Background:

Myocarditis is an inflammatory disease of the myocardium indicated by mononuclear cell infiltration. It is predominantly caused by infectious agents such as coxsackievirus B3 (CVB3). The clinical course of myocarditis has a broad spectrum of outcomes, ranging from complete recovery to cardiac dysfunction and dilated cardiomyopathy. G protein-coupled receptor 15 (GPR15) was identified as a T cell homing receptor in the context of inflammatory intestine and skin diseases.

Purpose:

We focused on the following questions: Does GPR15 deficiency (i) leads to differences in immune cell infiltrates in the acute phase of myocarditis; (ii) affects the efficient elimination of CVB3; or (iii) has an impact on tissue remodelling and thus on cardiac function and outcome after acute myocarditis? To elucidate the function of GPR15 in the molecular pathomechanism caused by cardiac infection, we further utilized RNA sequencing (RNA seq) and in vitro assays.

Methods:

Gpr15 deficient (Gpr15^gfp/gfp) and WT mice were infected with CVB3 to investigate the acute (5, 6, 7 days post infection (p.i.)) and the subacute phase (16 days p.i.) of myocarditis. To study differentially expressed genes, we used TaqMan analysis and RNA-sequencing. Inflammation and fibrosis were evaluated on histological level. For functional characterization, mice were hemodynamically characterized. Furthermore, we investigated the interactions of GPR15 and its two known ligands GPR15L and the EGF-like domain 5 of Thrombomodulin (TME5) in vitro.  Therefore, chemotaxis assays and adhesion assays were used.

Results:

In the subacute phase of myocarditis 16 days p.i., viral persistence was observed in more than 85 % of Gpr15^gfp/gfp mice, while more than 70 % of WT mice cleared the virus successfully. Furthermore, Gpr15^gfp/gfp mice demonstrated a decreased cardiac function accompanied by increased fibrosis in comparison to WT mice. Based on these findings, we investigated the acute phase of myocarditis in more detail.

While similar on day 6, infected Gpr15^gfp/gfp mice exhibited higher upregulation of immune response related genes 7 days p.i.. Especially T cell marker were significantly higher in infected Gpr15^gfp/gfp compared to infected WT mice. Bulk RNA-sequencing confirmed that the response to virus did not decline from day 6 to 7 in infected GPR15-deficient mice as observed in infected WT mice. Gene ontology (GO) term analyses reveled enhanced chemotaxis and cytotoxic T cell-related GO terms in GPR15-deficient mice on day 7. Regarding the insufficient virus clearance and further increased immune response 7 days p.i., we assumed later immune cell infiltration and therefore investigated the early acute phase of myocarditis 5 days p.i. Especially various T-cell subsets were reduced in GPR15-deficient mice. GPR15 might influence the chemotaxis or invasion of T cells. Its deficiency abolished chemotaxis of T cells towards GPR15 ligand in vitro, but the adhesion to its ligand TME5 expressed on endothelial cells was not altered.

Conclusion:

Our findings indicate that the scant virus elimination was caused by decelerated recruitment of T cells leading to impaired outcome in the GPR15-deficient mice. Regarding our in vitro assays, GPR15 recruits T-cells towards inflammation rather than influencing the adhesion of T cells to endothelial cells.