CX3CR1 knockout aggravates Coxsackievirus B3-induced myocarditis

RESEARCH ARTICLE CX3CR1 knockout aggravates Coxsackievirus B3-induced myocarditis Irene Müller1,2,3☯, Kathleen Pappritz1,2,3☯, Konstantinos Savvatis4,5, Kerstin Puhl1,3, Fengquan Dong1,3, Muhammad El-Shafeey1,3, Nazha Hamdani6, Isabell Hamann7, Michel Noutsias8, Carmen Infante-Duarte7, Wolfgang A. Linke6, Sophie Van Linthout1,2,3☯, Carsten Tschöpe1,2,3☯* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Charité –Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Internal Medicine and Cardiology, Campus Virchow Klinikum, Berlin, Germany, 2 DZHK (German Center for Cardiovascular Research), partner site Berlin, Germany, 3 Charité –Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, HumboldtUniversität zu Berlin, and Berlin Institute of Health, Berlin-Brandenburg Center for Regenerative Therapies, Campus Virchow Klinikum, Berlin, Germany, 4 Inherited Cardiovascular Diseases Unit, Barts Health NHS Trust, Barts Heart Centre, London, United Kingdom, 5 William Harvey Research Institute, Queen Mary University London, London, United Kingdom, 6 Department of Cardiovascular Physiology, Ruhr University Bochum, Bochum, Germany, 7 Charité –Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute for Medical Immunology, Campus Virchow Klinikum, Berlin, Germany, 8 Department of Internal Medicine III, Division of Cardiology, Angiology and Intensive Medical Care, University Hospital Halle, Halle (Saale), Germany ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Müller I, Pappritz K, Savvatis K, Puhl K, Dong F, El-Shafeey M, et al. (2017) CX3CR1 knockout aggravates Coxsackievirus B3-induced myocarditis. PLoS ONE 12(8): e0182643. https:// doi.org/10.1371/journal.pone.0182643 Editor: Nikolaos Frangogiannis, Albert Einstein College of Medicine, UNITED STATES Received: February 16, 2017 Accepted: July 22, 2017 Published: August 11, 2017 Copyright: © 2017 Müller et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Studies on inflammatory disorders elucidated the pivotal role of the CX3CL1/CX3CR1 axis with respect to the pathophysiology and diseases progression. Coxsackievirus B3 (CVB3)-induced myocarditis is associated with severe cardiac inflammation, which may progress to heart failure. We therefore investigated the influence of CX3CR1 ablation in the model of acute myocarditis, which was induced by inoculation with 5x105 plaque forming units of CVB3 (Nancy strain) in either CX3CR1-/- or C57BL6/j (WT) mice. Seven days after infection, myocardial inflammation, remodeling, and titin expression and phosphorylation were examined by immunohistochemistry, real-time PCR and Pro-Q diamond stain. Cardiac function was assessed by tip catheter. Compared to WT CVB3 mice, CX3CR1-/- CVB3 mice exhibited enhanced left ventricular expression of inflammatory cytokines and chemokines, which was associated with an increase of immune cell infiltration/presence. This shift towards a pro-inflammatory immune response further resulted in increased cardiac fibrosis and cardiomyocyte apoptosis, which was reflected by an impaired cardiac function in CX3CR1-/- CVB3 compared to WT CVB3 mice. These findings demonstrate a cardioprotective role of CX3CR1 in CVB3-infected mice and indicate the relevance of the CX3CL1/CX3CR1 system in CVB3-induced myocarditis. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the DZHK German Center for Cardiovascular Research. Introduction Competing interests: The authors have declared that no competing interests exist. Viral myocarditis is a cardiac disorder characterized by cardiac inflammation, which is often caused by cardiotropic viruses like Coxsackievirus group B type 3 (CVB3) and can progress to PLOS ONE | https://doi.org/10.1371/journal.pone.0182643 August 11, 2017 1 / 17 CX3CR1 CVB3-induced myocarditis dilated cardiomyopathy (DCM) and congestive heart failure [1]. With respect to CVB3, a direct cardiomyocyte injury and subsequent long-term inflammatory reaction belong to the discussed mechanisms involved in the CVB3-induced pathogenesis [2,3]. However, the exact pathogenesis of CVB3-induced myocarditis needs still a better understanding [4] to find efficient therapeutic options counteracting the virus-induced inflammatory response [5]. Chemokine-induced migration of inflammatory cells plays a crucial role during cardiac inflammation [6–8]. Among the chemokine super-family, fractalkine (CX3CL1) exists in two distinct forms. The membrane-bound form serves as an adhesion protein. The soluble molecule has chemoattractant properties and is proteolytically cleaved from the cell membraneanchored form of fractalkine [9,10]. Cellular sources of CX3CL1 include endothelial cells, epithelial cells, dendritic cells, macrophages and cardiomyocytes [9,11]. Both chemotaxis and adhesion are mediated by the G-protein-coupled receptor CX3CR1 [12], which is mainly expressed on natural killer cells, some T cell populations, dendritic cells and monocytes [10]. Interestingly, soluble CX3CL1 attracts natural killer cells, T cells, and dendritic cells and inhibits the function of the monocyte chemoattractant protein-1 (MCP-1) [10,13]. In previous studies, the CX3CL1/CX3CR1 system has been shown to be involved in the pathophysiology of cardiovascular disorders including heart failure [14,15] and inflammatory cardiomyopathy [9]. CX3CL1/CX3CR1 is also of relevance in the pathogenesis of other inflammatory disorders such as glomerulonephritis [16], rheumatoid arthritis [17] and cardiac allograft rejection [18]. Fractalkine and its receptor CX3CR1 have been shown to exert detrimental effects, since neutralization of the chemokine improved cardiac function after myocardial infarction [19] and inhibition of the respective receptor reduced atherosclerosis in mice [20]. Besides these findings, there are also data indicating a protective role of CX3CR1 since the loss of this receptor results in higher liver fibrosis in a model of hepatic fibrosis [21] and increased accumulation of inflammatory monocytes in gliomagenesis [22]. Abovementioned findings indicate the complexity of the CX3CL1/CX3CR1 system. Since CX3CR1 is involved in inflammatory disorders, cardiovascular diseases and viral infection, and due to the lack of data regarding the role of CX3CR1 in viral experimental myocarditis, we aimed to investigate the pathophysiological role of CX3CR1 in experimental CVB3-induced acute myocarditis. Materials and methods Induction of myocarditis and hemodynamic measurements Six week-old male C57 (...truncated)