Inhibition of NETosis via PAD4 could alleviate the inflammation of giant cell myocarditis

Giant cell myocarditis (GCM) is a rare, often rapidly progressive and potentially fatal, disease caused by T-cell-mediated cardiac inflammation that typically affects young and middle-aged adults. The detailed inflammatory response remained unknown, especially the formation of multinucleate giant cells.Methods and Results: Rats were treated with myosin heavy-chain-α peptides to generate a GCM model. We performed single-cell RNA sequencing analysis of 15,714 Cd45 + cells extracted from hearts from GCM rat model and normal rats. We identified 24 subclusters including macrophages, neutrophils, T cells and so on. The GCM was characteristic with macrophage cluster 1, neutrophil cluster 3, macrophage cluster 2, neutrophil cluster 1 and Th17 cells. Two specific macrophage clusters involved in the GCM have roles in phagocytosis, and NETosis was found to contribute into the process of GCM. Its receptor, Ccdc25 , expressed in the macrophage cluster 1 and Th17, suggesting that neutrophils could regulate other immune cells in GCM. Importantly, inhibitor of NETosis, GSK484, could alleviate the inflammation of GCM in vivo. Finally, MPO (a marker of neutrophils) and H3cit (a marker of NETosis) were expressed at higher levels in GCM patients than in DCM patients and healthy controls and neutrophils were part of multinucleated giant cells. IMC analysis revealed that the formation of multinucleate giant cells of GCM.Conclusions: We herein present a comprehensive single-cell landscape of the cardiac immune cells in GCM. In addition, we elucidated the contribution of NETosis to the pathogenesis of GCM, which may serve as a potential therapeutic target in the clinic.巨细胞性心肌炎（Giant cell myocarditis, GCM）是一种罕见、多呈快速进展性且具有潜在致命性的疾病，由T细胞介导的心脏炎症引发，通常累及青壮年人群。其具体的炎症反应机制尚未明确，尤其是多核巨细胞的形成过程。方法与结果：本研究通过给大鼠注射肌球蛋白重链-α肽段构建GCM大鼠模型，对从GCM模型大鼠及正常大鼠心脏中提取的15714个Cd45+细胞开展单细胞RNA测序分析。共鉴定出包括巨噬细胞、中性粒细胞、T细胞在内的24个细胞亚群。GCM的特征性免疫亚群包括巨噬细胞簇1、中性粒细胞簇3、巨噬细胞簇2、中性粒细胞簇1以及Th17细胞。两个参与GCM进程的特异性巨噬细胞亚群具备吞噬功能，且研究发现中性粒细胞胞外陷阱形成（NETosis）参与了GCM的发病过程。其受体CCDC25在巨噬细胞簇1和Th17细胞中表达，提示中性粒细胞可在GCM中调控其他免疫细胞。值得注意的是，中性粒细胞胞外陷阱形成抑制剂GSK484可在体内减轻GCM的炎症反应。最后，相较于扩张型心肌病（DCM）患者及健康对照者，GCM患者的中性粒细胞标志物髓过氧化物酶（MPO）和中性粒细胞胞外陷阱形成标志物瓜氨酸化组蛋白H3（H3cit）表达水平显著升高，且中性粒细胞可参与多核巨细胞的形成。免疫细胞互作（IMC）分析揭示了GCM中多核巨细胞的形成机制。结论：本研究首次绘制了GCM心脏免疫细胞的全面单细胞图谱。此外，本研究阐明了中性粒细胞胞外陷阱形成在GCM发病机制中的作用，该靶点有望成为临床潜在的治疗靶点。