A longitudinal study of antibody responses to selected host antigens in rheumatic fever and rheumatic heart disease.

Introduction. Group A streptococci can trigger autoimmune responses that lead to acute rheumatic fever (ARF) and rheumatic heart disease (RHD).Gap Statement. Some autoantibodies generated in ARF/RHD target antigens in the S2 subfragment region of cardiac myosin. However, little is known about the kinetics of these antibodies during the disease process.Aim. To determine the antibody responses over time in patients and healthy controls against host tissue proteins - cardiac myosin and peptides from its S2 subfragment, tropomyosin, laminin and keratin.Methodology. We used enzyme-linked immunosorbent assays (ELISA) to determine antibody responses in: (1) healthy controls; (2) patients with streptococcal pharyngitis; (3) patients with ARF with carditis and (4) patients with RHD on penicillin prophylaxis.Results. We observed significantly higher antibody responses against extracellular proteins - laminin and keratin in pharyngitis group, patients with ARF and patients with RHD when compared to healthy controls. The antibody responses against intracellular proteins - cardiac myosin and tropomyosin were elevated only in the group of patients with ARF with active carditis. While the reactivity to S2 peptides S2-1-3, 8-11, 14, 16-18, 21-22 and 32 was higher in patients with ARF, the reactivity in the RHD group was high only against S2-1, 9, 11, 12 when compared to healthy controls. The reactivity against S2 peptides reduced as the disease condition stabilized in the ARF group whereas the reactivity remained unaltered in the RHD group. By contrast antibodies against laminin and keratin persisted in patients with RHD.Conclusion. Our findings of antibody responses against host proteins support the multistep hypothesis in the development of rheumatic carditis. The differential kinetics of serum antibody responses against S2 peptides may have potential use as markers of ongoing cardiac damage that can be used to monitor patients with ARF/RHD.

Galectin-3 deficiency enhances type 2 immune cell-mediated myocarditis in mice.

Experimental autoimmune myocarditis (EAM) is a mouse model of immune-mediated myocarditis and cardiomyopathy. The role of Galectin-3 (Gal-3), a ß-galactoside-binding lectin, in autoimmune myocarditis has not been studied. Therefore, the aim of this study was to delineate the role of Gal-3 in myosin peptide-induced autoimmune myocarditis in mice. EAM was induced in relatively resistant C57BL/6J mice (wild type, WT) and in mice with a targeted deletion of Gal-3 gene (Gal-3KO) by immunization with myosin peptide MyHCα334-352. Gal-3KO mice developed more severe myocarditis and more pronounced heart hypertrophy than WT mice. Increased infiltration of CD45+ leucocytes, CD3+ T cells, F4/80+ macrophages, and eosinophils was observed in hearts of Gal-3KO mice compared to WT mice on day 21 after EAM induction. Moreover, hearts of Gal-3KO mice had more T helper type 2 (Th2) cells, alternatively activated M2 macrophages, higher amounts of IgG deposits, and higher serum levels of IL-4 and IL-33 than WT mice. Ablation of Gal-3 in Th1-dominant C57BL/6J mice that are relatively resistant to EAM resulted in more severe disease characterized by type 2 cardiac inflammation. The complex effects of Gal-3 on EAM progression might be important in the consideration of therapeutic options for the treatment of EAM.

HMGB1 silencing in macrophages prevented their functional skewing and ameliorated EAM development: Nuclear HMGB1 may be a checkpoint molecule of macrophage reprogramming.

High-mobility group box 1 (HMGB1), an important inflammatory factor, plays significant roles in CD4+T cell differentiation, cancer and autoimmune disease development. Our previous data have demonstrated that HMGB1 contributes to macrophage reprogramming and is involved in experimental autoimmune myocarditis (EAM) development. In contrast to the well-explored function of HMGB1, little is known about the nuclear function. Whether HMGB1 can serve as an architectural factor and control functional skewing of macrophages remains unclear. Therefore, the present work was performed to address the above speculation. The adenovirus-mediated shRNA (Ad-shRNA) was employed to knock down HMGB1 in RAW264.7 and monocytes/macrophages of EAM mice. Our data showed that in vitro HMGB1 silencing limited functional skewing of macrophages and down-regulated inflammatory factors secretion, which can't be reversed by the exogenous HMGB1. In M1 polarization system, the phosphorylations of NF-κB, p38 and Erk1/2 were inhibited following HMGB1 silencing. In vivo, HMGB1 silencing could effectively ameliorate EAM development. Our data suggest that HMGB1 may be a checkpoint nuclear factor of macrophage reprogramming. Our findings also provide an exciting therapeutic method for inflammatory disorders.

Exosomes expressing the self-antigens myosin and vimentin play an important role in syngeneic cardiac transplant rejection induced by antibodies to cardiac myosin.

Long-term success of heart transplantation is hindered by humoral and cell-mediated immune responses. We studied preexisting antibodies to cardiac self-antigens, myosin and vimentin, and exosomes induced by antibodies to self-antigens in eliciting immune responses to cardiac grafts. After syngeneic heterotopic murine heart transplantation, rabbit anti-myosin or normal rabbit immunoglobulin was administered at day 0 or 7. Sera were collected after heartbeat cessation, cellular infiltration was analyzed, and exosomes were isolated from sera. Histopathologic examination of the controls' transplanted hearts demonstrated normal architecture, and their sera demonstrated neither antibodies to self-antigens nor exosomes expressing self-antigens. Administration of antibodies to cardiac myosin immediately posttransplantation (day 0) but not on day 7 triggered graft failure on day 7, and histopathologic examination revealed marked cellular infiltration with neutrophils and lymphocytes. Histopathologic examination of rejected hearts also demonstrated myocyte damage as sera had increased antibodies to myosin and vimentin and development of exosomes expressing self-antigens. Administration of exosomes isolated from failed grafts containing self-antigens induced graft dysfunction; exosomes isolated from stable mice did not induce graft failure. Antibodies to self-antigens can induce exosomes containing self-antigens, initiating an immune response and causing graft failure after cardiac transplantation.

FGL2 knockdown improves heart function through regulation of TLR9 signaling in the experimental autoimmune myocarditis rats.

Fibrinogen-like protein 2 (FGL2) is an important immune regulator of both innate and adaptive response. It is present on the surface of macrophages and endothelial cells, and can be constitutively secreted by CD4+CD8+ T cells. Previous studies showed that FGL2 is a potential target for the treatment of experimental autoimmune myocarditis. However, the molecular mechanism of the roles of FGL2 in experimental autoimmune myocarditis is poorly understood. Here, we silenced FGL2 gene by using FGL2-RNAi lentivirus to reveal the heart function in experimental autoimmune myocarditis rats. We found that the cardiac myosin of pigs' hearts induced Lewis rats to come into being as autoimmune myocarditis. TLR9 was upregulated in the heart of experimental autoimmune myocarditis rats. After primary immunization (21 day), the cardiac function of the myocarditis model group improved (P < 0.05). Significantly, the levels of INF-α and NF-κB in the FGL2-RNAi-treated group were lower compared to those in the myocarditis model (EAM) group (P < 0.05). Notably, the inflammation score correspondence with the protein and mRNA levels of TLR9 in myocardial tissues was markedly reduced compared to that in the EAM group (P < 0.05). These results support a role of FGL2 to alleviate inflammatory situation in the myocardium through regulation of the TLR9 signaling pathway in the experimental autoimmune myocarditis rats.

Combined analysis of cross-reacting antibodies anti-ß1AR and anti-B13 in advanced stages of Chagas heart disease.

OBJECTIVE: Autoantibodies cross-reacting with the ß1 adrenergic receptor (anti-ß1AR and anti-p2ß) and cardiac myosin antigens (anti-B13) have been related to the pathogenesis of chronic Chagas heart disease (CCHD). Studies exploring their levels in different stages are scarce. We aimed to evaluate the relationship of these autoantibodies with the clinical profile of chronic patients, especially regarding their classificatory accuracy in severe presentation with heart failure. METHODS AND RESULTS: We conducted a cross-sectional study of 155 T. cruzi-seropositive patients and 26 age- and gender-matched healthy controls. They were categorised in three stages of CCHD. Serum antibodies were measured by specific immunoassays. Symptomatic individuals showed increased levels of anti-ß1AR and anti-B13, while anti-p2ß antibodies were similar between groups. A composite logistic regression model including anti-B13, anti-ß1AR antibody levels and age was able to predict systolic heart failure yielding an area under the curve of 83% (sensitivity of 67% and specificity of 89%). CONCLUSIONS: In our study, anti-ß1AR and anti-B13 antibodies were higher in individuals with chronic Chagas heart disease stage III, mainly in those with dilated cardiomyopathy associated with systolic heart failure. Logistic regression analysis showed that both antibodies were good predictors of severe CCHD. As well as being involved in disease progression, anti-ß1AR and anti-B13 antibodies may be used as a serum marker of poor prognosis in terms of heart compromise.

Macrophages and cardiac fibroblasts are the main producers of eotaxins and regulate eosinophil trafficking to the heart.

Cardiac manifestations are a major cause of morbidity and mortality in patients with eosinophil-associated diseases. Eosinophils are thought to play a pathogenic role in myocarditis. We investigated the pathways that recruit eosinophils to the heart using a model of eosinophilic myocarditis, in which experimental autoimmune myocarditis (EAM) is induced in IFNγ-/- IL-17A-/- mice. Two conditions are necessary for efficient eosinophil trafficking to the heart: high eotaxin (CCL11, CCL24) expression in the heart and expression of the eotaxin receptor CCR3 by eosinophils. We identified cardiac fibroblasts as the source of CCL11 in the heart interstitium. CCL24 is produced by F4/80+ macrophages localized at inflammatory foci in the heart. Expression of CCL11 and CCL24 is controlled by Th2 cytokines, IL-4 and IL-13. To determine the relevance of this pathway in humans, we analyzed endomyocardial biopsy samples from myocarditis patients. Expression of CCL11 and CCL26 was significantly increased in eosinophilic myocarditis compared to chronic lymphocytic myocarditis and positively correlated with the number of eosinophils. Thus, eosinophil trafficking to the heart is dependent on the eotaxin-CCR3 pathway in a mouse model of EAM and associated with cardiac eotaxin expression in patients with eosinophilic myocarditis. Blocking this pathway may prevent eosinophil-mediated cardiac damage.

Repeat exposure to group A streptococcal M protein exacerbates cardiac damage in a rat model of rheumatic heart disease.

Rheumatic fever and rheumatic heart disease (RF/RHD) develop following repeated infection with group A streptococci (GAS). We used the Rat Autoimmune Valvulitis (RAV) model of RF/RHD to demonstrate that repetitive booster immunization with GAS-derived recombinant M protein (rM5) resulted in an enhanced anti-cardiac myosin antibody response that may contribute to the breaking of immune tolerance leading to RF/RHD and increased infiltration of heart valves by mononuclear cells. With each boost, more inflammatory cells were observed infiltrating heart tissue which could lead to severe cardiac damage. We also found evidence that both complement and anti-M protein antibodies in serum from rM5-immunized rats have the potential to contribute to inflammation in heart valves by activating cardiac endothelium. More importantly, we have demonstrated by electrocardiography for the first time in the RAV model that elongation of P-R interval follows repetitive boost with rM5. Our observations provide experimental evidence for cardiac alterations following repeated exposure to GAS M protein with immunological and electrophysiological features resembling that seen in humans following recurrent GAS infection.

Cardiac Myosin Epitopes Recognized by Autoantibody in Acute and Convalescent Rheumatic Fever.

BACKGROUND: Acute rheumatic fever (ARF) is an autoimmune disorder associated with Streptococcus pyogenes infection. A prevailing hypothesis to account for this disease is that epitopes of self-antigens, such as cardiac myosin react to antibodies against S. pyogenes. The goal of our study was to confirm disease epitopes of cardiac myosin, identify immunodominant epitopes and to monitor the epitope response pattern in acute and convalescent rheumatic fever. METHODS: Enzyme-linked immunosorbant assays were used to determine epitopes immunodominant in acute disease and to track the immune response longitudinally to document any changes in the epitope pattern in convalescent sera. Multiplex fluorescence immunoassay was used to correlate anti-streptolysin O (ASO) and anti-human cardiac myosin antibodies. RESULTS: Disease-specific epitopes in rheumatic fever were identified as S2-1, 4 and 8. Epitopes S2-1, 4, 8 and 9 were found to be immunodominant in acute sera and S2-1, 8, 9, 29 and 30 in the convalescent sera. Frequency analysis showed that 50% of the ARF subjects responded to S2-8. S2-8 responders tended to maintain their epitope pattern throughout the convalescent period, whereas the S2-8 nonresponders tended to spread their responses to other epitopes later in the immune response. There was a significant correlation between anti-cardiac myosin and ASO titers. In addition, S2-8 responders showed elevated ASO titers compared with S2-8 non responders. CONCLUSION: Our studies confirm the existence of S2-1, 4 and 8 as disease-specific epitopes. We provide evidence that cardiac myosin S2-8 responders remain epitope stable in convalescence, whereas S2-8 nonresponders shift to neoepitopes. Multiplex data indicated a correlation between elevated ASO and anti-human cardiac myosin antibody titers. Mapping of cardiac myosin epitopes recognized in rheumatic fever sera may identify immunophenotypes of rheumatic fever.

FTY720 (Gilenya) treatment prevents spontaneous autoimmune myocarditis and dilated cardiomyopathy in transgenic HLA-DQ8-BALB/c mice.

Although dilated cardiomyopathy (DCM) is often caused by viral infections, it frequently involves autoimmune mechanisms associated with particular HLA-DR and DQ alleles. Our homozygous HLA-DQ8Ab(0) transgenic mice in the BALB/c background (HLA-DQ8(BALB/c)-Tg) developed early and progressive fatal heart failure from 4 to 5 weeks of age. Clinical signs of the disease included cyanotic eyes, tachycardia with dyspnea (from pale to cyanotic limbs), and terminal whole body edema. Sick mice had extremely dilated hearts, enlarged liver and spleen, and pleural/peritoneal effusion. Histology of the heart showed extensive heart muscle destruction with signs of fibrosis. The autoimmune nature of the disease was shown by high titers of antimyosin antibodies in the sera and IgG deposits in sick heart muscles, as well as focal neutrophil, T cell, and macrophage infiltration of the heart muscle. The sera of the sick mice showed a granular staining pattern on sections of healthy heart muscle. Quantitative analyses of DCM-specific gene expression studies revealed that sets of genes are involved in inflammation, hypoxia, and fibrosis. Treatment with FTY720 (Fingolimod/Gilenya) protected animals from the development of cardiomyopathy. HLA-DQ8(BALB/c)-Tg mice represent a spontaneous autoimmune myocarditis model that may provide a useful tool for studying the autoimmune mechanism of DCM and testing immunosuppressive drugs.

Autoimmunity Against the Heart and Cardiac Myosin in Children With Myocarditis.

BACKGROUND: Host autoimmune activity in myocarditis has been proposed to play a role in development of cardiac disease, but evidence of autoimmunity and relationship to outcomes have not been evaluated in pediatric myocarditis. METHODS: We performed a multi-institutional study of children with clinical myocarditis. Newly diagnosed patients were followed for up to 12 months and previously diagnosed patients at a single follow-up for serum levels of autoantibodies to human cardiac myosin, beta-adrenergic receptors 1 and 2, muscarinic-2 receptors, and antibody-mediated protein kinase A (PKA) activation in heart cells in culture. Results were compared with those of healthy control children. RESULTS: Both previously diagnosed patient at follow-up (P = .0061) and newly diagnosed patients at presentation (P = .0127) had elevated cardiac myosin antibodies compared with control subjects. Antibody levels were not associated with recovery status at follow-up in either group. PKA activation was higher at presentation in the newly diagnosed patients who did not recovery normal function (P = .042). CONCLUSIONS: Children with myocarditis have evidence of autoantibodies against human cardiac myosin at diagnosis and follow-up compared with control subjects. Differences in antibody-mediated cell signaling may contribute to differences in patient outcomes, as suggested by elevated antibody-mediated PKA activation in heart cells by the serum from nonrecovered patients.

Congenital heart disease linked to maternal autoimmunity against cardiac myosin.

Structural congenital heart disease (CHD) has not previously been linked to autoimmunity. In our study, we developed an autoimmune model of structural CHD that resembles hypoplastic left heart syndrome (HLHS), a life-threatening CHD primarily affecting the left ventricle. Because cardiac myosin (CM) is a dominant autoantigen in autoimmune heart disease, we hypothesized that immunization with CM might lead to transplacental passage of maternal autoantibodies and a prenatal HLHS phenotype in exposed fetuses. Elevated anti-CM autoantibodies in maternal and fetal sera, as well as IgG reactivity in fetal myocardium, were correlated with structural CHD that included diminished left ventricular cavity dimensions in the affected progeny. Further, fetuses that developed a marked HLHS phenotype had elevated serum titers of anti-ß-adrenergic receptor Abs, as well as increased protein kinase A activity, suggesting a potential mechanism for the observed pathological changes. Our maternal-fetal model presents a new concept linking autoimmunity against CM and cardiomyocyte proliferation with cardinal features of HLHS. To our knowledge, this report shows the first evidence in support of a novel immune-mediated mechanism for pathogenesis of structural CHD that may have implications in its future diagnosis and treatment.

Effects of hepatocyte growth factor in myocarditis rats induced by immunization with porcine cardiac myosin.

OBJECTIVES: Myocarditis is considered one of the major causes of dilated cardiomyopathy. Hepatocyte growth factor (HGF) has pleiotropic activities that promote tissue regeneration and facilitate functional improvement of injured tissue. We investigated whether the epicardial sustained-release of HGF, using gelatin hydrogel sheets, improves cardiac function in a chronic myocarditis rat model. METHODS: Six weeks after Lewis rats were immunized with porcine cardiac myosin to establish autoimmune myocarditis, HGF- or normal saline (NS)-incorporated gelatin hydrogel sheets were applied to the epicardium (G-HGF and G-NS, respectively). At either 2 or 4 weeks after treatment, these were compared with the Control myocarditis group. Cardiac function was evaluated by echocardiography and cardiac catheterization. Development of fibrosis was determined by histological study and expression of transforming growth factor-ß1 (TGF-ß1). Bax and Bcl-2 levels were measured to evaluate apoptotic activity. RESULTS: At both points, fractional shortening and end-systolic elastance were higher in the G-HGF group than in the Control and G-NS groups (P < 0.01). Fractional shortening at 2 weeks of each group were as follows: 31.0 ± 0.9%, 24.8 ± 2.7% and 48.6 ± 2.6% (Control, G-NS and G-HGF, respectively). The ratio of the fibrotic area of the myocardium was lower in the G-HGF group than in the Control and G-NS groups at 2 weeks (G-HGF, 8.8 ± 0.9%; Control, 17.5 ± 0.2%; G-NS, 15.6 ± 0.7%; P < 0.01). The ratio at 4 weeks was lower in the G-HGF group than in the G-NS group (10.9 ± 1.4% vs 18.5 ± 1.3%; P < 0.01). The mRNA expression of TGF-ß1 in the G-HGF group was lower than in the Control group at 2 weeks (0.6 ± 0.1 vs 1.1 ± 0.2) and lower than that in the G-NS group at 4 weeks (0.7 ± 0.1 vs 1.3 ± 0.2). The Bax-to-Bcl-2 ratios at both points were lower in the G-HGF group than in the Control group. CONCLUSIONS: Sustained-released HGF markedly improves cardiac function in chronic myocarditis rats. The antifibrotic and antiapoptotic actions of HGF may contribute to the improvement. HGF-incorporated gelatin hydrogel sheet can be a new therapeutic modality for myocarditis.

Myosin-primed tolerogenic dendritic cells ameliorate experimental autoimmune myocarditis.

AIMS: Autoimmunity plays an important role in the pathogenesis of viral myocarditis and giant cell myocarditis. Experimental autoimmune myocarditis (EAM) is a mouse model of myocarditis that is induced by cardiac myosin. Tolerogenic dendritic cells (tDCs) are used as anti-inflammatory and immunosuppressive targets in a number of autoimmune disease models, but their effect on EAM has not been addressed. The aim of this study was to investigate whether tDC therapy in an EAM mouse model can suppress inflammatory myocarditis, a potential precursor of dilated cardiomyopathy. METHODS AND RESULTS: tDCs were generated by treating immature DCs (imDCs) with TNF-α and cardiac myosin. Mice with EAM were injected twice with tDCs (with a 1-week interval) at three doses (2 × 10(5), 1 × 10(6), or 2 × 10(6)). The severity of myocarditis was histopathologically assessed. The phenotypes of the DC and regulatory T (Treg) cell populations were determined by flow cytometry and the effect of tDCs on autoimmunity-inducing cytokines was examined by ELISA. Myosin-pulsed tDCs displayed lower levels of DC-related surface markers and expressed higher levels of indoleamine 2, 3-dioxygenase (IDO) than mature DCs (mDCs). Histopathological examination revealed that hearts from tDC-treated mice showed markedly reduced myocardial inflammation compared with those of untreated EAM mice. These therapeutic effects by tDCs were mediated at least by enhanced myosin-specific Treg cell induction and anti-inflammatory cytokine secretion. CONCLUSION: Taken together, these results show for the first time that myosin-pulsed tDCs ameliorate EAM, and that this occurs most likely via the induction of antigen-specific Treg cells.

Effects of preexisting autoimmunity on heart graft prolongation after donor-specific transfusion and anti-CD154.

BACKGROUND: Alloreactive memory T cells prevent costimulatory blockade-induced heart graft survival in mice, but whether and how preexisting autoreactive T cells affect solid-organ transplants under these conditions is unknown. METHODS: We tested the impact of preexisting cardiac myosin (CM)-specific immunity on murine heart transplant recipients treated with donor-specific transfusion (DST) plus anti-CD154 monoclonal antibody MR1. RESULTS: Preimmunization with CM but not control ovalbumin abrogated the graft prolonging effects of DST/MR1, whether administered 2 weeks or more than 6 weeks before transplantation. Adoptive transfer of spleen cells from CM-immunized mice into naïve recipients had similar effects. CM-specific immunity did not cross-react with donor antigens and preimmunization with CM had no impact on the survival or histology of DST/MR1-treated syngeneic heart grafts, the latter indicating that persistent autoimmunity is insufficient to cause rejection in the context of costimulatory blockade. We observed that the CM preimmunized mice produced higher frequencies of donor-reactive T cells with higher ratios of CD8/CD4Foxp3 cells, suggesting that the autoreactive memory T cells provide help for activation of alloreactive T cells despite the costimulatory blockade. CONCLUSIONS: These mechanistic insights linking autoimmunity and alloimmunity in a model of murine heart transplantation have clinical relevance to the known association between autoimmunity and an elevated risk of acute and chronic heart transplant injury in humans.

Fatal eosinophilic myocarditis develops in the absence of IFN-γ and IL-17A.

CD4(+) T cells play a central role in inflammatory heart disease, implicating a cytokine product associated with Th cell effector function as a necessary mediator of this pathophysiology. IFN-γ-deficient mice developed severe experimental autoimmune myocarditis (EAM), in which mice are immunized with cardiac myosin peptide, whereas IL-17A-deficient mice were protected from progression to dilated cardiomyopathy. We generated IFN-γ(-/-)IL-17A(-/-) mice to assess whether IL-17 signaling was responsible for the severe EAM of IFN-γ(-/-) mice. Surprisingly, IFN-γ(-/-)IL-17A(-/-) mice developed a rapidly fatal EAM. Eosinophils constituted a third of infiltrating leukocytes, qualifying this disease as eosinophilic myocarditis. We found increased cardiac production of CCL11/eotaxin, as well as Th2 deviation, among heart-infiltrating CD4(+) cells. Ablation of eosinophil development improved survival of IFN-γ(-/-)IL-17A(-/-) mice, demonstrating the necessity of eosinophils in fatal heart failure. The severe and rapidly fatal autoimmune inflammation that developed in the combined absence of IFN-γ and IL-17A constitutes a novel model of eosinophilic heart disease in humans. This is also, to our knowledge, the first demonstration that eosinophils have the capacity to act as necessary mediators of morbidity in an autoimmune process.

The heart of the matter: protection of the myocardium from T cells.

Myocardial inflammation and damage can lead to lethal acute or chronic cardiac failure. A variety of regulatory mechanisms limit the magnitude and duration of T cell responses in the heart. Insights into these regulatory mechanisms have come from studies of specific deficiencies in central or peripheral T cell tolerance which cause or enhance the severity of myocarditis. Under non-inflammatory conditions, constitutive DC presentation of cardiac peptides to naïve T cells in cardiac draining lymph nodes tolerizes recirculating naïve T cells specific for these antigens. Cardiac antigen-specific naïve T cells, especially those specific of α-myosin heavy chain peptides, become activated and differentiate into expanded clones of effector T cells under various conditions, such as cardiac infection and/or genetic variations in peripheral tolerance. The pathology that these effector cells cause in the myocardium is limited by PD-L1 expressed on myocardial cells in response to inflammatory cytokines, and by CTLA-4 dependent mechanisms. The PD-1:PD-L1 pathway works together with other control mechanisms to keep the heart safe from T cells, and combined impairment of this pathway along with other regulatory mechanisms synergize to cause myocarditis. T cell derived IFNγ contributes to the inflammatory damage to the heart in autoimmune myocarditis, but it also engages regulatory mechanisms that limit disease, including upregulation of PD-L1, and differentiation of TNF and iNOS expressing DCs from monocytes. iNOS derived from these DCs and other IFNγ stimulated cells inhibits expansion of T cells that cause myocarditis. Regulatory T cells also appear to be critical for suppression of effector T cells specific for myocardial antigens.

Autoimmune myocarditis, valvulitis, and cardiomyopathy.

Myocarditis and valvulitis are inflammatory diseases affecting myocardium and valve. Myocarditis, a viral-induced disease of myocardium, may lead to dilated cardiomyopathy and loss of heart function. Valvulitis leads to deformed heart valves and altered blood flow in rheumatic heart disease. Animal models recapitulating these diseases are important in understanding the human condition. Cardiac myosin is a major autoantigen in heart, and antibodies and T cells to cardiac myosin are evident in inflammatory heart diseases. This unit is a practical guide to induction and evaluation of experimental autoimmune myocarditis (EAM) in several mouse strains and the Lewis rat. Purification protocols for cardiac myosin and protocols for induction of EAM by cardiac myosin and its myocarditis-producing peptides, and coxsackievirus CVB3, are defined. Protocols for assessment of myocarditis and valvulitis in humans and animal models provide methods to define functional autoantibodies targeting cardiac myosin, ß-adrenergic, and muscarinic receptors, and their deposition in tissues.

Coxsackievirus B3 infection leads to the generation of cardiac myosin heavy chain-α-reactive CD4 T cells in A/J mice.

Enteroviruses like coxsackievirus B3 (CVB3) are common suspects in myocarditis/dilated cardiomyopathy patients. Autoimmunity has been proposed as an underlying mechanism, but direct evidence of its role is lacking. To delineate autoimmune response in CVB3 myocarditis, we used IA(k) dextramers for cardiac myosin heavy chain (Myhc)-α 334-352. We have demonstrated that myocarditis-susceptible A/J mice infected with CVB3 generate Myhc-α-reactive CD4 T cells and such a repertoire was absent in naïve mice as measured by proliferative response to Myhc-α 334-352 and IA(k) dextramer staining. We also detected Myhc-α 334-352 dextramer(+) cells in the hearts of CVB3-infected mice. The autoreactive T cell repertoire derived from infected mice contained a high frequency of interleukin-17-producing cells capable of inducing myocarditis in naïve recipients. The data suggest that CVB3, a bona fide pathogen of cardiovascular system that primarily infects the heart can lead to the secondary generation of autoreactive T cells and contribute to cardiac pathology.

Olmesartan medoxomil treatment potently improves cardiac myosin-induced dilated cardiomyopathy via the modulation of ACE-2 and ANG 1-7 mas receptor.

Angiotensin converting enzyme-2 (ACE-2) is a monocarboxypeptidase that metabolises angiotensin (ANG)-II into angiotensin 1-7 (ANG 1-7), thereby functioning as a negative regulator of the renin-angiotensin system. We investigated whether treatment with ANG-II type 1 receptor blocker, olmesartan medoxomil is associated with the attenuation of cardiac myosin-induced dilated cardiomyopathy (DCM) through recently established new axis of ACE-2/ANG 1-7 mas receptor. DCM was elicited in Lewis rats by immunisation with cardiac myosin, and 28 days after immunisation, the surviving Lewis rats were divided into two groups and treated with either olmesartan medoxomil (10 mg/kg/day) or vehicle. Myocardial protein and mRNA levels of ACE-2, ANG 1-7 mas receptor were upregulated in the olmesartan-treated group compared with those of vehicle-treated DCM rats. In contrast, Olmesartan treatment effectively suppressed the myocardial protein and mRNA expressions of inflammatory markers in comparison to the vehicle-treated DCM rats. Olmesartan treatment significantly reduced fibrosis, hypertrophy and their marker molecules (OPN, CTGF, ANP and GATA-4, respectively), as well as matrix metalloproteinases compared with those of vehicle-treated DCM rats. Enhanced myocardial protein levels of phospho-p38 MAPK, phospho-JNK and phospho MAPKAPK-2 in the vehicle-treated DCM rats were prevented by olmesartan treatment. In addition, olmesartan treatment significantly lowered the protein expressions (Nitrotyrosine, p47phox and p67phox) and superoxide radical production compared with those of vehicle-treated DCM rats. Our present study might serve as a new therapeutic target of DCM in cardiovascular diseases and cardiac myosin-induced DCM via the modulation of ACE-2/ANG 1-7 mas receptor axis in rats with DCM after myosin-immunisation.