GPR15-dependent T cell recruitment in the acute phase of viral myocarditis is associated with improved virus elimination and outcome

Hanna Wolf (Freiburg im Breisgau)1, B. Stoffers (Freiburg im Breisgau)1, L. Bacmeister (Freiburg im Breisgau)2, S. Kupsch (Freiburg im Breisgau)1, T. A. Vico (Freiburg)3, T. Marchini (Freiburg im Breisgau)4, M. A. Brehm (Siegen)5, I. Yan (Hamburg)6, P. M. Becher (Hamburg)6, A. Ardeshirdavani (Freiburg im Breisgau)1, F. Escher (Berlin)7, S. V. Kim (Philadelphia)8, K. Klingel (Tübingen)9, P. Kirchhof (Hamburg)6, S. Blankenberg (Hamburg)6, T. Zeller (Hamburg)6, D. Wolf (Freiburg im Breisgau)2, I. Hilgendorf (Freiburg im Breisgau)2, D. Westermann (Freiburg im Breisgau)10, D. Lindner (Freiburg im Breisgau)1

1Universitäts-Herzzentrum Freiburg - Bad Krozingen Klinik für Kardiologie und Angiologie Freiburg im Breisgau, Deutschland; 2Universitäts-Herzzentrum Freiburg - Bad Krozingen Klinik für Kardiologie und Angiologie I Freiburg im Breisgau, Deutschland; 3University Heart Center Freiburg-Bad Krozingen Cardiology and Angiology Freiburg, Deutschland; 4Universitäts-Herzzentrum Freiburg - Bad Krozingen GmbH Klinik für Kardiologie und Angiologie I Freiburg im Breisgau, Deutschland; 5School of Life Sciences University of Siegen Department Digital Health Sciences and Biomedicine Siegen, Deutschland; 6Universitäres Herz- und Gefäßzentrum Hamburg Klinik für Kardiologie Hamburg, Deutschland; 7Deutsches Herzzentrum der Charite (DHZC) Klinik für Kardiologie, Angiologie und Intensivmedizin | CBF Berlin, Deutschland; 8Sidney Kimmel Medical College of Thomas Jefferson University Department of Microbiology and Immunology Philadelphia, USA; 9Universitätsklinikum Tübingen Kardiopathologie Tübingen, Deutschland; 10Universitäts-Herzzentrum Freiburg - Bad Krozingen Innere Medizin III, Kardiologie und Angiologie Freiburg im Breisgau, Deutschland



Myocarditis is an inflammatory heart disease that is primarily caused by viral pathogens such as Coxsackievirus B3 (CVB3). The clinical course of myocarditis has a wide spectrum of outcomes, ranging from complete recovery to cardiac dysfunction and dilated cardiomyopathy. The G protein-coupled receptor 15 (GPR15) has been identified as a T cell homing receptor associated with inflammatory bowel and skin diseases.



The aim of this study was to address the following questions: Does GPR15 deficiency (i) lead to differences in immune cell infiltration or inflammation in the acute phase of myocarditis; (ii) does it affect elimination of CVB3 and thus outcome; (iii) does it affect immune cell chemotaxis or functionality? Therefore, GPR15-deficient (Gpr15gfp/gfp) and WT mice were infected with CVB3 to study the acute (5, 6, 7 days post-infection (p.i.)) and subacute phase (16 days p.i.) of myocarditis. Genes differentially expressed between GPR15-deficient and WT mice were analysed using RNASeq and TaqMan. Furthermore, immune cell infiltration and fibrosis were quantified via histology. For functional characterisation, the mice were evaluated haemodynamically. In addition, we analysed the interaction of GPR15 and its ligand GPR15L in vitro using chemotaxis assays.



In the subacute phase of myocarditis 16 days p.i., virus persistence was observed in more than 85% of Gpr15gfp/gfp mice, while more than 70% of WT mice successfully cleared the virus. In addition, Gpr15gfp/gfp mice showed reduced cardiac function accompanied by increased fibrosis compared to WT mice. Based on these findings, we analysed the acute phase of myocarditis in more detail. Comparing the infected genotypes, no significant differences were found at 6 days p.i. in contrast to 5 and 7 days p.i.. After 5 days p.i., Gpr15gfp/gfp mice showed significantly lower gene expression of inflammatory cytokines and immune cell markers, especially regulatory T cells. In contrast, after 7 days p.i., inflammatory cytokines and markers for regulatory and cytotoxic T cells were significantly increased in infected Gpr15gfp/gfp mice compared to infected WT mice. RNASeq confirmed that the response to the virus did not decrease from day 6 to 7 in infected Gpr15gfp/gfp mice, as observed in infected WT mice. Furthermore, gene ontology (GO) analyses revealed altered chemotaxis and increased cytotoxic T cell-related GO terms in Gpr15gfp/gfp mice 7 days p.i.. Consistent with these in vivo data, we demonstrated in vitro that chemotaxis of both regulatory and cytotoxic T cells depends on the interaction between GPR15 and its ligand GPR15L and that loss of GPR15 abrogates chemotaxis of T cells towards GPR15L.



Here we show that GPR15 deficiency leads to reduced virus elimination in the heart and thus to adverse remodelling and impaired cardiac function. Delayed recruitment of regulatory T cells was associated with prolonged persistence of cytotoxic and regulatory T cells in Gpr15gfp/gfp mice. In addition, RNASeq revealed a prolonged inflammatory response and altered chemotaxis in Gpr15gfp/gfp mice. Through in vitro studies, we identified GPR15 and its ligand GPR15L as an important chemokine-receptor-ligand pair for the recruitment of regulatory and cytotoxic T cells. In conclusion, insufficient virus elimination could be caused by delayed recruitment of T cells, leading to a prolonged inflammatory response and an adverse outcome in GPR15-deficient mice.

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