inhibit TNF-120572 signal-ing in intestinal epithelial cells and reduce inflammation in amouse model of colitis via allosteric activation of the calcium-sensing receptorrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1852 no 5 pp 792ndash804 2015
[34] M L Diamond J A Boles A C Ritter et al ldquoIn response tocomments on IL-1120573 associations with posttraumatic epilepsydevelopment a genetics and biomarker cohort studyrdquo Epilepsiavol 55 no 8 pp 1313ndash1314 2014
[35] A Vezzani J French T Bartfai and T Z Baram ldquoThe role ofinflammation in epilepsyrdquo Nature Reviews Neurology vol 7 no1 pp 31ndash40 2011
[36] B Fournier and D J Philpott ldquoRecognition of Staphylococcusaureus by the innate immune systemrdquo Clinical MicrobiologyReviews vol 18 no 3 pp 521ndash540 2005
[37] J E Wang P F Joslashrgensen M Almlof et al ldquoPeptidoglycanand lipoteichoic acid from Staphylococcus aureus induce tumornecrosis factor alpha interleukin 6 (IL-6) and IL-10 productionin both T cells and monocytes in a human whole blood modelrdquoInfection and Immunity vol 68 no 7 pp 3965ndash3970 2000
[38] D Zhu D-Y Yang Y-Y Guo et al ldquoIntracameral interleukin1120573 6 8 10 12p tumor necrosis factor 120572 and vascular endothelialgrowth factor and axial length in patients with cataractrdquo PLoSONE vol 10 no 2 Article ID e0117777 2015
[39] P Lichte R Pfeifer P Kobbe et al ldquoInhalative IL-10 treatmentafter bilateral femoral fractures affect pulmonary inflammationin micerdquo Annals of Anatomy vol 200 pp 73ndash78 2015
[40] K Hara K Shirasuna F Usui et al ldquoInterferon-tau attenuatesuptake of nanoparticles and secretion of interleukin-1120573 inmacrophagesrdquoPLoSONE vol 9 no 12 Article ID e113974 2014
[41] A Mansson Kvarnhammar L Tengroth M Adner and L-OCardell ldquoInnate immune receptors in human airway smoothmuscle cells activation by TLR12 TLR3 TLR4 TLR7 andNOD1 agonistsrdquo PLoSONE vol 8 no 7 Article ID e68701 2013
[42] B Fournier ldquoThe function of TLR2 during staphylococcaldiseasesrdquo Frontiers in Cellular and Infection Microbiology vol2 article 167 2013
[43] L-H Ding D Liu M Xu et al ldquoTLR2ndashMyD88ndashNF-120581Bpathway is involved in tubulointerstitial inflammation causedby proteinuriardquoThe International Journal of Biochemistry amp CellBiology vol 69 pp 114ndash120 2015
[44] Z Zhang X Gao Y Cao et al ldquoSelenium deficiency facilitatesinflammation through the regulation of TLR4 and TLR4-related signaling pathways in the mice uterusrdquo Inflammationvol 38 no 3 pp 1347ndash1356 2015
[45] C Xu G Shen C Chen C Gelinas and A-N T KongldquoSuppression of NF-120581B and NF-120581B-regulated gene expressionby sulforaphane and PEITC through I120581B120572 IKK pathway inhuman prostate cancer PC-3 cellsrdquo Oncogene vol 24 no 28pp 4486ndash4495 2005
[46] W Zhang R Zhang T Wang et al ldquoSelenium inhibits LPS-induced pro-inflammatory gene expression by modulatingMAPK and NF-120581B signaling pathways in mouse mammaryepithelial cells in primary culturerdquo Inflammation vol 37 no 2pp 478ndash485 2014
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
4 BioMed Research International
(a) (b) (c)
(d) (e) (f)
Figure 2 Histopathological changes (HE times200) HampE staining of the harvested uterine tissues (a) Control group (CG) in which the micewere not treated (b) S aureus-infected group (S aureus) in which the mice were infected with S aureus (c) Dexamethasone group (DEX)in which the mice inoculated with S aureus and developed endometritis were treated with DEX (d)ndash(f) IFN-120591 groups in which the miceinoculated with S aureus and developed endometritis were treated with different concentrations of IFN-120591 (2 4 8mgkg)
Representative pictures are shown to demonstrate the effectsdose-dependent effects of IFN-120591The degree of inflammatoryinjury was also very slight in the DEX group as shown inFigure 2(f)
33 Effects of IFN-120591 onMPO Activity in Uterus Tissues MPOactivity has been used as a quantitative index of inflammationand can illustrate the level of neutrophil infiltrationThe effectof IFN-120591 on MPO activity was examined in mice with Saureus-induced endometritis Immunohistochemical stain-ing showed that MPO specific immunolabelling was scarcelyfound in the neutrophil andmononuclear cells of lamina pro-pria of mucosa in normal uterus in CG (Figure 3(a)) whereasthis expression was found elevated in cells of surface epithe-lium and in cells of the inflammatory infiltrate in S aureus-infected group (Figure 3(b)) The MPO activity was veryslight in DEX group as shown in Figure 3(c) In contrastmice treated with different doses of IFN-120591 uterus showed alower level of MPO activity the immunoreactivity of whichshowed the suppressive effects of IFN-120591 increased in a dose-dependent manner in uterus samples of mice (Figures 3(d)3(e) and 3(f)) The activity of MPO from different micegroups was also measured by MPO assay kits The resultsillustrates that the activity of MPO was low in CG and dra-matically increased in S aureus group (Figure 3(g)) while theS aureus-infected mice treated with different concentrationsof IFN-120591 (2 4 8mgkg) significantly inhibited the activity ofMPO (compare with S aureus group) The results indicatedthat reduction of neutrophil infiltration in uterine tissuescould be one mechanism underlying the protective effect ofIFN-120591
34 Effects of IFN-120591 on Inflammatory Cytokines To deter-mine the effect of IFN-120591 on the S aureus endometritis thelevels of TNF-120572 IL-1120573 IL-6 and IL-10 were measured byqPCR and ELISA The results are shown in Figure 4 Theexpressions of TNF-120572 IL-1120573 and IL-6 were increased signif-icantly in the mice inoculated with S aureus compared tothe CG The increases in TNF-120572 IL-1120573 and IL-6 expressionwhich were induced by S aureus were significantly inhibitedby IFN-120591 Compared with themice inoculated with S aureusthe mRNA and protein expression of TNF-120572 IL-1120573 and IL-6 was significantly decreased following IFN-120591 treatment Theeffects of IFN-120591 increased in a dose-dependent mannerWithincreasing concentrations of IFN-120591 more significant effectswere observed However the effect on IL-10 was differentThe mRNA and protein expression of IL-10 was increasedin the mice inoculated with S aureus compared to the CGCompared with the mice inoculated with S aureus theexpression of IL-10 further increased upon IFN-120591 treatmentWith increasing concentrations of IFN-120591 more significanteffects on IL-10 were observed
35 Effects of IFN-120591 on TLR2 Expression TLR2 signalingplays an important role in the innate inflammatory responseThe secretion of TLR2 by S aureus-infected can induce theactivation of the NF-120581B and MAPK pathways and subse-quently result in the release of proinflammatory cytokinesCompared to the CG TLR2 mRNA levels were significantlyincreased in the S aureus groupThe results showed that IFN-120591 had a suppressive effect on the expression of TLR2 mRNAwhich was originally upregulated by S aureus in the uterinetissues (Figure 5(a))
BioMed Research International 5
CG S aureus DEX 2 4 8
lowast
lowast
lowast
lowast lowast
(a) (b) (c)
(d) (e) (f)
(g)
02468
10121416
MPO
activ
ity (U
mg)
Figure 3 Effects of IFN-120591 onMPO activity in uterus tissues (HE times200) Immunohistochemical illustratesMPO activity in sections of uterinetissues (a) Control group (CG) the mice were without any treatment (b) S aureus-infected group (S aureus) the mice were infected withS aureus (c) Dexamethasone group (DEX) the mice of S aureus endometritis were treatment with DEX (d)ndash(f) IFN-120591 groups the miceof S aureus endometritis were treatment with different concentrations of IFN-120591 (2 4 8mgkg) (g) Effects of different doses of IFN-120591 (2 48mgkg) and dexamethasone (5mgkg) on myeloperoxidase activity in mice with S aureus-infected uterine tissues Data are expressed asthe means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
To further confirm that IFN-120591 inhibited the inflammatoryresponse via TLR2 the effect of IFN-120591 on the productionof IL-8 in S aureus-stimulated HEK293-mTLR2 cells wasassessedThe results showed that IFN-120591 inhibited the expres-sion of TLR2 and IL-8 (Figure 5(b)) in S aureus-stimulatedHEK293-mTLR2 cells With increasing doses of IFN-120591 theeffect became increasingly significant
36 IFN-120591 Affected the Activation of the NF-120581B PathwayNF-120581B is an important signaling molecule in the devel-opment of inflammatory diseases Once activated NF-120581Binduces the production of proinflammatory cytokines In thepresent study we examined NF-120581B activation by westernblot analysis As shown in Figure 6 the phosphorylation ofthe I120581B120572 and p65 proteins was activated in mice inoculatedwith S aureus but not in CG The phosphorylation levelin mice inoculated with S aureus was significantly higher
than that in the IFN-120591 treatment groups These observationsindicate that IFN-120591 can suppress the activation of NF-120581B through the inhibition of the phosphorylation of I120581B120572and p65 Compared to the mice inoculated with S aureusthe level of phosphorylation was significantly reduced withincreasing IFN-120591 concentrations in the treatment groupsTheresults showed that IFN-120591 can inhibit NF-120581B I120581B120572 and p65phosphorylation in a dose-dependent manner
37 IFN-120591 Inhibits the Activation of the MAPK PathwayMAPK is an important signaling molecule in the develop-ment of inflammatory diseases The activation of p38 JNKand ERK can induce MyD88-dependent signaling pathwayswhich in turn induce MAPK activation We found that Saureus-infected uterine tissues showed increased phospho-rylation of three MAPKs In the mice inoculated with Saureus the phosphorylation levels of p38 ERK and JNK
6 BioMed Research International
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
IL-1120573
mRN
A le
vel
02468
1012141618202224
0
2
4
6
8
10
12
14
16
TNF-
120572m
RNA
leve
l
0123456789
10
IL-6
mRN
A le
vel
0
2
4
6
8
10
12
14
16
IL-10
mRN
A le
vel
lowast
lowastlowast
lowast
lowast
lowastlowast
lowast lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
0
500
1000
1500
2000
2500
3000
3500
4000
IL-10
(pg
mL)
0
500
1000
1500
2000
2500
IL-6
(pg
mL)
0500
1000150020002500300035004000450050005500
TNF-
120572(p
gm
L)
0500
100015002000250030003500400045005000550060006500700075008000
IL-1120573
(pg
mL) lowast
lowast
lowastlowast
lowast lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowastlowast
lowast
(b)
Figure 4 Cytokine concentrations (a) TNF-120572 IL-1120573 IL-6 and IL-10 mRNA levels in the uterine tissue (b) TNF-120572 IL-1120573 IL-6 and IL-10protein levels in the uterine tissueThe data represent the contents of 1mL supernatant of uterine homogenate and are presented as the meansplusmn SD (119899 = 10) The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that were not given anydrug treatment 2 4 and 8 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasone treatmentgroup lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
BioMed Research International 7
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
0
1
2
3
4
5
6
7
8
9
TLR2
mRN
A le
vel
(a)CG S aureus248
CG S aureus248
lowast
lowast
lowast
lowast
TLR2
120573-Actin
0
500
1000
1500
2000
2500
3000
3500
4000
IL-8
(pg
mL)
(b)
Figure 5 Effects of IFN-120591 on TLR2 (a) The TLR2 mRNA levels in the uterine tissues (b) The TLR2 and IL-8 protein levels in HEK293-mTLR2 cells qPCR was performed to assess the mRNA levels of TLR2 Western blotting was performed to detect the protein levels of TLR2120573-Actin was used as a control The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that werenot given any drug treatment 8 4 and 2 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasonetreatment group lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
were significantly increased compared with CG (Figure 7)Additionally the phosphorylation levels of the three MAPKsin themice inoculatedwith S aureuswere significantly higherthan in the mice treated with IFN-120591 These data indicate thatIFN-120591 can suppress the phosphorylation of p38 JNK andERK Compared to the mice inoculated with S aureus in thetreatment groups the level of phosphorylation was reducedsignificantly with increasing concentrations of IFN-120591 Theseresults showed that IFN-120591 can inhibit MAPK p38 JNK andERK phosphorylation in a dose-dependent manner
4 Discussion
IFN-120591 a novel Type I IFN is secreted by the trophoblastectoderm of ruminants in the early stages of pregnancy[22] IFN-120591 regulates the expression of numerous genes inthe endometrium and has been shown to play an impor-tant biological role in the implantation stage of pregnancy[24] Endometritis is a local inflammatory condition whichdelays uterine degeneration and has a significant economicimpact on dairy production [25 26] However infection withmicrobes such as S aureus can cause the destruction of theuterine horn structure subsequently resulting in the loss inthe ability to conceive This has been found to be due toinflammation and necrosis as determined by studies utilizingmorphological and image measurement tools [27ndash29] In thepresent study based on histopathological observations wefound that IFN-120591 has anti-inflammatory functions as well
as a significant protective effect on uterine tissues followinginfection with S aureus
A key component of the host immune response toinfection is the upregulation of cytokine production [30] Tofurther study the effect of IFN-120591 on S aureus endometritisthe levels of TNF-120572 IL-1120573 IL-6 and IL-10 production wereevaluatedThe results showed that the levels of TNF-120572 IL-1120573and IL-6 which are induced by S aureus were significantlyinhibited by IFN-120591 treatment in a dose-dependent mannerDuring the inflammatory response TNF-120572 is induced byregulated genes acting as effective inducers and subsequentlyregulates the expression of cytokines chemokines and celladhesion molecules [31 32] In vivo TNF-120572 can activatethe intracellular I120581B120572 and JNK signaling pathways throughTLRs thus contributing to the production of proinflamma-tory cytokines [33] The proinflammatory cytokine IL-1120573 isa key regulator of acute inflammatory processes in the cen-tral nervous system [34 35] Pathogen-associated molecularpatterns of S aureus can activate inflammatory pathwaysand when bound to innate monocytesmacrophages theyresult in a significant increase in the secretion of TNF-120572 andIL-6 [36 37] IL-6 is a pleiotropic cytokine that regulatesmultiple biological processes including the developmentof inflammation immune responses and the acute phasereaction [38] IL-10 has been shown to be beneficial inimproving systemic immunity by having anti-inflammatoryeffects and promoting the repair of immune dysfunction afterthe invasion of different pathogens [39] Our experimental
8 BioMed Research International
CG S aureusDEX 248
I120581B
p-I120581B
p65
p-p65
120573-Actin
(a)CG S aureusDEX 248
CG S aureusDEX 248
lowastlowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
pNF-
120581B
120573-a
ctin
ratio
0
02
04
06
08
1
12
14
16
18
2
22
24
0
02
04
06
08
1
12
14
p-I120581
B120573
-act
in ra
tio
(b)
Figure 6 Effect of IFN-120591 on NF-120581B activation (a) The NF-120581B and I-120581B protein levels in uterine tissues (b) The ratio of phosphorylationproteins levels as well as 120573-actin levels Western blot was performed to detect the levels of total and phosphorylated NF-120581B and I-120581B proteinsin the control group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups(2 4 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
results showed that the expression of IL-10 mRNA andprotein was increased in mice inoculated with S aureusand the expression of IL-10 was further increased with IFN-120591 treatment With increasing concentrations of IFN-120591 theeffect on IL-10 was more significant IFN-120591 can promotethe secretion of inflammatory cytokines including IL-10thereby inhibiting the subsequent secretion of TNF-120572 IL-1120573and IL-6 [40] TLRs are transmembrane pattern recognitionreceptors which are part of the innate immune systemand are the key element in identifying viral and bacterialcomponents [37 41] TLR2 senses the inflammatory reactionscaused by S aureusmost rapidly and sensitively and presentspathogen-derived antigens to various types of cells [42]Our results showed TLR2 increased in mice infected withS aureus and its secretion was significantly reduced afterIFN-120591 treatment TLR2 plays a key role in the responseto both innate and adaptive immunity as it can identifyexogenous pathogen-associated molecular patterns (PAMPs)
and induce proinflammatory signaling pathways [43] In thisstudy IFN-120591 inhibited the secretion of TLR2 in a dose-dependent manner
NF-120581B and MAPKs are two important inflammation-associated pathways and they canmodulate the developmentof inflammation by mediating downstream signaling net-works [44] To further investigate the mechanism by whichIFN-120591 affects inflammation we analyzed the NF-120581B andMAPK pathways The NF-120581B signaling pathway is involvedin the regulation of genes that mediate inflammation as wellas various cytokines and is associatedwith numerous chronicinflammatory diseases [45] Normally theNF-120581Bp65 subunitis bound to I120581B in the cytosol When stimulated by variouspathogens NF-120581B p65 is released due to the phosphorylationof I120581B leading to its activation and nuclear translocationwhich then triggers inflammation [46] Western blottinganalysis showed the phosphorylation levels of I120581B120572 and p65in mice infected with S aureus were significantly higher than
BioMed Research International 9
CG S aureusDEX 248
p38
p-p38
120573-Actin
ERK
p-ERK
JNK
p-JNK
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
p-JN
K120573
-act
in ra
tio
0
02
04
06
08
1
p-p3
8120573
-act
in ra
tio
0
02
04
06
08
1
12
0
02
04
06
08
1
12
14
16
18
p-ER
K120573
-act
in ra
tio
(b)
Figure 7 Effect of IFN-120591 on MAPK activation (a) The protein levels of MAPKs in uterine tissues (b) The ratio of phosphorylation proteinsas well as 120573-actin levels Western blotting was performed to measure the levels of total and phosphorylated p38 ERK and JNK protein in theControl group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups (24 and 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
10 BioMed Research International
that in the IFN-120591 groups These results indicated that IFN-120591 can inhibit the phosphorylation of I120581B120572 and p65 therebyblocking NF-120581B activation In addition to NF-120581B MAPKsare another key regulator of signal transduction pathwaysthat can modulate the levels of inflammatory mediators [47]Specifically p38 JNK and ERK are the major effectors of theMAPK pathwayThe phosphorylation of these three proteinscan cause the translocation of Activator Protein-1 (AP-1) tothe nucleus thereby promoting the inflammatory response[48 49] In the current study we found an increase in thephosphorylation levels of p38 JNK and ERK in the miceinoculated with S aureus However in the groups treatedwith IFN-120591 the phosphorylation of these proteins was signif-icantly inhibited Recent studies have indicated that the inhi-bition of phosphorylation of MAPKs with specific inhibitorscould significantly attenuate inflammation by decreasingthe number of neutrophils Similar effects were observedfollowing the inhibition of proinflammatory cytokines andchemokines [50] In our study we demonstrated that IFN-120591 significantly inhibited the phosphorylation of proteinsinvolved in MAPK pathway activation Taken together ourresults showed that blocking the NF-120581B andMAPK signalingpathways and inhibiting the secretion of proinflammatorycytokines are the main mechanisms by which IFN-120591 abatesendometritis
5 Conclusion
Our study showed that S aureus increases the expression ofTNF-120572 IL-1120573 and IL-6 thereby activating TLR2 signalingpathways including NF-120581B and MAPK The experimentspresented herein show that IFN-120591 reduces the secretion ofproinflammatory cytokines such as TNF-120572 IL-1120573 and IL-6 while promoting the production of the anti-inflammatorycytokine IL-10 This reduction inhibits inflammation andenhances the repair of uterine tissues In addition IFN-120591 inhibited the expression of TLR2 thereby blocking theactivation of the NF-120581B and MAPK signaling pathwaysand abating endometritis Additional studies are requiredto validate the efficacy of IFN-120591 as a treatment for uterineinfection due to S aureus or other infectious pathogens
Competing Interests
The authors have no conflict of interest to declare
Acknowledgments
This work was supported by a grant from the Funda-mental Research Funds for the Central Universities (no2662014BQ024) the National Natural Science Foundationof China (nos 31272631 31472254 and 31502130) andUndergraduate Special Science and Technology Innovationof Huazhong Agricultural University (no 2015BC009)
References
[1] B Polat M Cengiz O Cannazik et al ldquoEndometrial echotex-ture variables in postpartum cows with subclinical endometri-tisrdquo Animal Reproduction Science vol 155 pp 50ndash55 2015
[2] J Roberson D Moll and G Saunders ldquoChronic Staphylococcusaureus endometritis in a virgin giltrdquo Veterinary Record vol 161no 24 pp 821ndash822 2007
[3] M Guo G Wang T Lv et al ldquoEndometrial inflammation andabnormal expression of extracellular matrix proteins inducedbyMycoplasma bovis in dairy cowsrdquoTheriogenology vol 81 no5 pp 669ndash674 2014
[4] I M Sheldon J Cronin L Goetze G Donofrio and H-J Schuberth ldquoDefining postpartum uterine disease and themechanisms of infection and immunity in the female reproduc-tive tract in cattlerdquo Biology of Reproduction vol 81 no 6 pp1025ndash1032 2009
[5] P W Farin L Ball J D Olson et al ldquoEffect of Actinomyces pyo-genes and gram-negative anaerobic bacteria on the developmentof bovine pyometrardquoTheriogenology vol 31 no 5 pp 979ndash9891989
[6] M Guo Y Cao TWang et al ldquoBaicalin inhibits Staphylococcusaureus-induced apoptosis by regulating TLR2 andTLR2-relatedapoptotic factors in the mouse mammary glandsrdquo EuropeanJournal of Pharmacology vol 723 no 1 pp 481ndash488 2014
[7] A K Syed T J Reed K L Clark B R Boles and J MKahlenberg ldquoStaphlyococcus aureus phenol-soluble modulinsstimulate the release of proinflammatory cytokines from ker-atinocytes and are required for induction of skin inflammationrdquoInfection and Immunity vol 83 no 9 pp 3428ndash3437 2015
[8] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[9] T W Chon and S Bixler ldquoInterferon-tau current applicationsand potential in antiviral therapyrdquo Journal of Interferon ampCytokine Research vol 30 no 7 pp 477ndash485 2010
[10] C H Pontzer T L Ott F W Bazer and H M JohnsonldquoStructurefunction studies with interferon tau evidence formultiple active sitesrdquo Journal of Interferon Research vol 14 no3 pp 133ndash141 1994
[11] R M Roberts ldquoInterferon-tau a Type 1 interferon involved inmaternal recognition of pregnancyrdquo Cytokine amp Growth FactorReviews vol 18 no 5-6 pp 403ndash408 2007
[12] A P Alexenko AD Ealy andRM Roberts ldquoThe cross-speciesantiviral activities of different IFN-tau subtypes on bovinemurine and human cells contradictory evidence for therapeu-tic potentialrdquo Journal of Interferon amp Cytokine Research vol 19no 12 pp 1335ndash1341 1999
[13] J A Neira D Tainturier R M LrsquoHaridon and J Martal ldquoCom-parative IFN-tau secretion after hatching by bovine blastocystsderived ex vivo and completely produced in vitrordquoReproductionin Domestic Animals vol 42 no 1 pp 68ndash75 2007
[14] A D Ealy J A Green A P Alexenko D H Keisler and R MRoberts ldquoDifferent ovine interferon-tau genes are not expressedidentically and their protein products display different activi-tiesrdquo Biology of Reproduction vol 58 no 2 pp 566ndash573 1998
[15] K A Naivar S K Ward K J Austin D W Moore and T RHansen ldquoSecretion of bovine uterine proteins in response toType I interferonsrdquo Biology of Reproduction vol 52 no 4 pp848ndash854 1995
[16] M G Teixeira K J Austin D J Perry et al ldquoBovine granulo-cyte chemotactic protein-2 is secreted by the endometrium inresponse to interferon-tau (IFN-tau)rdquo Endocrine vol 6 no 1pp 31ndash37 1997
BioMed Research International 11
[17] G Guarda M Braun F Staehli et al ldquoType I interferon inhibitsinterleukin-1 production and inflammasome activationrdquo Immu-nity vol 34 no 2 pp 213ndash223 2011
[18] T J Foster J A Geoghegan V K Ganesh and M HookldquoAdhesion invasion and evasion the many functions of thesurface proteins of Staphylococcus aureusrdquo Nature ReviewsMicrobiology vol 12 no 1 pp 49ndash62 2014
[19] B A Diep S R Gill R F Chang et al ldquoComplete genomesequence of USA300 an epidemic clone of community-acquiredmeticillin-resistant Staphylococcus aureusrdquoTheLancetvol 367 no 9512 pp 731ndash739 2006
[20] D Wang N Xu Z Zhang et al ldquoSophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstreampathways on LPS-induced mastitis in the mammary gland ofmicerdquo International Immunopharmacology vol 35 pp 111ndash1182016
[21] J-L Zhao Y-X Ding H-X Zhao et al ldquoPresence of super-antigen genes and antimicrobial resistance in Staphylococcusisolates obtained from the uteri of dairy cows with clinicalendometritisrdquo Veterinary Record vol 175 no 14 article 3522014
[22] D K Tennakoon R Smith M D Stewart T E Spencer MNayak and C J Welsh ldquoOvine IFN-tau modulates the expres-sion of MHC antigens on murine cerebrovascular endothelialcells and inhibits replication of Theilerrsquos virusrdquo Journal ofInterferonampCytokine Research vol 21 no 10 pp 785ndash792 2001
[23] J M Soos P S Subramaniam A C Hobeika J Schiffen-bauer and H M Johnson ldquoThe IFN pregnancy recognitionhormone IFN-tau blocks both development and superantigenreactivation of experimental allergic encephalomyelitis withoutassociated toxicityrdquo The Journal of Immunology vol 155 no 5pp 2747ndash2753 1995
[24] G Song T E Spencer and FW Bazer ldquoCathepsins in the ovineuterus regulation by pregnancy progesterone and interferontaurdquo Endocrinology vol 146 no 11 pp 4825ndash4833 2005
[25] R Grio C Giobbe A Cellula et al ldquoInflammation of theuterine corpus endometritisrdquoMinervaGinecologica vol 42 no4 pp 99ndash102 1990
[26] I M Sheldon and H Dobson ldquoPostpartum uterine health incattlerdquo Animal Reproduction Science vol 82-83 pp 295ndash3062004
[27] S A Kurganov ldquoUterine eosinophils and infertility in theratrdquo Rossiiskii Fiziologicheskii Zhurnal Imeni IM Sechen-ovaRossiiskaia Akademiia Nauk vol 96 no 2 pp 138ndash1462010
[28] K Shirasuna F Usui T Karasawa et al ldquoNanosilica-inducedplacental inflammation and pregnancy complications differentroles of the inflammasome components NLRP3 and ASCrdquoNanotoxicology vol 9 no 5 pp 554ndash567 2015
[29] Y Liu C QiuW Li WMu C Li andM Guo ldquoSelenium playsa protective role in Staphylococcus aureus-induced endometritisin the uterine tissue of ratsrdquo Biological Trace Element Researchvol 173 no 2 pp 345ndash353 2016
[30] A A M Lima L G Spınola G Baccan et al ldquoEvaluation ofcorticosterone and IL-1120573 IL-6 IL-10 and TNF-120572 expressionafter 670-nm laser photobiomodulation in ratsrdquo Lasers inMedical Science vol 29 no 2 pp 709ndash715 2014
[31] C-J Liang S-H Wang Y-H Chen et al ldquoViscolin reducesVCAM-1 expression in TNF-120572-treated endothelial cells viathe JNKNF-120581B and ROS pathwayrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1337ndash1346 2011
[32] H-K Tsou H-T Chen C-H Chang W-Y Yang and C-HTang ldquoApoptosis signal-regulating kinase 1 is mediated in TNF-120572-induced CCL2 expression in human synovial fibroblastsrdquoJournal of Cellular Biochemistry vol 113 no 11 pp 3509ndash35192012
[33] H Zhang J Kovacs-Nolan T Kodera Y Eto and Y Mine ldquo120574-Glutamyl cysteine and 120574-glutamyl valine inhibit TNF-120572 signal-ing in intestinal epithelial cells and reduce inflammation in amouse model of colitis via allosteric activation of the calcium-sensing receptorrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1852 no 5 pp 792ndash804 2015
[34] M L Diamond J A Boles A C Ritter et al ldquoIn response tocomments on IL-1120573 associations with posttraumatic epilepsydevelopment a genetics and biomarker cohort studyrdquo Epilepsiavol 55 no 8 pp 1313ndash1314 2014
[35] A Vezzani J French T Bartfai and T Z Baram ldquoThe role ofinflammation in epilepsyrdquo Nature Reviews Neurology vol 7 no1 pp 31ndash40 2011
[36] B Fournier and D J Philpott ldquoRecognition of Staphylococcusaureus by the innate immune systemrdquo Clinical MicrobiologyReviews vol 18 no 3 pp 521ndash540 2005
[37] J E Wang P F Joslashrgensen M Almlof et al ldquoPeptidoglycanand lipoteichoic acid from Staphylococcus aureus induce tumornecrosis factor alpha interleukin 6 (IL-6) and IL-10 productionin both T cells and monocytes in a human whole blood modelrdquoInfection and Immunity vol 68 no 7 pp 3965ndash3970 2000
[38] D Zhu D-Y Yang Y-Y Guo et al ldquoIntracameral interleukin1120573 6 8 10 12p tumor necrosis factor 120572 and vascular endothelialgrowth factor and axial length in patients with cataractrdquo PLoSONE vol 10 no 2 Article ID e0117777 2015
[39] P Lichte R Pfeifer P Kobbe et al ldquoInhalative IL-10 treatmentafter bilateral femoral fractures affect pulmonary inflammationin micerdquo Annals of Anatomy vol 200 pp 73ndash78 2015
[40] K Hara K Shirasuna F Usui et al ldquoInterferon-tau attenuatesuptake of nanoparticles and secretion of interleukin-1120573 inmacrophagesrdquoPLoSONE vol 9 no 12 Article ID e113974 2014
[41] A Mansson Kvarnhammar L Tengroth M Adner and L-OCardell ldquoInnate immune receptors in human airway smoothmuscle cells activation by TLR12 TLR3 TLR4 TLR7 andNOD1 agonistsrdquo PLoSONE vol 8 no 7 Article ID e68701 2013
[42] B Fournier ldquoThe function of TLR2 during staphylococcaldiseasesrdquo Frontiers in Cellular and Infection Microbiology vol2 article 167 2013
[43] L-H Ding D Liu M Xu et al ldquoTLR2ndashMyD88ndashNF-120581Bpathway is involved in tubulointerstitial inflammation causedby proteinuriardquoThe International Journal of Biochemistry amp CellBiology vol 69 pp 114ndash120 2015
[44] Z Zhang X Gao Y Cao et al ldquoSelenium deficiency facilitatesinflammation through the regulation of TLR4 and TLR4-related signaling pathways in the mice uterusrdquo Inflammationvol 38 no 3 pp 1347ndash1356 2015
[45] C Xu G Shen C Chen C Gelinas and A-N T KongldquoSuppression of NF-120581B and NF-120581B-regulated gene expressionby sulforaphane and PEITC through I120581B120572 IKK pathway inhuman prostate cancer PC-3 cellsrdquo Oncogene vol 24 no 28pp 4486ndash4495 2005
[46] W Zhang R Zhang T Wang et al ldquoSelenium inhibits LPS-induced pro-inflammatory gene expression by modulatingMAPK and NF-120581B signaling pathways in mouse mammaryepithelial cells in primary culturerdquo Inflammation vol 37 no 2pp 478ndash485 2014
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
BioMed Research International 5
CG S aureus DEX 2 4 8
lowast
lowast
lowast
lowast lowast
(a) (b) (c)
(d) (e) (f)
(g)
02468
10121416
MPO
activ
ity (U
mg)
Figure 3 Effects of IFN-120591 onMPO activity in uterus tissues (HE times200) Immunohistochemical illustratesMPO activity in sections of uterinetissues (a) Control group (CG) the mice were without any treatment (b) S aureus-infected group (S aureus) the mice were infected withS aureus (c) Dexamethasone group (DEX) the mice of S aureus endometritis were treatment with DEX (d)ndash(f) IFN-120591 groups the miceof S aureus endometritis were treatment with different concentrations of IFN-120591 (2 4 8mgkg) (g) Effects of different doses of IFN-120591 (2 48mgkg) and dexamethasone (5mgkg) on myeloperoxidase activity in mice with S aureus-infected uterine tissues Data are expressed asthe means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
To further confirm that IFN-120591 inhibited the inflammatoryresponse via TLR2 the effect of IFN-120591 on the productionof IL-8 in S aureus-stimulated HEK293-mTLR2 cells wasassessedThe results showed that IFN-120591 inhibited the expres-sion of TLR2 and IL-8 (Figure 5(b)) in S aureus-stimulatedHEK293-mTLR2 cells With increasing doses of IFN-120591 theeffect became increasingly significant
36 IFN-120591 Affected the Activation of the NF-120581B PathwayNF-120581B is an important signaling molecule in the devel-opment of inflammatory diseases Once activated NF-120581Binduces the production of proinflammatory cytokines In thepresent study we examined NF-120581B activation by westernblot analysis As shown in Figure 6 the phosphorylation ofthe I120581B120572 and p65 proteins was activated in mice inoculatedwith S aureus but not in CG The phosphorylation levelin mice inoculated with S aureus was significantly higher
than that in the IFN-120591 treatment groups These observationsindicate that IFN-120591 can suppress the activation of NF-120581B through the inhibition of the phosphorylation of I120581B120572and p65 Compared to the mice inoculated with S aureusthe level of phosphorylation was significantly reduced withincreasing IFN-120591 concentrations in the treatment groupsTheresults showed that IFN-120591 can inhibit NF-120581B I120581B120572 and p65phosphorylation in a dose-dependent manner
37 IFN-120591 Inhibits the Activation of the MAPK PathwayMAPK is an important signaling molecule in the develop-ment of inflammatory diseases The activation of p38 JNKand ERK can induce MyD88-dependent signaling pathwayswhich in turn induce MAPK activation We found that Saureus-infected uterine tissues showed increased phospho-rylation of three MAPKs In the mice inoculated with Saureus the phosphorylation levels of p38 ERK and JNK
6 BioMed Research International
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
IL-1120573
mRN
A le
vel
02468
1012141618202224
0
2
4
6
8
10
12
14
16
TNF-
120572m
RNA
leve
l
0123456789
10
IL-6
mRN
A le
vel
0
2
4
6
8
10
12
14
16
IL-10
mRN
A le
vel
lowast
lowastlowast
lowast
lowast
lowastlowast
lowast lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
0
500
1000
1500
2000
2500
3000
3500
4000
IL-10
(pg
mL)
0
500
1000
1500
2000
2500
IL-6
(pg
mL)
0500
1000150020002500300035004000450050005500
TNF-
120572(p
gm
L)
0500
100015002000250030003500400045005000550060006500700075008000
IL-1120573
(pg
mL) lowast
lowast
lowastlowast
lowast lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowastlowast
lowast
(b)
Figure 4 Cytokine concentrations (a) TNF-120572 IL-1120573 IL-6 and IL-10 mRNA levels in the uterine tissue (b) TNF-120572 IL-1120573 IL-6 and IL-10protein levels in the uterine tissueThe data represent the contents of 1mL supernatant of uterine homogenate and are presented as the meansplusmn SD (119899 = 10) The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that were not given anydrug treatment 2 4 and 8 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasone treatmentgroup lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
BioMed Research International 7
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
0
1
2
3
4
5
6
7
8
9
TLR2
mRN
A le
vel
(a)CG S aureus248
CG S aureus248
lowast
lowast
lowast
lowast
TLR2
120573-Actin
0
500
1000
1500
2000
2500
3000
3500
4000
IL-8
(pg
mL)
(b)
Figure 5 Effects of IFN-120591 on TLR2 (a) The TLR2 mRNA levels in the uterine tissues (b) The TLR2 and IL-8 protein levels in HEK293-mTLR2 cells qPCR was performed to assess the mRNA levels of TLR2 Western blotting was performed to detect the protein levels of TLR2120573-Actin was used as a control The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that werenot given any drug treatment 8 4 and 2 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasonetreatment group lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
were significantly increased compared with CG (Figure 7)Additionally the phosphorylation levels of the three MAPKsin themice inoculatedwith S aureuswere significantly higherthan in the mice treated with IFN-120591 These data indicate thatIFN-120591 can suppress the phosphorylation of p38 JNK andERK Compared to the mice inoculated with S aureus in thetreatment groups the level of phosphorylation was reducedsignificantly with increasing concentrations of IFN-120591 Theseresults showed that IFN-120591 can inhibit MAPK p38 JNK andERK phosphorylation in a dose-dependent manner
4 Discussion
IFN-120591 a novel Type I IFN is secreted by the trophoblastectoderm of ruminants in the early stages of pregnancy[22] IFN-120591 regulates the expression of numerous genes inthe endometrium and has been shown to play an impor-tant biological role in the implantation stage of pregnancy[24] Endometritis is a local inflammatory condition whichdelays uterine degeneration and has a significant economicimpact on dairy production [25 26] However infection withmicrobes such as S aureus can cause the destruction of theuterine horn structure subsequently resulting in the loss inthe ability to conceive This has been found to be due toinflammation and necrosis as determined by studies utilizingmorphological and image measurement tools [27ndash29] In thepresent study based on histopathological observations wefound that IFN-120591 has anti-inflammatory functions as well
as a significant protective effect on uterine tissues followinginfection with S aureus
A key component of the host immune response toinfection is the upregulation of cytokine production [30] Tofurther study the effect of IFN-120591 on S aureus endometritisthe levels of TNF-120572 IL-1120573 IL-6 and IL-10 production wereevaluatedThe results showed that the levels of TNF-120572 IL-1120573and IL-6 which are induced by S aureus were significantlyinhibited by IFN-120591 treatment in a dose-dependent mannerDuring the inflammatory response TNF-120572 is induced byregulated genes acting as effective inducers and subsequentlyregulates the expression of cytokines chemokines and celladhesion molecules [31 32] In vivo TNF-120572 can activatethe intracellular I120581B120572 and JNK signaling pathways throughTLRs thus contributing to the production of proinflamma-tory cytokines [33] The proinflammatory cytokine IL-1120573 isa key regulator of acute inflammatory processes in the cen-tral nervous system [34 35] Pathogen-associated molecularpatterns of S aureus can activate inflammatory pathwaysand when bound to innate monocytesmacrophages theyresult in a significant increase in the secretion of TNF-120572 andIL-6 [36 37] IL-6 is a pleiotropic cytokine that regulatesmultiple biological processes including the developmentof inflammation immune responses and the acute phasereaction [38] IL-10 has been shown to be beneficial inimproving systemic immunity by having anti-inflammatoryeffects and promoting the repair of immune dysfunction afterthe invasion of different pathogens [39] Our experimental
8 BioMed Research International
CG S aureusDEX 248
I120581B
p-I120581B
p65
p-p65
120573-Actin
(a)CG S aureusDEX 248
CG S aureusDEX 248
lowastlowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
pNF-
120581B
120573-a
ctin
ratio
0
02
04
06
08
1
12
14
16
18
2
22
24
0
02
04
06
08
1
12
14
p-I120581
B120573
-act
in ra
tio
(b)
Figure 6 Effect of IFN-120591 on NF-120581B activation (a) The NF-120581B and I-120581B protein levels in uterine tissues (b) The ratio of phosphorylationproteins levels as well as 120573-actin levels Western blot was performed to detect the levels of total and phosphorylated NF-120581B and I-120581B proteinsin the control group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups(2 4 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
results showed that the expression of IL-10 mRNA andprotein was increased in mice inoculated with S aureusand the expression of IL-10 was further increased with IFN-120591 treatment With increasing concentrations of IFN-120591 theeffect on IL-10 was more significant IFN-120591 can promotethe secretion of inflammatory cytokines including IL-10thereby inhibiting the subsequent secretion of TNF-120572 IL-1120573and IL-6 [40] TLRs are transmembrane pattern recognitionreceptors which are part of the innate immune systemand are the key element in identifying viral and bacterialcomponents [37 41] TLR2 senses the inflammatory reactionscaused by S aureusmost rapidly and sensitively and presentspathogen-derived antigens to various types of cells [42]Our results showed TLR2 increased in mice infected withS aureus and its secretion was significantly reduced afterIFN-120591 treatment TLR2 plays a key role in the responseto both innate and adaptive immunity as it can identifyexogenous pathogen-associated molecular patterns (PAMPs)
and induce proinflammatory signaling pathways [43] In thisstudy IFN-120591 inhibited the secretion of TLR2 in a dose-dependent manner
NF-120581B and MAPKs are two important inflammation-associated pathways and they canmodulate the developmentof inflammation by mediating downstream signaling net-works [44] To further investigate the mechanism by whichIFN-120591 affects inflammation we analyzed the NF-120581B andMAPK pathways The NF-120581B signaling pathway is involvedin the regulation of genes that mediate inflammation as wellas various cytokines and is associatedwith numerous chronicinflammatory diseases [45] Normally theNF-120581Bp65 subunitis bound to I120581B in the cytosol When stimulated by variouspathogens NF-120581B p65 is released due to the phosphorylationof I120581B leading to its activation and nuclear translocationwhich then triggers inflammation [46] Western blottinganalysis showed the phosphorylation levels of I120581B120572 and p65in mice infected with S aureus were significantly higher than
BioMed Research International 9
CG S aureusDEX 248
p38
p-p38
120573-Actin
ERK
p-ERK
JNK
p-JNK
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
p-JN
K120573
-act
in ra
tio
0
02
04
06
08
1
p-p3
8120573
-act
in ra
tio
0
02
04
06
08
1
12
0
02
04
06
08
1
12
14
16
18
p-ER
K120573
-act
in ra
tio
(b)
Figure 7 Effect of IFN-120591 on MAPK activation (a) The protein levels of MAPKs in uterine tissues (b) The ratio of phosphorylation proteinsas well as 120573-actin levels Western blotting was performed to measure the levels of total and phosphorylated p38 ERK and JNK protein in theControl group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups (24 and 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
10 BioMed Research International
that in the IFN-120591 groups These results indicated that IFN-120591 can inhibit the phosphorylation of I120581B120572 and p65 therebyblocking NF-120581B activation In addition to NF-120581B MAPKsare another key regulator of signal transduction pathwaysthat can modulate the levels of inflammatory mediators [47]Specifically p38 JNK and ERK are the major effectors of theMAPK pathwayThe phosphorylation of these three proteinscan cause the translocation of Activator Protein-1 (AP-1) tothe nucleus thereby promoting the inflammatory response[48 49] In the current study we found an increase in thephosphorylation levels of p38 JNK and ERK in the miceinoculated with S aureus However in the groups treatedwith IFN-120591 the phosphorylation of these proteins was signif-icantly inhibited Recent studies have indicated that the inhi-bition of phosphorylation of MAPKs with specific inhibitorscould significantly attenuate inflammation by decreasingthe number of neutrophils Similar effects were observedfollowing the inhibition of proinflammatory cytokines andchemokines [50] In our study we demonstrated that IFN-120591 significantly inhibited the phosphorylation of proteinsinvolved in MAPK pathway activation Taken together ourresults showed that blocking the NF-120581B andMAPK signalingpathways and inhibiting the secretion of proinflammatorycytokines are the main mechanisms by which IFN-120591 abatesendometritis
5 Conclusion
Our study showed that S aureus increases the expression ofTNF-120572 IL-1120573 and IL-6 thereby activating TLR2 signalingpathways including NF-120581B and MAPK The experimentspresented herein show that IFN-120591 reduces the secretion ofproinflammatory cytokines such as TNF-120572 IL-1120573 and IL-6 while promoting the production of the anti-inflammatorycytokine IL-10 This reduction inhibits inflammation andenhances the repair of uterine tissues In addition IFN-120591 inhibited the expression of TLR2 thereby blocking theactivation of the NF-120581B and MAPK signaling pathwaysand abating endometritis Additional studies are requiredto validate the efficacy of IFN-120591 as a treatment for uterineinfection due to S aureus or other infectious pathogens
Competing Interests
The authors have no conflict of interest to declare
Acknowledgments
This work was supported by a grant from the Funda-mental Research Funds for the Central Universities (no2662014BQ024) the National Natural Science Foundationof China (nos 31272631 31472254 and 31502130) andUndergraduate Special Science and Technology Innovationof Huazhong Agricultural University (no 2015BC009)
References
[1] B Polat M Cengiz O Cannazik et al ldquoEndometrial echotex-ture variables in postpartum cows with subclinical endometri-tisrdquo Animal Reproduction Science vol 155 pp 50ndash55 2015
[2] J Roberson D Moll and G Saunders ldquoChronic Staphylococcusaureus endometritis in a virgin giltrdquo Veterinary Record vol 161no 24 pp 821ndash822 2007
[3] M Guo G Wang T Lv et al ldquoEndometrial inflammation andabnormal expression of extracellular matrix proteins inducedbyMycoplasma bovis in dairy cowsrdquoTheriogenology vol 81 no5 pp 669ndash674 2014
[4] I M Sheldon J Cronin L Goetze G Donofrio and H-J Schuberth ldquoDefining postpartum uterine disease and themechanisms of infection and immunity in the female reproduc-tive tract in cattlerdquo Biology of Reproduction vol 81 no 6 pp1025ndash1032 2009
[5] P W Farin L Ball J D Olson et al ldquoEffect of Actinomyces pyo-genes and gram-negative anaerobic bacteria on the developmentof bovine pyometrardquoTheriogenology vol 31 no 5 pp 979ndash9891989
[6] M Guo Y Cao TWang et al ldquoBaicalin inhibits Staphylococcusaureus-induced apoptosis by regulating TLR2 andTLR2-relatedapoptotic factors in the mouse mammary glandsrdquo EuropeanJournal of Pharmacology vol 723 no 1 pp 481ndash488 2014
[7] A K Syed T J Reed K L Clark B R Boles and J MKahlenberg ldquoStaphlyococcus aureus phenol-soluble modulinsstimulate the release of proinflammatory cytokines from ker-atinocytes and are required for induction of skin inflammationrdquoInfection and Immunity vol 83 no 9 pp 3428ndash3437 2015
[8] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[9] T W Chon and S Bixler ldquoInterferon-tau current applicationsand potential in antiviral therapyrdquo Journal of Interferon ampCytokine Research vol 30 no 7 pp 477ndash485 2010
[10] C H Pontzer T L Ott F W Bazer and H M JohnsonldquoStructurefunction studies with interferon tau evidence formultiple active sitesrdquo Journal of Interferon Research vol 14 no3 pp 133ndash141 1994
[11] R M Roberts ldquoInterferon-tau a Type 1 interferon involved inmaternal recognition of pregnancyrdquo Cytokine amp Growth FactorReviews vol 18 no 5-6 pp 403ndash408 2007
[12] A P Alexenko AD Ealy andRM Roberts ldquoThe cross-speciesantiviral activities of different IFN-tau subtypes on bovinemurine and human cells contradictory evidence for therapeu-tic potentialrdquo Journal of Interferon amp Cytokine Research vol 19no 12 pp 1335ndash1341 1999
[13] J A Neira D Tainturier R M LrsquoHaridon and J Martal ldquoCom-parative IFN-tau secretion after hatching by bovine blastocystsderived ex vivo and completely produced in vitrordquoReproductionin Domestic Animals vol 42 no 1 pp 68ndash75 2007
[14] A D Ealy J A Green A P Alexenko D H Keisler and R MRoberts ldquoDifferent ovine interferon-tau genes are not expressedidentically and their protein products display different activi-tiesrdquo Biology of Reproduction vol 58 no 2 pp 566ndash573 1998
[15] K A Naivar S K Ward K J Austin D W Moore and T RHansen ldquoSecretion of bovine uterine proteins in response toType I interferonsrdquo Biology of Reproduction vol 52 no 4 pp848ndash854 1995
[16] M G Teixeira K J Austin D J Perry et al ldquoBovine granulo-cyte chemotactic protein-2 is secreted by the endometrium inresponse to interferon-tau (IFN-tau)rdquo Endocrine vol 6 no 1pp 31ndash37 1997
BioMed Research International 11
[17] G Guarda M Braun F Staehli et al ldquoType I interferon inhibitsinterleukin-1 production and inflammasome activationrdquo Immu-nity vol 34 no 2 pp 213ndash223 2011
[18] T J Foster J A Geoghegan V K Ganesh and M HookldquoAdhesion invasion and evasion the many functions of thesurface proteins of Staphylococcus aureusrdquo Nature ReviewsMicrobiology vol 12 no 1 pp 49ndash62 2014
[19] B A Diep S R Gill R F Chang et al ldquoComplete genomesequence of USA300 an epidemic clone of community-acquiredmeticillin-resistant Staphylococcus aureusrdquoTheLancetvol 367 no 9512 pp 731ndash739 2006
[20] D Wang N Xu Z Zhang et al ldquoSophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstreampathways on LPS-induced mastitis in the mammary gland ofmicerdquo International Immunopharmacology vol 35 pp 111ndash1182016
[21] J-L Zhao Y-X Ding H-X Zhao et al ldquoPresence of super-antigen genes and antimicrobial resistance in Staphylococcusisolates obtained from the uteri of dairy cows with clinicalendometritisrdquo Veterinary Record vol 175 no 14 article 3522014
[22] D K Tennakoon R Smith M D Stewart T E Spencer MNayak and C J Welsh ldquoOvine IFN-tau modulates the expres-sion of MHC antigens on murine cerebrovascular endothelialcells and inhibits replication of Theilerrsquos virusrdquo Journal ofInterferonampCytokine Research vol 21 no 10 pp 785ndash792 2001
[23] J M Soos P S Subramaniam A C Hobeika J Schiffen-bauer and H M Johnson ldquoThe IFN pregnancy recognitionhormone IFN-tau blocks both development and superantigenreactivation of experimental allergic encephalomyelitis withoutassociated toxicityrdquo The Journal of Immunology vol 155 no 5pp 2747ndash2753 1995
[24] G Song T E Spencer and FW Bazer ldquoCathepsins in the ovineuterus regulation by pregnancy progesterone and interferontaurdquo Endocrinology vol 146 no 11 pp 4825ndash4833 2005
[25] R Grio C Giobbe A Cellula et al ldquoInflammation of theuterine corpus endometritisrdquoMinervaGinecologica vol 42 no4 pp 99ndash102 1990
[26] I M Sheldon and H Dobson ldquoPostpartum uterine health incattlerdquo Animal Reproduction Science vol 82-83 pp 295ndash3062004
[27] S A Kurganov ldquoUterine eosinophils and infertility in theratrdquo Rossiiskii Fiziologicheskii Zhurnal Imeni IM Sechen-ovaRossiiskaia Akademiia Nauk vol 96 no 2 pp 138ndash1462010
[28] K Shirasuna F Usui T Karasawa et al ldquoNanosilica-inducedplacental inflammation and pregnancy complications differentroles of the inflammasome components NLRP3 and ASCrdquoNanotoxicology vol 9 no 5 pp 554ndash567 2015
[29] Y Liu C QiuW Li WMu C Li andM Guo ldquoSelenium playsa protective role in Staphylococcus aureus-induced endometritisin the uterine tissue of ratsrdquo Biological Trace Element Researchvol 173 no 2 pp 345ndash353 2016
[30] A A M Lima L G Spınola G Baccan et al ldquoEvaluation ofcorticosterone and IL-1120573 IL-6 IL-10 and TNF-120572 expressionafter 670-nm laser photobiomodulation in ratsrdquo Lasers inMedical Science vol 29 no 2 pp 709ndash715 2014
[31] C-J Liang S-H Wang Y-H Chen et al ldquoViscolin reducesVCAM-1 expression in TNF-120572-treated endothelial cells viathe JNKNF-120581B and ROS pathwayrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1337ndash1346 2011
[32] H-K Tsou H-T Chen C-H Chang W-Y Yang and C-HTang ldquoApoptosis signal-regulating kinase 1 is mediated in TNF-120572-induced CCL2 expression in human synovial fibroblastsrdquoJournal of Cellular Biochemistry vol 113 no 11 pp 3509ndash35192012
[33] H Zhang J Kovacs-Nolan T Kodera Y Eto and Y Mine ldquo120574-Glutamyl cysteine and 120574-glutamyl valine inhibit TNF-120572 signal-ing in intestinal epithelial cells and reduce inflammation in amouse model of colitis via allosteric activation of the calcium-sensing receptorrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1852 no 5 pp 792ndash804 2015
[34] M L Diamond J A Boles A C Ritter et al ldquoIn response tocomments on IL-1120573 associations with posttraumatic epilepsydevelopment a genetics and biomarker cohort studyrdquo Epilepsiavol 55 no 8 pp 1313ndash1314 2014
[35] A Vezzani J French T Bartfai and T Z Baram ldquoThe role ofinflammation in epilepsyrdquo Nature Reviews Neurology vol 7 no1 pp 31ndash40 2011
[36] B Fournier and D J Philpott ldquoRecognition of Staphylococcusaureus by the innate immune systemrdquo Clinical MicrobiologyReviews vol 18 no 3 pp 521ndash540 2005
[37] J E Wang P F Joslashrgensen M Almlof et al ldquoPeptidoglycanand lipoteichoic acid from Staphylococcus aureus induce tumornecrosis factor alpha interleukin 6 (IL-6) and IL-10 productionin both T cells and monocytes in a human whole blood modelrdquoInfection and Immunity vol 68 no 7 pp 3965ndash3970 2000
[38] D Zhu D-Y Yang Y-Y Guo et al ldquoIntracameral interleukin1120573 6 8 10 12p tumor necrosis factor 120572 and vascular endothelialgrowth factor and axial length in patients with cataractrdquo PLoSONE vol 10 no 2 Article ID e0117777 2015
[39] P Lichte R Pfeifer P Kobbe et al ldquoInhalative IL-10 treatmentafter bilateral femoral fractures affect pulmonary inflammationin micerdquo Annals of Anatomy vol 200 pp 73ndash78 2015
[40] K Hara K Shirasuna F Usui et al ldquoInterferon-tau attenuatesuptake of nanoparticles and secretion of interleukin-1120573 inmacrophagesrdquoPLoSONE vol 9 no 12 Article ID e113974 2014
[41] A Mansson Kvarnhammar L Tengroth M Adner and L-OCardell ldquoInnate immune receptors in human airway smoothmuscle cells activation by TLR12 TLR3 TLR4 TLR7 andNOD1 agonistsrdquo PLoSONE vol 8 no 7 Article ID e68701 2013
[42] B Fournier ldquoThe function of TLR2 during staphylococcaldiseasesrdquo Frontiers in Cellular and Infection Microbiology vol2 article 167 2013
[43] L-H Ding D Liu M Xu et al ldquoTLR2ndashMyD88ndashNF-120581Bpathway is involved in tubulointerstitial inflammation causedby proteinuriardquoThe International Journal of Biochemistry amp CellBiology vol 69 pp 114ndash120 2015
[44] Z Zhang X Gao Y Cao et al ldquoSelenium deficiency facilitatesinflammation through the regulation of TLR4 and TLR4-related signaling pathways in the mice uterusrdquo Inflammationvol 38 no 3 pp 1347ndash1356 2015
[45] C Xu G Shen C Chen C Gelinas and A-N T KongldquoSuppression of NF-120581B and NF-120581B-regulated gene expressionby sulforaphane and PEITC through I120581B120572 IKK pathway inhuman prostate cancer PC-3 cellsrdquo Oncogene vol 24 no 28pp 4486ndash4495 2005
[46] W Zhang R Zhang T Wang et al ldquoSelenium inhibits LPS-induced pro-inflammatory gene expression by modulatingMAPK and NF-120581B signaling pathways in mouse mammaryepithelial cells in primary culturerdquo Inflammation vol 37 no 2pp 478ndash485 2014
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
6 BioMed Research International
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
IL-1120573
mRN
A le
vel
02468
1012141618202224
0
2
4
6
8
10
12
14
16
TNF-
120572m
RNA
leve
l
0123456789
10
IL-6
mRN
A le
vel
0
2
4
6
8
10
12
14
16
IL-10
mRN
A le
vel
lowast
lowastlowast
lowast
lowast
lowastlowast
lowast lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
0
500
1000
1500
2000
2500
3000
3500
4000
IL-10
(pg
mL)
0
500
1000
1500
2000
2500
IL-6
(pg
mL)
0500
1000150020002500300035004000450050005500
TNF-
120572(p
gm
L)
0500
100015002000250030003500400045005000550060006500700075008000
IL-1120573
(pg
mL) lowast
lowast
lowastlowast
lowast lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowastlowast
lowast
(b)
Figure 4 Cytokine concentrations (a) TNF-120572 IL-1120573 IL-6 and IL-10 mRNA levels in the uterine tissue (b) TNF-120572 IL-1120573 IL-6 and IL-10protein levels in the uterine tissueThe data represent the contents of 1mL supernatant of uterine homogenate and are presented as the meansplusmn SD (119899 = 10) The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that were not given anydrug treatment 2 4 and 8 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasone treatmentgroup lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
BioMed Research International 7
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
0
1
2
3
4
5
6
7
8
9
TLR2
mRN
A le
vel
(a)CG S aureus248
CG S aureus248
lowast
lowast
lowast
lowast
TLR2
120573-Actin
0
500
1000
1500
2000
2500
3000
3500
4000
IL-8
(pg
mL)
(b)
Figure 5 Effects of IFN-120591 on TLR2 (a) The TLR2 mRNA levels in the uterine tissues (b) The TLR2 and IL-8 protein levels in HEK293-mTLR2 cells qPCR was performed to assess the mRNA levels of TLR2 Western blotting was performed to detect the protein levels of TLR2120573-Actin was used as a control The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that werenot given any drug treatment 8 4 and 2 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasonetreatment group lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
were significantly increased compared with CG (Figure 7)Additionally the phosphorylation levels of the three MAPKsin themice inoculatedwith S aureuswere significantly higherthan in the mice treated with IFN-120591 These data indicate thatIFN-120591 can suppress the phosphorylation of p38 JNK andERK Compared to the mice inoculated with S aureus in thetreatment groups the level of phosphorylation was reducedsignificantly with increasing concentrations of IFN-120591 Theseresults showed that IFN-120591 can inhibit MAPK p38 JNK andERK phosphorylation in a dose-dependent manner
4 Discussion
IFN-120591 a novel Type I IFN is secreted by the trophoblastectoderm of ruminants in the early stages of pregnancy[22] IFN-120591 regulates the expression of numerous genes inthe endometrium and has been shown to play an impor-tant biological role in the implantation stage of pregnancy[24] Endometritis is a local inflammatory condition whichdelays uterine degeneration and has a significant economicimpact on dairy production [25 26] However infection withmicrobes such as S aureus can cause the destruction of theuterine horn structure subsequently resulting in the loss inthe ability to conceive This has been found to be due toinflammation and necrosis as determined by studies utilizingmorphological and image measurement tools [27ndash29] In thepresent study based on histopathological observations wefound that IFN-120591 has anti-inflammatory functions as well
as a significant protective effect on uterine tissues followinginfection with S aureus
A key component of the host immune response toinfection is the upregulation of cytokine production [30] Tofurther study the effect of IFN-120591 on S aureus endometritisthe levels of TNF-120572 IL-1120573 IL-6 and IL-10 production wereevaluatedThe results showed that the levels of TNF-120572 IL-1120573and IL-6 which are induced by S aureus were significantlyinhibited by IFN-120591 treatment in a dose-dependent mannerDuring the inflammatory response TNF-120572 is induced byregulated genes acting as effective inducers and subsequentlyregulates the expression of cytokines chemokines and celladhesion molecules [31 32] In vivo TNF-120572 can activatethe intracellular I120581B120572 and JNK signaling pathways throughTLRs thus contributing to the production of proinflamma-tory cytokines [33] The proinflammatory cytokine IL-1120573 isa key regulator of acute inflammatory processes in the cen-tral nervous system [34 35] Pathogen-associated molecularpatterns of S aureus can activate inflammatory pathwaysand when bound to innate monocytesmacrophages theyresult in a significant increase in the secretion of TNF-120572 andIL-6 [36 37] IL-6 is a pleiotropic cytokine that regulatesmultiple biological processes including the developmentof inflammation immune responses and the acute phasereaction [38] IL-10 has been shown to be beneficial inimproving systemic immunity by having anti-inflammatoryeffects and promoting the repair of immune dysfunction afterthe invasion of different pathogens [39] Our experimental
8 BioMed Research International
CG S aureusDEX 248
I120581B
p-I120581B
p65
p-p65
120573-Actin
(a)CG S aureusDEX 248
CG S aureusDEX 248
lowastlowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
pNF-
120581B
120573-a
ctin
ratio
0
02
04
06
08
1
12
14
16
18
2
22
24
0
02
04
06
08
1
12
14
p-I120581
B120573
-act
in ra
tio
(b)
Figure 6 Effect of IFN-120591 on NF-120581B activation (a) The NF-120581B and I-120581B protein levels in uterine tissues (b) The ratio of phosphorylationproteins levels as well as 120573-actin levels Western blot was performed to detect the levels of total and phosphorylated NF-120581B and I-120581B proteinsin the control group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups(2 4 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
results showed that the expression of IL-10 mRNA andprotein was increased in mice inoculated with S aureusand the expression of IL-10 was further increased with IFN-120591 treatment With increasing concentrations of IFN-120591 theeffect on IL-10 was more significant IFN-120591 can promotethe secretion of inflammatory cytokines including IL-10thereby inhibiting the subsequent secretion of TNF-120572 IL-1120573and IL-6 [40] TLRs are transmembrane pattern recognitionreceptors which are part of the innate immune systemand are the key element in identifying viral and bacterialcomponents [37 41] TLR2 senses the inflammatory reactionscaused by S aureusmost rapidly and sensitively and presentspathogen-derived antigens to various types of cells [42]Our results showed TLR2 increased in mice infected withS aureus and its secretion was significantly reduced afterIFN-120591 treatment TLR2 plays a key role in the responseto both innate and adaptive immunity as it can identifyexogenous pathogen-associated molecular patterns (PAMPs)
and induce proinflammatory signaling pathways [43] In thisstudy IFN-120591 inhibited the secretion of TLR2 in a dose-dependent manner
NF-120581B and MAPKs are two important inflammation-associated pathways and they canmodulate the developmentof inflammation by mediating downstream signaling net-works [44] To further investigate the mechanism by whichIFN-120591 affects inflammation we analyzed the NF-120581B andMAPK pathways The NF-120581B signaling pathway is involvedin the regulation of genes that mediate inflammation as wellas various cytokines and is associatedwith numerous chronicinflammatory diseases [45] Normally theNF-120581Bp65 subunitis bound to I120581B in the cytosol When stimulated by variouspathogens NF-120581B p65 is released due to the phosphorylationof I120581B leading to its activation and nuclear translocationwhich then triggers inflammation [46] Western blottinganalysis showed the phosphorylation levels of I120581B120572 and p65in mice infected with S aureus were significantly higher than
BioMed Research International 9
CG S aureusDEX 248
p38
p-p38
120573-Actin
ERK
p-ERK
JNK
p-JNK
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
p-JN
K120573
-act
in ra
tio
0
02
04
06
08
1
p-p3
8120573
-act
in ra
tio
0
02
04
06
08
1
12
0
02
04
06
08
1
12
14
16
18
p-ER
K120573
-act
in ra
tio
(b)
Figure 7 Effect of IFN-120591 on MAPK activation (a) The protein levels of MAPKs in uterine tissues (b) The ratio of phosphorylation proteinsas well as 120573-actin levels Western blotting was performed to measure the levels of total and phosphorylated p38 ERK and JNK protein in theControl group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups (24 and 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
10 BioMed Research International
that in the IFN-120591 groups These results indicated that IFN-120591 can inhibit the phosphorylation of I120581B120572 and p65 therebyblocking NF-120581B activation In addition to NF-120581B MAPKsare another key regulator of signal transduction pathwaysthat can modulate the levels of inflammatory mediators [47]Specifically p38 JNK and ERK are the major effectors of theMAPK pathwayThe phosphorylation of these three proteinscan cause the translocation of Activator Protein-1 (AP-1) tothe nucleus thereby promoting the inflammatory response[48 49] In the current study we found an increase in thephosphorylation levels of p38 JNK and ERK in the miceinoculated with S aureus However in the groups treatedwith IFN-120591 the phosphorylation of these proteins was signif-icantly inhibited Recent studies have indicated that the inhi-bition of phosphorylation of MAPKs with specific inhibitorscould significantly attenuate inflammation by decreasingthe number of neutrophils Similar effects were observedfollowing the inhibition of proinflammatory cytokines andchemokines [50] In our study we demonstrated that IFN-120591 significantly inhibited the phosphorylation of proteinsinvolved in MAPK pathway activation Taken together ourresults showed that blocking the NF-120581B andMAPK signalingpathways and inhibiting the secretion of proinflammatorycytokines are the main mechanisms by which IFN-120591 abatesendometritis
5 Conclusion
Our study showed that S aureus increases the expression ofTNF-120572 IL-1120573 and IL-6 thereby activating TLR2 signalingpathways including NF-120581B and MAPK The experimentspresented herein show that IFN-120591 reduces the secretion ofproinflammatory cytokines such as TNF-120572 IL-1120573 and IL-6 while promoting the production of the anti-inflammatorycytokine IL-10 This reduction inhibits inflammation andenhances the repair of uterine tissues In addition IFN-120591 inhibited the expression of TLR2 thereby blocking theactivation of the NF-120581B and MAPK signaling pathwaysand abating endometritis Additional studies are requiredto validate the efficacy of IFN-120591 as a treatment for uterineinfection due to S aureus or other infectious pathogens
Competing Interests
The authors have no conflict of interest to declare
Acknowledgments
This work was supported by a grant from the Funda-mental Research Funds for the Central Universities (no2662014BQ024) the National Natural Science Foundationof China (nos 31272631 31472254 and 31502130) andUndergraduate Special Science and Technology Innovationof Huazhong Agricultural University (no 2015BC009)
References
[1] B Polat M Cengiz O Cannazik et al ldquoEndometrial echotex-ture variables in postpartum cows with subclinical endometri-tisrdquo Animal Reproduction Science vol 155 pp 50ndash55 2015
[2] J Roberson D Moll and G Saunders ldquoChronic Staphylococcusaureus endometritis in a virgin giltrdquo Veterinary Record vol 161no 24 pp 821ndash822 2007
[3] M Guo G Wang T Lv et al ldquoEndometrial inflammation andabnormal expression of extracellular matrix proteins inducedbyMycoplasma bovis in dairy cowsrdquoTheriogenology vol 81 no5 pp 669ndash674 2014
[4] I M Sheldon J Cronin L Goetze G Donofrio and H-J Schuberth ldquoDefining postpartum uterine disease and themechanisms of infection and immunity in the female reproduc-tive tract in cattlerdquo Biology of Reproduction vol 81 no 6 pp1025ndash1032 2009
[5] P W Farin L Ball J D Olson et al ldquoEffect of Actinomyces pyo-genes and gram-negative anaerobic bacteria on the developmentof bovine pyometrardquoTheriogenology vol 31 no 5 pp 979ndash9891989
[6] M Guo Y Cao TWang et al ldquoBaicalin inhibits Staphylococcusaureus-induced apoptosis by regulating TLR2 andTLR2-relatedapoptotic factors in the mouse mammary glandsrdquo EuropeanJournal of Pharmacology vol 723 no 1 pp 481ndash488 2014
[7] A K Syed T J Reed K L Clark B R Boles and J MKahlenberg ldquoStaphlyococcus aureus phenol-soluble modulinsstimulate the release of proinflammatory cytokines from ker-atinocytes and are required for induction of skin inflammationrdquoInfection and Immunity vol 83 no 9 pp 3428ndash3437 2015
[8] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[9] T W Chon and S Bixler ldquoInterferon-tau current applicationsand potential in antiviral therapyrdquo Journal of Interferon ampCytokine Research vol 30 no 7 pp 477ndash485 2010
[10] C H Pontzer T L Ott F W Bazer and H M JohnsonldquoStructurefunction studies with interferon tau evidence formultiple active sitesrdquo Journal of Interferon Research vol 14 no3 pp 133ndash141 1994
[11] R M Roberts ldquoInterferon-tau a Type 1 interferon involved inmaternal recognition of pregnancyrdquo Cytokine amp Growth FactorReviews vol 18 no 5-6 pp 403ndash408 2007
[12] A P Alexenko AD Ealy andRM Roberts ldquoThe cross-speciesantiviral activities of different IFN-tau subtypes on bovinemurine and human cells contradictory evidence for therapeu-tic potentialrdquo Journal of Interferon amp Cytokine Research vol 19no 12 pp 1335ndash1341 1999
[13] J A Neira D Tainturier R M LrsquoHaridon and J Martal ldquoCom-parative IFN-tau secretion after hatching by bovine blastocystsderived ex vivo and completely produced in vitrordquoReproductionin Domestic Animals vol 42 no 1 pp 68ndash75 2007
[14] A D Ealy J A Green A P Alexenko D H Keisler and R MRoberts ldquoDifferent ovine interferon-tau genes are not expressedidentically and their protein products display different activi-tiesrdquo Biology of Reproduction vol 58 no 2 pp 566ndash573 1998
[15] K A Naivar S K Ward K J Austin D W Moore and T RHansen ldquoSecretion of bovine uterine proteins in response toType I interferonsrdquo Biology of Reproduction vol 52 no 4 pp848ndash854 1995
[16] M G Teixeira K J Austin D J Perry et al ldquoBovine granulo-cyte chemotactic protein-2 is secreted by the endometrium inresponse to interferon-tau (IFN-tau)rdquo Endocrine vol 6 no 1pp 31ndash37 1997
BioMed Research International 11
[17] G Guarda M Braun F Staehli et al ldquoType I interferon inhibitsinterleukin-1 production and inflammasome activationrdquo Immu-nity vol 34 no 2 pp 213ndash223 2011
[18] T J Foster J A Geoghegan V K Ganesh and M HookldquoAdhesion invasion and evasion the many functions of thesurface proteins of Staphylococcus aureusrdquo Nature ReviewsMicrobiology vol 12 no 1 pp 49ndash62 2014
[19] B A Diep S R Gill R F Chang et al ldquoComplete genomesequence of USA300 an epidemic clone of community-acquiredmeticillin-resistant Staphylococcus aureusrdquoTheLancetvol 367 no 9512 pp 731ndash739 2006
[20] D Wang N Xu Z Zhang et al ldquoSophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstreampathways on LPS-induced mastitis in the mammary gland ofmicerdquo International Immunopharmacology vol 35 pp 111ndash1182016
[21] J-L Zhao Y-X Ding H-X Zhao et al ldquoPresence of super-antigen genes and antimicrobial resistance in Staphylococcusisolates obtained from the uteri of dairy cows with clinicalendometritisrdquo Veterinary Record vol 175 no 14 article 3522014
[22] D K Tennakoon R Smith M D Stewart T E Spencer MNayak and C J Welsh ldquoOvine IFN-tau modulates the expres-sion of MHC antigens on murine cerebrovascular endothelialcells and inhibits replication of Theilerrsquos virusrdquo Journal ofInterferonampCytokine Research vol 21 no 10 pp 785ndash792 2001
[23] J M Soos P S Subramaniam A C Hobeika J Schiffen-bauer and H M Johnson ldquoThe IFN pregnancy recognitionhormone IFN-tau blocks both development and superantigenreactivation of experimental allergic encephalomyelitis withoutassociated toxicityrdquo The Journal of Immunology vol 155 no 5pp 2747ndash2753 1995
[24] G Song T E Spencer and FW Bazer ldquoCathepsins in the ovineuterus regulation by pregnancy progesterone and interferontaurdquo Endocrinology vol 146 no 11 pp 4825ndash4833 2005
[25] R Grio C Giobbe A Cellula et al ldquoInflammation of theuterine corpus endometritisrdquoMinervaGinecologica vol 42 no4 pp 99ndash102 1990
[26] I M Sheldon and H Dobson ldquoPostpartum uterine health incattlerdquo Animal Reproduction Science vol 82-83 pp 295ndash3062004
[27] S A Kurganov ldquoUterine eosinophils and infertility in theratrdquo Rossiiskii Fiziologicheskii Zhurnal Imeni IM Sechen-ovaRossiiskaia Akademiia Nauk vol 96 no 2 pp 138ndash1462010
[28] K Shirasuna F Usui T Karasawa et al ldquoNanosilica-inducedplacental inflammation and pregnancy complications differentroles of the inflammasome components NLRP3 and ASCrdquoNanotoxicology vol 9 no 5 pp 554ndash567 2015
[29] Y Liu C QiuW Li WMu C Li andM Guo ldquoSelenium playsa protective role in Staphylococcus aureus-induced endometritisin the uterine tissue of ratsrdquo Biological Trace Element Researchvol 173 no 2 pp 345ndash353 2016
[30] A A M Lima L G Spınola G Baccan et al ldquoEvaluation ofcorticosterone and IL-1120573 IL-6 IL-10 and TNF-120572 expressionafter 670-nm laser photobiomodulation in ratsrdquo Lasers inMedical Science vol 29 no 2 pp 709ndash715 2014
[31] C-J Liang S-H Wang Y-H Chen et al ldquoViscolin reducesVCAM-1 expression in TNF-120572-treated endothelial cells viathe JNKNF-120581B and ROS pathwayrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1337ndash1346 2011
[32] H-K Tsou H-T Chen C-H Chang W-Y Yang and C-HTang ldquoApoptosis signal-regulating kinase 1 is mediated in TNF-120572-induced CCL2 expression in human synovial fibroblastsrdquoJournal of Cellular Biochemistry vol 113 no 11 pp 3509ndash35192012
[33] H Zhang J Kovacs-Nolan T Kodera Y Eto and Y Mine ldquo120574-Glutamyl cysteine and 120574-glutamyl valine inhibit TNF-120572 signal-ing in intestinal epithelial cells and reduce inflammation in amouse model of colitis via allosteric activation of the calcium-sensing receptorrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1852 no 5 pp 792ndash804 2015
[34] M L Diamond J A Boles A C Ritter et al ldquoIn response tocomments on IL-1120573 associations with posttraumatic epilepsydevelopment a genetics and biomarker cohort studyrdquo Epilepsiavol 55 no 8 pp 1313ndash1314 2014
[35] A Vezzani J French T Bartfai and T Z Baram ldquoThe role ofinflammation in epilepsyrdquo Nature Reviews Neurology vol 7 no1 pp 31ndash40 2011
[36] B Fournier and D J Philpott ldquoRecognition of Staphylococcusaureus by the innate immune systemrdquo Clinical MicrobiologyReviews vol 18 no 3 pp 521ndash540 2005
[37] J E Wang P F Joslashrgensen M Almlof et al ldquoPeptidoglycanand lipoteichoic acid from Staphylococcus aureus induce tumornecrosis factor alpha interleukin 6 (IL-6) and IL-10 productionin both T cells and monocytes in a human whole blood modelrdquoInfection and Immunity vol 68 no 7 pp 3965ndash3970 2000
[38] D Zhu D-Y Yang Y-Y Guo et al ldquoIntracameral interleukin1120573 6 8 10 12p tumor necrosis factor 120572 and vascular endothelialgrowth factor and axial length in patients with cataractrdquo PLoSONE vol 10 no 2 Article ID e0117777 2015
[39] P Lichte R Pfeifer P Kobbe et al ldquoInhalative IL-10 treatmentafter bilateral femoral fractures affect pulmonary inflammationin micerdquo Annals of Anatomy vol 200 pp 73ndash78 2015
[40] K Hara K Shirasuna F Usui et al ldquoInterferon-tau attenuatesuptake of nanoparticles and secretion of interleukin-1120573 inmacrophagesrdquoPLoSONE vol 9 no 12 Article ID e113974 2014
[41] A Mansson Kvarnhammar L Tengroth M Adner and L-OCardell ldquoInnate immune receptors in human airway smoothmuscle cells activation by TLR12 TLR3 TLR4 TLR7 andNOD1 agonistsrdquo PLoSONE vol 8 no 7 Article ID e68701 2013
[42] B Fournier ldquoThe function of TLR2 during staphylococcaldiseasesrdquo Frontiers in Cellular and Infection Microbiology vol2 article 167 2013
[43] L-H Ding D Liu M Xu et al ldquoTLR2ndashMyD88ndashNF-120581Bpathway is involved in tubulointerstitial inflammation causedby proteinuriardquoThe International Journal of Biochemistry amp CellBiology vol 69 pp 114ndash120 2015
[44] Z Zhang X Gao Y Cao et al ldquoSelenium deficiency facilitatesinflammation through the regulation of TLR4 and TLR4-related signaling pathways in the mice uterusrdquo Inflammationvol 38 no 3 pp 1347ndash1356 2015
[45] C Xu G Shen C Chen C Gelinas and A-N T KongldquoSuppression of NF-120581B and NF-120581B-regulated gene expressionby sulforaphane and PEITC through I120581B120572 IKK pathway inhuman prostate cancer PC-3 cellsrdquo Oncogene vol 24 no 28pp 4486ndash4495 2005
[46] W Zhang R Zhang T Wang et al ldquoSelenium inhibits LPS-induced pro-inflammatory gene expression by modulatingMAPK and NF-120581B signaling pathways in mouse mammaryepithelial cells in primary culturerdquo Inflammation vol 37 no 2pp 478ndash485 2014
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
BioMed Research International 7
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
0
1
2
3
4
5
6
7
8
9
TLR2
mRN
A le
vel
(a)CG S aureus248
CG S aureus248
lowast
lowast
lowast
lowast
TLR2
120573-Actin
0
500
1000
1500
2000
2500
3000
3500
4000
IL-8
(pg
mL)
(b)
Figure 5 Effects of IFN-120591 on TLR2 (a) The TLR2 mRNA levels in the uterine tissues (b) The TLR2 and IL-8 protein levels in HEK293-mTLR2 cells qPCR was performed to assess the mRNA levels of TLR2 Western blotting was performed to detect the protein levels of TLR2120573-Actin was used as a control The CG refers to the control group S aureus refers to the mice with S aureus-induced endometritis that werenot given any drug treatment 8 4 and 2 refer to the IFN-120591 (2 4 and 8mgkg) administration groups and DEX refers to the dexamethasonetreatment group lowast119901 lt 005 significantly different from the CG 119901 lt 005 significantly different from the S aureus group
were significantly increased compared with CG (Figure 7)Additionally the phosphorylation levels of the three MAPKsin themice inoculatedwith S aureuswere significantly higherthan in the mice treated with IFN-120591 These data indicate thatIFN-120591 can suppress the phosphorylation of p38 JNK andERK Compared to the mice inoculated with S aureus in thetreatment groups the level of phosphorylation was reducedsignificantly with increasing concentrations of IFN-120591 Theseresults showed that IFN-120591 can inhibit MAPK p38 JNK andERK phosphorylation in a dose-dependent manner
4 Discussion
IFN-120591 a novel Type I IFN is secreted by the trophoblastectoderm of ruminants in the early stages of pregnancy[22] IFN-120591 regulates the expression of numerous genes inthe endometrium and has been shown to play an impor-tant biological role in the implantation stage of pregnancy[24] Endometritis is a local inflammatory condition whichdelays uterine degeneration and has a significant economicimpact on dairy production [25 26] However infection withmicrobes such as S aureus can cause the destruction of theuterine horn structure subsequently resulting in the loss inthe ability to conceive This has been found to be due toinflammation and necrosis as determined by studies utilizingmorphological and image measurement tools [27ndash29] In thepresent study based on histopathological observations wefound that IFN-120591 has anti-inflammatory functions as well
as a significant protective effect on uterine tissues followinginfection with S aureus
A key component of the host immune response toinfection is the upregulation of cytokine production [30] Tofurther study the effect of IFN-120591 on S aureus endometritisthe levels of TNF-120572 IL-1120573 IL-6 and IL-10 production wereevaluatedThe results showed that the levels of TNF-120572 IL-1120573and IL-6 which are induced by S aureus were significantlyinhibited by IFN-120591 treatment in a dose-dependent mannerDuring the inflammatory response TNF-120572 is induced byregulated genes acting as effective inducers and subsequentlyregulates the expression of cytokines chemokines and celladhesion molecules [31 32] In vivo TNF-120572 can activatethe intracellular I120581B120572 and JNK signaling pathways throughTLRs thus contributing to the production of proinflamma-tory cytokines [33] The proinflammatory cytokine IL-1120573 isa key regulator of acute inflammatory processes in the cen-tral nervous system [34 35] Pathogen-associated molecularpatterns of S aureus can activate inflammatory pathwaysand when bound to innate monocytesmacrophages theyresult in a significant increase in the secretion of TNF-120572 andIL-6 [36 37] IL-6 is a pleiotropic cytokine that regulatesmultiple biological processes including the developmentof inflammation immune responses and the acute phasereaction [38] IL-10 has been shown to be beneficial inimproving systemic immunity by having anti-inflammatoryeffects and promoting the repair of immune dysfunction afterthe invasion of different pathogens [39] Our experimental
8 BioMed Research International
CG S aureusDEX 248
I120581B
p-I120581B
p65
p-p65
120573-Actin
(a)CG S aureusDEX 248
CG S aureusDEX 248
lowastlowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
pNF-
120581B
120573-a
ctin
ratio
0
02
04
06
08
1
12
14
16
18
2
22
24
0
02
04
06
08
1
12
14
p-I120581
B120573
-act
in ra
tio
(b)
Figure 6 Effect of IFN-120591 on NF-120581B activation (a) The NF-120581B and I-120581B protein levels in uterine tissues (b) The ratio of phosphorylationproteins levels as well as 120573-actin levels Western blot was performed to detect the levels of total and phosphorylated NF-120581B and I-120581B proteinsin the control group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups(2 4 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
results showed that the expression of IL-10 mRNA andprotein was increased in mice inoculated with S aureusand the expression of IL-10 was further increased with IFN-120591 treatment With increasing concentrations of IFN-120591 theeffect on IL-10 was more significant IFN-120591 can promotethe secretion of inflammatory cytokines including IL-10thereby inhibiting the subsequent secretion of TNF-120572 IL-1120573and IL-6 [40] TLRs are transmembrane pattern recognitionreceptors which are part of the innate immune systemand are the key element in identifying viral and bacterialcomponents [37 41] TLR2 senses the inflammatory reactionscaused by S aureusmost rapidly and sensitively and presentspathogen-derived antigens to various types of cells [42]Our results showed TLR2 increased in mice infected withS aureus and its secretion was significantly reduced afterIFN-120591 treatment TLR2 plays a key role in the responseto both innate and adaptive immunity as it can identifyexogenous pathogen-associated molecular patterns (PAMPs)
and induce proinflammatory signaling pathways [43] In thisstudy IFN-120591 inhibited the secretion of TLR2 in a dose-dependent manner
NF-120581B and MAPKs are two important inflammation-associated pathways and they canmodulate the developmentof inflammation by mediating downstream signaling net-works [44] To further investigate the mechanism by whichIFN-120591 affects inflammation we analyzed the NF-120581B andMAPK pathways The NF-120581B signaling pathway is involvedin the regulation of genes that mediate inflammation as wellas various cytokines and is associatedwith numerous chronicinflammatory diseases [45] Normally theNF-120581Bp65 subunitis bound to I120581B in the cytosol When stimulated by variouspathogens NF-120581B p65 is released due to the phosphorylationof I120581B leading to its activation and nuclear translocationwhich then triggers inflammation [46] Western blottinganalysis showed the phosphorylation levels of I120581B120572 and p65in mice infected with S aureus were significantly higher than
BioMed Research International 9
CG S aureusDEX 248
p38
p-p38
120573-Actin
ERK
p-ERK
JNK
p-JNK
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
p-JN
K120573
-act
in ra
tio
0
02
04
06
08
1
p-p3
8120573
-act
in ra
tio
0
02
04
06
08
1
12
0
02
04
06
08
1
12
14
16
18
p-ER
K120573
-act
in ra
tio
(b)
Figure 7 Effect of IFN-120591 on MAPK activation (a) The protein levels of MAPKs in uterine tissues (b) The ratio of phosphorylation proteinsas well as 120573-actin levels Western blotting was performed to measure the levels of total and phosphorylated p38 ERK and JNK protein in theControl group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups (24 and 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
10 BioMed Research International
that in the IFN-120591 groups These results indicated that IFN-120591 can inhibit the phosphorylation of I120581B120572 and p65 therebyblocking NF-120581B activation In addition to NF-120581B MAPKsare another key regulator of signal transduction pathwaysthat can modulate the levels of inflammatory mediators [47]Specifically p38 JNK and ERK are the major effectors of theMAPK pathwayThe phosphorylation of these three proteinscan cause the translocation of Activator Protein-1 (AP-1) tothe nucleus thereby promoting the inflammatory response[48 49] In the current study we found an increase in thephosphorylation levels of p38 JNK and ERK in the miceinoculated with S aureus However in the groups treatedwith IFN-120591 the phosphorylation of these proteins was signif-icantly inhibited Recent studies have indicated that the inhi-bition of phosphorylation of MAPKs with specific inhibitorscould significantly attenuate inflammation by decreasingthe number of neutrophils Similar effects were observedfollowing the inhibition of proinflammatory cytokines andchemokines [50] In our study we demonstrated that IFN-120591 significantly inhibited the phosphorylation of proteinsinvolved in MAPK pathway activation Taken together ourresults showed that blocking the NF-120581B andMAPK signalingpathways and inhibiting the secretion of proinflammatorycytokines are the main mechanisms by which IFN-120591 abatesendometritis
5 Conclusion
Our study showed that S aureus increases the expression ofTNF-120572 IL-1120573 and IL-6 thereby activating TLR2 signalingpathways including NF-120581B and MAPK The experimentspresented herein show that IFN-120591 reduces the secretion ofproinflammatory cytokines such as TNF-120572 IL-1120573 and IL-6 while promoting the production of the anti-inflammatorycytokine IL-10 This reduction inhibits inflammation andenhances the repair of uterine tissues In addition IFN-120591 inhibited the expression of TLR2 thereby blocking theactivation of the NF-120581B and MAPK signaling pathwaysand abating endometritis Additional studies are requiredto validate the efficacy of IFN-120591 as a treatment for uterineinfection due to S aureus or other infectious pathogens
Competing Interests
The authors have no conflict of interest to declare
Acknowledgments
This work was supported by a grant from the Funda-mental Research Funds for the Central Universities (no2662014BQ024) the National Natural Science Foundationof China (nos 31272631 31472254 and 31502130) andUndergraduate Special Science and Technology Innovationof Huazhong Agricultural University (no 2015BC009)
References
[1] B Polat M Cengiz O Cannazik et al ldquoEndometrial echotex-ture variables in postpartum cows with subclinical endometri-tisrdquo Animal Reproduction Science vol 155 pp 50ndash55 2015
[2] J Roberson D Moll and G Saunders ldquoChronic Staphylococcusaureus endometritis in a virgin giltrdquo Veterinary Record vol 161no 24 pp 821ndash822 2007
[3] M Guo G Wang T Lv et al ldquoEndometrial inflammation andabnormal expression of extracellular matrix proteins inducedbyMycoplasma bovis in dairy cowsrdquoTheriogenology vol 81 no5 pp 669ndash674 2014
[4] I M Sheldon J Cronin L Goetze G Donofrio and H-J Schuberth ldquoDefining postpartum uterine disease and themechanisms of infection and immunity in the female reproduc-tive tract in cattlerdquo Biology of Reproduction vol 81 no 6 pp1025ndash1032 2009
[5] P W Farin L Ball J D Olson et al ldquoEffect of Actinomyces pyo-genes and gram-negative anaerobic bacteria on the developmentof bovine pyometrardquoTheriogenology vol 31 no 5 pp 979ndash9891989
[6] M Guo Y Cao TWang et al ldquoBaicalin inhibits Staphylococcusaureus-induced apoptosis by regulating TLR2 andTLR2-relatedapoptotic factors in the mouse mammary glandsrdquo EuropeanJournal of Pharmacology vol 723 no 1 pp 481ndash488 2014
[7] A K Syed T J Reed K L Clark B R Boles and J MKahlenberg ldquoStaphlyococcus aureus phenol-soluble modulinsstimulate the release of proinflammatory cytokines from ker-atinocytes and are required for induction of skin inflammationrdquoInfection and Immunity vol 83 no 9 pp 3428ndash3437 2015
[8] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[9] T W Chon and S Bixler ldquoInterferon-tau current applicationsand potential in antiviral therapyrdquo Journal of Interferon ampCytokine Research vol 30 no 7 pp 477ndash485 2010
[10] C H Pontzer T L Ott F W Bazer and H M JohnsonldquoStructurefunction studies with interferon tau evidence formultiple active sitesrdquo Journal of Interferon Research vol 14 no3 pp 133ndash141 1994
[11] R M Roberts ldquoInterferon-tau a Type 1 interferon involved inmaternal recognition of pregnancyrdquo Cytokine amp Growth FactorReviews vol 18 no 5-6 pp 403ndash408 2007
[12] A P Alexenko AD Ealy andRM Roberts ldquoThe cross-speciesantiviral activities of different IFN-tau subtypes on bovinemurine and human cells contradictory evidence for therapeu-tic potentialrdquo Journal of Interferon amp Cytokine Research vol 19no 12 pp 1335ndash1341 1999
[13] J A Neira D Tainturier R M LrsquoHaridon and J Martal ldquoCom-parative IFN-tau secretion after hatching by bovine blastocystsderived ex vivo and completely produced in vitrordquoReproductionin Domestic Animals vol 42 no 1 pp 68ndash75 2007
[14] A D Ealy J A Green A P Alexenko D H Keisler and R MRoberts ldquoDifferent ovine interferon-tau genes are not expressedidentically and their protein products display different activi-tiesrdquo Biology of Reproduction vol 58 no 2 pp 566ndash573 1998
[15] K A Naivar S K Ward K J Austin D W Moore and T RHansen ldquoSecretion of bovine uterine proteins in response toType I interferonsrdquo Biology of Reproduction vol 52 no 4 pp848ndash854 1995
[16] M G Teixeira K J Austin D J Perry et al ldquoBovine granulo-cyte chemotactic protein-2 is secreted by the endometrium inresponse to interferon-tau (IFN-tau)rdquo Endocrine vol 6 no 1pp 31ndash37 1997
BioMed Research International 11
[17] G Guarda M Braun F Staehli et al ldquoType I interferon inhibitsinterleukin-1 production and inflammasome activationrdquo Immu-nity vol 34 no 2 pp 213ndash223 2011
[18] T J Foster J A Geoghegan V K Ganesh and M HookldquoAdhesion invasion and evasion the many functions of thesurface proteins of Staphylococcus aureusrdquo Nature ReviewsMicrobiology vol 12 no 1 pp 49ndash62 2014
[19] B A Diep S R Gill R F Chang et al ldquoComplete genomesequence of USA300 an epidemic clone of community-acquiredmeticillin-resistant Staphylococcus aureusrdquoTheLancetvol 367 no 9512 pp 731ndash739 2006
[20] D Wang N Xu Z Zhang et al ldquoSophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstreampathways on LPS-induced mastitis in the mammary gland ofmicerdquo International Immunopharmacology vol 35 pp 111ndash1182016
[21] J-L Zhao Y-X Ding H-X Zhao et al ldquoPresence of super-antigen genes and antimicrobial resistance in Staphylococcusisolates obtained from the uteri of dairy cows with clinicalendometritisrdquo Veterinary Record vol 175 no 14 article 3522014
[22] D K Tennakoon R Smith M D Stewart T E Spencer MNayak and C J Welsh ldquoOvine IFN-tau modulates the expres-sion of MHC antigens on murine cerebrovascular endothelialcells and inhibits replication of Theilerrsquos virusrdquo Journal ofInterferonampCytokine Research vol 21 no 10 pp 785ndash792 2001
[23] J M Soos P S Subramaniam A C Hobeika J Schiffen-bauer and H M Johnson ldquoThe IFN pregnancy recognitionhormone IFN-tau blocks both development and superantigenreactivation of experimental allergic encephalomyelitis withoutassociated toxicityrdquo The Journal of Immunology vol 155 no 5pp 2747ndash2753 1995
[24] G Song T E Spencer and FW Bazer ldquoCathepsins in the ovineuterus regulation by pregnancy progesterone and interferontaurdquo Endocrinology vol 146 no 11 pp 4825ndash4833 2005
[25] R Grio C Giobbe A Cellula et al ldquoInflammation of theuterine corpus endometritisrdquoMinervaGinecologica vol 42 no4 pp 99ndash102 1990
[26] I M Sheldon and H Dobson ldquoPostpartum uterine health incattlerdquo Animal Reproduction Science vol 82-83 pp 295ndash3062004
[27] S A Kurganov ldquoUterine eosinophils and infertility in theratrdquo Rossiiskii Fiziologicheskii Zhurnal Imeni IM Sechen-ovaRossiiskaia Akademiia Nauk vol 96 no 2 pp 138ndash1462010
[28] K Shirasuna F Usui T Karasawa et al ldquoNanosilica-inducedplacental inflammation and pregnancy complications differentroles of the inflammasome components NLRP3 and ASCrdquoNanotoxicology vol 9 no 5 pp 554ndash567 2015
[29] Y Liu C QiuW Li WMu C Li andM Guo ldquoSelenium playsa protective role in Staphylococcus aureus-induced endometritisin the uterine tissue of ratsrdquo Biological Trace Element Researchvol 173 no 2 pp 345ndash353 2016
[30] A A M Lima L G Spınola G Baccan et al ldquoEvaluation ofcorticosterone and IL-1120573 IL-6 IL-10 and TNF-120572 expressionafter 670-nm laser photobiomodulation in ratsrdquo Lasers inMedical Science vol 29 no 2 pp 709ndash715 2014
[31] C-J Liang S-H Wang Y-H Chen et al ldquoViscolin reducesVCAM-1 expression in TNF-120572-treated endothelial cells viathe JNKNF-120581B and ROS pathwayrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1337ndash1346 2011
[32] H-K Tsou H-T Chen C-H Chang W-Y Yang and C-HTang ldquoApoptosis signal-regulating kinase 1 is mediated in TNF-120572-induced CCL2 expression in human synovial fibroblastsrdquoJournal of Cellular Biochemistry vol 113 no 11 pp 3509ndash35192012
[33] H Zhang J Kovacs-Nolan T Kodera Y Eto and Y Mine ldquo120574-Glutamyl cysteine and 120574-glutamyl valine inhibit TNF-120572 signal-ing in intestinal epithelial cells and reduce inflammation in amouse model of colitis via allosteric activation of the calcium-sensing receptorrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1852 no 5 pp 792ndash804 2015
[34] M L Diamond J A Boles A C Ritter et al ldquoIn response tocomments on IL-1120573 associations with posttraumatic epilepsydevelopment a genetics and biomarker cohort studyrdquo Epilepsiavol 55 no 8 pp 1313ndash1314 2014
[35] A Vezzani J French T Bartfai and T Z Baram ldquoThe role ofinflammation in epilepsyrdquo Nature Reviews Neurology vol 7 no1 pp 31ndash40 2011
[36] B Fournier and D J Philpott ldquoRecognition of Staphylococcusaureus by the innate immune systemrdquo Clinical MicrobiologyReviews vol 18 no 3 pp 521ndash540 2005
[37] J E Wang P F Joslashrgensen M Almlof et al ldquoPeptidoglycanand lipoteichoic acid from Staphylococcus aureus induce tumornecrosis factor alpha interleukin 6 (IL-6) and IL-10 productionin both T cells and monocytes in a human whole blood modelrdquoInfection and Immunity vol 68 no 7 pp 3965ndash3970 2000
[38] D Zhu D-Y Yang Y-Y Guo et al ldquoIntracameral interleukin1120573 6 8 10 12p tumor necrosis factor 120572 and vascular endothelialgrowth factor and axial length in patients with cataractrdquo PLoSONE vol 10 no 2 Article ID e0117777 2015
[39] P Lichte R Pfeifer P Kobbe et al ldquoInhalative IL-10 treatmentafter bilateral femoral fractures affect pulmonary inflammationin micerdquo Annals of Anatomy vol 200 pp 73ndash78 2015
[40] K Hara K Shirasuna F Usui et al ldquoInterferon-tau attenuatesuptake of nanoparticles and secretion of interleukin-1120573 inmacrophagesrdquoPLoSONE vol 9 no 12 Article ID e113974 2014
[41] A Mansson Kvarnhammar L Tengroth M Adner and L-OCardell ldquoInnate immune receptors in human airway smoothmuscle cells activation by TLR12 TLR3 TLR4 TLR7 andNOD1 agonistsrdquo PLoSONE vol 8 no 7 Article ID e68701 2013
[42] B Fournier ldquoThe function of TLR2 during staphylococcaldiseasesrdquo Frontiers in Cellular and Infection Microbiology vol2 article 167 2013
[43] L-H Ding D Liu M Xu et al ldquoTLR2ndashMyD88ndashNF-120581Bpathway is involved in tubulointerstitial inflammation causedby proteinuriardquoThe International Journal of Biochemistry amp CellBiology vol 69 pp 114ndash120 2015
[44] Z Zhang X Gao Y Cao et al ldquoSelenium deficiency facilitatesinflammation through the regulation of TLR4 and TLR4-related signaling pathways in the mice uterusrdquo Inflammationvol 38 no 3 pp 1347ndash1356 2015
[45] C Xu G Shen C Chen C Gelinas and A-N T KongldquoSuppression of NF-120581B and NF-120581B-regulated gene expressionby sulforaphane and PEITC through I120581B120572 IKK pathway inhuman prostate cancer PC-3 cellsrdquo Oncogene vol 24 no 28pp 4486ndash4495 2005
[46] W Zhang R Zhang T Wang et al ldquoSelenium inhibits LPS-induced pro-inflammatory gene expression by modulatingMAPK and NF-120581B signaling pathways in mouse mammaryepithelial cells in primary culturerdquo Inflammation vol 37 no 2pp 478ndash485 2014
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
8 BioMed Research International
CG S aureusDEX 248
I120581B
p-I120581B
p65
p-p65
120573-Actin
(a)CG S aureusDEX 248
CG S aureusDEX 248
lowastlowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
pNF-
120581B
120573-a
ctin
ratio
0
02
04
06
08
1
12
14
16
18
2
22
24
0
02
04
06
08
1
12
14
p-I120581
B120573
-act
in ra
tio
(b)
Figure 6 Effect of IFN-120591 on NF-120581B activation (a) The NF-120581B and I-120581B protein levels in uterine tissues (b) The ratio of phosphorylationproteins levels as well as 120573-actin levels Western blot was performed to detect the levels of total and phosphorylated NF-120581B and I-120581B proteinsin the control group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups(2 4 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
results showed that the expression of IL-10 mRNA andprotein was increased in mice inoculated with S aureusand the expression of IL-10 was further increased with IFN-120591 treatment With increasing concentrations of IFN-120591 theeffect on IL-10 was more significant IFN-120591 can promotethe secretion of inflammatory cytokines including IL-10thereby inhibiting the subsequent secretion of TNF-120572 IL-1120573and IL-6 [40] TLRs are transmembrane pattern recognitionreceptors which are part of the innate immune systemand are the key element in identifying viral and bacterialcomponents [37 41] TLR2 senses the inflammatory reactionscaused by S aureusmost rapidly and sensitively and presentspathogen-derived antigens to various types of cells [42]Our results showed TLR2 increased in mice infected withS aureus and its secretion was significantly reduced afterIFN-120591 treatment TLR2 plays a key role in the responseto both innate and adaptive immunity as it can identifyexogenous pathogen-associated molecular patterns (PAMPs)
and induce proinflammatory signaling pathways [43] In thisstudy IFN-120591 inhibited the secretion of TLR2 in a dose-dependent manner
NF-120581B and MAPKs are two important inflammation-associated pathways and they canmodulate the developmentof inflammation by mediating downstream signaling net-works [44] To further investigate the mechanism by whichIFN-120591 affects inflammation we analyzed the NF-120581B andMAPK pathways The NF-120581B signaling pathway is involvedin the regulation of genes that mediate inflammation as wellas various cytokines and is associatedwith numerous chronicinflammatory diseases [45] Normally theNF-120581Bp65 subunitis bound to I120581B in the cytosol When stimulated by variouspathogens NF-120581B p65 is released due to the phosphorylationof I120581B leading to its activation and nuclear translocationwhich then triggers inflammation [46] Western blottinganalysis showed the phosphorylation levels of I120581B120572 and p65in mice infected with S aureus were significantly higher than
BioMed Research International 9
CG S aureusDEX 248
p38
p-p38
120573-Actin
ERK
p-ERK
JNK
p-JNK
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
p-JN
K120573
-act
in ra
tio
0
02
04
06
08
1
p-p3
8120573
-act
in ra
tio
0
02
04
06
08
1
12
0
02
04
06
08
1
12
14
16
18
p-ER
K120573
-act
in ra
tio
(b)
Figure 7 Effect of IFN-120591 on MAPK activation (a) The protein levels of MAPKs in uterine tissues (b) The ratio of phosphorylation proteinsas well as 120573-actin levels Western blotting was performed to measure the levels of total and phosphorylated p38 ERK and JNK protein in theControl group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups (24 and 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
10 BioMed Research International
that in the IFN-120591 groups These results indicated that IFN-120591 can inhibit the phosphorylation of I120581B120572 and p65 therebyblocking NF-120581B activation In addition to NF-120581B MAPKsare another key regulator of signal transduction pathwaysthat can modulate the levels of inflammatory mediators [47]Specifically p38 JNK and ERK are the major effectors of theMAPK pathwayThe phosphorylation of these three proteinscan cause the translocation of Activator Protein-1 (AP-1) tothe nucleus thereby promoting the inflammatory response[48 49] In the current study we found an increase in thephosphorylation levels of p38 JNK and ERK in the miceinoculated with S aureus However in the groups treatedwith IFN-120591 the phosphorylation of these proteins was signif-icantly inhibited Recent studies have indicated that the inhi-bition of phosphorylation of MAPKs with specific inhibitorscould significantly attenuate inflammation by decreasingthe number of neutrophils Similar effects were observedfollowing the inhibition of proinflammatory cytokines andchemokines [50] In our study we demonstrated that IFN-120591 significantly inhibited the phosphorylation of proteinsinvolved in MAPK pathway activation Taken together ourresults showed that blocking the NF-120581B andMAPK signalingpathways and inhibiting the secretion of proinflammatorycytokines are the main mechanisms by which IFN-120591 abatesendometritis
5 Conclusion
Our study showed that S aureus increases the expression ofTNF-120572 IL-1120573 and IL-6 thereby activating TLR2 signalingpathways including NF-120581B and MAPK The experimentspresented herein show that IFN-120591 reduces the secretion ofproinflammatory cytokines such as TNF-120572 IL-1120573 and IL-6 while promoting the production of the anti-inflammatorycytokine IL-10 This reduction inhibits inflammation andenhances the repair of uterine tissues In addition IFN-120591 inhibited the expression of TLR2 thereby blocking theactivation of the NF-120581B and MAPK signaling pathwaysand abating endometritis Additional studies are requiredto validate the efficacy of IFN-120591 as a treatment for uterineinfection due to S aureus or other infectious pathogens
Competing Interests
The authors have no conflict of interest to declare
Acknowledgments
This work was supported by a grant from the Funda-mental Research Funds for the Central Universities (no2662014BQ024) the National Natural Science Foundationof China (nos 31272631 31472254 and 31502130) andUndergraduate Special Science and Technology Innovationof Huazhong Agricultural University (no 2015BC009)
References
[1] B Polat M Cengiz O Cannazik et al ldquoEndometrial echotex-ture variables in postpartum cows with subclinical endometri-tisrdquo Animal Reproduction Science vol 155 pp 50ndash55 2015
[2] J Roberson D Moll and G Saunders ldquoChronic Staphylococcusaureus endometritis in a virgin giltrdquo Veterinary Record vol 161no 24 pp 821ndash822 2007
[3] M Guo G Wang T Lv et al ldquoEndometrial inflammation andabnormal expression of extracellular matrix proteins inducedbyMycoplasma bovis in dairy cowsrdquoTheriogenology vol 81 no5 pp 669ndash674 2014
[4] I M Sheldon J Cronin L Goetze G Donofrio and H-J Schuberth ldquoDefining postpartum uterine disease and themechanisms of infection and immunity in the female reproduc-tive tract in cattlerdquo Biology of Reproduction vol 81 no 6 pp1025ndash1032 2009
[5] P W Farin L Ball J D Olson et al ldquoEffect of Actinomyces pyo-genes and gram-negative anaerobic bacteria on the developmentof bovine pyometrardquoTheriogenology vol 31 no 5 pp 979ndash9891989
[6] M Guo Y Cao TWang et al ldquoBaicalin inhibits Staphylococcusaureus-induced apoptosis by regulating TLR2 andTLR2-relatedapoptotic factors in the mouse mammary glandsrdquo EuropeanJournal of Pharmacology vol 723 no 1 pp 481ndash488 2014
[7] A K Syed T J Reed K L Clark B R Boles and J MKahlenberg ldquoStaphlyococcus aureus phenol-soluble modulinsstimulate the release of proinflammatory cytokines from ker-atinocytes and are required for induction of skin inflammationrdquoInfection and Immunity vol 83 no 9 pp 3428ndash3437 2015
[8] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[9] T W Chon and S Bixler ldquoInterferon-tau current applicationsand potential in antiviral therapyrdquo Journal of Interferon ampCytokine Research vol 30 no 7 pp 477ndash485 2010
[10] C H Pontzer T L Ott F W Bazer and H M JohnsonldquoStructurefunction studies with interferon tau evidence formultiple active sitesrdquo Journal of Interferon Research vol 14 no3 pp 133ndash141 1994
[11] R M Roberts ldquoInterferon-tau a Type 1 interferon involved inmaternal recognition of pregnancyrdquo Cytokine amp Growth FactorReviews vol 18 no 5-6 pp 403ndash408 2007
[12] A P Alexenko AD Ealy andRM Roberts ldquoThe cross-speciesantiviral activities of different IFN-tau subtypes on bovinemurine and human cells contradictory evidence for therapeu-tic potentialrdquo Journal of Interferon amp Cytokine Research vol 19no 12 pp 1335ndash1341 1999
[13] J A Neira D Tainturier R M LrsquoHaridon and J Martal ldquoCom-parative IFN-tau secretion after hatching by bovine blastocystsderived ex vivo and completely produced in vitrordquoReproductionin Domestic Animals vol 42 no 1 pp 68ndash75 2007
[14] A D Ealy J A Green A P Alexenko D H Keisler and R MRoberts ldquoDifferent ovine interferon-tau genes are not expressedidentically and their protein products display different activi-tiesrdquo Biology of Reproduction vol 58 no 2 pp 566ndash573 1998
[15] K A Naivar S K Ward K J Austin D W Moore and T RHansen ldquoSecretion of bovine uterine proteins in response toType I interferonsrdquo Biology of Reproduction vol 52 no 4 pp848ndash854 1995
[16] M G Teixeira K J Austin D J Perry et al ldquoBovine granulo-cyte chemotactic protein-2 is secreted by the endometrium inresponse to interferon-tau (IFN-tau)rdquo Endocrine vol 6 no 1pp 31ndash37 1997
BioMed Research International 11
[17] G Guarda M Braun F Staehli et al ldquoType I interferon inhibitsinterleukin-1 production and inflammasome activationrdquo Immu-nity vol 34 no 2 pp 213ndash223 2011
[18] T J Foster J A Geoghegan V K Ganesh and M HookldquoAdhesion invasion and evasion the many functions of thesurface proteins of Staphylococcus aureusrdquo Nature ReviewsMicrobiology vol 12 no 1 pp 49ndash62 2014
[19] B A Diep S R Gill R F Chang et al ldquoComplete genomesequence of USA300 an epidemic clone of community-acquiredmeticillin-resistant Staphylococcus aureusrdquoTheLancetvol 367 no 9512 pp 731ndash739 2006
[20] D Wang N Xu Z Zhang et al ldquoSophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstreampathways on LPS-induced mastitis in the mammary gland ofmicerdquo International Immunopharmacology vol 35 pp 111ndash1182016
[21] J-L Zhao Y-X Ding H-X Zhao et al ldquoPresence of super-antigen genes and antimicrobial resistance in Staphylococcusisolates obtained from the uteri of dairy cows with clinicalendometritisrdquo Veterinary Record vol 175 no 14 article 3522014
[22] D K Tennakoon R Smith M D Stewart T E Spencer MNayak and C J Welsh ldquoOvine IFN-tau modulates the expres-sion of MHC antigens on murine cerebrovascular endothelialcells and inhibits replication of Theilerrsquos virusrdquo Journal ofInterferonampCytokine Research vol 21 no 10 pp 785ndash792 2001
[23] J M Soos P S Subramaniam A C Hobeika J Schiffen-bauer and H M Johnson ldquoThe IFN pregnancy recognitionhormone IFN-tau blocks both development and superantigenreactivation of experimental allergic encephalomyelitis withoutassociated toxicityrdquo The Journal of Immunology vol 155 no 5pp 2747ndash2753 1995
[24] G Song T E Spencer and FW Bazer ldquoCathepsins in the ovineuterus regulation by pregnancy progesterone and interferontaurdquo Endocrinology vol 146 no 11 pp 4825ndash4833 2005
[25] R Grio C Giobbe A Cellula et al ldquoInflammation of theuterine corpus endometritisrdquoMinervaGinecologica vol 42 no4 pp 99ndash102 1990
[26] I M Sheldon and H Dobson ldquoPostpartum uterine health incattlerdquo Animal Reproduction Science vol 82-83 pp 295ndash3062004
[27] S A Kurganov ldquoUterine eosinophils and infertility in theratrdquo Rossiiskii Fiziologicheskii Zhurnal Imeni IM Sechen-ovaRossiiskaia Akademiia Nauk vol 96 no 2 pp 138ndash1462010
[28] K Shirasuna F Usui T Karasawa et al ldquoNanosilica-inducedplacental inflammation and pregnancy complications differentroles of the inflammasome components NLRP3 and ASCrdquoNanotoxicology vol 9 no 5 pp 554ndash567 2015
[29] Y Liu C QiuW Li WMu C Li andM Guo ldquoSelenium playsa protective role in Staphylococcus aureus-induced endometritisin the uterine tissue of ratsrdquo Biological Trace Element Researchvol 173 no 2 pp 345ndash353 2016
[30] A A M Lima L G Spınola G Baccan et al ldquoEvaluation ofcorticosterone and IL-1120573 IL-6 IL-10 and TNF-120572 expressionafter 670-nm laser photobiomodulation in ratsrdquo Lasers inMedical Science vol 29 no 2 pp 709ndash715 2014
[31] C-J Liang S-H Wang Y-H Chen et al ldquoViscolin reducesVCAM-1 expression in TNF-120572-treated endothelial cells viathe JNKNF-120581B and ROS pathwayrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1337ndash1346 2011
[32] H-K Tsou H-T Chen C-H Chang W-Y Yang and C-HTang ldquoApoptosis signal-regulating kinase 1 is mediated in TNF-120572-induced CCL2 expression in human synovial fibroblastsrdquoJournal of Cellular Biochemistry vol 113 no 11 pp 3509ndash35192012
[33] H Zhang J Kovacs-Nolan T Kodera Y Eto and Y Mine ldquo120574-Glutamyl cysteine and 120574-glutamyl valine inhibit TNF-120572 signal-ing in intestinal epithelial cells and reduce inflammation in amouse model of colitis via allosteric activation of the calcium-sensing receptorrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1852 no 5 pp 792ndash804 2015
[34] M L Diamond J A Boles A C Ritter et al ldquoIn response tocomments on IL-1120573 associations with posttraumatic epilepsydevelopment a genetics and biomarker cohort studyrdquo Epilepsiavol 55 no 8 pp 1313ndash1314 2014
[35] A Vezzani J French T Bartfai and T Z Baram ldquoThe role ofinflammation in epilepsyrdquo Nature Reviews Neurology vol 7 no1 pp 31ndash40 2011
[36] B Fournier and D J Philpott ldquoRecognition of Staphylococcusaureus by the innate immune systemrdquo Clinical MicrobiologyReviews vol 18 no 3 pp 521ndash540 2005
[37] J E Wang P F Joslashrgensen M Almlof et al ldquoPeptidoglycanand lipoteichoic acid from Staphylococcus aureus induce tumornecrosis factor alpha interleukin 6 (IL-6) and IL-10 productionin both T cells and monocytes in a human whole blood modelrdquoInfection and Immunity vol 68 no 7 pp 3965ndash3970 2000
[38] D Zhu D-Y Yang Y-Y Guo et al ldquoIntracameral interleukin1120573 6 8 10 12p tumor necrosis factor 120572 and vascular endothelialgrowth factor and axial length in patients with cataractrdquo PLoSONE vol 10 no 2 Article ID e0117777 2015
[39] P Lichte R Pfeifer P Kobbe et al ldquoInhalative IL-10 treatmentafter bilateral femoral fractures affect pulmonary inflammationin micerdquo Annals of Anatomy vol 200 pp 73ndash78 2015
[40] K Hara K Shirasuna F Usui et al ldquoInterferon-tau attenuatesuptake of nanoparticles and secretion of interleukin-1120573 inmacrophagesrdquoPLoSONE vol 9 no 12 Article ID e113974 2014
[41] A Mansson Kvarnhammar L Tengroth M Adner and L-OCardell ldquoInnate immune receptors in human airway smoothmuscle cells activation by TLR12 TLR3 TLR4 TLR7 andNOD1 agonistsrdquo PLoSONE vol 8 no 7 Article ID e68701 2013
[42] B Fournier ldquoThe function of TLR2 during staphylococcaldiseasesrdquo Frontiers in Cellular and Infection Microbiology vol2 article 167 2013
[43] L-H Ding D Liu M Xu et al ldquoTLR2ndashMyD88ndashNF-120581Bpathway is involved in tubulointerstitial inflammation causedby proteinuriardquoThe International Journal of Biochemistry amp CellBiology vol 69 pp 114ndash120 2015
[44] Z Zhang X Gao Y Cao et al ldquoSelenium deficiency facilitatesinflammation through the regulation of TLR4 and TLR4-related signaling pathways in the mice uterusrdquo Inflammationvol 38 no 3 pp 1347ndash1356 2015
[45] C Xu G Shen C Chen C Gelinas and A-N T KongldquoSuppression of NF-120581B and NF-120581B-regulated gene expressionby sulforaphane and PEITC through I120581B120572 IKK pathway inhuman prostate cancer PC-3 cellsrdquo Oncogene vol 24 no 28pp 4486ndash4495 2005
[46] W Zhang R Zhang T Wang et al ldquoSelenium inhibits LPS-induced pro-inflammatory gene expression by modulatingMAPK and NF-120581B signaling pathways in mouse mammaryepithelial cells in primary culturerdquo Inflammation vol 37 no 2pp 478ndash485 2014
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
BioMed Research International 9
CG S aureusDEX 248
p38
p-p38
120573-Actin
ERK
p-ERK
JNK
p-JNK
(a)CG S aureusDEX 248
CG S aureusDEX 248
CG S aureusDEX 248
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast
p-JN
K120573
-act
in ra
tio
0
02
04
06
08
1
p-p3
8120573
-act
in ra
tio
0
02
04
06
08
1
12
0
02
04
06
08
1
12
14
16
18
p-ER
K120573
-act
in ra
tio
(b)
Figure 7 Effect of IFN-120591 on MAPK activation (a) The protein levels of MAPKs in uterine tissues (b) The ratio of phosphorylation proteinsas well as 120573-actin levels Western blotting was performed to measure the levels of total and phosphorylated p38 ERK and JNK protein in theControl group (CG) the S aureus-induced endometritis without drug treatment group (S aureus) and the IFN-120591 administration groups (24 and 8mgkg) 120573-Actin was used as a control The values are presented as the means plusmn SD (119899 = 10) lowast119901 lt 005 significantly different fromthe CG 119901 lt 005 significantly different from the S aureus group
10 BioMed Research International
that in the IFN-120591 groups These results indicated that IFN-120591 can inhibit the phosphorylation of I120581B120572 and p65 therebyblocking NF-120581B activation In addition to NF-120581B MAPKsare another key regulator of signal transduction pathwaysthat can modulate the levels of inflammatory mediators [47]Specifically p38 JNK and ERK are the major effectors of theMAPK pathwayThe phosphorylation of these three proteinscan cause the translocation of Activator Protein-1 (AP-1) tothe nucleus thereby promoting the inflammatory response[48 49] In the current study we found an increase in thephosphorylation levels of p38 JNK and ERK in the miceinoculated with S aureus However in the groups treatedwith IFN-120591 the phosphorylation of these proteins was signif-icantly inhibited Recent studies have indicated that the inhi-bition of phosphorylation of MAPKs with specific inhibitorscould significantly attenuate inflammation by decreasingthe number of neutrophils Similar effects were observedfollowing the inhibition of proinflammatory cytokines andchemokines [50] In our study we demonstrated that IFN-120591 significantly inhibited the phosphorylation of proteinsinvolved in MAPK pathway activation Taken together ourresults showed that blocking the NF-120581B andMAPK signalingpathways and inhibiting the secretion of proinflammatorycytokines are the main mechanisms by which IFN-120591 abatesendometritis
5 Conclusion
Our study showed that S aureus increases the expression ofTNF-120572 IL-1120573 and IL-6 thereby activating TLR2 signalingpathways including NF-120581B and MAPK The experimentspresented herein show that IFN-120591 reduces the secretion ofproinflammatory cytokines such as TNF-120572 IL-1120573 and IL-6 while promoting the production of the anti-inflammatorycytokine IL-10 This reduction inhibits inflammation andenhances the repair of uterine tissues In addition IFN-120591 inhibited the expression of TLR2 thereby blocking theactivation of the NF-120581B and MAPK signaling pathwaysand abating endometritis Additional studies are requiredto validate the efficacy of IFN-120591 as a treatment for uterineinfection due to S aureus or other infectious pathogens
Competing Interests
The authors have no conflict of interest to declare
Acknowledgments
This work was supported by a grant from the Funda-mental Research Funds for the Central Universities (no2662014BQ024) the National Natural Science Foundationof China (nos 31272631 31472254 and 31502130) andUndergraduate Special Science and Technology Innovationof Huazhong Agricultural University (no 2015BC009)
References
[1] B Polat M Cengiz O Cannazik et al ldquoEndometrial echotex-ture variables in postpartum cows with subclinical endometri-tisrdquo Animal Reproduction Science vol 155 pp 50ndash55 2015
[2] J Roberson D Moll and G Saunders ldquoChronic Staphylococcusaureus endometritis in a virgin giltrdquo Veterinary Record vol 161no 24 pp 821ndash822 2007
[3] M Guo G Wang T Lv et al ldquoEndometrial inflammation andabnormal expression of extracellular matrix proteins inducedbyMycoplasma bovis in dairy cowsrdquoTheriogenology vol 81 no5 pp 669ndash674 2014
[4] I M Sheldon J Cronin L Goetze G Donofrio and H-J Schuberth ldquoDefining postpartum uterine disease and themechanisms of infection and immunity in the female reproduc-tive tract in cattlerdquo Biology of Reproduction vol 81 no 6 pp1025ndash1032 2009
[5] P W Farin L Ball J D Olson et al ldquoEffect of Actinomyces pyo-genes and gram-negative anaerobic bacteria on the developmentof bovine pyometrardquoTheriogenology vol 31 no 5 pp 979ndash9891989
[6] M Guo Y Cao TWang et al ldquoBaicalin inhibits Staphylococcusaureus-induced apoptosis by regulating TLR2 andTLR2-relatedapoptotic factors in the mouse mammary glandsrdquo EuropeanJournal of Pharmacology vol 723 no 1 pp 481ndash488 2014
[7] A K Syed T J Reed K L Clark B R Boles and J MKahlenberg ldquoStaphlyococcus aureus phenol-soluble modulinsstimulate the release of proinflammatory cytokines from ker-atinocytes and are required for induction of skin inflammationrdquoInfection and Immunity vol 83 no 9 pp 3428ndash3437 2015
[8] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[9] T W Chon and S Bixler ldquoInterferon-tau current applicationsand potential in antiviral therapyrdquo Journal of Interferon ampCytokine Research vol 30 no 7 pp 477ndash485 2010
[10] C H Pontzer T L Ott F W Bazer and H M JohnsonldquoStructurefunction studies with interferon tau evidence formultiple active sitesrdquo Journal of Interferon Research vol 14 no3 pp 133ndash141 1994
[11] R M Roberts ldquoInterferon-tau a Type 1 interferon involved inmaternal recognition of pregnancyrdquo Cytokine amp Growth FactorReviews vol 18 no 5-6 pp 403ndash408 2007
[12] A P Alexenko AD Ealy andRM Roberts ldquoThe cross-speciesantiviral activities of different IFN-tau subtypes on bovinemurine and human cells contradictory evidence for therapeu-tic potentialrdquo Journal of Interferon amp Cytokine Research vol 19no 12 pp 1335ndash1341 1999
[13] J A Neira D Tainturier R M LrsquoHaridon and J Martal ldquoCom-parative IFN-tau secretion after hatching by bovine blastocystsderived ex vivo and completely produced in vitrordquoReproductionin Domestic Animals vol 42 no 1 pp 68ndash75 2007
[14] A D Ealy J A Green A P Alexenko D H Keisler and R MRoberts ldquoDifferent ovine interferon-tau genes are not expressedidentically and their protein products display different activi-tiesrdquo Biology of Reproduction vol 58 no 2 pp 566ndash573 1998
[15] K A Naivar S K Ward K J Austin D W Moore and T RHansen ldquoSecretion of bovine uterine proteins in response toType I interferonsrdquo Biology of Reproduction vol 52 no 4 pp848ndash854 1995
[16] M G Teixeira K J Austin D J Perry et al ldquoBovine granulo-cyte chemotactic protein-2 is secreted by the endometrium inresponse to interferon-tau (IFN-tau)rdquo Endocrine vol 6 no 1pp 31ndash37 1997
BioMed Research International 11
[17] G Guarda M Braun F Staehli et al ldquoType I interferon inhibitsinterleukin-1 production and inflammasome activationrdquo Immu-nity vol 34 no 2 pp 213ndash223 2011
[18] T J Foster J A Geoghegan V K Ganesh and M HookldquoAdhesion invasion and evasion the many functions of thesurface proteins of Staphylococcus aureusrdquo Nature ReviewsMicrobiology vol 12 no 1 pp 49ndash62 2014
[19] B A Diep S R Gill R F Chang et al ldquoComplete genomesequence of USA300 an epidemic clone of community-acquiredmeticillin-resistant Staphylococcus aureusrdquoTheLancetvol 367 no 9512 pp 731ndash739 2006
[20] D Wang N Xu Z Zhang et al ldquoSophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstreampathways on LPS-induced mastitis in the mammary gland ofmicerdquo International Immunopharmacology vol 35 pp 111ndash1182016
[21] J-L Zhao Y-X Ding H-X Zhao et al ldquoPresence of super-antigen genes and antimicrobial resistance in Staphylococcusisolates obtained from the uteri of dairy cows with clinicalendometritisrdquo Veterinary Record vol 175 no 14 article 3522014
[22] D K Tennakoon R Smith M D Stewart T E Spencer MNayak and C J Welsh ldquoOvine IFN-tau modulates the expres-sion of MHC antigens on murine cerebrovascular endothelialcells and inhibits replication of Theilerrsquos virusrdquo Journal ofInterferonampCytokine Research vol 21 no 10 pp 785ndash792 2001
[23] J M Soos P S Subramaniam A C Hobeika J Schiffen-bauer and H M Johnson ldquoThe IFN pregnancy recognitionhormone IFN-tau blocks both development and superantigenreactivation of experimental allergic encephalomyelitis withoutassociated toxicityrdquo The Journal of Immunology vol 155 no 5pp 2747ndash2753 1995
[24] G Song T E Spencer and FW Bazer ldquoCathepsins in the ovineuterus regulation by pregnancy progesterone and interferontaurdquo Endocrinology vol 146 no 11 pp 4825ndash4833 2005
[25] R Grio C Giobbe A Cellula et al ldquoInflammation of theuterine corpus endometritisrdquoMinervaGinecologica vol 42 no4 pp 99ndash102 1990
[26] I M Sheldon and H Dobson ldquoPostpartum uterine health incattlerdquo Animal Reproduction Science vol 82-83 pp 295ndash3062004
[27] S A Kurganov ldquoUterine eosinophils and infertility in theratrdquo Rossiiskii Fiziologicheskii Zhurnal Imeni IM Sechen-ovaRossiiskaia Akademiia Nauk vol 96 no 2 pp 138ndash1462010
[28] K Shirasuna F Usui T Karasawa et al ldquoNanosilica-inducedplacental inflammation and pregnancy complications differentroles of the inflammasome components NLRP3 and ASCrdquoNanotoxicology vol 9 no 5 pp 554ndash567 2015
[29] Y Liu C QiuW Li WMu C Li andM Guo ldquoSelenium playsa protective role in Staphylococcus aureus-induced endometritisin the uterine tissue of ratsrdquo Biological Trace Element Researchvol 173 no 2 pp 345ndash353 2016
[30] A A M Lima L G Spınola G Baccan et al ldquoEvaluation ofcorticosterone and IL-1120573 IL-6 IL-10 and TNF-120572 expressionafter 670-nm laser photobiomodulation in ratsrdquo Lasers inMedical Science vol 29 no 2 pp 709ndash715 2014
[31] C-J Liang S-H Wang Y-H Chen et al ldquoViscolin reducesVCAM-1 expression in TNF-120572-treated endothelial cells viathe JNKNF-120581B and ROS pathwayrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1337ndash1346 2011
[32] H-K Tsou H-T Chen C-H Chang W-Y Yang and C-HTang ldquoApoptosis signal-regulating kinase 1 is mediated in TNF-120572-induced CCL2 expression in human synovial fibroblastsrdquoJournal of Cellular Biochemistry vol 113 no 11 pp 3509ndash35192012
[33] H Zhang J Kovacs-Nolan T Kodera Y Eto and Y Mine ldquo120574-Glutamyl cysteine and 120574-glutamyl valine inhibit TNF-120572 signal-ing in intestinal epithelial cells and reduce inflammation in amouse model of colitis via allosteric activation of the calcium-sensing receptorrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1852 no 5 pp 792ndash804 2015
[34] M L Diamond J A Boles A C Ritter et al ldquoIn response tocomments on IL-1120573 associations with posttraumatic epilepsydevelopment a genetics and biomarker cohort studyrdquo Epilepsiavol 55 no 8 pp 1313ndash1314 2014
[35] A Vezzani J French T Bartfai and T Z Baram ldquoThe role ofinflammation in epilepsyrdquo Nature Reviews Neurology vol 7 no1 pp 31ndash40 2011
[36] B Fournier and D J Philpott ldquoRecognition of Staphylococcusaureus by the innate immune systemrdquo Clinical MicrobiologyReviews vol 18 no 3 pp 521ndash540 2005
[37] J E Wang P F Joslashrgensen M Almlof et al ldquoPeptidoglycanand lipoteichoic acid from Staphylococcus aureus induce tumornecrosis factor alpha interleukin 6 (IL-6) and IL-10 productionin both T cells and monocytes in a human whole blood modelrdquoInfection and Immunity vol 68 no 7 pp 3965ndash3970 2000
[38] D Zhu D-Y Yang Y-Y Guo et al ldquoIntracameral interleukin1120573 6 8 10 12p tumor necrosis factor 120572 and vascular endothelialgrowth factor and axial length in patients with cataractrdquo PLoSONE vol 10 no 2 Article ID e0117777 2015
[39] P Lichte R Pfeifer P Kobbe et al ldquoInhalative IL-10 treatmentafter bilateral femoral fractures affect pulmonary inflammationin micerdquo Annals of Anatomy vol 200 pp 73ndash78 2015
[40] K Hara K Shirasuna F Usui et al ldquoInterferon-tau attenuatesuptake of nanoparticles and secretion of interleukin-1120573 inmacrophagesrdquoPLoSONE vol 9 no 12 Article ID e113974 2014
[41] A Mansson Kvarnhammar L Tengroth M Adner and L-OCardell ldquoInnate immune receptors in human airway smoothmuscle cells activation by TLR12 TLR3 TLR4 TLR7 andNOD1 agonistsrdquo PLoSONE vol 8 no 7 Article ID e68701 2013
[42] B Fournier ldquoThe function of TLR2 during staphylococcaldiseasesrdquo Frontiers in Cellular and Infection Microbiology vol2 article 167 2013
[43] L-H Ding D Liu M Xu et al ldquoTLR2ndashMyD88ndashNF-120581Bpathway is involved in tubulointerstitial inflammation causedby proteinuriardquoThe International Journal of Biochemistry amp CellBiology vol 69 pp 114ndash120 2015
[44] Z Zhang X Gao Y Cao et al ldquoSelenium deficiency facilitatesinflammation through the regulation of TLR4 and TLR4-related signaling pathways in the mice uterusrdquo Inflammationvol 38 no 3 pp 1347ndash1356 2015
[45] C Xu G Shen C Chen C Gelinas and A-N T KongldquoSuppression of NF-120581B and NF-120581B-regulated gene expressionby sulforaphane and PEITC through I120581B120572 IKK pathway inhuman prostate cancer PC-3 cellsrdquo Oncogene vol 24 no 28pp 4486ndash4495 2005
[46] W Zhang R Zhang T Wang et al ldquoSelenium inhibits LPS-induced pro-inflammatory gene expression by modulatingMAPK and NF-120581B signaling pathways in mouse mammaryepithelial cells in primary culturerdquo Inflammation vol 37 no 2pp 478ndash485 2014
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
10 BioMed Research International
that in the IFN-120591 groups These results indicated that IFN-120591 can inhibit the phosphorylation of I120581B120572 and p65 therebyblocking NF-120581B activation In addition to NF-120581B MAPKsare another key regulator of signal transduction pathwaysthat can modulate the levels of inflammatory mediators [47]Specifically p38 JNK and ERK are the major effectors of theMAPK pathwayThe phosphorylation of these three proteinscan cause the translocation of Activator Protein-1 (AP-1) tothe nucleus thereby promoting the inflammatory response[48 49] In the current study we found an increase in thephosphorylation levels of p38 JNK and ERK in the miceinoculated with S aureus However in the groups treatedwith IFN-120591 the phosphorylation of these proteins was signif-icantly inhibited Recent studies have indicated that the inhi-bition of phosphorylation of MAPKs with specific inhibitorscould significantly attenuate inflammation by decreasingthe number of neutrophils Similar effects were observedfollowing the inhibition of proinflammatory cytokines andchemokines [50] In our study we demonstrated that IFN-120591 significantly inhibited the phosphorylation of proteinsinvolved in MAPK pathway activation Taken together ourresults showed that blocking the NF-120581B andMAPK signalingpathways and inhibiting the secretion of proinflammatorycytokines are the main mechanisms by which IFN-120591 abatesendometritis
5 Conclusion
Our study showed that S aureus increases the expression ofTNF-120572 IL-1120573 and IL-6 thereby activating TLR2 signalingpathways including NF-120581B and MAPK The experimentspresented herein show that IFN-120591 reduces the secretion ofproinflammatory cytokines such as TNF-120572 IL-1120573 and IL-6 while promoting the production of the anti-inflammatorycytokine IL-10 This reduction inhibits inflammation andenhances the repair of uterine tissues In addition IFN-120591 inhibited the expression of TLR2 thereby blocking theactivation of the NF-120581B and MAPK signaling pathwaysand abating endometritis Additional studies are requiredto validate the efficacy of IFN-120591 as a treatment for uterineinfection due to S aureus or other infectious pathogens
Competing Interests
The authors have no conflict of interest to declare
Acknowledgments
This work was supported by a grant from the Funda-mental Research Funds for the Central Universities (no2662014BQ024) the National Natural Science Foundationof China (nos 31272631 31472254 and 31502130) andUndergraduate Special Science and Technology Innovationof Huazhong Agricultural University (no 2015BC009)
References
[1] B Polat M Cengiz O Cannazik et al ldquoEndometrial echotex-ture variables in postpartum cows with subclinical endometri-tisrdquo Animal Reproduction Science vol 155 pp 50ndash55 2015
[2] J Roberson D Moll and G Saunders ldquoChronic Staphylococcusaureus endometritis in a virgin giltrdquo Veterinary Record vol 161no 24 pp 821ndash822 2007
[3] M Guo G Wang T Lv et al ldquoEndometrial inflammation andabnormal expression of extracellular matrix proteins inducedbyMycoplasma bovis in dairy cowsrdquoTheriogenology vol 81 no5 pp 669ndash674 2014
[4] I M Sheldon J Cronin L Goetze G Donofrio and H-J Schuberth ldquoDefining postpartum uterine disease and themechanisms of infection and immunity in the female reproduc-tive tract in cattlerdquo Biology of Reproduction vol 81 no 6 pp1025ndash1032 2009
[5] P W Farin L Ball J D Olson et al ldquoEffect of Actinomyces pyo-genes and gram-negative anaerobic bacteria on the developmentof bovine pyometrardquoTheriogenology vol 31 no 5 pp 979ndash9891989
[6] M Guo Y Cao TWang et al ldquoBaicalin inhibits Staphylococcusaureus-induced apoptosis by regulating TLR2 andTLR2-relatedapoptotic factors in the mouse mammary glandsrdquo EuropeanJournal of Pharmacology vol 723 no 1 pp 481ndash488 2014
[7] A K Syed T J Reed K L Clark B R Boles and J MKahlenberg ldquoStaphlyococcus aureus phenol-soluble modulinsstimulate the release of proinflammatory cytokines from ker-atinocytes and are required for induction of skin inflammationrdquoInfection and Immunity vol 83 no 9 pp 3428ndash3437 2015
[8] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[9] T W Chon and S Bixler ldquoInterferon-tau current applicationsand potential in antiviral therapyrdquo Journal of Interferon ampCytokine Research vol 30 no 7 pp 477ndash485 2010
[10] C H Pontzer T L Ott F W Bazer and H M JohnsonldquoStructurefunction studies with interferon tau evidence formultiple active sitesrdquo Journal of Interferon Research vol 14 no3 pp 133ndash141 1994
[11] R M Roberts ldquoInterferon-tau a Type 1 interferon involved inmaternal recognition of pregnancyrdquo Cytokine amp Growth FactorReviews vol 18 no 5-6 pp 403ndash408 2007
[12] A P Alexenko AD Ealy andRM Roberts ldquoThe cross-speciesantiviral activities of different IFN-tau subtypes on bovinemurine and human cells contradictory evidence for therapeu-tic potentialrdquo Journal of Interferon amp Cytokine Research vol 19no 12 pp 1335ndash1341 1999
[13] J A Neira D Tainturier R M LrsquoHaridon and J Martal ldquoCom-parative IFN-tau secretion after hatching by bovine blastocystsderived ex vivo and completely produced in vitrordquoReproductionin Domestic Animals vol 42 no 1 pp 68ndash75 2007
[14] A D Ealy J A Green A P Alexenko D H Keisler and R MRoberts ldquoDifferent ovine interferon-tau genes are not expressedidentically and their protein products display different activi-tiesrdquo Biology of Reproduction vol 58 no 2 pp 566ndash573 1998
[15] K A Naivar S K Ward K J Austin D W Moore and T RHansen ldquoSecretion of bovine uterine proteins in response toType I interferonsrdquo Biology of Reproduction vol 52 no 4 pp848ndash854 1995
[16] M G Teixeira K J Austin D J Perry et al ldquoBovine granulo-cyte chemotactic protein-2 is secreted by the endometrium inresponse to interferon-tau (IFN-tau)rdquo Endocrine vol 6 no 1pp 31ndash37 1997
BioMed Research International 11
[17] G Guarda M Braun F Staehli et al ldquoType I interferon inhibitsinterleukin-1 production and inflammasome activationrdquo Immu-nity vol 34 no 2 pp 213ndash223 2011
[18] T J Foster J A Geoghegan V K Ganesh and M HookldquoAdhesion invasion and evasion the many functions of thesurface proteins of Staphylococcus aureusrdquo Nature ReviewsMicrobiology vol 12 no 1 pp 49ndash62 2014
[19] B A Diep S R Gill R F Chang et al ldquoComplete genomesequence of USA300 an epidemic clone of community-acquiredmeticillin-resistant Staphylococcus aureusrdquoTheLancetvol 367 no 9512 pp 731ndash739 2006
[20] D Wang N Xu Z Zhang et al ldquoSophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstreampathways on LPS-induced mastitis in the mammary gland ofmicerdquo International Immunopharmacology vol 35 pp 111ndash1182016
[21] J-L Zhao Y-X Ding H-X Zhao et al ldquoPresence of super-antigen genes and antimicrobial resistance in Staphylococcusisolates obtained from the uteri of dairy cows with clinicalendometritisrdquo Veterinary Record vol 175 no 14 article 3522014
[22] D K Tennakoon R Smith M D Stewart T E Spencer MNayak and C J Welsh ldquoOvine IFN-tau modulates the expres-sion of MHC antigens on murine cerebrovascular endothelialcells and inhibits replication of Theilerrsquos virusrdquo Journal ofInterferonampCytokine Research vol 21 no 10 pp 785ndash792 2001
[23] J M Soos P S Subramaniam A C Hobeika J Schiffen-bauer and H M Johnson ldquoThe IFN pregnancy recognitionhormone IFN-tau blocks both development and superantigenreactivation of experimental allergic encephalomyelitis withoutassociated toxicityrdquo The Journal of Immunology vol 155 no 5pp 2747ndash2753 1995
[24] G Song T E Spencer and FW Bazer ldquoCathepsins in the ovineuterus regulation by pregnancy progesterone and interferontaurdquo Endocrinology vol 146 no 11 pp 4825ndash4833 2005
[25] R Grio C Giobbe A Cellula et al ldquoInflammation of theuterine corpus endometritisrdquoMinervaGinecologica vol 42 no4 pp 99ndash102 1990
[26] I M Sheldon and H Dobson ldquoPostpartum uterine health incattlerdquo Animal Reproduction Science vol 82-83 pp 295ndash3062004
[27] S A Kurganov ldquoUterine eosinophils and infertility in theratrdquo Rossiiskii Fiziologicheskii Zhurnal Imeni IM Sechen-ovaRossiiskaia Akademiia Nauk vol 96 no 2 pp 138ndash1462010
[28] K Shirasuna F Usui T Karasawa et al ldquoNanosilica-inducedplacental inflammation and pregnancy complications differentroles of the inflammasome components NLRP3 and ASCrdquoNanotoxicology vol 9 no 5 pp 554ndash567 2015
[29] Y Liu C QiuW Li WMu C Li andM Guo ldquoSelenium playsa protective role in Staphylococcus aureus-induced endometritisin the uterine tissue of ratsrdquo Biological Trace Element Researchvol 173 no 2 pp 345ndash353 2016
[30] A A M Lima L G Spınola G Baccan et al ldquoEvaluation ofcorticosterone and IL-1120573 IL-6 IL-10 and TNF-120572 expressionafter 670-nm laser photobiomodulation in ratsrdquo Lasers inMedical Science vol 29 no 2 pp 709ndash715 2014
[31] C-J Liang S-H Wang Y-H Chen et al ldquoViscolin reducesVCAM-1 expression in TNF-120572-treated endothelial cells viathe JNKNF-120581B and ROS pathwayrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1337ndash1346 2011
[32] H-K Tsou H-T Chen C-H Chang W-Y Yang and C-HTang ldquoApoptosis signal-regulating kinase 1 is mediated in TNF-120572-induced CCL2 expression in human synovial fibroblastsrdquoJournal of Cellular Biochemistry vol 113 no 11 pp 3509ndash35192012
[33] H Zhang J Kovacs-Nolan T Kodera Y Eto and Y Mine ldquo120574-Glutamyl cysteine and 120574-glutamyl valine inhibit TNF-120572 signal-ing in intestinal epithelial cells and reduce inflammation in amouse model of colitis via allosteric activation of the calcium-sensing receptorrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1852 no 5 pp 792ndash804 2015
[34] M L Diamond J A Boles A C Ritter et al ldquoIn response tocomments on IL-1120573 associations with posttraumatic epilepsydevelopment a genetics and biomarker cohort studyrdquo Epilepsiavol 55 no 8 pp 1313ndash1314 2014
[35] A Vezzani J French T Bartfai and T Z Baram ldquoThe role ofinflammation in epilepsyrdquo Nature Reviews Neurology vol 7 no1 pp 31ndash40 2011
[36] B Fournier and D J Philpott ldquoRecognition of Staphylococcusaureus by the innate immune systemrdquo Clinical MicrobiologyReviews vol 18 no 3 pp 521ndash540 2005
[37] J E Wang P F Joslashrgensen M Almlof et al ldquoPeptidoglycanand lipoteichoic acid from Staphylococcus aureus induce tumornecrosis factor alpha interleukin 6 (IL-6) and IL-10 productionin both T cells and monocytes in a human whole blood modelrdquoInfection and Immunity vol 68 no 7 pp 3965ndash3970 2000
[38] D Zhu D-Y Yang Y-Y Guo et al ldquoIntracameral interleukin1120573 6 8 10 12p tumor necrosis factor 120572 and vascular endothelialgrowth factor and axial length in patients with cataractrdquo PLoSONE vol 10 no 2 Article ID e0117777 2015
[39] P Lichte R Pfeifer P Kobbe et al ldquoInhalative IL-10 treatmentafter bilateral femoral fractures affect pulmonary inflammationin micerdquo Annals of Anatomy vol 200 pp 73ndash78 2015
[40] K Hara K Shirasuna F Usui et al ldquoInterferon-tau attenuatesuptake of nanoparticles and secretion of interleukin-1120573 inmacrophagesrdquoPLoSONE vol 9 no 12 Article ID e113974 2014
[41] A Mansson Kvarnhammar L Tengroth M Adner and L-OCardell ldquoInnate immune receptors in human airway smoothmuscle cells activation by TLR12 TLR3 TLR4 TLR7 andNOD1 agonistsrdquo PLoSONE vol 8 no 7 Article ID e68701 2013
[42] B Fournier ldquoThe function of TLR2 during staphylococcaldiseasesrdquo Frontiers in Cellular and Infection Microbiology vol2 article 167 2013
[43] L-H Ding D Liu M Xu et al ldquoTLR2ndashMyD88ndashNF-120581Bpathway is involved in tubulointerstitial inflammation causedby proteinuriardquoThe International Journal of Biochemistry amp CellBiology vol 69 pp 114ndash120 2015
[44] Z Zhang X Gao Y Cao et al ldquoSelenium deficiency facilitatesinflammation through the regulation of TLR4 and TLR4-related signaling pathways in the mice uterusrdquo Inflammationvol 38 no 3 pp 1347ndash1356 2015
[45] C Xu G Shen C Chen C Gelinas and A-N T KongldquoSuppression of NF-120581B and NF-120581B-regulated gene expressionby sulforaphane and PEITC through I120581B120572 IKK pathway inhuman prostate cancer PC-3 cellsrdquo Oncogene vol 24 no 28pp 4486ndash4495 2005
[46] W Zhang R Zhang T Wang et al ldquoSelenium inhibits LPS-induced pro-inflammatory gene expression by modulatingMAPK and NF-120581B signaling pathways in mouse mammaryepithelial cells in primary culturerdquo Inflammation vol 37 no 2pp 478ndash485 2014
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
BioMed Research International 11
[17] G Guarda M Braun F Staehli et al ldquoType I interferon inhibitsinterleukin-1 production and inflammasome activationrdquo Immu-nity vol 34 no 2 pp 213ndash223 2011
[18] T J Foster J A Geoghegan V K Ganesh and M HookldquoAdhesion invasion and evasion the many functions of thesurface proteins of Staphylococcus aureusrdquo Nature ReviewsMicrobiology vol 12 no 1 pp 49ndash62 2014
[19] B A Diep S R Gill R F Chang et al ldquoComplete genomesequence of USA300 an epidemic clone of community-acquiredmeticillin-resistant Staphylococcus aureusrdquoTheLancetvol 367 no 9512 pp 731ndash739 2006
[20] D Wang N Xu Z Zhang et al ldquoSophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstreampathways on LPS-induced mastitis in the mammary gland ofmicerdquo International Immunopharmacology vol 35 pp 111ndash1182016
[21] J-L Zhao Y-X Ding H-X Zhao et al ldquoPresence of super-antigen genes and antimicrobial resistance in Staphylococcusisolates obtained from the uteri of dairy cows with clinicalendometritisrdquo Veterinary Record vol 175 no 14 article 3522014
[22] D K Tennakoon R Smith M D Stewart T E Spencer MNayak and C J Welsh ldquoOvine IFN-tau modulates the expres-sion of MHC antigens on murine cerebrovascular endothelialcells and inhibits replication of Theilerrsquos virusrdquo Journal ofInterferonampCytokine Research vol 21 no 10 pp 785ndash792 2001
[23] J M Soos P S Subramaniam A C Hobeika J Schiffen-bauer and H M Johnson ldquoThe IFN pregnancy recognitionhormone IFN-tau blocks both development and superantigenreactivation of experimental allergic encephalomyelitis withoutassociated toxicityrdquo The Journal of Immunology vol 155 no 5pp 2747ndash2753 1995
[24] G Song T E Spencer and FW Bazer ldquoCathepsins in the ovineuterus regulation by pregnancy progesterone and interferontaurdquo Endocrinology vol 146 no 11 pp 4825ndash4833 2005
[25] R Grio C Giobbe A Cellula et al ldquoInflammation of theuterine corpus endometritisrdquoMinervaGinecologica vol 42 no4 pp 99ndash102 1990
[26] I M Sheldon and H Dobson ldquoPostpartum uterine health incattlerdquo Animal Reproduction Science vol 82-83 pp 295ndash3062004
[27] S A Kurganov ldquoUterine eosinophils and infertility in theratrdquo Rossiiskii Fiziologicheskii Zhurnal Imeni IM Sechen-ovaRossiiskaia Akademiia Nauk vol 96 no 2 pp 138ndash1462010
[28] K Shirasuna F Usui T Karasawa et al ldquoNanosilica-inducedplacental inflammation and pregnancy complications differentroles of the inflammasome components NLRP3 and ASCrdquoNanotoxicology vol 9 no 5 pp 554ndash567 2015
[29] Y Liu C QiuW Li WMu C Li andM Guo ldquoSelenium playsa protective role in Staphylococcus aureus-induced endometritisin the uterine tissue of ratsrdquo Biological Trace Element Researchvol 173 no 2 pp 345ndash353 2016
[30] A A M Lima L G Spınola G Baccan et al ldquoEvaluation ofcorticosterone and IL-1120573 IL-6 IL-10 and TNF-120572 expressionafter 670-nm laser photobiomodulation in ratsrdquo Lasers inMedical Science vol 29 no 2 pp 709ndash715 2014
[31] C-J Liang S-H Wang Y-H Chen et al ldquoViscolin reducesVCAM-1 expression in TNF-120572-treated endothelial cells viathe JNKNF-120581B and ROS pathwayrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1337ndash1346 2011
[32] H-K Tsou H-T Chen C-H Chang W-Y Yang and C-HTang ldquoApoptosis signal-regulating kinase 1 is mediated in TNF-120572-induced CCL2 expression in human synovial fibroblastsrdquoJournal of Cellular Biochemistry vol 113 no 11 pp 3509ndash35192012
[33] H Zhang J Kovacs-Nolan T Kodera Y Eto and Y Mine ldquo120574-Glutamyl cysteine and 120574-glutamyl valine inhibit TNF-120572 signal-ing in intestinal epithelial cells and reduce inflammation in amouse model of colitis via allosteric activation of the calcium-sensing receptorrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1852 no 5 pp 792ndash804 2015
[34] M L Diamond J A Boles A C Ritter et al ldquoIn response tocomments on IL-1120573 associations with posttraumatic epilepsydevelopment a genetics and biomarker cohort studyrdquo Epilepsiavol 55 no 8 pp 1313ndash1314 2014
[35] A Vezzani J French T Bartfai and T Z Baram ldquoThe role ofinflammation in epilepsyrdquo Nature Reviews Neurology vol 7 no1 pp 31ndash40 2011
[36] B Fournier and D J Philpott ldquoRecognition of Staphylococcusaureus by the innate immune systemrdquo Clinical MicrobiologyReviews vol 18 no 3 pp 521ndash540 2005
[37] J E Wang P F Joslashrgensen M Almlof et al ldquoPeptidoglycanand lipoteichoic acid from Staphylococcus aureus induce tumornecrosis factor alpha interleukin 6 (IL-6) and IL-10 productionin both T cells and monocytes in a human whole blood modelrdquoInfection and Immunity vol 68 no 7 pp 3965ndash3970 2000
[38] D Zhu D-Y Yang Y-Y Guo et al ldquoIntracameral interleukin1120573 6 8 10 12p tumor necrosis factor 120572 and vascular endothelialgrowth factor and axial length in patients with cataractrdquo PLoSONE vol 10 no 2 Article ID e0117777 2015
[39] P Lichte R Pfeifer P Kobbe et al ldquoInhalative IL-10 treatmentafter bilateral femoral fractures affect pulmonary inflammationin micerdquo Annals of Anatomy vol 200 pp 73ndash78 2015
[40] K Hara K Shirasuna F Usui et al ldquoInterferon-tau attenuatesuptake of nanoparticles and secretion of interleukin-1120573 inmacrophagesrdquoPLoSONE vol 9 no 12 Article ID e113974 2014
[41] A Mansson Kvarnhammar L Tengroth M Adner and L-OCardell ldquoInnate immune receptors in human airway smoothmuscle cells activation by TLR12 TLR3 TLR4 TLR7 andNOD1 agonistsrdquo PLoSONE vol 8 no 7 Article ID e68701 2013
[42] B Fournier ldquoThe function of TLR2 during staphylococcaldiseasesrdquo Frontiers in Cellular and Infection Microbiology vol2 article 167 2013
[43] L-H Ding D Liu M Xu et al ldquoTLR2ndashMyD88ndashNF-120581Bpathway is involved in tubulointerstitial inflammation causedby proteinuriardquoThe International Journal of Biochemistry amp CellBiology vol 69 pp 114ndash120 2015
[44] Z Zhang X Gao Y Cao et al ldquoSelenium deficiency facilitatesinflammation through the regulation of TLR4 and TLR4-related signaling pathways in the mice uterusrdquo Inflammationvol 38 no 3 pp 1347ndash1356 2015
[45] C Xu G Shen C Chen C Gelinas and A-N T KongldquoSuppression of NF-120581B and NF-120581B-regulated gene expressionby sulforaphane and PEITC through I120581B120572 IKK pathway inhuman prostate cancer PC-3 cellsrdquo Oncogene vol 24 no 28pp 4486ndash4495 2005
[46] W Zhang R Zhang T Wang et al ldquoSelenium inhibits LPS-induced pro-inflammatory gene expression by modulatingMAPK and NF-120581B signaling pathways in mouse mammaryepithelial cells in primary culturerdquo Inflammation vol 37 no 2pp 478ndash485 2014
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
12 BioMed Research International
[47] E K Kim and E-J Choi ldquoPathological roles ofMAPK signalingpathways in human diseasesrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1802 no 4 pp 396ndash4052010
[48] S Ogata Y Kubota T Yamashiro et al ldquoSignaling pathwaysregulating IL-1120572-induced COX-2 expressionrdquo Journal of DentalResearch vol 86 no 2 pp 186ndash191 2007
[49] E Saba B R Jeon D-H Jeong et al ldquoA novel Korean redginseng compound gintonin inhibited inflammation by MAPKand NF-120581B pathways and recovered the levels of mir-34a andmir-93 in RAW 2647 cellsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 624132 11 pages2015
[50] C Y Choi K-R Park J-H Lee et al ldquoIsoeugenol suppressionof inducible nitric oxide synthase expression is mediated bydown-regulation of NF-120581B ERK12 and p38 kinaserdquo EuropeanJournal of Pharmacology vol 576 no 1ndash3 pp 151ndash159 2007
