RESUMO
Protease-activated receptor 1 (PAR1) is widely expressed in humans and mice, and is activated by a variety of proteases, including thrombin. Recently, we showed that PAR1 contributes to the innate immune response to viral infection. Mice with a global deficiency of PAR1 expressed lower levels of CXCL10 and had increased Coxsackievirus B3 (CVB3)-induced myocarditis compared with control mice. In this study, we determined the effect of cell type-specific deletion of PAR1 in cardiac myocytes (CMs) and cardiac fibroblasts (CFs) on CVB3-induced myocarditis. Mice lacking PAR1 in either CMs or CFs exhibited increased CVB3 genomes, inflammatory infiltrates, macrophages and inflammatory mediators in the heart and increased CVB3-induced myocarditis compared with wild-type controls. Interestingly, PAR1 enhanced poly I:C induction of CXCL10 in rat CFs but not in rat neonatal CMs. Importantly, activation of PAR1 reduced CVB3 replication in murine embryonic fibroblasts and murine embryonic cardiac myocytes. In addition, we showed that PAR1 reduced autophagy in murine embryonic fibroblasts and rat H9c2 cells, which may explain how PAR1 reduces CVB3 replication. These data suggest that PAR1 on CFs protects against CVB3-induced myocarditis by enhancing the anti-viral response whereas PAR1 on both CMs and fibroblasts inhibits viral replication.
Assuntos
Quimiocina CXCL10/metabolismo , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/metabolismo , Fibroblastos/metabolismo , Miocardite/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Ativados por Proteinase/metabolismo , Animais , Autofagia , Linhagem Celular , Deleção de Genes , Humanos , Imunidade Inata , Inflamação , Mediadores da Inflamação , Macrófagos/imunologia , Masculino , Camundongos , Miocárdio/imunologia , Ratos , Trombina/metabolismo , Replicação ViralRESUMO
OBJECTIVES: This study sought to evaluate the role of protease-activated receptor-2 (PAR2) in coxsackievirus B3 (CVB3)-induced myocarditis. BACKGROUND: An infection with CVB3 leads to myocarditis. PAR2 modulates the innate immune response. Toll-like receptor-3 (TLR3) is crucial for the innate immune response by inducing the expression of the antiviral cytokine interferon-beta (IFNß). METHODS: To induce myocarditis, wild-type (wt) and PAR2 knockout (ko) mice were infected with 10(5) plaque-forming units CVB3. Mice underwent hemodynamic measurements with a 1.2-F microconductance catheter. Wt and PAR2ko hearts and cardiac cells were analyzed for viral replication and immune response with plaque assay, quantitative polymerase chain reaction, Western blot, and immunohistochemistry. RESULTS: Compared with wt mice, PAR2ko mice and cardiomyocytes exhibited a reduced viral load and developed no myocarditis after infection with CVB3. Hearts and cardiac fibroblasts from PAR2ko mice expressed higher basal levels of IFNß than wt mice did. Treatment with CVB3 and polyinosinic:polycytidylic acid led to higher IFNß expression in PAR2ko than in wt fibroblasts and reduced virus replication in PAR2ko fibroblasts was abrogated by neutralizing IFNß antibody. Overexpression of PAR2 reduced the basal IFNß expression. Moreover, a direct interaction between PAR2 and Toll-like receptor 3 was observed. PAR2 expression in endomyocardial biopsies of patients with nonischemic cardiomyopathy was positively correlated with myocardial inflammation and negatively with IFNß expression and left ventricular ejection fraction. CONCLUSIONS: PAR2 negatively regulates the innate immune response to CVB3 infection and contributes to myocardial dysfunction. The antagonism of PAR2 is of therapeutic interest to strengthen the antiviral response after an infection with a cardiotropic virus.
Assuntos
Enterovirus Humano B/imunologia , Infecções por Enterovirus/imunologia , Imunidade Inata , Miocardite/imunologia , Miocárdio/enzimologia , Receptor PAR-2/biossíntese , Animais , Anticorpos Antivirais/análise , Biópsia , Western Blotting , Modelos Animais de Doenças , Infecções por Enterovirus/virologia , Regulação da Expressão Gênica , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocardite/metabolismo , Miocardite/virologia , Miocárdio/patologia , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor PAR-2/genética , Receptor 3 Toll-Like/biossíntese , Receptor 3 Toll-Like/genéticaRESUMO
Systemic application of mesenchymal stromal cells (MSCs) in inflammatory cardiomyopathy exerts cardiobeneficial effects. The mode of action is unclear since a sufficient and long-acting cardiac homing of MSCs is unlikely. We therefore investigated the regulation of the immune response in coxsackievirus B3 (CVB3)-induced acute myocarditis after intravenous application of MSCs. Wildtype mice were infected with CVB3 and treated with either PBS, human MSCs or human cardiac fibroblasts intravenously 1 day after infection. Seven days after infection, MSCs could be detected in the spleen, heart, pancreas, liver, lung and kidney, whereby the highest presence was observed in the lung. MSCs increased significantly the myocardial expression of HGF and decreased the expression of the proinflammatory cytokines TNFα, IL1ß and IL6 as well as the severity of myocarditis and ameliorated the left ventricular dysfunction measured by conductance catheter. MSCs upregulated the production of IFNγ in CD4+ and CD8+ cells, the number of IL10-producing regulatory T cells and the apoptosis rate of T cells in the spleen. An increased number of CD4+CD25+FoxP3 could be found in the spleen as well as in the circulation. In contrast, application of human cardiac fibroblasts had no effect on the severity of myocarditis and the systemic immune response observed after MSCs-administration. In conclusion, modulation of the immune response in extracardiac organs is associated with cardiobeneficial effects in experimental inflammatory cardiomyopathy after systemic application of MSCs.
Assuntos
Fibroblastos/citologia , Fatores de Transcrição Forkhead/metabolismo , Células-Tronco Mesenquimais/citologia , Miocardite/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Cardiomiopatias/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Citometria de Fluxo/métodos , Hemodinâmica , Humanos , Sistema Imunitário , Hibridização In Situ , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocardite/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Patients with diabetes mellitus (DM) suffer from an increased risk of cardiovascular events caused by thrombotic conditions. Adipose tissue might play a crucial role in this pathogenesis by synthesis of procoagulant mediators. This study was performed to elucidate the role of the adipocytokines leptin and resistin in the development of hypercoagulability and hypofibrinolysis under diabetic conditions. METHODS: Sixty two patients with or without DM were included in our study to measure leptin, resistin and tissue factor (TF) plasma concentrations. Moreover, flow chamber experiments were performed to assess factor Xa and plasmin activity on the surface of HUVECs. Western blot and real-time PCR were performed to determine mRNA and protein expression of main factors of the coagulation and fibrinolytic system. RESULTS: Patients with diabetes showed increased levels of leptin and resistin (leptin: 25.69±13.9 vs. 15.98±17.5 ng/mL, p<0.05; resistin: 2.61±0.6 vs. 1.19±0.7 ng/mL, p<0.05), which were positively correlated with TF. In vitro, leptin and resistin induced increased factor Xa activity (leptin: 4.29±0.57-fold, p<0.05; resistin 4.19±0.7-fold, p<0.05 vs. control) on HUVECs as also reflected by elevated TF mRNA and protein expression. Moreover, stimulatory (plasminogen activator inhibitor 1) and inhibitory (tissue plasminogen activator) mediators of the fibrinolytic cascade were induced by leptin and resistin, leading to a balanced plasmin activity regulation. CONCLUSIONS: Leptin and resistin lead to a procoagulant state in HUVECs by inducing TF expression. This mechanism might be one explanation for the prothrombotic state observed under diabetic conditions.