Research Article Potential of Inducible Nitric Oxide ...

Research ArticlePotential of Inducible Nitric Oxide Synthase as a TherapeuticTarget for Allergen-Induced Airway HyperresponsivenessA Critical Connection to Nitric Oxide Levels and PARP Activity

Salomersquo V Ibba12 Mohamed A Ghonim13 Kusma Pyakurel1 Matthew R Lammi4

Anil Mishra5 and A Hamid Boulares1

1The Stanley Scott Cancer Center School of Medicine Louisiana State University Health Sciences Center New Orleans LA 70112 USA2Department of Biomedical and Clinical Sciences University of Milan Milan Italy3Microbiology and Immunology Department Faculty of Pharmacy Al-Azhar University Cairo Egypt4PulmonaryCritical Care and AllergyImmunology Section School of Medicine Louisiana State UniversityNew Orleans LA 70112 USA5Eosinophilic Disorder Center in the Section of Pulmonary Diseases Tulane University School of MedicineNew Orleans LA 70112 USA

Correspondence should be addressed to A Hamid Boulares hboulrlsuhscedu

Received 5 February 2016 Revised 11 May 2016 Accepted 22 May 2016

Academic Editor Alex Kleinjan

Copyright copy 2016 Salomersquo V Ibba et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Although expression of inducible NO synthase (iNOS) in the lungs of asthmatics and associated nitrosative damage are establishediNOS failed as a therapeutic target for blocking airway hyperresponsiveness (AHR) and inflammation in asthmaticsThis dichotomycalls for better strategieswithwhich the enzyme is adequately targetedHere we confirm iNOS expression in the asthmatic lungwithconcomitant protein nitration and poly(ADP-ribose) polymerase (PARP) activationWe show for the first time that iNOS is highlyexpressed in peripheral blood mononuclear cells (PBMCs) of asthmatics with uncontrolled disease which did not correspond toprotein nitration Selective iNOS inhibition with L-NIL protected against AHR upon acute but not chronic exposure to ovalbuminor house dust mite (HDM) in mice Supplementation of NO by nitrite administration significantly blocked AHR in chronicallyHDM-exposed mice that were treated with L-NIL Protection against chronic HDM exposure-induced AHR by olaparib-mediatedPARP inhibitionmay be associatedwith the partial but not the complete blockade of iNOS expression Indeed L-NIL administrationprevented olaparib-mediated protection against AHR in chronically HDM-exposed mice Our study suggests that the amount ofiNOS and NO are critical determinants in the modulation of AHR by selective iNOS inhibitors and renews the potential of iNOSas a therapeutic target for asthma

1 Introduction

Asthma is a chronic disease characterized by airway inflam-mation and hyperresponsiveness (AHR) overproduction ofmucus and airway and vascular wall remodeling [1ndash4]These manifestations lead to repeated periods of shortness ofbreath wheezing and chest tightness whichmay incapacitateaffected individualsThe incidence of the disease is increasingat an alarming rate affecting 1 in 10 children and 1 in 12adults with a total of 300 million worldwide [5] Worldwidedeaths from asthma have reached over 250000 annually

Asthma can be controlled by a combination of an inhaledcorticosteroid (anti-inflammatory) and a short- or long-acting 1205732-adrenergic agonist However a sizable portionof these patients (sim10) do not respond to the availabletherapies [5 6] Thus new therapies that target all or someof the symptoms of asthma are urgently needed

An increasing number of conflicting reports have demon-strated detrimental protective and sometimes neutral rolesfor inducible NO synthase (iNOS) in the pathogenesis ofasthma [7 8] However it is undoubtedly established thatiNOS is expressed in lungs of asthmatics with a subsequent

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 1984703 10 pageshttpdxdoiorg10115520161984703

2 Mediators of Inflammation

production of NO and generation of the reactive metaboliteONOOminus [9ndash12] It appears that expression of iNOS is evenhigher in sputum cells from asthmatics compared to thosefrom patients with controlled disease or healthy individuals[13]Thus inhibition of iNOS appears to be a very viable ther-apeutic target to prevent manifestation of asthma symptomsupon exposure to allergens [14ndash16] This potential has beenchallenged by the observation that a selective iNOS inhibitordid not affect airway inflammatory cell numbers or AHRafter allergen challenge in steroid-naıve human asthmatics[17] However it is difficult to ignore the fact that asthmaprotection and susceptibility are associated with polymor-phisms in the iNOS gene [18] We recently showed that iNOSgene deletionwas associated with a reduction in eosinophiliamucus hypersecretion andTh2 cytokine production upon anacute exposure to ovalbumin (OVA) [19] Such protectionwascompletely abolished upon a chronic exposure to the allergenInterestingly pulmonary fibrosis observed in wild type miceunder the chronic protocol was completely absent in iNOSminusminusmice despite persistent IL-5 and IL-13 production The pub-lished results exemplified the complexity of the role of iNOSin asthma and the preservation of its potential as a therapeutictarget We also showed that poly(ADP-ribose) polymerase-(PARP-) 1 is required for iNOS expression [20] and that PARPinhibition protects against AHR upon an acute or chronicexposure to allergens [21 22] Overall we believe that it ispremature to conclude that targeting iNOS in asthma is futileand that more studies should be geared toward exploringnew avenues to take advantage of such an important clinicaltarget Accordingly the goal of the present study was toexamine whether pharmacological inhibition of iNOS couldbe manipulated to provide protection against AHR uponchronic OVA or house dust mite extracts (HDM) exposureand whether the protection conferred by PARP inhibitionwas related to its control of iNOS expression level L-N6-(1-Iminoethyl)lysine dihydrochloride (L-NIL) and AZD2281(olaparib) two clinically tested iNOS and PARP inhibitorsrespectively were used to conduct the following study

2 Methods

21 Human Subjects Immunohistochemistry and ImmunoblotAnalysis Five healthy and eight asthmatic individuals wererecruited under a protocol (8450) approved by institutionalreview board of Louisiana State University Health SciencesCenter Subjects were included in the study if they were ge18years of age with a physician diagnosis of asthma Subjectswere excluded if they were diagnosed with another lungdisease other than asthma had active malignancy or inflam-matory condition or smokedge10 pack-years Asthma controlwas assessed by the Asthma Control Test (ACT) with a scorele19 indicating uncontrolled asthma and ge20 demonstratingthat asthma is well controlled [23] Human PBMCs isolatedfrom peripheral blood of donors were subjected to proteinextractions followed by immunoblot analysis with antibodiesto nitrotyrosine (EMD Millipore Bedford MA) humaniNOS (Abcam Cambridge MA) the poly(ADP-ribose) moi-ety (PAR) of PARP-modified proteins (ENZO Life Sciences

Farmingdale NY) or GAPDH (Abcam) Paraffin-embeddedtissue sections from two deidentified lung specimens fromindividuals who died from severe asthma were subjected toimmunohistochemistry with antibodies to PAR iNOS ornitrotyrosine The sections were then counterstained withhematoxylin and mounted prior to examination by lightmicroscopy

22 Animals OVA and HDM Challenge and AHR Measure-ments Six-week-old to eight-week-old C57BL6J male micewere purchased from Jackson Laboratories (Bar Harbor MEUSA) C57BL6 iNOSminusminus mice were bred at the LSUHSCvivarium and allowed unlimited access to sterilized chow andwater Husbandry experimental protocols and procedureswere all approved by the LSUHSC Animal Care and UseCommittee

Mice were sensitized to chicken (3mgkg) OVA (Sigma-Aldrich St LouisMO) or (05120583gkg)HDM(Dermatophago-ides pteronyssinus) extract (Greer Labs Lenoir NC) Thechallenge protocols with OVA or HDM are outlined inSupplementary Figure S1 in Supplementary Martial availableonline at httpdxdoiorg10115520161984703 Briefly micewere challenged with aerosolized 3 OVA for 30min threetimes on days 14 16 and 18 for the acute asthma model orthree times a week for three weeks (chronic asthma model)Other groups of mice were challenged intranasally with125 120583gkg whole HDM extract on days 24 25 and 26 forthe acute asthma model or 3 times per week for a totalof 4 weeks for the chronic asthma model Control groupswere not sensitized or challenged Additional challengedgroups of mice were administered ip 5mgkg L-N6-(1-Iminoethyl)lysine dihydrochloride (L-NIL) (Sigma-Aldrich)andor olaparib (Selleckchem Pittsburgh PA) in saline30 minutes after each challenge Some groups of micealso received ip injections of 20 120583gkg of nitrite (NaNO

2

)(Sigma-Aldrich) as NO source 30min after each challenge

AHR to inhaled methacholine was measured in unre-strained conscious mice 24 h after the last challenge byrecording ldquoenhanced pauserdquo (Penh) by whole body plethys-mography (EMKA Systems Falls Church VA or DSI BuxcoSt Paul MN) In brief the baseline readings were taken andaveraged for 3min after animals were placed in a barometricplethysmographic chamber Normal saline or increasingconcentrations of aerosolized methacholine (Sigma-Aldrich)(125ndash100mgmL) were nebulized and readings were takenand averaged for 3min after each nebulization and enhancedpause (Penh) representing AHR was calculated Mice weresacrificed 24 h after the last challenge and lungs were sub-jected to RNA extraction as described below

23 Cell Culture Real Time PCR Wild type or PARP-1minusminusmouse embryonic fibroblasts (MEFs) were serum starved for24 hours and then treated with 1 120583gmL LPS (Sigma-Aldrich)and 10 ngmL IFN-120574 (RD System Minneapolis MN) for 6hours The collected cells as well as homogenized lungs fromthe abovementioned experimental groups were subjectedto RNA extraction using the RNeasy mini kit (QIAGENValencia CA) The extracted RNA was reverse-transcribed

Mediators of Inflammation 3

and the resulting cDNA was subjected to quantitative realtime PCR using primer sets (IDT San Jose CA USA)specific to mouse iNOS (F 51015840-GTG TTG CAA GCT GATGGT CA-31015840 and R 51015840-TGT TGT AGC GCT GTG TGT CA-31015840) IL-5 (F 51015840-GGGCTTCCTGCTCCTATCTA-31015840 and R 51015840-CAGTCATGGCA CAGTCTGAT-31015840) or 120573-actin (F 51015840-TACAGC TTC ACC ACC ACA GC-31015840 and R 51015840-TCT CCA GGGAGG AAG AGG AT-31015840)

24 Data Analysis All data are expressed as means plusmn SDof values from at least 5 mice per group PRISM software(GraphPad San Diego CA USA) was used to analyze thedifferences between experimental groups by t-test or one wayANOVA followed by Tukeyrsquos multiple comparison tests

3 Results

31 PARP Activation iNOS Expression and Protein Nitra-tion Are Elevated in Lung and PBMCs of Asthmatics Werecently showed that PARP is activated in lung tissues andPBMCs of human asthmatics [21] Figure 1(a) confirms theactivation of PARP in lung tissues by immunohistochemistryusing antibodies to the poly(ADP-ribose) moiety of mod-ified proteins The figure also shows that PARP activationoccurred in epithelial and a subpopulation of immune cellsFigure 1(b) shows that iNOS expression is prominent inepithelial and endothelial cells and macrophages Proteinnitration as assessed by IHC with antibodies to nitrotyrosineappears to be distributed throughout the lung tissue includingthe matrix but was more prominent in epithelial cells (Fig-ure 1(c))

PBMCs collected from asthmatics or healthy individualswere subjected to immunoblot analysis with antibodies tonitrotyrosine iNOS orGAPDH Figure 1(d) shows that iNOSis highly expressed in PBMCs from asthmatics compared tocells from healthy individuals However the expression ofiNOS did not strictly correspond to protein nitration Indeedsome PBMCs exhibited high levels of iNOS but showedprotein nitration levels comparable to those detected incells from nonasthmatics Conversely PBMCs that exhibitedextensive protein nitration displayed low levels of iNOSInterestingly the two samples (6 and 7) that displayed highlevels of protein nitration were collected from patients whoseasthma was under control according to their ACT score of21 However samples from uncontrolled asthma (10 and 12with ACT scores = 16) displayed levels of protein nitrationcomparable to those from nonasthmatics but with high levelsof iNOS Overall these results exemplify the known com-plexity of the relationship between iNOS protein nitrationand asthma in humans Nevertheless it is noteworthy that thepresence of these factors does not necessarily mean a criticalfunction in the disease but results from our laboratory andmany others have suggested a potentially direct role for PARPand iNOS in some or most aspects of asthma

32 Differential Protection of iNOS Inhibition against AHRManifestation upon Acute and Chronic Exposure to OVA inMice Our laboratory has shown that iNOS gene deletion

is protective against airway inflammation upon acute butnot chronic exposures to OVA [19] Interestingly such genedeletion prevented lung fibrosis in the chronic model of thedisease Given the potential connection between and thecoexistence of lung fibrosis and AHR in chronic asthma[24] we explored the possibility that administration of L-NIL a clinically tested iNOS inhibitor may be protectiveagainst AHR upon both acute and chronic exposures toOVA in mice L-NIL is a selective and long acting inhibitorof iNOS with IC

50

= 33 120583M for mouse iNOS [25] Aclinical trial conducted by Barnes group [14] showed thatadministration of 200mg of L-NIL reduced exhaled NO inpatients with mild-to-moderate asthma to levels lower thanthose detected in placebo-administered healthy subjects asearly as 30min after administration Mice were subjectedto the acute or chronic model of asthma as described inSupplementary Figure S1 followed by an assessment of AHRusing full body plethysmography Figure 2(a) shows that L-NIL administration at a dose of 5mgkg was very effectivein blocking the manifestation of AHR upon acute exposureto OVA Surprisingly however the protection achieved byL-NIL administration was completely lost when mice werechronically exposed toOVA (Figure 2(b)) Similar differentialresults were achieved using iNOSminusminus mice that were sensi-tized and acutely (Supplementary Figure S2A) or chronically(Supplementary Figure S2B) challenged to OVA The effectsof iNOS inhibition on AHR were similar to the differentialprotection conferred by iNOS gene deletion against acuteversus chronic airway inflammation reported by us [19]

33 Inhibition of iNOS by L-NIL Failed to Protect against AHRInduced by a Chronic Exposure to HDM Which Is Reversedupon NO Supplementation by Nitrite Administration Giventhe clinical relevance of the present studies and the limitationof the OVAmodels we elected to use HDM to induce asthmain mice due to its characteristic as a major allergen forhumans [21] To this end mice were sensitized to HDM andthen subjected to intranasal exposures to the allergen eitheracutely constituted by simultaneous daily exposures for 3days or chronically by challenging the animals three times aweek for four weeks as described in Supplementary FigureS1 Figure 3(a) shows that similar to the acute OVA modelL-NIL administration was extremely efficient in blockingHDM-induced AHR in fact AHR of HDM-treated micethat received the drug was identical to animals that werenot exposed to HDM Contrary to the acute HDM exposuremodel iNOS inhibition by L-NIL did not provide a signif-icant protection against AHR upon a chronic exposure toHDM Altogether the differential effects of iNOS inhibitionon AHR induced by acute or chronic HDM exposure werevery similar to those observed using the acute and chronicOVAmodels of allergic lung inflammationThese results alsodemonstrate that the role of iNOS in AHR manifestation isnot specific to a given model and may be considered as ageneral phenomenon

We next examined whether the failure of iNOS inhi-bition by L-NIL administration can be reversed by mereadministration of NO source such as nitrite Figure 3(b)


Poly(ADP-ribose)

Ep

Im

(a)

iNOS

MQ

Ep

Ed

(b)

Nitrotyrosine

(c)

Negative control

(d)

Nitr

otyr

osin

e

Nonasthmatic Asthmatic

GAPDH

NA NA NA NA NA

iNOS

2521 21 18 22 16 1516ACT score1 2 3 4 5 116 7 8 9 10 1312Subject number

(e)

Figure 1 PARP activation iNOS expression and protein nitration in human asthmatics Lung sections from a deidentified individual whodied from severe asthma were subjected to immunohistochemistry with antibodies to PAR (a) iNOS (b) nitrotyrosine (c) or (d) control IgGfollowed by visualization with light microscopy Bars 20120583m The lower panels represent magnifications of the boxed areas Ed endothelialcells Ep epithelial cells Im immune cells MQ macrophages (e) PBMCs collected from asthmatics with controlled (ACT score ge 20) oruncontrolled (ACT scorele 19) disease or healthy nonasthmatic (NA) volunteers were subjected to protein extraction followed by immunoblotanalysis with antibodies to human iNOS nitrotyrosine or GAPDH

Mediators of Inflammation 5Pe

nh

0 125 25 50 100MeCh (mgmL)

lowastlowastlowast

12

9

6

3

0

WT (OVA+L-NIL)

WT (control)WT (OVA)

Acute asthma model

(a)

Penh

ns

0 125 25 50 100MeCh (mgmL)

12

9

6

3

0

WT (OVA+L-NIL)


Chronic asthma model

(b)

Figure 2 Effect of iNOS inhibition by L-NIL on AHR manifestation upon acute or chronic exposure to OVA in mice C57BL6 mice weresubjected to OVA sensitization followed by an acute (a) or chronic (b) challenge to aerosolized OVA or left unchallenged A group of WTmice were administered ip 5mgkg of the specific iNOS inhibitor (L-NIL) or saline 30min after each OVA challenge Penhwas recorded 24 hafter the last challenge using a whole body plethysmograph system before and after the indicated concentrations of aerosolized methacholine(MeCh) Results are plotted as maximal fold increase of Penh relative to baseline (0120583MMeCh) and expressed as mean plusmn SEM where 119899 = 5mice per group lowast lowast lowast difference from OVA-challenged mice 119901 le 0001 difference from control unchallenged mice 119901 le 001 ns nosignificant difference

shows that administration of 20120583gkg nitrite ip 30min aftereach challenge with HDM exerted a remarkable protectionagainst AHR manifestation in response to increasing dosesof methacholine Concomitantly nitrite supplementationdecreasedTh2 inflammation as assessed by IL-5mRNA in thelung (Figure 3(c)) Given the fact that nitrite administrationprovidesmoderate levels ofNO [26] these results suggest thatNO levels are an important determinant for the protectionagainst AHR andTh2 inflammation

34 PARP-1 Inhibition-Mediated Protection against AHR inChronically HDM-Exposed Mice is Lost upon L-NIL Admin-istration We previously demonstrated that PARP inhibitiongenetically or by olaparib protects against asthma mani-festation including airway inflammation and AHR [21 22]We also showed that PARP-1 regulates iNOS expression inan animal model of asthma and in cultured cells [20] Themost fascinating aspect of the connection between PARP-1 and iNOS is that PARP-1 inhibition does not completelyinhibit expression of iNOS [20] as demonstrated in resultsshown in Figure 4(a) using lung fibroblasts isolated fromWT and PARP-1minusminus mice and treated with a combinationof LPS and IFN-120574 Such incomplete inhibition was alsoobserved in endothelial cells (data not shown) We thushypothesized that the beneficial effect of PARP-1 inhibitiononAHRmay be associatedwith the partial reduction in iNOSexpression which presumably leads to a production of low-to-moderate levels of NO Accordingly inhibition of iNOSby L-NIL treatment was predicted to abrogate the protectiveeffect of PARP inhibition To test this hypothesis mice

were chronically exposed to HDM and were administereda combination of 5mgkg olaparib and 5mgkg L-NIL aftereach challenge Figure 4(b) shows that olaparib treatmentprovided an excellent protection against AHR which wassignificantly abolished by L-NIL administration Unlike itseffect on AHR L-NIL administration only partially reversedolaparib-mediated protection against Th2 inflammation asassessed by IL-5 mRNA in lungs (Figure 4(c)) These resultssuggest that the low expression of iNOS observed uponPARP inhibition was protective against both AHR and Th2inflammation

4 Discussion

The viability of iNOS as a therapeutic target for the treatmentof asthma was hindered by the contradictory reports ofa number of encouraging animal studies and the negativeresults of a clinical trial The results of the trial showed thata selective iNOS inhibitor similar to the one used in thecurrent study provided no protection against AHR or airwayinflammation after allergen challenge in steroid-naıve humanasthmatics [14] A detailed study from our laboratory showedthat iNOS exerted a differential effect on acute and chronicasthma [19] iNOS inhibition was effective in blocking onlyacute allergic inflammation but failed to provide any protec-tion against chronic inflammation [19]The effects on chronicinflammationwere consistent with those reported by Singh etal using a selective iNOS inhibitor (GW274150) [17] Inter-estingly however iNOS inhibition protected against airwayremodeling potentially by preventing an increase in TIMP-2 expression [19] despite the persistence of inflammation


Penh

WT (HDM+L-NIL)

WT (control)

0 125 25 50 100

WT (HDM)

MeCh (mgmL)

lowast

12

9

6

3

0

Acute asthma model

(a)

Penh

0 125 25 50 100

ns

MeCh (mgmL)WT (HDM+L-NIL)WT (control)

WT (HDM) WT (HDM+L-NIL+NaNO2)

lowastlowast

12

9

6

3

0


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

WT

(HD

M+L

-NIL

)

WT

(con

trol)

WT

(HD

M)

WT

(HD

M+L

-NIL

+NaN

O2)

lowastlowast p = 0002

p = 00031

ns p = 04086

(c)

Figure 3 Effect of iNOS inhibition by L-NIL onAHRmanifestation upon exposure toHDMinmice and the influence ofNO supplementationby nitrite administration C57BL6 mice were subjected to HDM sensitization followed by acute (a) or chronic (b) intranasal challenge withHDM or were left unchallenged Challenged mice were administered ip 5mgkg L-NIL with or without 20mgkg of nitrite (NaNO

2

) as NOsource 30min after each HDM challenge Penh was recorded 24 h after the last HDM challenge using a whole body plethysmograph beforeand after the indicated concentrations of aerosolized MeCh Results are plotted as maximal fold increase of Penh relative to baseline andexpressed as mean plusmn SEM where 119899 = 5 mice per group lowast and lowastlowast difference from HDM challenged mice with 119901 le 005 and 119875 le 001respectively difference from control unchallenged mice with 119901 lt 005 RNA isolated from the lungs of the different experimental groupswas reverse-transcribed and the resulting cDNA was subjected to real-time PCR with primer sets specific to mouse IL-5 or 120573-actin Datais expressed as fold change of values detected in lungs of control mice lowastlowast difference from control unchallenged mice with 119901 = 0002 difference from HDM-exposed mice receiving L-NIL with 119901 = 00031 ns nonsignificant difference with 119901 = 04086

These results suggest that iNOS inhibition may be protectiveagainst some aspects of asthma but not others The presentstudy represents a continuation of our effort to understandthe role of iNOS in asthma and explore new mechanisms bywhich the enzyme can be targeted for therapy against thedisease What is certain is that the enzyme plays important

roles in the disease what is lacking however is the strategy bywhich it can be adequately targeted to achieve an efficaciousclinical outcome We believe that the results of the presentstudy renew the potential of iNOS as a therapeutic target forasthma with a specific consideration to the levels of NO inpatients treated with selective iNOS inhibitors


WT

iNO

S ex

pres

sion

(fold

chan

ge)

LPS+IFN-120574 + +minus minus

PARP-1minusminus

40

30

20

10

0

lowast

lowastlowastlowast

(a)

Penh

WT (HDM)WT (HDM+olaparib)WT (HDM+olaparib+L-NIL)

0 125 25 50 100

ns

12

9

6

3

0

MeCh (mgmL)

lowastlowast


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

Con

HD

M

HD

M+o

lapa

rib

HD

M+o

lapar

ib+

L-N

IL


ns p = 00510

p = 00016

(c)

Figure 4 Effects of PARP inhibition on iNOS expression in LPSIFN-120574-treated MEFs and of iNOS inhibition on the protection conferred bythe PARP inhibitor olaparib against AHR in chronicallyHDM-exposedmice (a)MEFs derived fromWTor PARP-1minusminusmicewere treatedwitha combination of 1 120583gmL LPS and 10 ngmL IFN-120574 RNA was isolated after 6 hours of treatment and reverse-transcribed to generate cDNAwhich was subjected to quantitative PCR with primers specific to mouse iNOS or 120573-actinThe data is expressed as fold change normalized tolevels of 120573-actin lowast and lowast lowast lowast difference from nonstimulated cells with 119901 le 005 and 119901 le 0001 respectively difference from LPSIFN-120574-treated WT cells with 119901 le 0001 (b) WT mice were subjected to HDM sensitization followed by a chronic in challenge with HDM or leftunchallenged Challenged mice were administered ip 5mgkg olaparib with or without 5mgkg of L-NIL 30min after each HDM challengePenh was recorded 24 h after the last challenge in response to increasing doses of MeCh Results are plotted as maximal fold increase of Penhrelative to baseline and expressed asmeanplusmn SEMwhere 119899 = 5mice per grouplowastlowast difference fromHDMchallengedmice difference fromthe group that received 5mgkg olaparib 119901 le 001 RNA isolated from the lungs of the different experimental groups was reverse-transcribedand the resulting cDNAwas subjected to real time PCR with primer sets specific to mouse IL-5 or 120573-actin Data is expressed as fold change ofvalues detected in lungs of control mice lowastlowast difference from control unchallenged mice difference from HDM-exposed mice receivingolaparib ns no significant difference Note that values of control and HDM-exposed mice are the same as those described for Figure 3(c)

High levels of iNOS and ONOOminus the reactive byproductof NO are undoubtedly deleterious and participate in thepathology of asthma [19 27 28] as well as many chronicinflammatory diseases [9ndash12] Sugiura et al showed thatpatients with refractory asthma have even higher levels of

iNOS and protein nitration in cells collected from theirsputum than those from patients with well-controlled asthma[29] This connection was recently strengthened by a studydemonstrating that an iNOS-Dual oxidase-2-thyroid per-oxidase metabolome is the basis of nitrogen radicals and


subsequent protein nitration in human severe asthma [30]Our results show that the elevated iNOS expression is alsoobserved in PBMCs of asthmatics and potentially evenhigher in cells from asthmatics with uncontrolled diseaseSurprisingly this is the first report examining the levels ofiNOS and protein nitration in PBMCs of asthmatics Ourresults suggest that the role of iNOS in the pathogenesisof the disease may stem from circulating cells in additionto those in the lung Although the cohort size is small inthe present study our results show a potential disconnectbetween expression of iNOS and protein nitration withinthe same cells suggesting that PBMCs may not be the majortarget of NO and its byproducts This disconnect does notappear to exist in cells derived from either bronchial lavagefluids or sputum as shown by Yamamoto et al and Sugiuraet al respectively [13 29] Altogether the results of thesestudies and many others including ours [19 20] predict thatiNOS may be an ideal target for the treatment of asthmaSurprisingly targeting this enzyme has been unsuccessful[17] We speculated that our understanding of the role of NOand iNOS in asthma may not be sufficient to allow a betterapproach to achieve the desirable clinical outcomes usingselective iNOS inhibitors

In the current study we show that iNOS inhibition bythe selective inhibitor L-NIL and confirmed by the use ofa gene knockout approach provided an excellent protectionagainst AHR upon an acute exposure but not upon a chronicexposure to allergens in two experimental asthma modelsThese results are rather similar to the differential protectionprovided by iNOS inhibition against airway inflammationIt is noteworthy that NO supplementation aggravated Th2inflammation in HDM-exposed mice as assessed by lungIL-5 mRNA levels At this stage it is not clear whetherthe protection and loss of protection in the acute andchronic models respectively are associated with the statusof inflammation as manifestation of AHR is not alwaysstrictly related to inflammation in human asthmatics [31]The interesting aspect of this study is that the protectionwas recovered in the chronic HDM asthma model after NOsupplementation by nitrite administrationThe dose of nitriteused in this study was shown to deliver low-to-moderatelevels of NO and protects against several oxidative stress-related conditions including hypoxic vasodilation ischemiaof the heart and liver and postoperative ileus [26 32 33]It is important to acknowledge that the failure of iNOSinhibition to protect against AHR in chronic asthma maybe related to other factors besides NO however the level ofthe gas may constitute a major determinant in the protectionagainst AHR It is also important to acknowledge that oneof the limitations of the current study is that the levels ofNO upon nitrate supplementation were not measured Wepredict that these levels might not have reached those inHDM-exposed mice otherwise we would expect a failureof protection against AHR manifestation An additionallimitation is the quantification of Penh using whole bodyplethysmography to measure AHR Although measuringlung resistance is regarded as a better means to assess lungfunction our previous studies demonstrate for instance asimilar protection against AHR by PARP inhibitors using

either method [22 34] Additionally Penh has been demon-strated to correlate well with invasive measures of AHR[35]

Our laboratory established a reciprocal relationshipbetween iNOS and PARP-1 [20] PARP-1 activity and expres-sion are required for iNOS expression [36] Such regulationis linked to the dependence of iNOS gene on NF-120581B andcontrol of the activity as well as the subcellular traffickingof the transcription factor by PARP-1 Interestingly PARPinhibition pharmacologically or by gene knockout pro-tected against inflammation and AHR both upon acute andchronic exposures to either OVA or HDM Such protectionoccurred despite the fact that iNOS expression is markedlyreduced upon PARP inhibition It is interesting howeverthat PARP-1 inhibition does not completely abrogate expres-sion of iNOS This partial inhibition may allow for onlylow-to-moderate level production of NO and constitutesthe basis for the persistent protection against AHR con-ferred by PARP inhibition even after a chronic exposure toallergens

5 Conclusions

Our results provide additional support for the hypothesisthat the amount of iNOS and NO are critical determinantsin the modulation of AHR by selective iNOS inhibitors andmay also help explain why the clinical study by Singh et al[17] found that GW274150 failed to protect against AHR Itis noteworthy that the levels of exhaled NO achieved uponadministration of the inhibitor reached amounts lower thanthose observed in healthy nonsmoker and nonatopic subjectsas comprehensively reviewed by Dweik et al [37] The severereduction in the levels of NO in the study by Singh et almay be detrimental and as such prevention of AHR mightnot have been possible Furthermore the drug was deliveredorally which may be another limitation of the study Thesespeculations and the positive results of our study on the role ofiNOS and the critical influence of NO levels on AHR shouldlead to reevaluation of the benefit of selective iNOS inhibitorsin blocking AHR in human asthmatics especially those withuncontrolled disease

Abbreviations

AHR Airway hyperresponsivenessHDM House dust mite extractiNOS Inducible NO synthaseL-NIL L-N6-(1-Iminoethyl)lysine dihydrochloridePARP-1 Poly(ADP-ribose) polymerase-1PBMCs Peripheral blood mononuclear cellsPenh Enhanced pause

Additional Points

The results of the present study suggest that the amount ofnitric oxide may be critical for the protection against airwayhyperresponsiveness and renew the potential of iNOS as atherapeutic target for the treatment of asthma


Competing Interests

All authors state that they have no competing interests todeclare

Authorsrsquo Contributions

Salomersquo V Ibba is the graduate student who led the studyand conductedmajority of the experiments and the statisticalanalyses Mohamed A Ghonim assisted in the animal studiesand helped with statistical analyses Kusma Pyakurel assistedin the animal studies and helped with statistical analysesMatthew R Lammi contributed to the human subjectsrsquo refer-ral assisted in the acquisition of human PBMCs edited thepaper and contributed to discussion Anil Mishra providedinstruments for AHR quantification and training A HamidBoulares is the principal investigator and contributed to thedesign of the experiment and the training of researchers andprovided the financial support for the conducted work

Acknowledgments

The authors would like to thank the clinical coordina-tors (Marie Sandi and Connie Romaine) for their effortin collecting peripheral blood from the human subjectsThey would like to thank the Pulmonary and Critical CareSection at LSUHSC for giving them access to the wholebody plethysmograph system They would also like to thankDr Luis Del Valle and the Stanley Scott Cancer Centerimaging core for their assistance with tissue processing andimmunohistochemistry and Zinaida Osipova for assistancewith immunoblotting analysis during her summer trainingThis work was supported by the National Institute of Health(NIH) [Grants no HL072889 and 1P30GM114732-01 (to AHamid Boulares)] the National Institute of General MedicalSciences of the National Institutes of Health which funds theLouisiana Clinical and Translational Science Center [Grantno 1 U54 GM104940 (to Matthew R Lammi)] the EgyptianCultural and Educational Bureau [Grant no JS-2634 (toMohamed A Ghonim)] the American Heart Association[Grant no 14PRE19630012 (to Kusma Pyakurel)] and theNational Institute of General Medical Sciences [Grant noCOBRE 5P30GM106392]

References

[1] R H Moreno J C Hogg and P D Pare ldquoMechanics of airwaynarrowingrdquo American Review of Respiratory Disease vol 133no 6 pp 1171ndash1180 1986

[2] Y Bosse P D Pare and C Y Seow ldquoAirway wall remodelingin asthma from the epithelial layer to the adventitiardquo CurrentAllergy and Asthma Reports vol 8 no 4 pp 357ndash366 2008

[3] R Halwani S Al-Muhsen and Q Hamid ldquoAirway remodelingin asthmardquo Current Opinion in Pharmacology vol 10 no 3 pp236ndash245 2010

[4] H S Park S Y Kim S R Kim and Y C Lee ldquoTargetingabnormal airway vascularity as a therapeutical strategy inasthmardquo Respirology vol 15 no 3 pp 459ndash471 2010

[5] C B Barnes and C S Ulrik ldquoAsthma and adherence toinhaled corticosteroids current status and future perspectivesrdquoRespiratory Care vol 60 no 3 pp 455ndash468 2015

[6] B N Lambrecht and H Hammad ldquoThe immunology ofasthmardquo Nature Immunology vol 16 no 1 pp 45ndash56 2015

[7] L R DeNicola N Kissoon L J Duckworth K V Blake S PMurphy andP E Silkoff ldquoExhaled nitric oxide as an indicator ofseverity of asthmatic inflammationrdquo Pediatric Emergency Carevol 16 no 4 pp 290ndash295 2000

[8] F L M Ricciardolo F P Nijkamp and G Folkerts ldquoNitricOxide synthase (NOS) as therapeutic target for asthma andchronic obstructive pulmonary diseaserdquo Current Drug Targetsvol 7 no 6 pp 721ndash735 2006

[9] P Kirkham and I Rahman ldquoOxidative stress in asthma andCOPD antioxidants as a therapeutic strategyrdquo PharmacologyandTherapeutics vol 111 no 2 pp 476ndash494 2006

[10] WMacNee ldquoOxidative stress and lung inflammation in airwaysdiseaserdquoEuropean Journal of Pharmacology vol 429 no 1ndash3 pp195ndash207 2001

[11] J S Beckman ldquoUnderstanding peroxynitrite biochemistry andits potential for treating human diseasesrdquo Archives of Biochem-istry and Biophysics vol 484 no 2 pp 114ndash116 2009

[12] H Sugiura and M Ichinose ldquoOxidative and nitrative stress inbronchial asthmardquoAntioxidants andRedox Signaling vol 10 no4 pp 785ndash797 2008

[13] M Yamamoto Y Tochino K Chibana J B Trudeau FHolguin and S E Wenzel ldquoNitric oxide and related enzymesin asthma relation to severity enzyme function and inflamma-tionrdquo Clinical and Experimental Allergy vol 42 no 5 pp 760ndash768 2012

[14] T T Hansel S A Kharitonov L E Donnelly et al ldquoA selectiveinhibitor of inducible nitric oxide synthase inhibits exhaledbreath nitric oxide in healthy volunteers and asthmaticsrdquo TheFASEB Journal vol 17 no 10 pp 1298ndash1300 2003

[15] S A Mulrennan and A E Redington ldquoNitric oxide synthaseinhibition therapeutic potential in asthmardquo Treatments inRespiratory Medicine vol 3 no 2 pp 79ndash88 2004

[16] C Brindicci K Ito P J Barnes and S A Kharitonov ldquoEffectof an inducible nitric oxide synthase inhibitor on differentialflow-exhaled nitric oxide in asthmatic patients and healthyvolunteersrdquo Chest vol 132 no 2 pp 581ndash588 2007

[17] D Singh D Richards R G Knowles et al ldquoSelective induciblenitric oxide synthase inhibition has no effect on allergenchallenge in asthmardquo American Journal of Respiratory andCritical Care Medicine vol 176 no 10 pp 988ndash993 2007

[18] T Islam C Breton M T Salam et al ldquoRole of inducible nitricoxide synthase in asthma risk and lung function growth duringadolescencerdquoThorax vol 65 no 2 pp 139ndash145 2010

[19] A S Naura M Zerfaoui H Kim et al ldquoRequirement forinducible nitric oxide synthase in chronic allergen exposure-induced pulmonary fibrosis but not inflammationrdquo Journal ofImmunology vol 185 no 5 pp 3076ndash3085 2010

[20] A S Naura R Datta C P Hans et al ldquoReciprocal regulationof iNOS and PARP-1 during allergen-induced eosinophiliardquoEuropean Respiratory Journal vol 33 no 2 pp 252ndash261 2009

[21] M A Ghonim K Pyakurel S V Ibba et al ldquoPARP is activatedin human asthma and its inhibition by olaparib blocks housedust mite-induced disease in micerdquo Clinical Science (LondonEngland) vol 129 no 11 pp 951ndash962 2015

[22] M A Ghonim K Pyakurel S V Ibba et al ldquoPARP inhibitionby olaparib or gene knockout blocks asthma-like manifestation


in mice by modulating CD4+ T cell functionrdquo Journal ofTranslational Medicine vol 13 no 1 article 225 2015

[23] R A Nathan C A Sorkness M Kosinski et al ldquoDevelopmentof the asthma control test a survey for assessing asthmacontrolrdquo Journal of Allergy andClinical Immunology vol 113 no1 pp 59ndash65 2004

[24] J Bousquet P K Jeffery W W Busse M Johnson and AM Vignola ldquoAsthma from bronchoconstriction to airwaysinflammation and remodelingrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 161 no 5 pp 1720ndash1745 2000

[25] W M Moore R K Webber G M Jerome F S TjoengT P Misko and M G Currie ldquoL-N6-(1-iminoethyl)lysine aselective inhibitor of inducible nitric oxide synthaserdquo Journal ofMedicinal Chemistry vol 37 no 23 pp 3886ndash3888 1994

[26] K Cosby K S Partovi J H Crawford et al ldquoNitrite reductionto nitric oxide by deoxyhemoglobin vasodilates the humancirculationrdquoNatureMedicine vol 9 no 12 pp 1498ndash1505 2003

[27] C Gerard ldquoAsthmatics breathe easier when itrsquos SNO-ingrdquoScience vol 308 no 5728 pp 1560ndash1561 2005

[28] C Lane D Knight S Burgess et al ldquoEpithelial inducible nitricoxide synthase activity is the major determinant of nitric oxideconcentration in exhaled breathrdquoThorax vol 59 no 9 pp 757ndash760 2004

[29] H Sugiura Y Komaki A Koarai and M Ichinose ldquoNitrativestress in refractory asthmardquo Journal of Allergy and ClinicalImmunology vol 121 no 2 pp 355ndash360 2008

[30] N Voraphani M T Gladwin A U Contreras et al ldquoAn air-way epithelial iNOS-DUOX2-thyroid peroxidase metabolomedrives Th1Th2 nitrative stress in human severe asthmardquoMucosal Immunology vol 7 no 5 pp 1175ndash1185 2014

[31] W W Busse ldquoThe relationship of airway hyperresponsive-ness and airway inflammation airway hyperresponsiveness inasthma its measurement and clinical significancerdquo Chest vol138 no 2 supplement pp 4Sndash10S 2010

[32] M R Duranski J J M Greer A Dejam et al ldquoCytoprotectiveeffects of nitrite during in vivo ischemia-reperfusion of theheart and liverrdquoThe Journal of Clinical Investigation vol 115 no5 pp 1232ndash1240 2005

[33] J Hamacher K Eichert C Braun et al ldquoMontelukast exerts noacute direct effect on NO synthasesrdquo Pulmonary PharmacologyandTherapeutics vol 20 no 5 pp 525ndash533 2007

[34] A S Naura C P Hans M Zerfaoui et al ldquoPost-allergenchallenge inhibition of poly(ADP-ribose) polymerase harborstherapeutic potential for treatment of allergic airway inflamma-tionrdquo Clinical and Experimental Allergy vol 38 no 5 pp 839ndash846 2008

[35] E Hamelmann J Schwarze K Takeda et al ldquoNoninvasivemeasurement of airway responsiveness in allergic mice usingbarometric plethysmographyrdquo American Journal of Respiratoryand Critical Care Medicine vol 156 no 3 pp 766ndash775 1997

[36] M Zerfaoui Y Errami A S Naura et al ldquoPoly(ADP-ribose)polymerase-1 is a determining factor in Crm1-mediated nuclearexport and retention of p65NF-120581BuponTLR4 stimulationrdquoTheJournal of Immunology vol 185 no 3 pp 1894ndash1902 2010

[37] R A Dweik P B Boggs S C Erzurum et al ldquoAn official ATSclinical practice guideline interpretation of exhaled nitric oxidelevels (FENO) for clinical applicationsrdquo American Journal ofRespiratory and Critical Care Medicine vol 184 no 5 pp 602ndash615 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


production of NO and generation of the reactive metaboliteONOOminus [9ndash12] It appears that expression of iNOS is evenhigher in sputum cells from asthmatics compared to thosefrom patients with controlled disease or healthy individuals[13]Thus inhibition of iNOS appears to be a very viable ther-apeutic target to prevent manifestation of asthma symptomsupon exposure to allergens [14ndash16] This potential has beenchallenged by the observation that a selective iNOS inhibitordid not affect airway inflammatory cell numbers or AHRafter allergen challenge in steroid-naıve human asthmatics[17] However it is difficult to ignore the fact that asthmaprotection and susceptibility are associated with polymor-phisms in the iNOS gene [18] We recently showed that iNOSgene deletionwas associated with a reduction in eosinophiliamucus hypersecretion andTh2 cytokine production upon anacute exposure to ovalbumin (OVA) [19] Such protectionwascompletely abolished upon a chronic exposure to the allergenInterestingly pulmonary fibrosis observed in wild type miceunder the chronic protocol was completely absent in iNOSminusminusmice despite persistent IL-5 and IL-13 production The pub-lished results exemplified the complexity of the role of iNOSin asthma and the preservation of its potential as a therapeutictarget We also showed that poly(ADP-ribose) polymerase-(PARP-) 1 is required for iNOS expression [20] and that PARPinhibition protects against AHR upon an acute or chronicexposure to allergens [21 22] Overall we believe that it ispremature to conclude that targeting iNOS in asthma is futileand that more studies should be geared toward exploringnew avenues to take advantage of such an important clinicaltarget Accordingly the goal of the present study was toexamine whether pharmacological inhibition of iNOS couldbe manipulated to provide protection against AHR uponchronic OVA or house dust mite extracts (HDM) exposureand whether the protection conferred by PARP inhibitionwas related to its control of iNOS expression level L-N6-(1-Iminoethyl)lysine dihydrochloride (L-NIL) and AZD2281(olaparib) two clinically tested iNOS and PARP inhibitorsrespectively were used to conduct the following study

2 Methods

21 Human Subjects Immunohistochemistry and ImmunoblotAnalysis Five healthy and eight asthmatic individuals wererecruited under a protocol (8450) approved by institutionalreview board of Louisiana State University Health SciencesCenter Subjects were included in the study if they were ge18years of age with a physician diagnosis of asthma Subjectswere excluded if they were diagnosed with another lungdisease other than asthma had active malignancy or inflam-matory condition or smokedge10 pack-years Asthma controlwas assessed by the Asthma Control Test (ACT) with a scorele19 indicating uncontrolled asthma and ge20 demonstratingthat asthma is well controlled [23] Human PBMCs isolatedfrom peripheral blood of donors were subjected to proteinextractions followed by immunoblot analysis with antibodiesto nitrotyrosine (EMD Millipore Bedford MA) humaniNOS (Abcam Cambridge MA) the poly(ADP-ribose) moi-ety (PAR) of PARP-modified proteins (ENZO Life Sciences

Farmingdale NY) or GAPDH (Abcam) Paraffin-embeddedtissue sections from two deidentified lung specimens fromindividuals who died from severe asthma were subjected toimmunohistochemistry with antibodies to PAR iNOS ornitrotyrosine The sections were then counterstained withhematoxylin and mounted prior to examination by lightmicroscopy

22 Animals OVA and HDM Challenge and AHR Measure-ments Six-week-old to eight-week-old C57BL6J male micewere purchased from Jackson Laboratories (Bar Harbor MEUSA) C57BL6 iNOSminusminus mice were bred at the LSUHSCvivarium and allowed unlimited access to sterilized chow andwater Husbandry experimental protocols and procedureswere all approved by the LSUHSC Animal Care and UseCommittee

Mice were sensitized to chicken (3mgkg) OVA (Sigma-Aldrich St LouisMO) or (05120583gkg)HDM(Dermatophago-ides pteronyssinus) extract (Greer Labs Lenoir NC) Thechallenge protocols with OVA or HDM are outlined inSupplementary Figure S1 in Supplementary Martial availableonline at httpdxdoiorg10115520161984703 Briefly micewere challenged with aerosolized 3 OVA for 30min threetimes on days 14 16 and 18 for the acute asthma model orthree times a week for three weeks (chronic asthma model)Other groups of mice were challenged intranasally with125 120583gkg whole HDM extract on days 24 25 and 26 forthe acute asthma model or 3 times per week for a totalof 4 weeks for the chronic asthma model Control groupswere not sensitized or challenged Additional challengedgroups of mice were administered ip 5mgkg L-N6-(1-Iminoethyl)lysine dihydrochloride (L-NIL) (Sigma-Aldrich)andor olaparib (Selleckchem Pittsburgh PA) in saline30 minutes after each challenge Some groups of micealso received ip injections of 20 120583gkg of nitrite (NaNO

2

)(Sigma-Aldrich) as NO source 30min after each challenge

AHR to inhaled methacholine was measured in unre-strained conscious mice 24 h after the last challenge byrecording ldquoenhanced pauserdquo (Penh) by whole body plethys-mography (EMKA Systems Falls Church VA or DSI BuxcoSt Paul MN) In brief the baseline readings were taken andaveraged for 3min after animals were placed in a barometricplethysmographic chamber Normal saline or increasingconcentrations of aerosolized methacholine (Sigma-Aldrich)(125ndash100mgmL) were nebulized and readings were takenand averaged for 3min after each nebulization and enhancedpause (Penh) representing AHR was calculated Mice weresacrificed 24 h after the last challenge and lungs were sub-jected to RNA extraction as described below

23 Cell Culture Real Time PCR Wild type or PARP-1minusminusmouse embryonic fibroblasts (MEFs) were serum starved for24 hours and then treated with 1 120583gmL LPS (Sigma-Aldrich)and 10 ngmL IFN-120574 (RD System Minneapolis MN) for 6hours The collected cells as well as homogenized lungs fromthe abovementioned experimental groups were subjectedto RNA extraction using the RNeasy mini kit (QIAGENValencia CA) The extracted RNA was reverse-transcribed




3 Results





50





Poly(ADP-ribose)

Ep

Im

(a)

iNOS

MQ

Ep

Ed

(b)

Nitrotyrosine

(c)

Negative control

(d)

Nitr

otyr

osin

e


GAPDH

NA NA NA NA NA

iNOS


(e)



nh

0 125 25 50 100MeCh (mgmL)

lowastlowastlowast

12

9

6

3

0

WT (OVA+L-NIL)


Acute asthma model

(a)

Penh

ns

0 125 25 50 100MeCh (mgmL)

12

9

6

3

0

WT (OVA+L-NIL)



(b)





4 Discussion



Penh

WT (HDM+L-NIL)

WT (control)

0 125 25 50 100

WT (HDM)

MeCh (mgmL)

lowast

12

9

6

3

0

Acute asthma model

(a)

Penh

0 125 25 50 100

ns



lowastlowast

12

9

6

3

0


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

WT

(HD

M+L

-NIL

)

WT

(con

trol)

WT

(HD

M)

WT

(HD

M+L

-NIL

+NaN

O2)


p = 00031

ns p = 04086

(c)


2





WT

iNO

S ex

pres

sion

(fold

chan

ge)


PARP-1minusminus

40

30

20

10

0

lowast

lowastlowastlowast

(a)

Penh


0 125 25 50 100

ns

12

9

6

3

0

MeCh (mgmL)

lowastlowast


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

Con

HD

M

HD

M+o

lapa

rib

HD

M+o

lapar

ib+

L-N

IL


ns p = 00510

p = 00016

(c)









5 Conclusions


Abbreviations


Additional Points



Competing Interests




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















3 Results





50





Poly(ADP-ribose)

Ep

Im

(a)

iNOS

MQ

Ep

Ed

(b)

Nitrotyrosine

(c)

Negative control

(d)

Nitr

otyr

osin

e


GAPDH

NA NA NA NA NA

iNOS


(e)



nh

0 125 25 50 100MeCh (mgmL)

lowastlowastlowast

12

9

6

3

0

WT (OVA+L-NIL)


Acute asthma model

(a)

Penh

ns

0 125 25 50 100MeCh (mgmL)

12

9

6

3

0

WT (OVA+L-NIL)



(b)





4 Discussion



Penh

WT (HDM+L-NIL)

WT (control)

0 125 25 50 100

WT (HDM)

MeCh (mgmL)

lowast

12

9

6

3

0

Acute asthma model

(a)

Penh

0 125 25 50 100

ns



lowastlowast

12

9

6

3

0


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

WT

(HD

M+L

-NIL

)

WT

(con

trol)

WT

(HD

M)

WT

(HD

M+L

-NIL

+NaN

O2)


p = 00031

ns p = 04086

(c)


2





WT

iNO

S ex

pres

sion

(fold

chan

ge)


PARP-1minusminus

40

30

20

10

0

lowast

lowastlowastlowast

(a)

Penh


0 125 25 50 100

ns

12

9

6

3

0

MeCh (mgmL)

lowastlowast


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

Con

HD

M

HD

M+o

lapa

rib

HD

M+o

lapar

ib+

L-N

IL


ns p = 00510

p = 00016

(c)









5 Conclusions


Abbreviations


Additional Points



Competing Interests




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Poly(ADP-ribose)

Ep

Im

(a)

iNOS

MQ

Ep

Ed

(b)

Nitrotyrosine

(c)

Negative control

(d)

Nitr

otyr

osin

e


GAPDH

NA NA NA NA NA

iNOS


(e)



nh

0 125 25 50 100MeCh (mgmL)

lowastlowastlowast

12

9

6

3

0

WT (OVA+L-NIL)


Acute asthma model

(a)

Penh

ns

0 125 25 50 100MeCh (mgmL)

12

9

6

3

0

WT (OVA+L-NIL)



(b)





4 Discussion



Penh

WT (HDM+L-NIL)

WT (control)

0 125 25 50 100

WT (HDM)

MeCh (mgmL)

lowast

12

9

6

3

0

Acute asthma model

(a)

Penh

0 125 25 50 100

ns



lowastlowast

12

9

6

3

0


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

WT

(HD

M+L

-NIL

)

WT

(con

trol)

WT

(HD

M)

WT

(HD

M+L

-NIL

+NaN

O2)


p = 00031

ns p = 04086

(c)


2





WT

iNO

S ex

pres

sion

(fold

chan

ge)


PARP-1minusminus

40

30

20

10

0

lowast

lowastlowastlowast

(a)

Penh


0 125 25 50 100

ns

12

9

6

3

0

MeCh (mgmL)

lowastlowast


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

Con

HD

M

HD

M+o

lapa

rib

HD

M+o

lapar

ib+

L-N

IL


ns p = 00510

p = 00016

(c)









5 Conclusions


Abbreviations


Additional Points



Competing Interests




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















nh

0 125 25 50 100MeCh (mgmL)

lowastlowastlowast

12

9

6

3

0

WT (OVA+L-NIL)


Acute asthma model

(a)

Penh

ns

0 125 25 50 100MeCh (mgmL)

12

9

6

3

0

WT (OVA+L-NIL)



(b)





4 Discussion



Penh

WT (HDM+L-NIL)

WT (control)

0 125 25 50 100

WT (HDM)

MeCh (mgmL)

lowast

12

9

6

3

0

Acute asthma model

(a)

Penh

0 125 25 50 100

ns



lowastlowast

12

9

6

3

0


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

WT

(HD

M+L

-NIL

)

WT

(con

trol)

WT

(HD

M)

WT

(HD

M+L

-NIL

+NaN

O2)


p = 00031

ns p = 04086

(c)


2





WT

iNO

S ex

pres

sion

(fold

chan

ge)


PARP-1minusminus

40

30

20

10

0

lowast

lowastlowastlowast

(a)

Penh


0 125 25 50 100

ns

12

9

6

3

0

MeCh (mgmL)

lowastlowast


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

Con

HD

M

HD

M+o

lapa

rib

HD

M+o

lapar

ib+

L-N

IL


ns p = 00510

p = 00016

(c)









5 Conclusions


Abbreviations


Additional Points



Competing Interests




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Penh

WT (HDM+L-NIL)

WT (control)

0 125 25 50 100

WT (HDM)

MeCh (mgmL)

lowast

12

9

6

3

0

Acute asthma model

(a)

Penh

0 125 25 50 100

ns



lowastlowast

12

9

6

3

0


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

WT

(HD

M+L

-NIL

)

WT

(con

trol)

WT

(HD

M)

WT

(HD

M+L

-NIL

+NaN

O2)


p = 00031

ns p = 04086

(c)


2





WT

iNO

S ex

pres

sion

(fold

chan

ge)


PARP-1minusminus

40

30

20

10

0

lowast

lowastlowastlowast

(a)

Penh


0 125 25 50 100

ns

12

9

6

3

0

MeCh (mgmL)

lowastlowast


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

Con

HD

M

HD

M+o

lapa

rib

HD

M+o

lapar

ib+

L-N

IL


ns p = 00510

p = 00016

(c)









5 Conclusions


Abbreviations


Additional Points



Competing Interests




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















WT

iNO

S ex

pres

sion

(fold

chan

ge)


PARP-1minusminus

40

30

20

10

0

lowast

lowastlowastlowast

(a)

Penh


0 125 25 50 100

ns

12

9

6

3

0

MeCh (mgmL)

lowastlowast


(b)

Lung

IL-5

expr

essio

n (fo

ld ch

ange

)

12

10

8

6

4

2

0

Con

HD

M

HD

M+o

lapa

rib

HD

M+o

lapar

ib+

L-N

IL


ns p = 00510

p = 00016

(c)









5 Conclusions


Abbreviations


Additional Points



Competing Interests




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusions


Abbreviations


Additional Points



Competing Interests




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Competing Interests




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Potential of Inducible Nitric Oxide ...

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Transcript of Research Article Potential of Inducible Nitric Oxide ...