| Viral myocarditis (VMC) is characterized by myocardialinflammation and can progress to chronic dilated cardiomyopathy (DCM).Coxsackievirus B (CVB), an enterovirus of Picornaviridae family, isbelieved to be the primary pathogen in human VMC. Previousexperiments had indicated the major mechanisms in the pathogenesis ofVMC and DCM were inflammation and autoimmune responses triggeredby the viral infection, mediated by T cells and their cytokines.Emergingevidence have demonstrated that several Th subsets, such asTh1,Th2,Th17and their effective cytokines such as IFN-γ,IL-12,IL-10,IL-17are involved in the pathogenesis of VMC. But thefundamental mechanisms responsible for VMC are not completelyclarified, and therapeutics for VMC are restricted to supportive careincluding basic medications.Recently, the Th22cell has been recognized as a novel Th cellsubset, which is characterized by abundant production of IL-22but notIL-17or IFN-γ. Further more, IL-22has either proinflammatory ortissue-protective properties, depending on the nature of the affected tissueand the local cytokine milieu, including the presence or absence ofIL-17A co-expression. Demonstrated by emerging literature in kinds ofdiseases such as rheumatoid arthritis, Th22cells were considered providing a unique contribution to tissue inflammation, virus infectionand immune responses.The existence of Th22cells in CVB3-induced mice VMC is stillunclear. The roles of IL-22in the pathogenesis of VMC, as well as theeffect of IL-17A on IL-22, remain to be clarified. Whether Th22participate in development of DCM remain to be determined. In order toelucidate the role of Th22cells in VMC, and explore the newtherapeutical target, VMC was induced in male BALB/c mice by CVB3peritoneal injection, then the following3parts researches were carriedout:Part1: The alteration of Th22cells and their related cytokines in murineviral myocarditis.In this part, two contents were included as below:1.Eastablish micemodel of viral myocarditis and evaluate the pathological changes ofmyocardium;2.The alteration of spleen Th22subsets and its relativecytokines(IL-22、IL-22R1、IL-10R2) in VMC mice.Objective: To investigate the alteration of Th22cells and its relatedcytokines such as IL-22in murine VMC, and to investigate their role inVMC.Methods: A total of32mice were randomly divided into VMC(n=20)and control groups(n=12). Specific pathogen-free male BALB/c miceaged6week were intraperitoneally (i.p) infected with CVB3forestablishing VMC models. Control mice were treated withphosphate-buffered saline (PBS) i.p. On day14after injection, myocardial histopathological changes were evaluated byhematoxylin-eosin staining. Frequencies of splenic Th22cells weredetermined, productions of IL-22and expressions of IL-22R (IL-22receptor) were measured. Results: The cardiac pathological scores ofVMC were dramatically increased on day14post infection (2.6±0.4). Incontrast, the pathological scores of heart sections in the control group was0（P<0.05）.The percentage of the pure Th22cell represented a highervalue in the VMC group (3.47±0.66%), showing a significant increase incomparison with that in the control group (0.84±0.33%, P <0.01).Muchhigher levels of the IL-22protein were detected in myocardium in VMCmice (236.24±43.26vs.17.45±5.21, p<0.01). Much higher levels of theIL-22gene transcripts were detected in myocardium in VMC mice(21.28±4.28vs.4.28±0.97, p<0.01). Meanwhile, the relative cardiacIL-22R1mRNA was obviously up-regulated in the VMC group,compared with that in the control group (3.01±0.78vs.1.05±0.29,p<0.01).In contrast, the relative cardiac transcripts of IL-10R2in theVMC group were less than those in the control group (43.28±9.14vs.58.5±9.01, p<0.05). The significantly increased level of the circulatingIL-22protein in the VMC group was observed compared with that in thecontrol group (20.21±3.81vs.3.02±0.83pg/ml, p<0.01).Conclusion：It was differentiated Th22cells and its related cytokinessignificantly elevated in CVB3-induced VMC. The microenvironment ofVMC seemed to contribute to the differentiation and proliferation ofTh22cells. Our preliminary data implied Th22cells and its relatedcytokines may involve in the pathogenesis of VMC. Part2: Treatment with a neutralizing anti-interleukin-22antibody incoxsackievirus b3-induced viral myocarditis exacerbates myocardiuminflammationIn this part, three contents were included as below:1. The effect ofAnti-IL-22Ab for the severity of viral myocarditis;2. The role ofAnti-IL-22Ab to the levels of IL-22--IL-22R1;3. The role of Anti-IL-22Ab to the inflammatory cytokines IFN-γ、IL-17、TNF-α、IL-6、IL-1β andviral replication.Objective: To investigate the role of IL-22in the coxsackievirusb3-induced viral myocarditis.Methods: For in vivo IL-22neutralization, a total of36BALB/cmice which infected with CVB3(VMC mice) were randomly divided intothree groups: administered either Anti-IL-22Ab (50μg per mouse; R&DSystems, Inc. Minneapolis, MN.AF582; n=12,Anti-IL-22Ab group); annormal IgG control (50μg per mouse; R&D Systems,Inc.Minneapolis,MN.AB-108-C; n=12, IgG control group); or PBS (n=12,50μg per mouse; PBS group) i.p., at day0. All surviving animals weresacrificed on day14after CVB3infection. VMC mice treated withAnti-IL-22neutralizing antibody were explored to investigate the effectsof IL-22. The severities of VMC were monitored; the frequencies of Th22cells, the expressions of IL-22and IL-22R were investigated; in additionto IFN-γ, inflammatory cytokines IL-17, TNF-α, IL-6as well as IL-1β,were evaluated. Cardiac viral replications were detected.Results: Treatment of VMC mice with Anti-IL-22Ab exacerbatedthe severity of viral myocarditis, verified by higher HW/BW ratios and cardiac pathological scores. Anti-IL-22Ab decreased the frequencies ofTh22cells and the levels of IL-22, and increased the expressions ofcardiac IL-22R1. Up-regulations of IL-17、 IL-6and TNF-α,down-regulations of IFN-γ proteins and gene expressions in the plasmaand myocardium, were observed in Anti-IL-22Ab group. Furthermore,neutralization of IL-22significantly promoted cardiac viral replication.Conclusions: Our data indicate that IL-22may act as anmyocardium-protective cytokine, and suggest that targeting the Th22celleffector cytokine IL-22could provide new therapeutic modalities for thetreatment of CVB3-induced VMC.Part3: IL-22exacerbates the severity of CVB3-induced viral myocarditisin IL-17A-deficient miceIn this part, two contents were included as below:1.The effect ofAnti-IL-22Ab for the severity of viral myocarditis in IL-17A-/-VMCmice;2. The role of Anti-IL-22Ab to the expressions of IL-6、TNF-α、IFN-γ、STAT3and viral replication in IL-17A-/-VMC mice.Objective: To explore whether IL-17A determines the function ofIL-22in VMC.Methods: A total of32IL-17A-/-mice were randomly divided intofour groups: Mice in the VMC group were injected with CVB3andPBS(n=8);in the anti-IL-22Ab group, mice were administered withCVB3and anti-IL-22Ab(n=8); IgG control group, mice were injectedwith CVB3and normal IgG control(n=8); and in the normal group,IL-17A-/-mice received no treatments(n=8). The day of intraperitoneal injection was defined as day0. All surviving animals were sacrificed onday14after CVB3infection. The ratios for heart weight/body weight(HW/BW) were recorded, pathological scores of heart sections wasanalysis by HE, CVB3、TNF-α、IL-6、IFN-γ、STAT3and IL-22的mRNA were measured by RT-PCR; Serum IL-22was recorded byELISA; Cardiac viral replication were detected by plaque-forming assay;cardia phosphorylated-STAT3(p-STAT3) was measured by Western-blot;for spleen lymphocytes and cell cultures, Cells were collected and usedRT-PCR to measure the IL-22and IL-6mRNA, the culture supernatantswere assayed by ELISA to detecte the protein of IL-22.Result: Our data demonstrated that the neutralization of IL-22inIL-17A-deficient mice alleviated the severity of myocarditis. This wasdemonstrated by the lower pathological scores of heart sections and ratiosof heart weight/body weight (HW/BW), reduced production of activatorof transcription3(STAT3) and proinflammatory cytokines TNF-α andIL-6, followed by increased viral replication and decreased levels of theantiviral cytokine IFN-γ. Furthermore, the correlation between cardiacCVB3RNA and IL-22mRNA or IFN-γ mRNA was negative. Inconclusion, IL-22exacerbated the severity of VMC and restrained viralreplication in the absence of IL-17A. Furthermore, spleen lymphocytescultured with recombinant IL-17(rIL-17) increased the production ofIL-22and IL-6.Conclusions: Combined with our previous data, these resultsindicate that IL-17A is not involved in regulating the antiviral role ofIL-22, however, may mediate the tissue-protective versus pathogenicproperties of IL-22in CVB3-induced VMC in mice. In summary, our research suggested that:Increased of Th22cells and its related cytokines IL-22, IL-22R1were observed in VMC group, indicating that Th22cells and its relatedcytokines may involve in the pathogenesis of VMC; in the present ofIL-17A, IL-22may act as an myocardium-protective and antiviralcytokine; while in the absent of IL-17A, IL-22may act as anmyocardium-destructive and antiviral cytokine, indicating that IL-17A isnot involved in regulating the antiviral role, however, may mediate thetissue-protective versus pathogenic properties of IL-22in CVB3-inducedVMC mice. In conclusion, our data suggest that Th22cells and itscytokine IL-22immune response may be implicated in the pathogenesisof VMC. |
PDF Full Text Request