CD40-TRAF2/3/5 Signaling Promotes Cardiac Repair by Mediating Macrophage Efferocytosis After Myocardial Infarction

Abstract

BACKGROUND: After myocardial infarction (MI), macrophage-mediated clearance of dead cells, a process known as efferocytosis, represents a pivotal role in tissue remodeling. Efficient efferocytosis contributes to rescuing neighboring viable cardiomyocytes, drives the phenotypic transition of reparative macrophages, and facilitates the resolution of inflammation. In this study, we explored the roles of CD40 and the signals transduced by its 2 downstream adaptor-protein binding sites (TRAF2/3/5 and TRAF6) in the cardiac macrophage efferocytosis after MI. METHODS: Systemic, myeloid- and macrophage-specific CD40-deficient mice were used to determine the functional significance of CD40 during post-MI repair. The effects of CD40 on macrophages functional states were evaluated with single-cell RNA sequencing (scRNA-seq). Flow cytometry, immunofluorescence staining, Western blot, and ELISA were used to assess the efferocytosis and inflammatory status of macrophages after MI. CD40-TRAF2/3/5 −/− and CD40-TRAF6 −/− mice were used to explore the roles of CD40 downstream signaling intermediates in MI and macrophage efferocytosis. RESULTS: The expression level of CD40 was increased remarkably from 3 to 7 days after MI. Myeloid-derived macrophages emerged as the dominant population expressing CD40. CD40 deficiency resulted in an augmented infarct size and compromised cardiac function after MI. Further investigations demonstrated that CD40 deficiency led to a notable decline in macrophage efferocytosis, which is associated with a reduced abundance of cluster 0 cells, identified by scRNA-seq, representing the precursor of reparative macrophages. Moreover, scRNA-seq indicated that CD40 + macrophages could be classified primarily into 2 distinct cell subsets: 1 subset was associated mainly with efferocytosis functions, and the other was involved predominantly in immune-inflammatory responses. Direct activation of CD40 failed to upregulate macrophage efferocytosis but instead induced a proinflammatory state. These implied differential effects of the signals transduced by the 2 TRAF binding sites (TRAF2/3/5 and TRAF6) downstream of CD40 on efferocytosis. Findings from CD40-TRAF2/3/5 −/− and CD40-TRAF6 −/− mice confirmed that the CD40-TRAF2/3/5 signaling served as a crucial determinant in mediating CD40-related efferocytosis. STAT6 was identified as a key downstream factor in this process. Adenovirus-mediated gene transfer to overexpress a CD40 variant retaining TRAF2/3/5 binding site but lacking the TRAF6 in cardiac macrophages led to improvements in cardiac function and macrophage efferocytosis after MI. CONCLUSIONS: Our study established a pivotal positive role of macrophage CD40 in post-MI repair by facilitating macrophage efferocytosis. Specifically, TRAF2/3/5 rather than TRAF6 serves as the crucial signaling pathway that mediates CD40-associated efferocytosis.