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

Rationale: Nlrx1, 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. Objective: To investigate the role of Nlrx1 in post-infarction inflammation and healing and to dissect the underlying mechanism. Methods and results: Single 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. Conclusions: CD9+ IL1rn+ macrophages modulated by Nlrx1 deficient cardiomyocytes-derived CCN2, is a novel subset of reparative macrophages contributing to infarct healing. ### Competing Interest Statement The authors have declared no competing interest.