| Virus myocarditis(VMC) is a common infectious disease of cardiovasclar system resulting from viral infection, featured by focal of diffuse cardiac inflammatory infiltration. The true incidence and prevalence of myocarditis are underestimated due to non-specific symptoms and lack of targeted diagnostic tests. Now the incidence of VMC estimates as 0.12%-12% worldwide, and 5%-15% in our country, which continues to rise. VMC accounts for one of the main causes of sudden death among adolescents since up to 20% of sudden cardiovascular deaths among young adults are attributed to myocarditis. Asymptomatic, and untreated VMC is easy to develop into deadly diseases including fibrosis, dilated cardiomyopathy(DCM) and heart failure. Viral myocarditis is an important cause of deadly cardiovasclar diseases for which no specific treatments are available. Therefore, it is of great significance to elucidate the mechanism of pathogenesis of VMC. Viruses are the most common cause of myocarditis. A wide spectrum of viruses, including enteroviruses, adenoviruses, influenza viruses, parvoviruses and cytomegaloviruses is associated with VMC. The enteroviral Coxsackievirus, particularly serotype B3(CVB3) has been the most prevalent pathogens found in myocarditis biopsies, which estimates as causes of ~30% viral myocarditis cases.CVB3 is a nonenveloped single-stranded RNA viruses belonging to the family of the Picornaviridae. CVB3 usually is taken into esophageal and gastrointestinal tracts via fecal-oral route, and then spreads into multiple organs such as heart, pancreas, brain, liver, lung and spleen. CVB3 infects and propagates in intestinal cells, pancreatic cells and cardiomyocytes through interacting with coxsackie-adenovirus receptor(CAR) and decay accelerating factor(DAF), finally causing myocarditis and pancreatitis. The development of viral myocarditis is generally divided into three distinct phases. The first acute phase(day0-3/4) is characterized by virus replication and interaction with myocyte signaling before cardiac infiltration by innate cells. Small amounts of proinflammatory cytokines including IL-1b, IL-6, IL-18, TNF-a and type I/II interferons(IFNs) are produced by cardiac myocytes and fibroblasts. The subacute phase(day 3-10), commences with infiltration of innate immune cells, including natural killer(NK) cells and macrophages together with elevated expression of proinflammatory cytokines. Within days, immune cells(T and B) from the adaptive immune system also accumulate in the infected heart with NK and CTLs contributing to elimination of infected cardiomyocytes by direct killing. During the subacute phase, the cardiac inflammation and damage reaches to peak. The third stage(chronic phase, day 10~) is characterized by resolution of immune response by TGF-b and IL-10 as well as cardiac repair and remodeling. Fibrotic scar and cardiac dilation may starts during this phase contributing to contractile dysfunction. Failure to completely clear virus from the heart may result in chronic inflammation and accelerated progression to DCMCVB3 infection causes cardiac injury through multiple mechanisms, including direct cardiocyte death through virus replication, virus protease cleavage of contractile proteins and induction of virus-specific and autoimmune responses resulting in cardiomyocyte death. However, there is consensus that that direct damage of myocytes by CVB3 may occur at very early phase(day1-3), still CVB3-induced cardiac inflammatory immune infiltration and so-caused inflammatory injury is the major cause of VMC. The primary infiltrate in mouse models of myocarditis consists mainly of macrophages, neutrophils, T cells and much less B, NK, mast cells and gdT cells. The most abundant infiltrated immune cells including macrophages and pro-inflammatory T helper(Th)1/Th17 cells are most extensively studied. Inflammatory cytokines secreted by these immune cells, such as IFN-γ, TNF-α, IL-6, IL-1b and IL-17 are thought most closely associated with pathogenesis of CVB3 myocarditis.Previous attention about the pathogenesis of VMC is mainly focused on adaptive immune response, with pro-inflammatory role of Th1, Th17 and protective role of Tregs mostly widely accepted. Innate immunity, first line of host defense against pathogens, gets more and more concern nowadays for its critical role to provide early control of infection and to prime and shape the type of subsequent adaptive T cell immunity. During early phase of infection, vigorous innate signalings are activated in various innate cells, such as macrophages and dendritic cells(DCs), by membrane or cytosol pattern-recognizing receptors(PRRs) after recognition of pathogen-assocoated molecular patterns(PAMPs) of pathogens; leading to proinflammatory cytokines production, type I interferon secretion and antigen presentation. Role of macrophages, NK cells, gdT cells as well as TLR1~9 and RIG-I mediated innate innate immunity in pathogenesis of VMC has been reported in recent years. Our group has paid long attention on innate immunity-mediated inflammation in the pathogenesis of VMC. We found that sex-differentially polarized macrophages and TLRs signals play critical roles for the susceptibility to viral myocarditis. All these findings suggest the significance of innate immunity in the development of CVB3 myocarditis.In terms of pro-inflammatory cytokines and their roles in CVB3-induced VMC, pro-inflammatory cytokine IL-17 A undoubtedly is among the most famous and extensively studied ones. Members of the IL-17 cytokine family(IL-17 A through IL-17F) are proinflammatory cytokines that possess a prominent role of local inflammation and tissue repair. Its detrimental role for susceptibility to VMC has been demonstrated by anti-IL-17 A neutralizing m Ab and in IL-17 A KO mice in which significantly alleviated cardiac inflammation was confirmed after blockade of IL-17 A. Since IL-17 A has been considered as the main effector molecule of CD4+T cells for lone time; it is concluded that Th17 play an indispensable role in development of VMC. However, CD4+ αβ Th cells would not be available during the crucial early phases of infection. Apart from CD4+ αβ Th cells, it is recently recognized that a number of other innate cells including γδ T cells, i NKT cells, paneth cells and neutrophils all are available of releasing IL-17 A. Unlike conventional αβT cells, γδ T cells have properties of both innate and adaptive immune cells expressing TCRs with a limited set of Vγ and Vδ genes. They are usually located in the body’s skin, mucus and abdominal cavity, recognizing nonpeptide compounds presented by CD1 and play an critical early control of infection and and tumor. γδ T cells are recently reported as the main source of IL-17 A in many tumor context(liver and colon cancer), and play detrimental role in promoting tumor growth and metastasis. In several infection models, γδ-17 cells produce IL-17 at an early stage in the immune response and are required for the recruitment of neutrophils and subsequent clearance of the pathogen. These research progresses have prompted us to re-survey the role of IL-17A+γδ T cells in the pathogenesis of VMC. Our preliminary experiments have found an early infiltration of γδT cells into heart upon CVB3 infection(day 3), which secrete high levels of local IL-17 A. These results give good evidence and significance for the deep investigation of γδT-derived IL-17 A in the development of VMC.In conclusion, we hypothesize in this study that innate γδT cells are another important source of IL-17 A, a key pro-inflammatory cytokines during CVB3-induced VMC apart from CD4+Th17 cells. gdT17 may play very prominent role in the initiation of acute VMC in that IL-17A-producing γδ-T17 cells and Th17 cells are both elicited in acute phase of VMC, but functional γδ-T17 cells appear at least 3 days before Th17 cells and seem to produce more abundant amounts of IL-17. So we have the reason to believe that the earlier cardiac infiltrated gdT may play a more important role for the initiation of local inflammation than the conventional CD4+Th17 cells in acute VMC. Using CVB3-induced C57BL/6 VMC model, we plan to first detected IL-17 A production kinetics in heart after CVB3 infection, then confirmed the role of IL-17 A by IL-17 A KO mice. Next, we plan to analyze the IL-17A-secrting T cell subsets during early(3 days) and acute phase(7 days) of VMC via flow cytometry. To confirm the critical role of gdT17 in pathogenesis of VMC, TCRd KO mice and anti-Vg4 neutralizing antibody would be used to blockade in vivo function of gdT17. Finally, we would examine the downstream signal and activities of IL-17A-IL-17 RA pathway including chemokine secretion and neutrophils recruitment to explain the mechanism of gdT17 cells in provoking the cardiac inflammation following viral infection.Part one: The key role of IL-17 A in CVB3-induced VMC1. Establishment of murine model of CVB3-induced myocarditisMurine model of CVB3-induced myocarditis is very similar in several levels to clinical VMC. C57BL/6 murine model of CVB3 myocarditis was established by intraperitoneal injection of 1-1.5 ╳103 TCID50 dosage of CVB3, Mice lose their weight from day 3 post-infection when viral replication reaches its peak, with 7 days-weight loss amounting to 20% and survival rate being 40%-70%. Compared to intact cardiomyocytes seen in non-treated mice, massive inflammatory infiltration and myocytes necrosis were found in CVB3-infected mice on day 7 by HE staining of paraffin section of heart tissue, indicating CVB3-VMC model was successfully established in C57BL/6 mice.2. IL-17 A expression dynamics in heart of VMC miceTo see local IL-17 A expression dynamics in acute phase of VMC, heart and intestines were harvested to detect IL-17 A level by ELISA assay.. IL-17 A is not detectable in untreated mice. On day 4~7 post-infection, levels of inflammatory cytokine including IL-17 A, TNFa, IL-6, and IFNg was significantly increased suggesting inflammatory cytokines like IL-17 A during VMC.3. VMC development in IL-17 A KO miceTo evaluate role of IL-17 A during acute phase of CVB3-induced myocarditis, IL-17 A KO mice were subjected to CVB3 infection. Compared to WT mice, IL-17 A KO mice were seen with significantly reduced weight loss and elevated survival rate; Cardiac inflammatory infiltration was significantly reduced on day day 7 together with significantly decreased CK-MB activity. ELISA assay of cardiac homogenates confirmed significantly reduced cardiac levels of TNF-α and IL-6 on day 7 in IL-17 A KO mice. IL-17 A was undetectable while IFN-g level was significantly elevated. CVB3 load and replication was somewhat elevated on day 3 post infection. All data suggest that IL-17 A knockout has protective effect against myocarditis, and IL-17 A plays critical inflammatory role in the development of VMC. Part two:T cells are the main source of cardiac IL-17 A in CVB3-induced VMC1. Both CD4+Th cells and γδT cells infiltrate into heart acute phase of CVB3-induced VMC.It is known that CD4+Th cells and γδT cells are both producers for IL-17 A. In CVB3-induced VMC model, we tested cardiac infiltration of T cells on day 0, 4, 7 post-infection by flow cytometry. There was very low infiltration in heart before infection; while both T cells were significantly enriched into heart from day 3 PI except γδT cells moved earlier reaching peak on day 4(5.35% among CD3+T cells, 104 cells/heart). CD4+Th cells infiltration increased with time,up to day 7(13.4% in CD3+T cells and almost 106 cells/heart). These data showed that CD4+Th cells and γδT cells both infiltrate into myocardial tissue upon CVB3 infection with different kinetics in acute phase of VMC.2. Both CD4+Th cells and γδT cells are source of IL-17 A in acute phaseBy FACS examination, cardiac infiltrated cells are gated on IL-17A+, and then analyzed proportion of CD4+Th and γδT cells. Both T cells were detected in IL-17A+CD3+T cells, with 60% being γδT and 35% being Th17 cells. It suggestes that in acute phase of VMC, both Th17 cells and γδT cells infiltrate into heart and produce IL-17 A. But γδT cells represents as the major producer for cardiac IL-17 A.3. γδT subsets in cardiac infiltrates during acute VMCOn day4 PI when γδT infiltrates reach a peak, we tested the proportion and numbers of Vγ1and Vγ4 subsets. We found an equal and similar enrichment dynamics of Vγ1 and Vγ4 on day4-7 PI. However, IL-17 A was 89% produced by Vγ4 T cells, not by Vγ1..Taken together, Th17 cells and Vγ4γδ T cells are both recruited into cardiac tissue during acute phase of VMC. But Vγ4γδ T(gdT17)cells are the major source of cardiac IL-17 A.Part three: Role of IL-17A+γδT cells in acute VMC1. VMC development in TCRδ KO miceTo investigate role of γδT cells during the development of VMC, TCRδ KO mice were i.p. infected with CVB3. Compared to control mice, γδT-deficient mice were seen with significantly reduced VMC, testified by significantly reduced body loss, cardiac CK-MB activity and less inflammatory infiltration. All cardiac inflammatory CKs including IL-17 A, TNF-α and IL-6 were remarkably decreased, while virus replication on day 3 seemed intact. These data directly indicate the critical pro-inflammatory role of γδ T cells in CVB3-induced VMC.2 Role of Vγ4γδ T cell in acute VMC by depletionTo further investigate the role of Vγ4γδ T cells in acute VMC, we use anti-Vγ4–specific m Ab to deplete Vγ4γδT cellsbefore CVB3 infection. We found that : 1) 2 intravenous injection of anti-Vγ4–specific m Ab on days-5 and 1 before CVB3 infection results in up to 95% Vγ4γδT cell depletion in spleen. 2) Vγ4γδ T depletion led to decreased mortality, cardiac CK-MB and inflammatory infiltration in the heart indicating alleviated VMC; 3) cardiac level of IL-17 A, IFNg, IL-6 and TNFa were significantly reduced; 4) CVB3 load on day 3 were robustly decreased. All data indicate that Vγ4γδT cells play a detrimental role in the development of VMC by promoting inflammatory response and viral replication.3.Transfer of IL-17 A KO mice-derived γδ T cells into TCRd KO mice to investigate role of γδT-derived IL-17 A in VMCBy in vitro culture of splenocytes by Vγ4-specific m Ab plus anti-CD28 m Ab and r IL-2 for 6 days, we acquired cells with 60% purity. 1x106 cells γδ T were intravenously transferred into each TCRδ KO mice 24 hours before CVB3 treatment. We found that IL-17-/-γδ T cells into TCRδ KO mice had no role on acute VMC; while.acute cardiac inflammation and IL-17 A was significantly reduced in TCRδ KO mice receiving IL-17 KO mice-derived γδ T cells. It indicates that the pro-inflammatory role of gdT or Vg4gdT is mediated by IL-17 A..4. Depletion of γδ T, Vγ4 or Vγ4 γδ T cells and CD4+Th cells parallelly to evaluate the contribution of γδ T and Th17 cells to acute VMC.We have shown that similar proportion and numbers of γδ T and CD4+Th17 were enriched in the heart on day 3~7 upon CVB3 infection and both secret IL-17 A, then how to assess the contribution of two T cells to the pathogenesis of VMC remains an important but difficult scientific problems. We plan to clear γδ T, Vγ1 γδ T, Vγ4 γδ T and CD4+Th cells by injection of specific neutralizing m Ab against TCRδ, Vγ1Vγ4, CD4 neutralizing antibodies parallelly to evaluate contribution of γδ T and Th17 cells to cardiac inflammation during acute VMC. Data not achieved.5. Mutual regulation of Vγ1 and Vγ4 subsets in the pathogenesis of VMCVγ1 T and Vγ4T cells are reported to play opposite role in infection by producing different CKs. And we have shown the two subsets both infiltrate into heart during the acute phase with equivalent pattern. Therefore it is worth to follow the phenotype and chacteristics of both Vγ4T and Vγ1 T cell during acute VMC. We found that: 1)When cardiac Vγ4T mainly produce IL-17 A, Vγ1 T cell in the spleen are activated to secret IFNg, the ability increasing with time from day3~7 after CVB3 infection comparable to CD4+Th1 cells; 2) By depletion of Vγ1 γδ T, cell by m Ab, we confirmed protective role of Vγ1 γδ T(data not shown); 3) During 1~7 day period after depletion of Vγ4 γδ T, cell by m Ab, we found an interesting 2 times up-regulation of Vγ1 γδ T indicating a mutual regulation of the two γδ T subsets, More experiments are needed. Together, these data indicate a critical pro-inflammatory role of cardiac infiltrated γδT cell in CVB3-induced acute VMC: the Vγ4 γδ T subsets exerts detrimental effect through producing vigorous amounts of IL-17A; while simultaneously infiltrated Vγ1γδ T subsets may represents as a regulatory cell by producing IFN-γ.Part Four:The mechansim of T17 In Promoting VMCIL-17 A can act on a variety of cells with IL-17 RA receptors, thus exerting a variety of effector function, the major one is demonstrated as neutrophils recruitment.1. γδT17-derived IL-17 A regulates the chemokine expression profiles on target cellsWe first detect the regulation of chemokine expression on cardiomyocytes and splenic cells by IL-17 A through Real-Time PCR. On stimulation with IL-17 A, significantly increased expression of CXCL1, CXCL2 and CXCL3 was observed in cardiomyocytes; Dose-dependent up-regulation of CXCL2 and CXCL3 was seen in splenic cells by IL-17 A. We plan to detect the CXCR2, CXCR5 recepter expression on splenocytes and neutrophils(data not achieved). Our data suggest that cardiac infiltrated Vγ4γδT produce IL-17 A at early phase of VMC, promoting the recruitment of peripheral immune cells into heart through upregulating the secretion of CXCL1, CXCL2,CXCL3 by cardiomyocytes, thus exacerbating the acute myocarditis.2. γδT17 promote the cardiac infiltration of neutrophilsAnother effect of IL-17A-IL-17 RA pathway is the recruitment and activation of neutrophils. To figure out these, we detected the cardiac neutrophil infiltration upon CVB3 infection. We found that: 1) Ly6G+CD11b+ neutrophils infiltrated into heart and spleen on day 3~ 7 post-infection. 2) We next purified neutrophils and splenocytes, and plan to perform Transwell assay to evaluate the chemotactic role of IL-17 A or Vγ4 γδ T on splenocytes or neutrophils(data not achieved).3.Influence of cardiac neutrophil infiltration by Vγ4γδ T cell depletionIn Vγ4γδ T cell depleted mice, we detected the cardiac neutrophil infiltration on day 7, and found the number of neutrophils dereased significantly, remaining less than 20% in the heart,.It suggests that γδT produce IL-17 A,effectively promotes neutrophil recruitment into the heart during acyte VMC. We next plan to detect roles of cardiac neutrophils including phagocytosis, ROS reaction and inflammatory CK secretion.Taken together, during acute phase of VMC induced by CVB3 infection, we have found that: 1) Both CD4+Th cell and gdT cells infiltrate into heart from day3~day7 and both produce IL-17A; but gdT show an early infiltration and represents as the major source of cardiac IL-17 A at acute phase; 2) Among gdT cells, 90% IL-17A-producing cells are Vg4gdT cells; while Vg1gdT cells increase production of IFNg with time. 3) Deficiency of gdT cells or Vg4gdT cells significantly protects against VMC, indicating Vg4gdT play critical pro-inflammatory role in acute VMC; 4) Transfer assay demonstrates that Vg4gdT cell exerts its pro-inflammatory role through secretion of IL-17A; 5) The detrimental role of IL-17+Vg4gdT cell may be mediated through cardiac recruitment of neutrophils by up-regulation cardiac CXCL1, CXCL2 and CXCL3. Our study finds and demonstrates the pro-inflammatory role of cardiac early infiltrated IL-17+Vg4gdT cell(gdT17) in the pathogenesis of CVB3 induced acute myocarditis and the possible mechanism. These data may provide important clue and targets for the future design of novel immune therapy for the treatment of viral myocarditis. |
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