Peripheral blood immune cells distribution in biopsy-proven myocarditis: etiology and prognostic correlates

Abstract Background Myocarditis is an inflammatory disease of the myocardium with viral or immune-mediated/autoimmune etiology. Definite etiological diagnosis relays on endomyocardial biopsy (EMB). High titre anti-heart autoantibodies (AHA) define severe autoimmune forms. Virus-negative myocarditis may require immunosuppression (IS) to reduce progression to dilated cardiomyopathy, heart transplant or death. Prognostic stratification is incomplete and IS is not always efficacious. Animal and human studies suggest a pathogenetic role of immune cells, but little is known on their peripheral distribution or prognostic role. Purpose Characterize EMB-proven myocarditis patients’ peripheral blood to identify new non-invasive etiological and prognostic biomarkers. Methods Seventy-eight EMB-proven myocarditis patients and 9 healthy controls (HC) were enrolled according to the Declaration of Helsinki and written informed consent was collected for each participant. Peripheral blood mononuclear cells were purified by Ficoll stratification and immune cells distribution was evaluated by flow cytometry. Variables were analysed by Mann-Whitney or Kruskall-Wallis and Dunn post-hoc tests clustering patients according to clinical features, EMB and AHA results. Results Compared to HC (figure 1), plasmacytoid dendritic cells were reduced in myocarditis patients: with autoimmune/lymphocytic forms (p=0.0091); without extra cardiac autoimmune diseases (AD, p=0.0075); in those unresponsive to IS (p=0.0379) or never treated because of spontaneous healing (p=0.0015). Moreover, several immunophenotypical features discriminated myocarditis type/IS-response from HC. Viral forms were characterized by reduced CD94+ NK cells and CCR2 over-expression in intermediate monocytes (p=0.029, p=0.013). Autoimmune cases were defined by: higher CD62L+ NK cells when AHA negative (p=0.019); increased Th1 if not requiring IS (p=0.03); higher γδ-TCR+ Th17 and reduced Treg cells if without other AD (both p=0.04), or higher CD4+/IL17+ cells with concurrent AD (p=0.02) and AHA positivity (p=0.036). Moreover, ongoing IS treatment influenced peripheral cells distribution: whole blood cell counts were more frequently altered and total NK cells were reduced (p=0.007); treg cells were reduced in patients actively treated but unresponsive to IS (p=0.036). Conclusion Biopsy-proven myocarditis patients showed a skewed peripheral blood distribution of either innate or adaptive immune cells according to different histology, etiology (viral vs autoimmune) and response to IS in autoimmune forms, unveiling potential new non-invasive immunological biomarkers for myocarditis. Figure: Plasmacytoid dendritic cells (pDCs) percentage was assessed by flow-cytometry and data were showed by boxplots clustering myocarditis patients for etiology (A), EMB histological result (B), presence of extracardiac AD (C) or IS response (D). Data were analysed by Kruskall-Wallis (graph bottom) and Dunn post-hoc tests.