Single-cell RNA-sequencing identifies a novel subset of CD9+ reparative macrophages modulated by CCN2 derived from Nlrx1 deficient cardiomyocytes in the infarcted heart

RationaleNlrx1, a pattern recognition receptor belonging to the NLR family, is highly expressed in the heart and is significantly increased after myocardial infarction in vivo, however, the role of Nlrx1 in post-infarction inflammation and healing is poorly understood. Although the effects of infiltrating macrophages on survival of cardiomyocytes and healing of injured hearts have been intensely studied, whether or not and how surviving cardiomyocytes might influence the phenotype and function of macrophages in the infarcted hearts was largely unexplored. ObjectiveTo investigate the role of Nlrx1 in post-infarction inflammation and healing and to dissect the underlying mechanism. Methods and resultsSingle cell RNA-sequencing of 23,805 cells isolated from infarcted hearts revealed 7 clusters of infarct macrophages including CCR2+, Isg+, Mki67+ proliferative, and LYVE1+ cardiac resident macrophages, and identified a novel subset of CD9+Il1rn+ macrophages, which were characterized by high expression of Cd9, Il1rn, Gpnmb and Spp1, and downregulation of proinflammatory cytokines and chemokines. CD9+Il1rn+ macrophages were largely found in infarcted hearts of Nlrx1KO mice, and were transcriptionally unique with reparative phenotype. Loss of Nlrx1 attenuated ventricular dysfunction and adverse remodeling, and decreased post-infarction mortality. Flow cytometry analysis showed markedly increased number of CD9+ macrophages and higher mean fluorescence intensity of CD9 in Nlrx1KO mice. RNA-seq analysis revealed that CD9+ macrophages of Nlrx1KO infarcted hearts exhibited a reparative and anti-inflammatory phenotype as featured by upregulation of wound healing-associated genes including CD9, Spp1 and Fn1, and downregulation of proinflammatory genes. Conditioned medium from Nlrx1 deficient cardiomyocytes upon hypoxia stimulation skewed macrophage polarization towards a reparative and inflammation-suppressed phenotype. Gain-and loss-of function of Nlrx1 combined with RNA-seq analysis identified CCN2, whose expression and secretion were significantly increased in Nlrx1 deficient cardiomyocytes upon hypoxia, as a cardiomyocytes-derived mediator responsible, at least partly, for reprograming macrophage towards a reparative phenotype. In vivo, CCN2 expression was markedly elevated in Nlrx1KO infarcted hearts. Moreover, exogenous CCN2 partially mediated macrophage transition towards a reparative phenotype. ConclusionsCD9+ IL1rn+ macrophages modulated by Nlrx1 deficient cardiomyocytes-derived CCN2, is a novel subset of reparative macrophages contributing to infarct healing. Inhibition of myocardial Nlrx1 and/or activation of CCN2 to boost CD9+ IL1rn+ reparative macrophages may represent new therapeutics for infarct healing following ischemic injury.