Role And Mechanism Of Innate IL-17A In The Pathogenesis Of CVB3-induced Viral Pancreatitis

Objective:Acute pancreatitis(AP)is an inflammation and injury of the pancreas caused by pancreatic enzyme activation by various etiologies.Among the common causes of AP,viral infection constitutes a minor but important leading agent.Hepatitis virus,Human immunodeficiency virus,cytomegalovirus and herpes simplex virus are reported to induce AP.Currently Coxsackie virus B3 and B4 are most extensively studied causing agents for viral pancreatitis.However,the mechanism underlying CVB3-induced pancreatitis remains unclarified.CVB3 is a non-capsid positive-strand RNA virus of the Picornaviridae family.It enters the host through the fecal-oral pathway,then infects hearts,pancreas,brains and livers of mice,leading to myocarditis,pancreatitis,encephalitis and viral hepatitis.Both virus-mediated myocyte injury(day0～3)and cardiac immune injury contribute to the pathogenesis of CVB3-induced viral myocarditis.In the innate immune phase,cardiac neutrophil and mono/macrophage infiltration and Toll-like receptors(TLRs)-triggered inflammatory cytokine(TNF-α,IL-1β,IL-6,IL-18,IL-17A)response play important roles in early antiviral defense and cardiac injury.The lately incardiac-recruited CD4+Th1 and Th17(IFNγ and IL-17A)cells act as main pathogenic cells for the development of VMC.The early cardiac up-regulated IL-17A and IL-17-IL-17RA signaling play a central role in the pathogennesis of viral myocarditis.The pancreas is the primary organ for CVB3 infection after peripheral dissemination,holding highest viral burden among allsusceptible organs.However,the immunological mechanism of viral pancreatitis remains unclear.Our preliminary data indicate that:1)CVB3 infects hearts after an effective and most robust replication in the pancreas;2)The pathology of viral pancreatitis is attributed more to acinar cell necrosis,less to inflammatory injury;3)The commen sense of viral myocarditis and pancreatitis lies in a robust up-regulation of loca IL-17A during early phase of infection(day3);4)IL-17A deficient mice suffered significantly reduced myocarditis and pancreatits,indicating the IL-17A pathway is involved in the pathogenesis of viral pancreatitis.IL-17 is a key pro-inflammatory cytokine originally reported to induce neutrophil-mediated inflammation and anti-microbial activity.It is involved in the development of various physiological and pathophysiological processes.Recent studies have revealed that IL-17A-producing γδT cells(γδT 17 cells),Innate Lymphoid-like cell 3(ILC3),and IL-17A-producing CD4+T cells(Thl7)cells are the main producers of IL-17A in mouse models of inflammatory diseases.IL-17A-producing γδT cells participate in the protective immune response at an early stage of infection with Listeria monocytogenes and Mycobacterium Tuberculosis in mice;but contribute to pathology and inflammatory injury in several chronic inflammatory disease including rheumatoid arthritis,asthma,and Psoriasis.Upon CVB3 infection,we found an early infiltration ofγδT cells in the pancreas,contributing more than 50%of local IL-17A at day 3 p.i..γδT-derived IL-17A has been suggested to play critical pro-inflammatory roles in the pathogenesis of CVB3-myocarditis,suggesting γδT-derivedIL-17Aalso play important roles in the pathogenesis of CVB3 pancreatitis.And IL-23 is critical for IL-17 production.In this study,we propose that early pancreatic y8T influx plays important roles in IL-17A-mediated pathogenesis of viral pancreatitis via promoting Th17 induction.We define the kinetics and cellular source of early pancreatic IL-17A upon CVB3 infection in a murine model of CVB3 pancreatitis.Then we elucidate the role of early pancreaticγδT17 in the pathogenesis of viral pancreatitis by infecting IL-17A-/-,TCRδ-/-and Vy4-depleted mice with CVB3 and by y8T 17-transfer experiment.And effect of IL-23-IL-17 axis on the induction of local y8T17 induction is studied.Our data indicate that innate γδT-derived IL-17A is indispensable in the pathogensis of viral pancreatitis.Methods:1.Establishment of C57BL/6 model of CVB3-induced pancreatitis.C57BL/6 mice were intraperitoneally injected with 2000 TCIDso CVB3.During a 7 days period,30～60%survival rate,25%weight loss and 106 PFU/g viral burden were confirmed.2.Hematoxylin/Eosin(HE)staining:The pancreatic tissue was fixed and embedded and made into 5μm sections before staining with Hematoxylin and Eosin.Acinar cell necrosis and immune infiltration were observed under micro-scopy.3.Determination of viral load:20 mg pancreatic tissue was homogenated in 0.5 ml PBS,supernatant was obtained and diluted to a gradient of 10-1-10-10.30 μl diluted pancreas homogenates was added onto Hela monolayer in 8 replicates.After incubating at 37℃ for 1 hour,cells were washed three times with PBS,and cultured in 2%FBS DMEM for 5 consecutive days to observe the cytopathic condition.More than 50%of cell shrinkage,refractive loss,and floating was regarded as CPE.The tissue virus TCID50/g was calculated and multiplied by 0.7 to convert PFU/g.4.Preparation of pancreatic,splenic and blood mononuclear cells:The mononuclear lymphocytes were obtained by digestion of pancreas tissues with collagenase IV and DNAse I,followed by 75%-40%Percoll density gradient centrifugation.Peripheral blood cells and splenic cells were lysed by ACK buffer and mershed through 70μm dish5.Flow cytometry analysis of immune cells.Single mononuclear cells were surface stained with CD45,CD11b,Ly6C,Ly6G,F4/80,TCRδ,Vγ4,CD3,CD4,CD8 to verify macrophage,monocyte,neutrophil,γδT,CD4+and CD8+T cells.6.Intracellular staining:cells pretreated with anti-FcγR mAb were stained with mAbs against surface markers,then cells were permeabilized-using the Cytofix/Cytoperm Plus kit and stained with PE-anti-IL-17A mAb.The data were acquired using FACS and analyzed by CellQuest software.7.In vivo depletion of Vγ4 γδT cells:mice were i.v.injected with Vy4-neutrolizing mAb(120μg)on day-1 and+3 and infected with CVB3 on day 0.Viral titer and acute pancreatitis were analyzed at 7 days post infection.8.In vitro preparation of Vγ4-γδT cells.Anti-Vγ4 mAb was coated overnight at 4℃.Fresh splenic cells were resuspended in medium containing anti-CD28(1 μg/ml)and rmIL-2(2 ng/ml),then seeded into Anti-Vy4-coated plates and incubated for 48 h Fresh medium containing rmIL-2(2 ng/ml)was relaced every 1.5 days until day 6.108 cells would be obtained,of which about 20%-60%were corresponding Vy4 y8T cells.9.Vy4-transfer experiment:WT and TCRδ-/-mice were i.v.with 106 Vy4 cells on day-1 and+3;and infected with CVB3 on day 0.10.Recombinant IL-23 injection.C57BL/6 male mice were injected with IL-23(4μg/mice)on day-1,and infected with CVB3 on day 0.Viral titer and acute pancreatitis analyzed at 7 days post-infection11.Detection of lipase activity.Serum samples,blank.(double distilled water)and calibration(standard)sample were mixed with the reagents,and incubated at 37℃ for 100s at the measurement wavelength(570 nm).The absorbance is measured and the sample LPS activity is calculated according to the formula.12.Elisa detection of inflammatory cytokines:Serum samples and pancreas homogenenate samples were detected protein levels of IFNy,IL-1β,IL-6,TNF-α,IL-17A by quantitative ELISA assay.13.Statistical analysis:data are expressed as mean plus standard deviation.Comparisons between groups were performed using one-way ANOVA with Bonferroni multiple comparison test.Normally distributed data on continuous parametric axes were analyzed with the Student’s t-test.Log-rank test was used to compare survival curves.Statistical analyses were performed using GraphPad Prism 5 software.P<0.05 was considered to be statistically different.Results:1.Intraperitoneal injection of 2000TCID50 induces acute pancreatitis in male C57BL/6 miceC57BL/6 mice were intraperitoneally injected with 2000TCID50 dosage of CVB3.During 7 days infection,mice succumbed to 20%of weight loss with survival rate being 30%-60%.Acinar cells necrosis and massive immune infiltration were found in pancreas of mice at day 7 p.i.,indicating establishment of CVB3-pancreatitis model.2.CVB3 infection significantly up-regulates pancreatic IL-17A expression at day3 p.i.and IL-17A significantly increases CVB3-induced viral pancreastitisWe analyzed the expression of IL-17 in the pancreas of CVB3-infected mice.Elisa,WB,IHC,IF and flow cytometry analysis all revealed that IL-17 protein or IL-17A-secreting cells were induced in the pancreas on day 1 postinfection,peaked on day 2～3,and then returned to a reduced but relatively high level by day 7.To evaluate role of IL-17A in CVB3-induced pancreatitis,IL-17A-/-mice were subjected to CVB3 infection.Compared to the WT mice,IL-17A-deficient mice showed an increased survival rate,reduced weight loss,decreased pancreatic acinar cells necrosis and inflammatory infiltration together with reduced IL-1β,TNF-α and IL-6 production after infection.Viral burden in the pancreas was not significantly changed.All data indicate that IL-17A plays a-detrimental role in the development of acute viral pancreatitis.3.γδT are one of important IL-17A-secreting cells in pancreas at early phase of CVB3 infection and Vy4 γδT cells are the main IL-17-aecreting γδT subset.Flow cytometry analysis of the IL-17A-secreting cells in pancreas at day 0,3,7 post-infection revealed that:1)Influx of γδT cells increased after infection,peaking on day 3(2.2%among CD3+T cells,104 cells/pancreas).While pancreatic CD4+Th cell infiltration increased with time,up to day 7(30%,106 cells/pancreas).2)Intracellular staining of IL-17A found that CD3+T cells,rather than CD3-cells(0.5%vs.0.2%,p<0.05),are major IL-17A producers.Within CD3+T cells,CD8+T(0.08%)and NK(0.02%)hardly secreted IL-17A,while CD4+T(0.24%)and y8T(0.31%)were main IL-17A+cells on day 3 p.i..More than 10%of the yST cells secreted IL-17A,while only 1%of CD4+T cells secreted IL-17A(p<0.05).On day 3 p.i.,an equal enrichment dynamic of Vyl and Vy4 was observed.However,IL-17A was 80%produced by Vy4γδT cells,not by Vγ1γδT cells.Taken together,Vγ4 γδT(γδT17)cells are a primary source of pancreatic IL-17A especially at the early stage of CVB3 infection.4.Deficiency of γδT cells reduces CVB3-induced pancreatitisTo investigate role of γδT cells in the development of pancreatitis,TCRδ-/-mice－were i.p.infected with CVB3.Compared to WT mice,γδT-deficient mice had a significantly reduced actue pancreatitis,with less inflammatory infiltration,less acinar cells necrosis and decreased pancreatic inflammatory CKs(IL-17A,TNF-α and IL-6)on day 7 p.i.Viral titer was elevated on day 3 p.i.but not significantly changed.These data indicate the detrimental role of γδT cells in CVB3-induced acute pancreastitis.5.mAb depletion of Vγ4 γδT cell significantly reduces CVB3-induced acute pancreatitisTo determine the role of Vγ4 γδT cells in CVB3-acute pancreatitis,Vγ4 γδT cells were depleted by two injections of anti-Vγ4-specific mAb before and after CVB3 infection.Peripheral 90%Vγ4 γδT deficiency led to increased survival rate,decreased serum lipase activity,reduced levels of pancreatic CKs(IL-17A,IFNγ,IL-1β and TNF-α)and pancreatic pathology indicating alleviated acute pancreatitis.Viral titer on day 3 were not changed.It indicates that Vγ4 γδT cells play a detrimental role in the course of acute pancreatitis by promoting inflammatory response.6.Transfer of IL-17A+Vy4 γδT cells into TCRδ-/-mice exacerbates CVB3-induced pancreatitis while IL-17A-/--derived γδT cells fails to get this effectIL-17A+Vγ4 γδ T cells with 60%purity were in vitro obtained by culturing splenocytes with anti-Vγ4 mAb plus anti-CD28 and rIL-2 for 6 days.Intravenously transfer of 1×106 γδT17 cells into TCRδ-/-mice 24 hrs before CVB3 infection significantly exacerbated CVB3-pancreatitis;while IL-17A-/--derived γδT cells can not afford this effect.It indicates that the pro-inflammatory and pathogenic role of Vγ4 γδT is mediated by IL-17A.7.γδT17 cells promote CVB3-pancreatitis via promoting neutrophil infiltration and Th17 polarizationTo elucidate the mechanism of y8T17-promoting CVB3-pancreatitis,intrapancre-atic neutrophil infiltration was accessed by FACS during CVB3 infection.The pancreatic neutrophil infiltration started from day 1 and peaked on day 3 p.i.,which was significantly blocked in TCRδ-/-,IL-17A-/-and Vy4-depleted mice.Meanwhile,depletion of IL-17A+γδT cells significantly increased peripheral IFNγ+Thl response,but reduced IL-17A+Th17 response.Thereafter,early local γδT-derived IL-17A,increases CVB3-induced pancreatic via promoting neutrophil infiltration and amplyfying inflammatory Th17 response.8.Pancreatic early IL-23-IL-17 signaling activates local γδT17 differentiation therefore accelerating pancreatitisTo elucidate how local γδT17 cells are activated,pancreatic IL-23 was detected and found up-regulated 1 day after CVB3 infection.After injecting rIL-23 into WT and TCRδ-/-mice,significantly increased susceptibility of mice to CVB3 infection,promoted pancreatic inflammatory infiltration and acinar cell necrosis and elevated serum lipase activity were observed in WT but not TCRδ-/-mice.Exogeneous IL-23 significantly enhanced pancreatic IL-17+y8T(not Th17)differentiation 12 hrs after rIL-23 injection.Therefore CVB3 infection activates Vγ4 γδT differentiation into γδT17 via up-regulating very early local IL-23-IL-23R pathway,thus promoting the induction of pathogenic Th17 response and viral pancreatitis.Conclusion:1.CVB3 infection causes acute viral pancreatitis in male C57BL/6 mice.2.Innate IL-17A is significantly up-regulated in pancreas of mice after CVB3 infection which increases CVB3-induced pancreatitis...3.γδT cells appear early(day3)during CVB3 infection and Vγ4 γδT subset are one of important early IL-17A-producers in pancreas of mice.The IL-17A-producing efficiency of γδT cells is higher than that of CD4+Th17 cells.4.γδT cells play a detrimental role in CVB3-induced pancreatitis.Peripheral depletion of Vy4 γδT cells decreased CVB3-pancreatitis.Transfer of IL-17+Vy4 y8T cells into TCRδ-/-mice exacerbates viral pancreatitis while IL-17A-/--derived Vy4 γδT cells fails to get this effect.All the data indicate that Vy4 γδT cell significantly increases CVB3-induced acute pancreatitis via IL-17.5.y8T17 cells promote CVB3-pancreatitis via promoting neutrophil infiltration and Th17 polarization..6.Pancreatic early IL-23 production and IL-23/IL-17 axis is requied for local activation of IL-17A-secreting γδT17 cells,which is important to induce pathogenic Thl7 response during CVB3-pancreatitis.In conclusion,IL-17A-producing Vy4 y8T cells,appearing early in the infected pancreas,play key roles during the course of CVB3-induced acute pancreatitis by promoting Th17 differentiation and exacerbating inflammatory injury by enhancing neutrophil infiltration.γδT cells are thus intensively involved in the pathogenesis of acute viral pancreatitis.