Mesenchymal stromal cell (MSC) application in Coxsackievirus B3 (CVB3)‐induced myocarditis reduces myocardial inflammation and fibrosis, exerts prominent extra‐cardiac immunomodulation, and improves heart function. Although the abovementioned findings demonstrate the benefit of MSC application, the mechanism of the MSC immunomodulatory effects leading to a final cardioprotective outcome in viral myocarditis remains poorly understood. Monocytes are known to be a trigger of myocardial tissue inflammation. The present study aims at investigating the direct effect of MSC on the mobilization and trafficking of monocytes to the heart in CVB3‐induced myocarditis. One day post CVB3 infection, C57BL/6 mice were intravenously injected with 1 x 106 MSC and sacrificed 6 days later for molecular biology and flow cytometry analysis. MSC application reduced the severity of myocarditis, and heart and blood pro‐inflammatory Ly6Chigh and Ly6Cmiddle monocytes, while those were retained in the spleen. Anti‐inflammatory Ly6Clow monocytes increased in the blood, heart, and spleen of MSC‐treated CVB3 mice. CVB3 infection induced splenic myelopoiesis, while MSC application slightly diminished the spleen myelopoietic activity in CVB3 mice. Left ventricular (LV) mRNA expression of the chemokines monocyte chemotactic protein‐1 (MCP)−1, MCP‐3, CCL5, the adhesion molecules intercellular adhesion molecule‐1, vascular cell adhesion molecule‐1, the pro‐inflammatory cytokines interleukin‐6, interleukin‐12, tumor necrosis factor‐α, the pro‐fibrotic transforming growth factorβ1, and circulating MCP‐1 and MCP‐3 levels decreased in CVB3 MSC mice, while LV stromal cell‐derived factor‐1α RNA expression and systemic levels of fractalkine were increased in CVB3 MSC mice. MSC application in CVB3‐induced myocarditis modulates monocytes trafficking to the heart and could be a promising strategy for the resolution of cardiac inflammation and prevention of the disease progression. Stem Cells Translational Medicine 2017;6:1249–1261

Inflammation in myocarditis induces cardiac injury and triggers disease progression to heart failure. NLRP3 inflammasome activation is a newly identified amplifying step in the pathogenesis of myocarditis. We previously have demonstrated that mesenchymal stromal cells (MSC) are cardioprotective in Coxsackievirus B3 (CVB3)-induced myocarditis. In this study, MSC markedly inhibited left ventricular (LV) NOD2, NLRP3, ASC, caspase-1, IL-1β, and IL-18 mRNA expression in CVB3-infected mice. ASC protein expression, essential for NLRP3 inflammasome assembly, increased upon CVB3 infection and was abrogated in MSC-treated mice. Concomitantly, CVB3 infection in vitro induced NOD2 expression, NLRP3 inflammasome activation and IL-1β secretion in HL-1 cells, which was abolished after MSC supplementation. The inhibitory effect of MSC on NLRP3 inflammasome activity in HL-1 cells was partly mediated via secretion of the anti-oxidative protein stanniocalcin-1. Furthermore, MSC application in CVB3-infected mice reduced the percentage of NOD2-, ASC-, p10- and/or IL-1β-positive splenic macrophages, natural killer cells, and dendritic cells. The suppressive effect of MSC on inflammasome activation was associated with normalized expression of prominent regulators of myocardial contractility and fibrosis to levels comparable to control mice. In conclusion, MSC treatment in myocarditis could be a promising strategy limiting the adverse consequences of cardiac and systemic NLRP3 inflammasome activation.