High-Fat Diet Induces Toll-Like Receptor 4-Dependent ... fileRetina High-Fat Diet Induces Toll-Like...

Retina

High-Fat Diet Induces Toll-Like Receptor 4-DependentMacrophage/Microglial Cell Activation and RetinalImpairment

Jong-Jer Lee,1,2 Pei-Wen Wang,3 I-Hui Yang,1 Hsiu-Mei Huang,1 Chia-Shiang Chang,1

Chia-Lin Wu,4 and Jiin-Haur Chuang2,4

1Department of Ophthalmology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine,Kaohsiung, Taiwan2The Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Taoyuan, Taiwan3Department of Internal Medicine and Nuclear Medicine, Division of Endocrinology and Metabolism, Kaohsiung Chang GungMemorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan4Department of Surgery, Division of Pediatric Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung UniversityCollege of Medicine, Kaohsiung, Taiwan

Correspondence: Jiin-Haur Chuang,Department of Pediatric Surgery,Kaohsiung Chang Gung MemorialHospital and Chang Gung UniversityCollege of Medicine, 123 Ta-PeiRoad, Niao-Song District, Kaohsiung833, Taiwan;[email protected].

Submitted: January 20, 2015Accepted: April 3, 2015

Citation: Lee J-J, Wang P-W, Yang I-H,et al. High-fat diet induces toll-likereceptor 4-dependent macrophage/microglial cell activation and retinalimpairment. Invest Ophthalmol Vis

Sci. 2015;56:3041–3050.DOI:10.1167/iovs.15-16504

PURPOSE. The toll-like receptor 4 (TLR4) signaling pathway is involved in chronic inflammationand insulin resistance, which are associated with obesity and diabetes mellitus. In the presentstudy, a model of high-fat diet (HFD) feeding of mice was used to investigate the role of TLR4in overnutrition- and obesity-associated inflammation and infiltration of macrophages andmicroglia in the retina.

METHODS. Wild-type C57BL/6 and TLR4 knockout (TLR4KO; B6.B10ScN-Tlr4lps-del/JthJ) micewere fed a HFD or control chow diet (CD) for 6 months. The TLR4 expression, the relativeincrease in macrophages/microglia (CD11bþ and CD45þ cells), the presence of markers ofoxidative stress (gp91phox and malondialdehyde; MDA), and DNA damage (phosphorylatedhistone H2AX; cH2AX) were assessed by real-time PCR and immunofluorescence studies.

RESULTS. The HFD for 6 months showed increased obesity, glucose intolerance, and insulinresistance in mice. Toll-like receptor 4 expression was found in vascular pericytes at the innerretina. Increased CD11bþ and CD45þ cells, phosphorylated NF-jB, interleukin-6, gp91phox,MDA, and cH2AX were observed in the retina of mice fed a HFD compared to CDcounterparts. TLR4KO mice did not show the adverse effects of HFD.

CONCLUSIONS. Our results indicate that HFD-induced macrophage/microglial cell activation andretinal impairment were reduced in the absence of TLR4. The findings suggest that TLR4 isimplicated in the pathogenesis of retinal diseases caused by metabolic disorders.

Keywords: CD11b, CD45, high-fat diet, oxidative stress, retina, toll-like receptor 4

Overnutrition and reduced exercise due to the modernlifestyle are often associated with obesity and systemic

diseases, including type 2 diabetes mellitus (T2DM), in humanpopulations worldwide.1 Obesity can lead to islet b-cell failure,increased insulin resistance, and subsequently T2DM.2 Chronicand poorly controlled T2DM may result in multiple systemicvascular complications including diabetic retinopathy (DR),which is the primary cause of visual loss in diabetic patientsbetween 20 and 64 years of age.3

The overexpression of proinflammatory cytokine in adiposetissue connects obesity with inflammation,4 leading to systemicinsulin resistance and metabolic syndrome through thesecretion of adipokines.5 In the central nervous system(CNS), obesity-induced metabolic alterations may synergizewith age to impair brain function and accelerate age-relateddiseases of the nervous system.6 High-fat diet (HFD) feeding isan important model for insulin resistance and T2DM.7 A HFDelicits increases in tumor necrosis factor-alpha (TNF-a), whichleads to the activation of microglia and macrophages in murinebrains.8 In mouse retina, 12 weeks of HFD induces the

differential expression of several stress-related genes,9 higherlevels of inducible nitric oxide synthase (iNOS), and 4-hydroxynonenal and retinal degeneration.10 However, themechanism underlying this effect has not been fully explored,particularly in the retina.

The toll-like receptor (TLR) family, composed of 13 membersin mammals, is the best-characterized class of pattern recogni-tion receptors capable of detecting conserved molecularpatterns present in a broad spectrum of pathogens andtriggering innate immune responses.11 Toll-like receptors arechronically activated in patients with T2DM.12,13 Nutrientmetabolism could elicit TLR-associated innate immune respons-es, triggering the release of inflammatory molecules frommonocytes or B cells into the circulation.14,15 Moreover,nutrients or other ligands may elicit TLR4 signaling to induceinflammasome activation in dendritic cells, leading to therelease of cytokines previously implicated in insulin resis-tance.16

In this study, C57BL/6 mice fed a HFD exhibited impairedglucose tolerance and insulin sensitivity that was physiologi-

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cally equivalent to T2DM. The effect of HFD on pathologicalchanges within the retina was examined in both wild-type(WT) and TLR4 knockout (TLR4KO) mice in order to elucidatethe involvement of TLR4 signaling in DR.

METHODS

Animals

Six- to eight-week old male mice with body weights of 16 to 20 gat the beginning of the study were used. Toll-like receptor 4knockout, Tlr4�/� (B6.B10ScN-Tlr4lps-del/JthJ), mice were pur-chased from Jackson Laboratory (Bar Harbor, ME, USA). Wild-type C57BL/6 mice (C57BL/6JNarl) were purchased from theNational Laboratory Animal Center, Taiwan. Both groups of miceshowed no Rd8 mutation in the Crb1 gene (Supplementary Fig.S3).17 Housing and all surgical procedures, analgesia, andassessments were implemented in the animal center ofKaohsiung Chang Gung Memorial Hospital, an Association forAssessment and Accreditation of Laboratory Animal Care-accredited, specific pathogen-free (SPF) facility followingnational and institutional guidelines. The study was approvedby the Institutional Animal Care and Use Committee in ourhospital. All animals were managed and experiments wereconducted according to the ARVO Statement for the Use ofAnimals in Ophthalmic and Vision Research.

HFD Feeding Induced Obesity, GlucoseIntolerance, and Insulin Resistance

Male WT and TLR4KO mice were maintained in a temperature-controlled room with a 12-hour light-dark cycle for 6 months.They were allowed access to water and diet ad libitum. Theexperimental group was fed a HFD (D12331; Research Diets,Inc., New Brunswick, NJ, USA), and the control group was feda chow diet (CD). The HFD (total 5.56 kcal/gm; 58 kcal% fat)was composed of protein 23.0 gm%, carbohydrate 35.5 gm%,and fat 35.8 gm%. The CD (total 4.07 kcal/gm; 11 kcal% fat)was composed of protein 16.8 gm%, carbohydrate 74.3 gm%,and fat 4.8 gm%. The diet contained approximately 20%protein and met the American Institute of Nutrition require-ments for mice with regard to mineral and vitamin content.Blood tests were conducted for plasma sugar levels as well asfor the insulin resistance index by using an intraperitonealglucose tolerance test (IPGTT) and an intraperitoneal insulintolerance test (IPITT). Animals were euthanized after 6 monthsof feeding. The eyes were extracted for studies.

Animal Glucose and Insulin Tolerance Tests

Intraperitoneal glucose tolerance tests and IPITT wereperformed (n ¼ 3 of each group) after the animals had fastedfor 16 hours. Mice were placed in restrainers, and bloodsamples were obtained by tail bleeding and analyzed byglucose meter (Optium Xceed XCN 289-2337; Abbott Taiwan,Taipei, Taiwan) immediately before (0 minutes) and at 60 and120 minutes after intraperitoneal glucose (1 g/kg in 0.9% NaCl)or 30, 60, and 90 minutes after insulin (0.75 U/kg) injection.

Preparation of Mouse Retina

Eye samples were prepared for immunofluorescence bymodifying a published procedure.18 Briefly, after extraction,the eyeballs were fixed in 4% paraformaldehyde in 0.1 Mphosphate buffer for 1 hour, soaked in 10% sucrose in 0.1 Mphosphate buffer overnight, and then shifted to 20% sucrose in0.1 M phosphate buffer for 8 hours. The tissues were

embedded with optimal cutting temperature compound(Sakura Finetek, Torrance, CA, USA) and stored at�808C untilfurther use.

Immunofluorescence Study

For the immunofluorescence study, mouse retinas (n¼4) werecut as 8-lm-thick sections in the region of 400 lm from thecenter of the eyeballs. Sections were blocked at roomtemperature with phosphate-buffered saline (PBS) containing5% normal goat serum for 1 hour and mouse on mouseblocking reagent (Vector, Burlingame, CA, USA) for 1 hour,when mouse-derived primary antibody was used. The sectionswere then incubated overnight at 48C with the followingprimary antibodies diluted in PBS: TLR4 (Santa Cruz Biotech-nology, Santa Cruz, CA, USA), isolectin GS-IB4 conjugate(Invitrogen, Carlsbad, CA, USA), a-smooth muscle actin (a-SMA; Abcam, Cambridge, UK), BRN3a (Abcam), monocytechemotactic protein-1 (MCP-1; Santa Cruz Biotechnology),CD11b (clone M1/70; BioLegend, San Diego, CA, USA), CD45(BioLegend), phospho-H2A histone family member X (cH2AX;Cell Signaling, Danvers, MA, USA), 8-hydroxy-20-deoxyguano-sine (8-OHdG; JaICA, Shizuoka, Japan), malondialdehyde(MDA; JaICA), nitrotryptophan (JaICA), and glutamine synthe-tase (GS; Abcam). Sections were next incubated withsecondary antibody conjugated with Alexa Fluor 488 or 594(Invitrogen) and with 40,6-diamidino-2-phenylindole (DAPI) fornuclear staining (Invitrogen). Coverslips were mounted onslides using fluorescence mounting medium (Dako, Glostrup,Denmark), and fluorescence images were captured andanalyzed using the Leica DMI3000 B fluorescent microscope(Leica, Singapore) at 3200 magnification unless otherwisespecified. To quantify the numbers of macrophage/microglia inretina, cells with CD11b- and CD45-positive cells were countedin three nonoverlapping views of 250- 3 100-lm size withinthe central one-third of the retina from each section by twoindependent observers.

RNA Expression Analysis Using Real-Time PCR

Individual retinas (n¼ 6 from each group) were obtained fromeyeballs of mice and homogenized with sonication. Total RNAin retina was extracted using the Qiagen RNeasy Plus UniversalMini Kit (Venlo, The Netherlands). After quantification, 2 lgtotal RNA was used to synthesize cDNA by reverse transcrip-tion with the PrimeScript RT Reagent Kit (Takara Bio, Otsu,Japan) according to the manufacturer’s recommendations.Real-time quantitative PCR (RT-qPCR) was performed using theApplied Biosystems 7500 Real-Time PCR system (Life Technol-ogies, Carlsbad, CA, USA) with SYBR green assays. We used thefollowing primers in RT-qPCR: b-actin forward 5 0-AGGCCCCTCTGAAACCTAAG-30, reverse 50-CAACACAGCCTGGATGGCTAC-3 0; TLR4 forward 5 0-ATGGCATGGCTTACACCACC-3 0, reverse 5 0-GAGGCCAATTTTGTCTCCACA-3 0; IL-6forward 5 0-TAGTCCTTCCTACCCCAATTTCC-3 0, reverse 5 0-TTGGTCCTTAGCCACTCCTTC-3 0; F4/80 forward 5 0-CTTCTGGGCCTGCTGTTCA-3 0, reverse 50-CCAGCCTACTCATTGGGATCA-3 019; gp91phox forward 5 0-TTGGGTCAGCACTGGCTCTG-30, reverse TGGCGGTGTGCAGTGCTATC-30.

Statistical Analysis

Statistical analyses were performed using the StatisticalPackage for Social Science program (SPSS for Windows, version13.0; IBM, Armonk, NY, USA). Statistical comparisons wereperformed using one-way ANOVA with post hoc Bonferronitest. Values were considered significant at a P value of <0.05,and the data are presented as means 6 SEM.

HFD Induces TLR4-Dependent Retinal Impairment IOVS j May 2015 j Vol. 56 j No. 5 j 3042


RESULTS

Animal Model

Wild-type and TLR4KO mice fed a HFD for 6 months exhibitedsystemic changes, including increased body weight (averageincrease of 45% in WT; 40% in TLR4KO) as well as impairedresponses to IPGTT and IPITT when compared to mice fedcontrol CD (Table). Interestingly, TLR4KO mice displayedhigher overall body weights than the WT mice regardless ofdiet (19% and 15% increase with CD and HFD, respectively). Nosignificant differences in response to IPGTT and IPITT wereobserved between WT and TLR4KO mice fed either CD or HFD.

HFD Induced TLR4-Dependent Nuclear Factor j-Light-Chain-Enhancer of Activated B Cells (NF-jB)Activation and IL-6 Expression in the Retina

Toll-like receptor 4-expressing cells were found from the outerplexiform layer (OPL) to the retinal ganglion cell layer (RGCL) ofthe inner retina by immunofluorescence (Fig. 1). Multiple celltypes with TLR4 expression were observed in retina. Theassociation between TLR4-expressing cells and large vascularstructures in the RGCL of WT mice fed the HFD was determinedusing isolectin GS-IB4 (Supplementary Fig. S1A). The TLR4-expressing cells that encircled a lumen in the RGCL andcolocalized with aSMA, a vascular pericyte marker, wereobserved only in HFD-fed WT mice (Supplementary Fig. S1B).Additionally, relatively weak TLR4 expression was also detectedin the BRN3aþ cells, presumed to be neuronal cells in the RGCL,and was less commonly found in the inner nuclear layer of bothCD- and HFD-fed mice (Supplementary Fig. S1C). Conversely, noTLR4 expression was detected in the glutamine synthetase-positive Muller cells (Supplementary Fig. S1D). The cells inRGCL with phosphorylated NF-jB, an indicator of NF-jBactivation, were significantly higher in HFD-fed WT mice ascompared to those fed a CD (Fig. 2A). However, the differencein cells with phosphorylated NF-jB was insignificant betweenCD and HFD-fed TLR4KO mice, whereas a significant reductionof HFD-induced phosphorylated NF-jB was observed in HFD-fedTLR4KO mice when compared to the WT counterparts.Furthermore, WT mice fed a HFD exhibited increased mRNAexpression of interleukin-6 (IL-6) in the retina when comparedto CD controls, yet no significant change in IL-6 expression wasobserved between CD- and HFD-fed TLR4KO mice (Fig. 2B). TheIL-6-expressing cells located in the RGCL colocalized with TLR4in WT mice fed a HFD (Fig. 2C).

HFD Induced TLR4-Dependent Macrophages and/or Microglial Activation in the Retina

The HFD-fed WT mice showed a 103% (P¼ 0.001) and 91% (P¼ 0.011) mean increase of CD11bþ and CD45þ cells,respectively, when quantified in the RGCL, inner plexiformlayer (IPL), and OPL of the retina as compared to CD-fedcontrols. In contrast, the HFD induced only a 20% increase (P¼0.404) in CD11bþ and a 33% increase (P ¼ 1.000) in CD45þ

cells in TLR4KO mice (Fig. 3). Despite same HFD feeding, theprevalence of CD11bþ and CD45þ cells in the retinas ofTLR4KO mice was only 68.3% (5.0 vs. 3.4, P ¼ 0.028) and41.0% (6.5 vs. 2.7, P¼ 0.001), respectively, of that observed inWT counterparts. This change in the macrophage and/ormicroglial cells was also verified with the expression of F4/80mRNA that increased significantly following HFD in WT, butnot the TLR4KO mice (Fig. 3C). Dual immunostaining of theretina from HFD-fed WT mice study showed ramified oramoeboid CD11bþ or CD45þ cells colocalized or closelyassociated with TLR4-expressing cells in RGCL of HFD-fedWT mice (Supplementary Fig. S2).

TLR4 Is Associated With Increased Oxidative Stressin the Inner Retinas of HFD-Fed Mice

Following 6 months of HFD, the mRNA expression ofgp91phox—a subunit of superoxide-generating nicotinamideadenine dinucleotide phosphate (NADPH) oxidase—in retinawas significantly higher than that of CD WT mice. This findingwas notably absent in TLR4KO mice (Fig. 4A). Furthermore, anincrease in markers of oxidative stress, including MDA and8OH-dG, was observed in the RGCL, IPL, and OPL, indicatingthat increased oxidative DNA and lipid peroxidation injurywere present in HFD WT animals compared to CD-fed controls.In contrast, HFD TLR4KO mice were relatively spared from thisHFD-induced oxidative stress (Figs. 4B, 4D). Interestingly, theCD11bþ cells were closely associated with cells expressingMDA and 8OH-dG in the inner retina of WT mice fed the HFD(Figs. 4C, 4E).

TLR4 Is Associated With Increased DNA Damage inthe Inner Retinas of HFD-Fed Mice

DNA damage incurred in the retinal cells was assessed usingan antibody against phosphorylated histone H2AX (cH2AX), amarker of DNA double-strand breaks. When compared tothose with CD, mice fed HFD showed an increased number of

TABLE. Comparison of Changes in Body Weight, Fasting Blood Glucose, Glucose Tolerance, and Insulin Tolerance in C57BL/6 Mice Fed EitherControl Chow Diet (CD) or High-Fat Diet (HFD) for 6 Months

N ¼ 3

A. CD

Wild-Type

B. HFD

Wild-Type

C. CD TLR4

Knockout

D. HFD TLR4

KnockoutP Value

Mean 6 SD Mean 6 SD Mean 6 SD Mean 6 SD A vs. B C vs. D A vs. C B vs. D

Body weight, g 26.4 6 2.3 38.2 6 4.3 31.4 6 1.0 44.1 6 4.5 <0.001 0.001 0.073 0.114

IPGTT, mg/dL

0 min 155 6 28.6 223 6 45.8 146 6 16.6 232 6 48.2 0.053 0.005 1.000 1.000

60 min 325 6 100 504 6 51.7 310 6 72.9 480 6 77.3 0.043 0.009 1.000 1.000

120 min 239 6 38.1 442 6 48.7 222 6 74.3 403 6 48.1 0.004 <0.001 1.000 1.000

IPITT, % of concentration of baseline blood glucose

Baseline 100 100 100 100

30 min 51 6 4 97 6 4 59 6 4 91 6 8 0.001 0.005 1.000 1.000

60 min 41 6 13 96 6 9 43 6 24 84 6 13 0.002 0.010 0.913 0.326

90 min 34 6 5 92 6 8 44 6 10 83 6 12 <0.001 0.022 1.000 1.000

SD, standard deviation.



cH2AXþ cells primarily in the RGCL and occasionally in the

INL. Notably, the percentage of cH2AXþ cells in RGCL was

11.8% in CD and 27.9% in HFD WT mice (P ¼ 0.009). In

contrast, no significant difference in the percentage of

cH2AXþ cells was observed in TLR4KO mice (Fig. 5A),

indicating that TLR4 is required for the DNA damage occurring

in cells in the RGCL. As previously noted, the CD11bþ cells

also showed a close spatial relationship with cH2AXþ cells in

the RGCL (Fig. 5B).

DISCUSSION

Animals fed a HFD have served as models for T2DM, metabolicsyndrome, and obesity for many years.7 In this study, C57BL/6mice fed a HFD for 6 months exhibited increases in bodyweight and systemic manifestations resembling T2DM inhumans, including a higher fasting blood glucose concentra-tion, glucose intolerance, and insulin resistance. Furthermore,the number of CD11bþ or CD45þ cells in the inner retina, fromthe OPL to the RGCL, was significantly higher in the HFD-fedmice than in the CD-fed controls. The activation of CD11bþ andCD45þ macrophages and/or microglia in the retina has beenreported in diabetic patients and in animal models of diabetesmellitus that do not use HFD feeding.20–22 Toll-like receptor 4could play a critical role in the activation of macrophages and/or microglia in the retina since the difference in diet-inducedincrease of CD11bþ and CD45þ cells, as well as the associatedoxidative stress and DNA damage, was not observed inTLR4KO mice. This increase of CD11bþ and CD45bþ cells inretina could be associated with oxidative stress and cell injury(Fig. 6).

The CD11bþ or CD45þ cells in the retina could beresidential immune microglial cells or bone marrow (BM)-derived macrophages. The majority of CD45þ retinal cells alsoexpress CD11b and are phenotypically similar to CNSmicroglia.23 Interestingly, another study investigating theturnover of monocyte-derived cells in the retina, using cellsisolated from enhanced green fluorescent protein (EGFP)transgenic mice, found that the EGFPþ cells within the ganglionlayer were amoeboid in shape and expressed high levels ofCD45.24 Therefore, the possibility also exists that HFDpromotes migration of BM-derived monocyte precursor cellsacross the blood–retinal barrier to replace residential microg-lial cells. The results of these studies indicate that two distinctpopulations of monocyte-derived cells, the parenchymalmicroglial cells and perivascular macrophages, may exist andplay specific roles in retinal homeostasis, neuroinflammation,and injury.

In our study, we found that aSMAþ cells, which arepresumably vascular pericytes, expressed TLR4 in the retinasof mice fed with HFD. Pericytes are adult pluripotent cells thatsurround capillaries and function as first-line immune defensefor the CNS vasculature.25,26 In our study, TLR4-expressingcells in RGCL may play a critical role in the induction of retinalmicroglia migration through TLR4-associated NFj-B activationand the induction of cytokines including IL-6. Previous reportshave shown that IL-6-stimulated microglia may impart acytotoxic effect on neurons in the CNS,27 and pericyte loss isan early event in experimental DR.28 This interaction betweeninflammatory cells and vascular pericytes could be associatedwith the loss of the latter in the early stage of DR.

Toll-like receptor 4 signaling is involved in diabetes mellitus-associated chronic inflammation and is also a molecular linkbetween nutrition, lipids, and inflammation in adipocytes andmacrophages.29 Obese and type 2 diabetic subjects havesignificantly elevated TLR4 gene and protein expression inmuscle.30 Toll-like receptor 4 activation leads to increasedtranscription of proinflammatory genes as well as increases incytokines, chemokines, reactive oxygen species, and eicosa-noid levels that promote further insulin desensitization in boththe target and adjacent cells via autocrine, paracrine, andsystemic effects.31 In type 1 diabetes mellitus animal models,the increase of inflammatory cytokines in serum and macro-phages was significantly attenuated in TLR4KO mice.32

Furthermore, increased TLR4 expression was also found inthe renal tubules of humans with diabetic nephropathy anddirectly correlated with interstitial macrophage infiltration andhemoglobin A1c level, whereas an inverse effect was observed

FIGURE 1. TLR4 expression was assessed in the inner retina of wild-type (WT) C57BL/6 mice. (A) Immunofluorescence study showedTLR4þ cells in the inner retina between outer plexiform layer (OPL)and retinal ganglion cell layer (RGCL) of the WT mice fed the chow diet(CD) and high-fat diet (HFD) but not in TLR4-knockout (TLR4KO) mice(scale bar: 50 lm). (B) In the magnified image of RGCL in HFD-fed WTmice, note multiple types of TLR4-expressing cells including the well-aligned small cells (arrow) and scattered large cells (scale bar: 20 lm).(C) TLR4 mRNA expression in HFD-fed WT mice was higher than inCD-fed controls.



with estimated glomerular filtration rate.33 The effect of TLR4deficiency in protecting HFD-induced insulin resistance at 16weeks of experimentation has been reported to be due toreduced inflammatory gene expression in liver and fat.34,35

However, in our study, insulin resistance and glucoseintolerance were instead observed in TLR4KO mice after 6months of HFD. Dissimilarity in study design and duration mayexplain the difference between the results of these twostudies, since it is possible that the protective effect of TLR4deficiency on insulin resistance decreases with longer dura-tion. Another possible explanation is that after the activation ofreceptors for advanced glycation end products (RAGE) andTLR2 induced by HFD in adipocytes, the systemic insulin

response may also change.36–38 Our data show that theTLR4KO mice insidiously gained more weight than the WTC57BL/6 after 4 months of HFD. These findings suggest thatthe lack of TLR4 may initially protect the mice from insulinresistance, but eventually the excessive weight of TLR4KOmice induces systemic metabolic disorder via other signalingpathways such as RAGE and TLR2.36–38

Although the role of TLR4 in DR has been previouslyunexplored, TLR4 signaling is involved in ischemia-inducedretinal damage and inflammation.39 Toll-like receptor 4 has apivotal role in the pathogenesis of ocular ischemic syndromecreated by ligation of the unilateral external carotid artery andthe pterygopalatine artery of mice.40 Notably, TLR4-mediated

FIGURE 2. TLR4 is associated with proinflammatory markers in the retinas of mice fed the HFD. (A) At RGCL of retina, the percentage of cells withphosphorylated NF-jB (p-NFjB) in HFD-fed mice was higher than that in CD-fed WT mice; however, this difference was insignificant between CD-and HFD-fed TLR4KO mice. (B) HFD induced an increased IL-6 mRNA expression in the retina of WT, but not TLR4KO mice. (C) The white arrow

shows the colocalization of IL-6 with TLR4 in large cells of the RGCL in HFD-fed WT mice (scale bar: 50 lm).



FIGURE 3. HFD induced a TLR4-dependent increase in macrophages and/or microglia in the retinas of HFD-fed mice. In a fixed area, the numbers ofCD11bþ (A) and CD45þ cells (B) in the inner retina of HFD-fed WT mice were significantly higher than in CD-fed WT mice. Additionally, HFD did notinduce significant difference in the numbers of CD11bþ and CD45þ cells between CD and HFD-fed TLR4 KO mice. (C) HFD elicited increased F4/80mRNA expression in the retinas of WT, but not the TLR4KO mice (scale bar: 50 lm).



FIGURE 4. TLR4KO inhibited oxidative damage in retinal cells following 6 months of HFD. (A) HFD induced an increase in gp91phox (NOX2)mRNA expression in the retinas of WT, but not TLR4KO mice. (B, D) HFD induced an increase of malondialdehyde (MDA [B]) and 8OH-dG (D) inthe inner retina of WT mice, but not TLR4KO. (C, E) Close spatial correlations between the CD11b and MDA (C) and 8OH-dG (E) were observed inthe inner retina (3400 images) (scale bar: 50 lm [A, C]; 20 lm [D, E]).



microglial activation by endogenous photoreceptor proteinsmay exacerbate retinal cell death and represent an underlyingcommon pathology in degenerative retinal disorders.41 Inhyperglycemic mice, systemic treatment with TLR4 ligand wasassociated with pathological changes in early backgroundDR.42 Consistent with the above studies, we found that TLR4 isimportant for macrophage and/or microglial activation in theretinas of mice fed a HFD for 6 months. In the CNS, reactiveoxidative species (ROS) could be produced by the microglia,which express the components of NADPH oxidase, includinggp91phox.43 Accordingly, the expression of NADPH oxidaseenzymes in the CNS and accompanying increase in ROS areassociated with a variety of diseases including experimentalbrain ischemia, multiple sclerosis, and traumatic brain inju-ry.44–46 In an experimental study of stroke, NADPH oxidase(NOX) was shown to mediate oxidative stress throughactivation of NOX2 and NOX4, and superoxide within theischemic core colocalized with the activated microglia andmacrophages.47 The oxidative burst generated by these celltypes is associated with demyelination and free radical-mediated tissue injury characteristic of multiple sclerosis.45

Altogether, the evidence supports that the increased levels ofoxidative stress in the perivascular region could be the result ofan overactivation of macrophages or microglia associated withTLR4 signaling.

In addition to vascular damage, close contact betweenramified CD11bþ/CD45þ cells was also observed in theneuronal cells lining the RGCL in HFD-fed mice. We speculatethat the observed DNA damage, demonstrated by the increasedcH2AX phosphorylation, was the result of interactionsbetween neuronal cells and macrophages or microglia.Microglia are known to be involved in both neuroprotectionand neurotoxicity depending on the level of activation. Thus,microglia may be activated to clear unwanted or toxic cellulardebris48; however, these cells may have an innate immunememory, resulting in prolonged activation after acute injuryand subsequent tissue degeneration.49 Neuronal inflammation

can induce the release of an ‘‘eat-me’’ signal exposure from

otherwise viable neurons, leading to their death through

phagocytosis by microglia.50 Therefore, it is plausible that

retinal microglia may initially respond to metabolic stress and

clear the debris from neuronal cells, but begin to generate

damaging effects in the presence of TLR4-dependent inflam-

matory signals. In contrast to the study by Ross et al.51 showing

that the decrease in TLR4 expression and function in the RPE

may contribute to retinal degeneration in the Ccl2�/�/Cx3cr1�/�

mice, degeneration of photoreceptors was not observed in

TLR4KO mice in our study. This suggested that knockout of

TLR4 expression alone may not be able to duplicate the effect

of double knockout of Ccl2/Cx3cr1 on the retina. Nevertheless,

the lack of TLR4 and its role in the scavenging of waste

products in retina by microglia, neuronal transduction, and

visual function could be a topic for future study.

FIGURE 5. TLR4 expression was associated with DNA damage in cells of retina after HFD feeding. (A) A significant increase in the number of cellswith phosphorylated histone H2AX (cH2AX, red) was observed in the RGCL of HFD-fed WT mice, but not TLR4KO mice (scale bar: 50 lm). (B) Theramified CD11bþ cells (green) were spatially associated with cH2AXþ cells (red) in HFD-fed WT mice (3400 images; scale bar: 20 lm).

FIGURE 6. Hypothetical diagram of the role of TLR4 in retinal injuriesafter HFD feeding. TLR4 signaling triggers the expression ofinflammatory cytokines that impart a chemotactic effect on macro-phages from the bloodstream or residential microglia in retina. Thesecells could be associated with the oxidative injury of blood vessels andDNA damage of neuronal cells observed in the retina.



In conclusion, 6 months of HFD resulted in obesity,impaired glucose tolerance, and insulin resistance in C57BL/6mice, which was associated with simultaneous TLR4-depen-dent macrophage or microglia activation in the inner retina andthe upregulation of oxidative stress markers at the perivascularzone. However, the involvement of multiple cell types andtheir complicated interactions within the retina make itdifficult to clarify the exact effects resulting from TLR4signaling on an individual cell layer basis, as well as thecrosstalk between neuronal, glial, and perivascular cells andthe subsequent retinal injury observed in the present study.Further studies are necessary to answer these questions.

Acknowledgments

The fluorescence microscope was kindly provided by Chia-WeiLiou, MD, Department of Neurology, and real-time PCR wasprovided by Stem Cell Research Core Laboratory Facilities(CLRPG8B0052), Kaohsiung Chang Gung Memorial Hospital andChang Gung University College of Medicine.

Supported by Grant CMRPG8D0461 from Kaohsiung Chang GungMemorial Hospital and Chang Gung University College of Medicineand NSC 101-2314-B-182A-123-MY3 from the National ScienceCouncil, Taiwan.

Disclosure: J.-J. Lee, None; P.-W. Wang, None; I-H. Yang, None;H.-M. Huang, None; C.-S. Chang, None; C.-L. Wu, None; J.-H.Chuang, None

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