suppression of th1 and th17 responses and induction of...

Research ArticleSuppression of Th1 and Th17 Responses and Induction ofTreg Responses by IL-18-Expressing Plasmid Gene Combinedwith IL-4 on Collagen-Induced Arthritis

Qiaomei Dai 12 Yang Li 1 Haiyue Yu3 and XiaoyanWang1

1Department of Rheumatology and Immunology The Second Affiliated Hospital of Harbin Medical University Harbin China2Department of Pathology Heilongjiang University of Chinese Medicine Harbin China3Department of Rheumatology Qiqihar First Hospital Qiqihar China

Correspondence should be addressed to Yang Li liyanghrbmueducn

Received 21 November 2017 Revised 22 February 2018 Accepted 29 March 2018 Published 8 May 2018

Academic Editor Henry Wong

Copyright copy 2018 Qiaomei Dai et al This 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

Objectives IL-18 is a proinflammatory cytokine with multiple immunoregulatory properties We studied the effect of IL-18 genetherapy on the development ofmurine collagen-induced arthritis (CIA)Methods Plasmid pCAGGS-IL-18 along or in combinationwith IL-10 or IL-4 was administered to CIA mice The incidence and severity of arthritis of the paws were determined by a visualscale Joint destruction was determined by histology The levels of a panel of cytokines and transcription factors in the synoviumwere determined by reverse transcription polymerase chain reaction and quantitative RT-PCRQuantitative RT-PCRwas employedto detect the mRNA expression of TLRs and their pathway on the surface of DCs Results IL-18 gene therapy had no therapeuticeffect on CIA mice Additional coadministration with low dosage of recombinant IL-4 ameliorated the disease progressionHistopathological examination of the joints showed intact cartilage surface in IL-18 gene combined with IL-4-treated mice Thesynovium of IL-18 gene combined with rIL4-treated mice had lower expression of TNF-120572 IFN-120574 and IL-17 and higher expressionof IL-10 The mechanism of this response appeared to involve modulation of transcription factors FoxP3 and GATA-3 The DCs inthe spleen and lymph nodes of IL-18 gene combined with rIL4-treated mice had lower expression of TLR2 MyD88 and NF-kBConclusions Our findings indicate that pIL-18 gene combined with IL-4 ameliorates arthritis in the CIA mouse by suppression ofTh1 and Th17 cytokines and increasing expression of FoxP3 and GATA-3 The plasmid backbone and multiple immunoregulatoryproperties of IL-18 appear to play a major role in the pIL-18 coadministration with rIL-4-mediated immunomodulation of arthritisthrough blocking the TLR2MyD88NF-kappa B signaling pathway

1 Introduction

Rheumatoid arthritis (RA) is an autoimmune disease char-acterized by chronic inflammation that results in cartilagedamage and bone destruction [1] The roles of T cells in thepathogenesis of RA are not fully understood However previ-ous studies reported thatTh1 cytokines (interferon-120574 [IFN-120574]tumor necrosis factor-120572 [TNF-120572] and interleukin-1 [IL-1])Th2 cytokines (IL-4 and IL-10)Th17 cytokines (IL-17 and IL-22) and CD4+CD25+FoxP3+ regulatory T cells (Tregs) affecteach other and together with cell surface molecules activatesynovial macrophages fibroblast-like cells and osteoclastsleading to chronic inflammation and joint destruction [2 3]

Dendritic cells are professional antigen presenting cells(APCs) that can initiate and regulate T-cell responses Toll-like receptor (TLR) as the surface molecule of DCs activatesinnate immunity and plays an important role in RA [4] TGF-120573 alone induces the Treg transcription factor Foxp3 and isessential for the development of Tregs in the periphery [5]However the presence of proinflammatory cytokines suchas IL-6 which is induced during infection inflammationand injury inhibits the induction of Foxp3+ Tregs and simul-taneously promotes Th17-cell differentiation [5 6]

IL-18 is a proinflammatory cytokine withmultiple immu-noregulatory properties IL-18 belongs to the IL-1 ligandsuperfamily and is structurally and functionally similar to

HindawiBioMed Research InternationalVolume 2018 Article ID 5164715 8 pageshttpsdoiorg10115520185164715

2 BioMed Research International

IL-1 IL-18 was originally described as an inducer of Th1responses [7] In addition IL-18 can also stimulate Th2development and in concert with IL-23 can activateTh17 cells[7 8] Other research indicates that IL-18 plays a prominentrole in the onset and maintenance of the inflammatoryresponse during RA [9] Local neutralisation of IL-18 bythe naturally occurring IL-18-binding protein (IL-18BP) orsystemic neutralisation by specific antibodies amelioratescollagen-induced arthritis (CIA) reduces inflammation andreduces cartilage erosion [10 11] Yao et al reported thata recombinant virus expressing the IL-18BPIL-4 fusionprotein (AD-IL-18BPIL-4) suppresses the production andexpression of inflammatory cytokines in lipopolysaccharide-(LPS-) stimulated synovial fibroblasts [12] Another studydemonstrated that soluble IL-18 receptor 120573 inhibited IL-18during experimental arthritis had a major effect on T-cellcytokine balances and led to aggravation of CIA [9] Ourprevious research suggested that recombinant murine IL-18(rmIL-18) treatment alone ameliorated the progression ofarthritis in mice with CIA and that coadministration withlow-dose rIL-10 reversed this effect [2]

In this study we used the murine CIA model to examinethe effect of gene transfer of an IL-18-expressing plasmid onsuppression of CIA and the possible roles of Treg Th2 Th1Th17 and TLRs in the pathogenesis of arthritis

2 Materials and Methods

21 Reagents LPS (Escherichia coli 011184) Bovine typeII collagen and Freundrsquos complete adjuvant (FCA) werepurchased from Sigma (St Louis MO) RNA PCRKit (AMVVer30 and EX TAQ R-PCR Version 21) was from TaKaRaBiotechnology (Dalian China) Trizol was from InvitrogenCorporation (San Diego CA) primers and probes werefrom Pharmacia Biotech (Roosendaal Netherlands) andmurine recombinant IL-10 and IL-4 were from Prospec-TanyTechnogene (Rehovot Israel) CD11c MicroBeads were fromMiltenyi Biotec Inc (Bergisch Gladbach Germany)

22 Induction of Collagen-Induced Arthritis (CIA) MaleDBA1 mice (8ndash12 weeks old purchased from Slac Labora-tories Shanghai China) were housed in a specific pathogen-free environment with a 12 h light-dark cycle For the CIAmodel 100 120583g of bovine type II collagen dissolved in 005Macetic acid was emulsified with an equal volume of FCA andadministered intradermally at the base of the tail On day 21the mice were boosted by intraperitoneal injection of 100 120583gof bovine type II collagen dissolved in phosphate bufferedsaline (PBS)On day 28 fortymicewith nomacroscopic signswere treated by an additional intraperitoneal injection of LPS(40 120583g) Fortymicewere sacrificed onday 60 for analysisThisstudy was reviewed and approved by the Ethics Committeefor Experimental Animals of Harbin Medical University andall animals were treated according to the guidelines of theanimal ethical committee

23 Assessment of Arthritis The onset of arthritis was con-sidered to be the day that erythema andor swelling werefirst observed Mice were considered to have arthritis when

significant changes in redness and or swelling were notedin the digits or in other parts of at least 2 paws At latertime points ankylosis was also included in the macro-scopic scoring Cumulative scoring depending on rednessswelling and in later stadium ankylosis was as follows 0mdashnochanges 025mdash1 to 2 toes red or swollen 05mdash3 to 5 toesred or swollen 05mdashswollen ankle 05mdashswollen footpad05mdashsevere swelling and ankylosis The macroscopic scorewas assessed by two independent blinded observers [10]

24 Histology After animal sacrifice whole knee joints wereremoved and specimens were fixed for 4 days in 4 formalindecalcified in 5 formic acid and processed for paraffinembedding Tissue sections were stained with hematoxylinand eosin (HampE) or Masson stain Histopathology was usedto assess the occurrence of cartilage destruction and boneerosion

25 Gene Therapy After animal sacrifice plasmid DNAexpressingmurine IL-18was prepared as previously described[13] On day 28 forty mice without signs of arthritis weregiven intramuscular injections of pIL-18 (10 120583g) pIL-18(10 120583g) combined with IL-4 (01120583g) pIL-18 (10 120583g) combinedwith IL-10 (01120583g) or PBS as control A booster injectionof the same dosage was given two weeks later The samevolume of hyaluronidase IV (Sigma St Louis MO) wasinjected 10min before these injections to improve the plasmidexpression In a repeat experiment fifty mice without signsof arthritis were given intramuscular injections of pIL-18(10 120583g) combined with IL-4 (01120583g) IL-4 (01 120583g) plasmid(pCAGGS) combined with IL-4 (01 120583g) or with plasmid(pCAGGS) and PBS as control

26 Isolation of Spleen and Lymph Nodes CD11c+ DCs Micewere sacrificed by cervical dislocation The spleens andpopliteal lymph nodes were removed any fatty tissue wastrimmed away and spleens were washed three times withPBS The spleens and popliteal lymph nodes were laid on a200-mesh pore size cell strainer and using the barrel froma 1mL syringe the spleens and lymph nodes were pressedthrough the strainer until only a small amount of fibroustissue remained in the strainer CD11c+ cells were isolatedwith the CD11c MicroBeads according to the manufacturerrsquosinstructions [14]

27 RNAPreparation Reverse Transcription Polymerase ChainReaction and Quantitative RT-PCR Thepatella and adjacentsynovium were dissected immediately after animal sacrificeThe synovium samples were immediately frozen in liquidnitrogen and ground into powder with a tissue grinder andtotal RNA was extracted in 1mL Trizol reagent similar to theprocedure used for cartilage samples The levels of a panelof cytokines and transcription factors in the synovium weredetermined by reverse transcription polymerase chain reac-tion and quantitative RT-PCR The CD11c+ cell suspensionwas counted and RNAwas extractedThe expression of TLRsand their pathway on the surface of DCs was determined byquantitative RT-PCR Primer sequences and conditions forPCR were from the paper described previously [11] Primer

BioMed Research International 3

sequences for PCR were as follows 120573-actin forward 51015840-AGCGGT TCC GAT GCC CT-31015840 reverse 51015840-AGA GGT CTTTAC GGA TGT CAA CG-31015840 T-bet forward 51015840-GCC AGGAAC CGC TTA TAT G-31015840 reverse 51015840-TTG TTG GAA GCCCCC TTG-31015840 GATA-3 forward 51015840-AGG TGG ACG TACTTT TTAACATCG reverse 51015840-GCTAGCCCTGACGGAGTT TTC-31015840 IFN-120574 forward 51015840-TGA ACG CTA CAC ACTGCA TCT TGG reverse 51015840-CGA CTC CTT TTC CGC TTCCTG AG-31015840 Foxp3 forward 51015840-GGC CCT TCT CCA GGACAG A reverse 51015840-GCT GAT CAT GGC TGG GTT GT-31015840 interleukin-17A forward 51015840-AGT GAA GGC AGC AGCGAT CAT-31015840 reverse 51015840-CGC CAA GGG AGT TAA AG-31015840 TNF-120572 forward 51015840-TCT CAT CAG TTC TAT GGC CC-31015840 reverse 51015840-GGG AGT AGA CAA GGT ACA AC-31015840 IL-18 forward 51015840-ACC GAA TTC ACT GTA CAA CCG CAGTAA TAC GGA 31015840 reverse 51015840 GCC TCT AGA GTG AACATT ACA GAT TTA TCC CCA 31015840 IL-10 forward 51015840GAAGAC CCT CAG GAT GCG 31015840 reverse 51015840 CCA AGG AGTTGT TTC CGT TA 31015840 TLR-2 forward 51015840-GCA AAC GCTGTTCTGCTCAG-31015840 reverse 51015840-AGGCGTCTCCCTCTATTG TAT T-31015840 TLR-4 forward 51015840-ATG GCA TGG CTTACA CCA CC-31015840 reverse 51015840-GAG GCC AAT TTT GTCTCC ACA-31015840 TLR-9 forward 51015840-ATG GTT CTC CGT CGAAGG ACT-31015840 reverse 51015840-GAG GCT TCA GCT CAC AGGG-31015840 MyD88 forward 51015840 TCA TGT TCT CCA TAC CCTTGGT-31015840 reverse 51015840-AAA CTG CGA GTG GGG TCA G-31015840 TRIF forward 51015840-TGT CTG TCA GGA GGT GCT CAA-31015840 reverse 51015840-CGT TCC GGA CAT GCT CTT TC-31015840 NF-120581B forward 51015840-GGA GGC ATG TTC GGT AGT GG-31015840reverse 51015840-CCCTGCGTTGGATTTCGTG-31015840 Transcriptswere quantified using the EX TAQ R-PCR The PCR wasinitiated for 2min at 95∘C continued with 40 cycles of 10 secat 95∘C and 40 sec at 60∘C The fold change in expression ofeach gene was calculated using the ΔΔCt method with thehousekeeping gene 120573-actin mRNA as an internal control

28 Statistical Analysis Data are presented as the means plusmnSDsThe statistical significance of differences was analyzed byStudentrsquos 119905-test and Wilcoxon rank test using SPSS software130 for Windows (SPSS USA) A 119901 value less than 005 wasconsidered significant

3 Results

31 IL-18 Gene Combined with IL-4 Inhibits CIA by Skewingthe Balance of Th1Th2Th17 to a Th2 Type Previous studiesdemonstrated that recombinant IL-18 coadministration withlow dosage rIL-4 does not prevent the progression of CIA[2] Thus we studied the role of IL-18 gene therapy inmurine CIA on day 28 after immunization and administereda boost two weeks later (Figure 1) Surprisingly IL-18 genetherapy combined with IL-4 significantly reduced the meanarthritis score of our murine model (Figure 1(a)) Additionalcoadministration with low dosage rIL-10 had same effectthat accord with our previous studies Consistent with theseresults histopathological examination of the joints indicatedthat mice given the control PBS had severe disruption ofthe cartilage surface but that mice given pIL-18 combinedwith IL-4 had intact cartilage surface (Figure 1(b)) Next

we measured gene expression in synovial specimens toinvestigate the possible mechanism of IL-18 gene combinedwith rIL4 therapy (Figures 1(c) and 1(d)) The synovium ofIL-18 gene combined with rIL4-treatedmice had significantlylower expression of TNF-120572 and IL-17A and significantlyhigher expression of IL-10 (Figures 1(c) and 1(d))

32 The Plasmid Backbone Contributes to the TherapeuticEffect of IL-18 Gene Combined with rIL-4 Previous researchhas shown that treatment with rIL-18 and rIL-4 had notherapeutic effects on murine CIA model [2] The presentstudy however showed that treatment with pIL-18 combinedwith rIL-4 was markedly protective We hypothesized thatthis difference was due to the plasmid vector in pIL-18 Thuswe injected rIL-4 into CIA mice with or without an emptyplasmid vector (Figure 2) The results indicate significantexacerbation of arthritis in mice treated with the controlvector or rIL-4 but that coadministration of the control vectorand rIL-4 had lower macroscopic arthritis score than theother three groups except pIL-18 combined with IL-4 group

33 Interaction of the P IL-18 with rIL-4 CIA mice treatedwith rIL-4 alone or plasmid vector alone had lower levels ofIL-10 and higher levels of IFN-120574 and TNF-120572 than mice givenempty plasmid + rIL-4 or pIL-18 with rIL-4 (Figures 3(a) and3(b)) Disruption of the balance of Th1 Th2 andTh17 cells isassociated with progression of RA [5] Thus we investigatedthe expression of different master regulators of these T-cellimmune responsesThe results indicated that pIL-18 and rIL-4 therapy dramatically increased the expression of GATA-3and FoxP3 and decreased the expression of IL-17A (Figures3(a) and 3(b))

34 Expression of TLRs and Their Pathways on DCs of Spleenand Lymph Nodes Finally we investigated the mechanismof the interaction of the PIL-18 and rIL-4 (Figure 4) Theexpression levels of TLR2 TLR4 MyD88 and NF-120581B in thespleenDCs of PIL-18 and rIL-4 groupwere significantly lowerthan those in the spleenDCs of vector + rIL-4 group howeverthere was no statistical difference in the expressions of TLR9and TRIF (Figure 4(a)) The expressions of TLR2 TLR9MyD88 and NF-120581B in the lymph nodes DCs of PIL-18 andrIL-4 group were significantly lower than those in the lymphnodes DCs of vector + rIL-4 group while TLR4 and TRIFwere not significantly different (Figure 4(b))

4 Discussion

Various animalmodels of autoimmune diseases have demon-strated the importance of IL-18 as an immune regulatorycytokine [15] A recent study reported that treatment ofexperimental autoimmune encephalomyelitis (EAE) micewith the eukaryotic plasmid DT390-IL-18-Sra (encodingrecombinant immunotoxinDT390-IL-18) suppressed clinicaland histopathological progression of disease [16] Our pre-vious report showed that combined treatment of low-doseIL-18 with IL-10 can prevent the progression of arthritis inCIA mice and that this is mediated by a GATA-3-dependentmechanism [2] In the present study we demonstrated for


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(a) (b)

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0IL-18 IL-17A TNF- IL-10

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(d)

Figure 1 Effect of pIL-18 combined with rIL4 on CIA mice Male DBA1 mice were intradermally immunized with type II collagen emulsifiedin an equal volume of Freundrsquos complete adjuvant (FCA) and given intraperitoneal booster injections with type II collagen in PBS after 21days At day 28 mice were given intramuscular injections of 10120583g of pIL-18 (alone or in combination with rIL4 or rIL10) or PBS as controlwith a booster at the same dosage 14 days later (a) Mean arthritis macroscopic scores of mice treated with pIL-18 + rIL4 and pIL-18 + rIL10were significantly lower than the control from day 44 onwards (lowast119901 lt 005 versus control by Wilcoxon rank test) Each point represents themean plusmn SD of ten mice (b) Representative histological findings (left HampE staining times40 right Masson staining times40) Mice were sacrificedon day 60 and ankle and knee joints were removed The upper two images show severe cartilage surface disruption in CIA mice given thePBS control the lower two images show intact cartilage surface in CIA mice given pIL-18 combined with rIL4 (c) Representative reversetranscription PCR results showing the expression of IL-18 TNF-120572 IL-17A and IL-10 in CIA mice given the control PBS or pIL-18 combinedwith rIL4 (60 days after immunization) (d) Changes in the expression of TNF-120572 IL-18 IL-10 and IL-17A were determined by real-time PCRlowast119901 lt 005 versus control by Studentrsquos 119905-test lowastlowast119901 lt 001 versus control by Studentrsquos 119905-test

the first time that IL-18-expressing plasmid gene combinedwith IL-4 skewed the balance of Th1Th2Th17 to a Th2 typeand increased GATA-3 and Foxp3 expression on collagen-induced arthritis

Previous research has described IL-18 as an inducer ofTh1responses and a stimulator of Th2 development in differentcytokine microenvironments [17] IL-18 can promoteTh1 cellproliferation and IFN-120574 production with no need for TCRactivation so it can be considered an endogenous activator

of Th1 cells [18] IL-18 can also induce IFN-120574 productionfrom NK cells and seems to play an early and nonredundantrole in priming NK cells for optimal production of IFN-120574 inresponse to IL-12 [19] In the absence of IL-12 IL-18 inducesTh2 cytokines from NK and NKT cells and induces innateallergic mediators such as basophils and in some cases mastcells and eosinophils [19] IL-18 can also drive Th2 effectorcytokine production from activated Th1 cells and these so-called ldquosuper Th1 cellsrdquo which produce IFN-120574 IL-9 and


0

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28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58

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Figure 2 Effect of various vectors on the mean arthritis macroscopicscore of CIAmice DBA1mice were immunizedwith type II collagenon day 0 and day 21 On day 28 mice were treated with PBS 10 120583gof control vector 10 120583g of vector + 01 120583g rIL-4 01 120583g of rIL-4 or10 120583g of pIL-18 + 01 120583g rIL-4 A booster treatment was given withthe same dosage after two weeks Each point represents the mean plusmnSD of ten mice Mean arthritis macroscopic scores of mice treatedwith pIL-18 + rIL4 were significantly lower than the control fromday 44 onwards lowast119901 lt 005 versus control by Wilcoxon rank test

IL-13 are implicated in the IL-18-mediated promotion ofallergic-like pathology [20]Other research has demonstratedthat IL-18 + IL-4 can reduce the differentiation of Th1 cellsIL-18 + IL-10 induces Th2 responses to secrete Th2 cytokinesIL-4 [21] IL-18 + IL-10 + IL-4 can reduceTh1 cell secretion ofTNF-120572 and IFN-120574 and the inhibition of TNF-120572 promotes thepopulation of Treg cells [22 23] In addition IL-18 in concertwith IL-23 can activate Th17 cells [8 24] Taken togetherthis indicates that IL-18 is a special cytokine with multipleimmune-regulating potentials

In this report the synovium of IL-18 gene combined withrIL4-treated mice had significantly lower expression of TNF-120572 IL-17A and IFN-120574 and significantly higher expressionof IL-10 Our previous report showed that IL-18 receptor(IL-18R) 120572 expression in patella with adjacent synovium inCIA mouse is downregulated by the combined treatmentof rIL-18 with IL-4 [2] This is expected to contribute tothe downregulation of Th1 immune responses AlthoughrIL-18IL-4 treatment had a slight suppressive effect on themacroscopic arthritis score and incidence of arthritis in ourprevious research it did not reach statistical significance[2] Moreover combined treatment of empty plasmid vectorand recombinant IL-4 had a slight suppressive effect on themacroscopic arthritis score higher than pIL-18 combinedwith IL-4 therapy As we show here pIL-18 combined withrIL-4 ameliorates arthritis in CIA mice Thus the differencesbetween pIL-18 and rIL-18 must be examined

Previous research has reported that pIL-18 containingCpG motifs can activate signaling of Toll-like receptor 9(TLR9) [13] Dendritic cells (DCs) express TLR9 intracel-lularly and specifically recognize the unmethylated CpGmotif to developTh1 immune responses in L major-infectedsusceptible BALBc mice [13 25] Thus TLR9 actuates theexpression of inflammatory cytokines via activation of NF-120581B The NF-120581B signaling pathway has an important role inthe induction andmodulation of suppressive function of nat-ural Treg when they are confronted with TLR4-stimulatingagents such as Gram-negative bacteria [26] A previous studyconfirmed that human plasmacytoid DCs activated by CpGoligodeoxynucleotides induce the generation of CD4+CD25+regulatory T cells [27] TLR9 signaling may protect againstlupus by modulating the activity of regulatory T cells[28 29]

Treg cells play an active role in preventing the sponta-neous development of systemic autoimmunity and previousresearch has focused on whether deficiencies in Treg cellsactivity might contribute to the development of autoimmunediseases such as RA [30] Treg cells are present in the synovialfluid of RA patients and are potent suppressors of responderT-cell proliferation and TNF and IFN-120574 production inaddition activated T cells in the synovium also seemed tobe suppressed by Treg cells [1 31] On the other hand IL-17 is a key driver of inflammation and it is present in therheumatoid synovium of the joints of arthritic mice [32 33]Mice deficient in IL-17 have reduced severity of arthritisand those with increased IL-17 level have exacerbated disease[34 35] At the molecular level Th17 and Treg transcriptionfactors ROR120574tROR120572 and FoxP3 can bind to each other andinhibit its function [32 33] IL-2 a growth factor for Tregsinhibits the differentiation ofTh17-cells whereas IL-21 whichpromotes Th17 differentiation inhibits Treg expansion [36]Microbial stimuli signaling through TLR2-6 induces Tregto secrete TGF-120573 and IL-10 and to activate dendritic cells(DCs) [21] TGF-120573 induces the FoxP3 expression but alsostable FoxP3 expression [1] Some evidence indicates thatinhibition of TNF in RA promotes the emergence of a Tregcell population that can suppress effector T cells throughTGF-120573 and IL-10-dependent pathways [37]

In the present study we found that IL-18 gene combinedwith rIL-4 and vector combined with rIL-4 increased theexpression of FoxP3 Thus the plasmid backbone whichcontains the CpG motif signaling with TLR9 appears to playa major role in stimulating Treg cells thereby slowing theprogression of arthritis in our CIA model The present studyof a CIA model indicates that pIL-18 coadministration withrIL-4 delivery can activate Treg cells by increasing expressionof FoxP3 which presumably leads to the inhibition ofTh1 andTh17 inflammatory cytokines

Although the plasmid backbone had the Treg cells effectthe use of the vector alone or in combination with IL-4had no therapeutic effect TNF and IFN-120574 (Th1 cytokines)production in synovium were found to decrease after thevector alone or in combination with IL-4 treatment but it didnot reach statistical significance whereas marked decreasesin TNF-120572 IFN-120574 and IL-17 expression were detected in PIL-18rIl-4-treated mice compared with that of control group


-actin -actinIL-18

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Figure 3 Effect of various vectors on cytokine expression in the synovia of CIA mice (a) Representative results showing changes in theexpression of IL-17A IL-18 IFN-120574 TNF-120572 IL-10 FoxP3 T-bet and GATA-3 determined by reverse transcription PCR analysis at 60 daysafter immunizationThe lower part displays the density histogram data from three separated RT-PCR analyses (mean plusmn SE) which representsthe relative expression of T-bet GATA-3 TNF-120572 IL-17A IL-18 IFN-120574 IL-10 and FoxP3 (b) Changes in the expression of T-bet GATA-3TNF-120572 IL-17A IL-18 IFN-120574 IL-10 and FoxP3 were determined by real-time PCR lowast119901 lt 005 versus control by Studentrsquos 119905-test lowastlowast119901 lt 001versus control by Studentrsquos 119905-test

48

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Figure 4 Effect of various vectors on expression of TLRs and their pathways on DCs of spleen and lymph nodes (a) Representative resultsshowing changes in the expression of TLR2 TLR4 MyD88 NF-120581B TLR9 and TRIF in spleen DCs determined by real-time PCR analysisat 60 days after immunization (b) Changes in the expression of TLR2 TLR9 MyD88 NF-120581B TLR4 and TRIF in lymph node DCs weredetermined by real-time PCR lowast119901 lt 005 versus control by Studentrsquos 119905-test lowastlowast119901 lt 001 versus control by Studentrsquos 119905-test

Enhanced levels for Foxp3 and IL-10 were more impressiveafter PIL-18rIl-4 treatment IL-18 can promote Th1 cellproliferation IL-18 receptor (IL-18R) 120572 expression in patellawith adjacent synovium in CIA mouse is downregulated bythe combined treatment of rIL-18 with rIL-4 in our previous

research [2] Th1 response can be inhibited by the effect ofrIL-18 + rIL-4 and Treg effect of plasmid backbone in thisresearch IL-18 gene combined with IL-4 inhibits CIA byskewing the balance of Th1Th2 to a Th2 type IL-10 is aTh2 cytokine The expression of IL-10 in synovium increased


which further induced the expression of Treg Th1 and Th17responses can be inhibited by Treg cells The transcriptionfactors GATA-3 and T-bet have been described as ldquomasterswitchesrdquo in the development ofTh1 andTh2 cells expressionof T-bet or GATA-3 not only determines Th differentiationbut also can actually override the influence of exogenouspolarizing stimuli or previous polarization [38 39] A sig-nificant increase in GATA-3 expression was demonstratedin PIL-18rIl-4-treated group compared with that in controlgroup Increased expression of GATA-3 indicated that IL-18gene combinedwith IL-4 inhibits CIA by skewing the balanceof Th1Th2Th17 to aTh2 type

To further understand the mechanism of pIL-18 + rIL-4 on CIA the expression levels of TLRs and their pathwayon DCs of the spleen and lymph nodes were detected Theresults indicated that pIL-18 and rIL-4 therapy dramaticallydecreased the expressions of TLR2 TLR4 MyD88 and NF-120581B in the DCs of spleen The expression levels of TLR2TLR9 MyD88 and NF-120581B in the lymph nodes DCs of PIL-18 and rIL-4 were significantly lower than those in vector+ rIL-4 group There were no statistical differences in theexpressions of TRIF in the spleen and lymph nodesTheTLRsinDCs activated can cause costimulatorymolecules increasesand proinflammatory cytokines secretion [40] The TLRssignaling involves theMyD88 pathway and the TRIF pathway[41] The results indicated that PIL-18 combined with rIL-4played a therapeutic role by blocking the TLR2MyD88NF-kappa B signaling pathway

In summary pIL-18 coadministration with rIL-4 treat-ment protected CIA mice by significantly reducing theseverity of arthritis and the production of proinflammatorycytokines On the one hand the effect appeared to bemediated by multiple immune-regulating potentials of IL-18[2] on the other hand the effect appeared to be mediated bymodulation of the activity of regulatory T cells The plasmidbackbone and multiple immune-regulating potentials of IL-18 appeared to play the major role in the pIL-18 coadminis-tration with rIL-4-mediated immunomodulation of arthritisresulting in inhibition ofTh17 andTh1 inflammatory immuneresponses and improving anti-inflammatory mediators by aGATA-3-dependent mechanism The plasmid backbone andmultiple immunoregulatory properties of IL-18 appeared toplay themajor role in the pIL-18 coadministrationwith rIL-4-mediated immunomodulation of arthritis through blockingthe TLR2MyD88NF-kappa B signaling pathway

Conflicts of Interest

No competing financial interests exist

Acknowledgments

This work was supported by the National NaturalScience Foundation of China (30600561 30972739 and81373202) Fund of Heilongjiang Science and TechnicalOffice (QC06C051 QC2011C059 and D201276) HarbinScience and Technology Bureau of Heilongjiang Province(2012RFQXS020) Fund of Heilongjiang Education Office(1055HQ018) and Postdoctoral Foundation of China and

Heilongjiang Province (2013M531081 LBH-Z11004 andLBH-Q15138)

References

[1] M A De Vera J Mailman and J S Galo ldquoEconomics of non-adherence to biologic therapies in rheumatoid arthritisrdquo Cur-rent Rheumatology Reports vol 16 no 11 p 460 2014

[2] Q Dai Y Li F Zhang H Yu and X Wang ldquoTherapeuticeffect of low-dose IL-18 combined with IL-10 on collagen-induced arthritis by down-regulation of inflammatory and Th1responses and induction ofTh2 responsesrdquoRheumatology Inter-national vol 29 no 6 pp 615ndash622 2009

[3] G Guggino A Giardina A Ferrante et al ldquoThe in vitro addi-tion of methotrexate andor methylprednisolone determinesperipheral reduction in Th17 and expansion of conventionalTreg and of IL-10 producingTh17 lymphocytes in patients withearly rheumatoid arthritisrdquo Rheumatology International vol 35no 1 pp 171ndash175 2015

[4] J A Carlsson A E Wold A-S Sandberg and S M OstmanldquoThe polyunsaturated fatty acids arachidonic acid and docosa-hexaenoic acid induce mouse dendritic cells maturation butreduce T-cell responses in vitrordquo PLoS ONE vol 10 no 11Article ID e0143741 2015

[5] E Gonzalo-Gil G Criado B Santiago J Dotor J L Pablos andM Galindo ldquoTransforming Growth Factor (TGF)-120573 signallingis increased in rheumatoid synovium but TGF-120573 blockade doesnot modify experimental arthritisrdquo Clinical amp ExperimentalImmunology vol 174 no 2 pp 245ndash255 2013

[6] T Korn E Bettelli M Oukka and V K Kuchroo ldquoIL-17 andTh17 cellsrdquo Annual Review of Immunology vol 27 pp 485ndash5172009

[7] M Kinoshita N Kuranaga A Matsumoto et al ldquoMultipleinterleukin-18 injections promote both mouse Th1 and Th2responses after sublethal Escherichia coli infectionrdquo Clinical ampExperimental Immunology vol 143 no 1 pp 41ndash49 2006

[8] C T Weaver L E Harrington P R Mangan M Gavrieli andK M Murphy ldquoTh17 an effector CD4 T cell lineage with regu-latory T cell tiesrdquo Immunity vol 24 no 6 pp 677ndash688 2006

[9] S Veenbergen R L Smeets M B Bennink et al ldquoThe naturalsoluble form of IL-18 receptor 120573 exacerbates collagen-inducedarthritis via modulation of T-cell immune responsesrdquo Annals ofthe Rheumatic Diseases vol 69 no 1 pp 276ndash283 2010

[10] R L Smeets F A J van de Loo O J Arntz M B Benninck LA B Joosten and W B van den Berg ldquoAdenoviral delivery ofIL-18 binding protein C ameliorates collagen-induced arthritisin micerdquo Gene Therapy vol 10 no 12 pp 1004ndash1011 2003

[11] J P van Hamburg P S Asmawidjaja N Davelaar et al ldquoTh17cells but not Th1 cells from patients with early rheumatoidarthritis are potent inducers of matrix metalloproteinases andproinflammatory cytokines upon synovial fibroblast interac-tion including autocrine interleukin-17A productionrdquo Arthritisamp Rheumatology vol 63 no 1 pp 73ndash83 2011

[12] H-P Yao Y Qian X-T Shao et al ldquoConstruction of a recom-binant adenovirus vector expressing IL-18BPIL-4 fusion geneand the anti-inflammatory effect induced by this gene on lipo-polysaccharide-stimulated synovial fibroblastsrdquo InflammationResearch vol 59 no 2 pp 97ndash104 2010

[13] Y Li K Ishii H Hisaeda et al ldquoIL-18 gene therapy devel-ops Th1-type immune responses in Leishmania major-infectedBALBc mice Is the effect mediated by the CpG signalingTLR9rdquo Gene Therapy vol 11 no 11 pp 941ndash948 2004


[14] M Liu P Wang M Zhao and D Y Liu ldquoIntestinal DendriticCells Are Altered inNumberMaturity andChemotactic Abilityin Fulminant Hepatic Failurerdquo PLoS ONE vol 11 no 11 ArticleID 0166165 2016

[15] C A Dinarello ldquoInterleukin-18 and the pathogenesis of inflam-matory diseasesrdquo Seminars in Nephrology vol 27 no 1 pp 98ndash114 2007

[16] J Jia H Li S Tai et al ldquoConstruction and preliminary investi-gation of a plasmid containing a novel immunotoxinDT390-IL-18 gene for the prevention ofmurine experimental autoimmuneencephalomyelitisrdquoDNAandCell Biology vol 27 no 5 pp 279ndash285 2008

[17] S-M Dai Z-Z Shan H Xu and K Nishioka ldquoCellular targetsof interleukin-18 in rheumatoid arthritisrdquo Annals of the Rheu-matic Diseases vol 66 no 11 pp 1411ndash1418 2007

[18] D E Smith ldquoThe biological paths of IL-1 family members IL-18and IL-33rdquo Journal of Leukocyte Biology vol 89 no 3 pp 383ndash392 2011

[19] Y Fujibayashi Y Fujimori I Kasumoto et al ldquoInterleukin-18regulates T helper 1 or 2 immune responses of human cordblood CD4+ V12057224+V12057311+ natural killer T cellsrdquo InternationalJournal of Molecular Medicine vol 20 no 2 pp 241ndash245 2007

[20] K Nakanishi H Tsutsui and T Yoshimoto ldquoImportance ofIL-18-induced super Th1 cells for the development of allergicinflammationrdquo Allergology International vol 59 no 2 pp 137ndash141 2010

[21] B Pulendran H Tang and S Manicassamy ldquoProgrammingdendritic cells to induce T(H)2 and tolerogenic responsesrdquoNature Immunology vol 11 no 8 pp 647ndash655 2010

[22] S F Ahmad KMA Zoheir H E Abdel-Hamied et al ldquoAmeli-oration of autoimmune arthritis by naringin through modu-lation of T regulatory cells and Th1Th2 cytokinesrdquo CellularImmunology vol 287 no 2 pp 112ndash120 2014

[23] S Nadkarni CMauri andM R Ehrenstein ldquoAnti-TNF-120572 ther-apy induces a distinct regulatory T cell population in patientswith rheumatoid arthritis via TGF-120573rdquo The Journal of Experi-mental Medicine vol 204 no 1 pp 33ndash39 2007

[24] S J Lalor L S Dungan C E Sutton S A Basdeo JM Fletcherand K H G Mills ldquoCaspase-1-processed cytokines IL-1120573 andIL-18 promote IL-17 production by 120574120575 and CD4 T cells thatmediate autoimmunityrdquoTheJournal of Immunology vol 186 no10 pp 5738ndash5748 2011

[25] V Lougaris M BaronioM Vitali et al ldquoBruton tyrosine kinasemediates TLR9-dependent human dendritic cell activationrdquoThe Journal of Allergy and Clinical Immunology vol 133 no 6pp 1644ndashe4 2014

[26] LMilkova V Voelcker I Forstreuter et al ldquoTheNF-120581B signall-ing pathway is involved in the LPSIL-2-induced upregulationof FoxP3 expression in human CD4+CD25high regulatory Tcellsrdquo Experimental Dermatology vol 19 no 1 pp 29ndash37 2010

[27] F Carranza C R Falcon N Nunez et al ldquoHelminth antigensenable CpG-activated dendritic cells to inhibit the symptoms ofcollagen-induced arthritis through Foxp3+ regulatory T cellsrdquoPLoS ONE vol 7 no 7 Article ID e40356 2012

[28] D Hackl J Loschko T Sparwasser W Reindl and A B KrugldquoActivation of dendritic cells via TLR7 reduces Foxp3 expressionand suppressive function in induced Tregsrdquo European Journal ofImmunology vol 41 no 5 pp 1334ndash1343 2011

[29] O Wu G P Chen H Chen et al ldquoThe expressions of Toll-like receptor 9 and T-bet in circulating B and T cells innewly diagnosed untreated systemic lupus erythematosus and

correlations with disease activity and laboratory data in aChinese populationrdquo Immunobiology vol 214 no 5 pp 392ndash402 2009

[30] F A H Cooles J D Isaacs and A E Anderson ldquoTreg cellsin rheumatoid arthritis an updaterdquo Current RheumatologyReports vol 15 no 9 article 352 2013

[31] J M R van Amelsfort K M G Jacobs J W J Bijlsma F PJ G Lafeber and L S Taams ldquoCD4+CD25+ regulatory T cellsin rheumatoid arthritis differences in the presence phenotypeand function between peripheral blood and synovial fluidrdquoArthritis amp Rheumatology vol 50 no 9 pp 2775ndash2785 2004

[32] L Zhou J E Lopes M M W Chong et al ldquoTGF-Β-inducedFoxp3 inhibits TH17 cell differentiation by antagonizing ROR120574tfunctionrdquo Nature vol 453 no 7192 pp 236ndash240 2008

[33] S Alzabin S M Abraham T E Taher et al ldquoIncompleteresponse of infl ammatory arthritis to blockade is associatedwith the Th17 pathwayrdquo Annals of the Rheumatic Diseases vol71 no 10 pp 1741ndash1748 2012

[34] S Nakae A Nambu K Sudo and Y Iwakura ldquoSuppressionof immune induction of collagen-induced arthritis in IL-17-deficient micerdquo The Journal of Immunology vol 171 no 11 pp6173ndash6177 2003

[35] E Lubberts L van den Bersselaar B Oppers-Walgreen etal ldquoIL-17 promotes bone erosion in murine collagen-inducedarthritis through loss of the receptor activator of NF-120581B lig-andosteoprotegerin balance1rdquoThe Journal of Immunology vol170 no 5 pp 2655ndash2662 2003

[36] A Laurence C M Tato T S Davidson et al ldquoInterleukin-2 signaling via STAT5 constrains T helper 17 cell generationrdquoImmunity vol 26 no 3 pp 371ndash381 2007

[37] B Ying Y Shi X Pan et al ldquoAssociation of polymorphismsin the human IL-10 and IL-18 genes with rheumatoid arthritisrdquoMolecular Biology Reports vol 38 no 1 pp 379ndash385 2011

[38] Y Tanriver and A Diefenbach ldquoTranscription factors con-trolling development and function of innate lymphoid cellsrdquoInternational Immunology vol 26 no 3 Article ID dxt063 pp119ndash128 2014

[39] Y R Dıaz R Rojas L Valderrama and N G Saravia ldquoT-betGATA-3 and Foxp3 expression and th1th2 cytokine produc-tion in the clinical outcome of human infection with leishmania(viannia) speciesrdquoTheJournal of InfectiousDiseases vol 202 no3 pp 406ndash415 2010

[40] S-J Kim Z Chen N D Chamberlain et al ldquoAngiogenesisin rheumatoid arthritis is fostered directly by toll-like receptor5 ligation and indirectly through interleukin-17 inductionrdquoArthritis amp Rheumatology vol 65 no 8 pp 2024ndash2036 2013

[41] D Voehringer L P Kane and L Teyton ldquoThe secondtouch hypot-hesis T cell activation homing and PolarizationrdquoResearch vol 3 pp 37ndash43 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


IL-1 IL-18 was originally described as an inducer of Th1responses [7] In addition IL-18 can also stimulate Th2development and in concert with IL-23 can activateTh17 cells[7 8] Other research indicates that IL-18 plays a prominentrole in the onset and maintenance of the inflammatoryresponse during RA [9] Local neutralisation of IL-18 bythe naturally occurring IL-18-binding protein (IL-18BP) orsystemic neutralisation by specific antibodies amelioratescollagen-induced arthritis (CIA) reduces inflammation andreduces cartilage erosion [10 11] Yao et al reported thata recombinant virus expressing the IL-18BPIL-4 fusionprotein (AD-IL-18BPIL-4) suppresses the production andexpression of inflammatory cytokines in lipopolysaccharide-(LPS-) stimulated synovial fibroblasts [12] Another studydemonstrated that soluble IL-18 receptor 120573 inhibited IL-18during experimental arthritis had a major effect on T-cellcytokine balances and led to aggravation of CIA [9] Ourprevious research suggested that recombinant murine IL-18(rmIL-18) treatment alone ameliorated the progression ofarthritis in mice with CIA and that coadministration withlow-dose rIL-10 reversed this effect [2]

In this study we used the murine CIA model to examinethe effect of gene transfer of an IL-18-expressing plasmid onsuppression of CIA and the possible roles of Treg Th2 Th1Th17 and TLRs in the pathogenesis of arthritis

2 Materials and Methods

21 Reagents LPS (Escherichia coli 011184) Bovine typeII collagen and Freundrsquos complete adjuvant (FCA) werepurchased from Sigma (St Louis MO) RNA PCRKit (AMVVer30 and EX TAQ R-PCR Version 21) was from TaKaRaBiotechnology (Dalian China) Trizol was from InvitrogenCorporation (San Diego CA) primers and probes werefrom Pharmacia Biotech (Roosendaal Netherlands) andmurine recombinant IL-10 and IL-4 were from Prospec-TanyTechnogene (Rehovot Israel) CD11c MicroBeads were fromMiltenyi Biotec Inc (Bergisch Gladbach Germany)

22 Induction of Collagen-Induced Arthritis (CIA) MaleDBA1 mice (8ndash12 weeks old purchased from Slac Labora-tories Shanghai China) were housed in a specific pathogen-free environment with a 12 h light-dark cycle For the CIAmodel 100 120583g of bovine type II collagen dissolved in 005Macetic acid was emulsified with an equal volume of FCA andadministered intradermally at the base of the tail On day 21the mice were boosted by intraperitoneal injection of 100 120583gof bovine type II collagen dissolved in phosphate bufferedsaline (PBS)On day 28 fortymicewith nomacroscopic signswere treated by an additional intraperitoneal injection of LPS(40 120583g) Fortymicewere sacrificed onday 60 for analysisThisstudy was reviewed and approved by the Ethics Committeefor Experimental Animals of Harbin Medical University andall animals were treated according to the guidelines of theanimal ethical committee

23 Assessment of Arthritis The onset of arthritis was con-sidered to be the day that erythema andor swelling werefirst observed Mice were considered to have arthritis when

significant changes in redness and or swelling were notedin the digits or in other parts of at least 2 paws At latertime points ankylosis was also included in the macro-scopic scoring Cumulative scoring depending on rednessswelling and in later stadium ankylosis was as follows 0mdashnochanges 025mdash1 to 2 toes red or swollen 05mdash3 to 5 toesred or swollen 05mdashswollen ankle 05mdashswollen footpad05mdashsevere swelling and ankylosis The macroscopic scorewas assessed by two independent blinded observers [10]

24 Histology After animal sacrifice whole knee joints wereremoved and specimens were fixed for 4 days in 4 formalindecalcified in 5 formic acid and processed for paraffinembedding Tissue sections were stained with hematoxylinand eosin (HampE) or Masson stain Histopathology was usedto assess the occurrence of cartilage destruction and boneerosion

25 Gene Therapy After animal sacrifice plasmid DNAexpressingmurine IL-18was prepared as previously described[13] On day 28 forty mice without signs of arthritis weregiven intramuscular injections of pIL-18 (10 120583g) pIL-18(10 120583g) combined with IL-4 (01120583g) pIL-18 (10 120583g) combinedwith IL-10 (01120583g) or PBS as control A booster injectionof the same dosage was given two weeks later The samevolume of hyaluronidase IV (Sigma St Louis MO) wasinjected 10min before these injections to improve the plasmidexpression In a repeat experiment fifty mice without signsof arthritis were given intramuscular injections of pIL-18(10 120583g) combined with IL-4 (01120583g) IL-4 (01 120583g) plasmid(pCAGGS) combined with IL-4 (01 120583g) or with plasmid(pCAGGS) and PBS as control

26 Isolation of Spleen and Lymph Nodes CD11c+ DCs Micewere sacrificed by cervical dislocation The spleens andpopliteal lymph nodes were removed any fatty tissue wastrimmed away and spleens were washed three times withPBS The spleens and popliteal lymph nodes were laid on a200-mesh pore size cell strainer and using the barrel froma 1mL syringe the spleens and lymph nodes were pressedthrough the strainer until only a small amount of fibroustissue remained in the strainer CD11c+ cells were isolatedwith the CD11c MicroBeads according to the manufacturerrsquosinstructions [14]

27 RNAPreparation Reverse Transcription Polymerase ChainReaction and Quantitative RT-PCR Thepatella and adjacentsynovium were dissected immediately after animal sacrificeThe synovium samples were immediately frozen in liquidnitrogen and ground into powder with a tissue grinder andtotal RNA was extracted in 1mL Trizol reagent similar to theprocedure used for cartilage samples The levels of a panelof cytokines and transcription factors in the synovium weredetermined by reverse transcription polymerase chain reac-tion and quantitative RT-PCR The CD11c+ cell suspensionwas counted and RNAwas extractedThe expression of TLRsand their pathway on the surface of DCs was determined byquantitative RT-PCR Primer sequences and conditions forPCR were from the paper described previously [11] Primer




3 Results






4 Discussion




lowast

Mea

n ar

thrit

is sc

ore

25

2

15

1

05

028 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58

PBSP18

P18IL4P18IL10

(a) (b)

TNF-

IL-18 IL-17A

IL-10

-actin -actin


(c)

lowastlowast

lowastlowast

lowast

Fold

chan

ge

16

12

8

4


controlpIL18rIL4

(d)






0

05

1

15

2

25

28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58

Mea

n ar

thrit

is sc

ore


PBSvectorrIL-4


lowast









-actin -actinIL-18

IL-17AIFN-

TNF-IL-10

PBS

vect

or

vec

rIL4

rIL4

pIL1

8r

IL4

PBS

vect

or

vec

rIL4

rIL4

pIL1

8r

IL4

T-betGATA-3

FoxP3

(a)

02468

101214

Relat

ive m

RNA

expr

essio

n


rIL4PIL18rIL4

IL-1

8

IL-1

7A

IFN

-

TNF-

IL-1

0

T-be

t

GAT

A-3

FOX-

P3

(b)

vecrIL4pIL18rIL4

FoxP

3

IL-1

8

IL-1

7A

IFN

-

TNF-

IL-1

0

T-be

t

GAT

A-3

0

control

16

12

8

4

Fold

chan

ge

lowast

lowastlowast

lowast

lowast


(c)


48

40

32

24

16

8


vecrIL4pIL18rIL4

lowast

lowast


(a)


36

30

24

18

12

6

0

vecrIL4pIL18rIL4

lowast lowastlowast


(b)










Acknowledgments



References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















3 Results






4 Discussion




lowast

Mea

n ar

thrit

is sc

ore

25

2

15

1

05

028 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58

PBSP18

P18IL4P18IL10

(a) (b)

TNF-

IL-18 IL-17A

IL-10

-actin -actin


(c)

lowastlowast

lowastlowast

lowast

Fold

chan

ge

16

12

8

4


controlpIL18rIL4

(d)






0

05

1

15

2

25

28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58

Mea

n ar

thrit

is sc

ore


PBSvectorrIL-4


lowast









-actin -actinIL-18

IL-17AIFN-

TNF-IL-10

PBS

vect

or

vec

rIL4

rIL4

pIL1

8r

IL4

PBS

vect

or

vec

rIL4

rIL4

pIL1

8r

IL4

T-betGATA-3

FoxP3

(a)

02468

101214

Relat

ive m

RNA

expr

essio

n


rIL4PIL18rIL4

IL-1

8

IL-1

7A

IFN

-

TNF-

IL-1

0

T-be

t

GAT

A-3

FOX-

P3

(b)

vecrIL4pIL18rIL4

FoxP

3

IL-1

8

IL-1

7A

IFN

-

TNF-

IL-1

0

T-be

t

GAT

A-3

0

control

16

12

8

4

Fold

chan

ge

lowast

lowastlowast

lowast

lowast


(c)


48

40

32

24

16

8


vecrIL4pIL18rIL4

lowast

lowast


(a)


36

30

24

18

12

6

0

vecrIL4pIL18rIL4

lowast lowastlowast


(b)










Acknowledgments



References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















lowast

Mea

n ar

thrit

is sc

ore

25

2

15

1

05

028 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58

PBSP18

P18IL4P18IL10

(a) (b)

TNF-

IL-18 IL-17A

IL-10

-actin -actin


(c)

lowastlowast

lowastlowast

lowast

Fold

chan

ge

16

12

8

4


controlpIL18rIL4

(d)






0

05

1

15

2

25

28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58

Mea

n ar

thrit

is sc

ore


PBSvectorrIL-4


lowast









-actin -actinIL-18

IL-17AIFN-

TNF-IL-10

PBS

vect

or

vec

rIL4

rIL4

pIL1

8r

IL4

PBS

vect

or

vec

rIL4

rIL4

pIL1

8r

IL4

T-betGATA-3

FoxP3

(a)

02468

101214

Relat

ive m

RNA

expr

essio

n


rIL4PIL18rIL4

IL-1

8

IL-1

7A

IFN

-

TNF-

IL-1

0

T-be

t

GAT

A-3

FOX-

P3

(b)

vecrIL4pIL18rIL4

FoxP

3

IL-1

8

IL-1

7A

IFN

-

TNF-

IL-1

0

T-be

t

GAT

A-3

0

control

16

12

8

4

Fold

chan

ge

lowast

lowastlowast

lowast

lowast


(c)


48

40

32

24

16

8


vecrIL4pIL18rIL4

lowast

lowast


(a)


36

30

24

18

12

6

0

vecrIL4pIL18rIL4

lowast lowastlowast


(b)










Acknowledgments



References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

05

1

15

2

25

28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58

Mea

n ar

thrit

is sc

ore


PBSvectorrIL-4


lowast









-actin -actinIL-18

IL-17AIFN-

TNF-IL-10

PBS

vect

or

vec

rIL4

rIL4

pIL1

8r

IL4

PBS

vect

or

vec

rIL4

rIL4

pIL1

8r

IL4

T-betGATA-3

FoxP3

(a)

02468

101214

Relat

ive m

RNA

expr

essio

n


rIL4PIL18rIL4

IL-1

8

IL-1

7A

IFN

-

TNF-

IL-1

0

T-be

t

GAT

A-3

FOX-

P3

(b)

vecrIL4pIL18rIL4

FoxP

3

IL-1

8

IL-1

7A

IFN

-

TNF-

IL-1

0

T-be

t

GAT

A-3

0

control

16

12

8

4

Fold

chan

ge

lowast

lowastlowast

lowast

lowast


(c)


48

40

32

24

16

8


vecrIL4pIL18rIL4

lowast

lowast


(a)


36

30

24

18

12

6

0

vecrIL4pIL18rIL4

lowast lowastlowast


(b)










Acknowledgments



References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















-actin -actinIL-18

IL-17AIFN-

TNF-IL-10

PBS

vect

or

vec

rIL4

rIL4

pIL1

8r

IL4

PBS

vect

or

vec

rIL4

rIL4

pIL1

8r

IL4

T-betGATA-3

FoxP3

(a)

02468

101214

Relat

ive m

RNA

expr

essio

n


rIL4PIL18rIL4

IL-1

8

IL-1

7A

IFN

-

TNF-

IL-1

0

T-be

t

GAT

A-3

FOX-

P3

(b)

vecrIL4pIL18rIL4

FoxP

3

IL-1

8

IL-1

7A

IFN

-

TNF-

IL-1

0

T-be

t

GAT

A-3

0

control

16

12

8

4

Fold

chan

ge

lowast

lowastlowast

lowast

lowast


(c)


48

40

32

24

16

8


vecrIL4pIL18rIL4

lowast

lowast


(a)


36

30

24

18

12

6

0

vecrIL4pIL18rIL4

lowast lowastlowast


(b)










Acknowledgments



References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























Acknowledgments



References
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















suppression of th1 and th17 responses and induction of...

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