Single-Cell RNA Sequencing to Dissect the Immunological Network of Autoimmune Myocarditis.

Abstract

Myocarditis can develop into dilated cardiomyopathy, which may require heart transplantation. The immunological network of myocarditis phases remains unknown. This study aimed to investigate the immunological network during the transition from myocarditis to cardiomyopathy and to identify the genes contributing to the inflammatory response to myocarditis. Mice were treated with myosin heavy chain-α peptides to generate an experimental autoimmune myocarditis (EAM) model. We performed single-cell RNA sequencing analysis of <i>Cd45</i>⁺ cells extracted from mouse hearts during different EAM phases, including normal control, acute inflammatory, subacute inflammatory, and myopathy phases. Human heart tissues were collected from the surgically removed hearts of patients who had undergone heart transplantation. We identified 26 cell subtypes among 34 665 cells. Macrophages constituted the main immune cell population at all disease phases (>60%), and an inflammation-associated macrophage cluster was identified in which the expression of <i>Hif1a</i>-regulated genes was upregulated. The neutrophil population was increased after the induction of EAM, and neutrophils then released <i>Il-1</i> to participate in the EAM process. T cells were observed at the highest percentage at the subacute inflammatory phase. T-helper 17 cells, in which the expression of <i>Hif1a</i>-regulated genes was upregulated, constituted the main T-cell population detected at the acute inflammatory phase, whereas regulatory T cells were the main T-cell population detected at the subacute inflammatory phase, and γδ T cells releasing <i>Il-17</i> were the main T-cell population observed at the myopathy phase. Moreover, the <i>Hif1a</i> expression level correlated with the extent of inflammation. In addition, PX-478 could alleviate the inflammatory responses of the different EAM phases. Last, <i>HIF1A</i> was expressed at higher levels in patients with acute autoimmune myocarditis than in patients with dilated cardiomyopathy and healthy control subjects. We present here a comprehensive single-cell landscape of the cardiac immune cells in different EAM phases. In addition, we elucidate the contribution of <i>Hif1a</i> to the inflammatory response through the regulation of immune cell activity, particularly of macrophage cluster 2 and T-helper 17 cells. Moreover, an <i>Hif1a</i> inhibitor alleviated inflammatory cell infiltration of the EAM model and may serve as a potential therapeutic target in the clinic.