MTHFD2 orchestrates monocyte-macrophage immune homeostasis by targeting Rap1 to protect against CVB3-induced viral myocarditis

Aims Pro-inflammatory macrophages are critical mediators of the viral myocarditis (VMC) pathological process. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a key enzyme involved in one-carbon metabolism, plays an essential regulatory role in macrophage function. However, the regulatory effect of MTHFD2 on macrophages in VMC remains unclear. Here, we investigated whether MTHFD2 regulates macrophage function to exert a protective effect against coxsackievirus B3 (CVB3)-induced myocarditis. Methods and results Six-week-old C57BL/6J and BALB/c mice were intraperitoneally injected with CVB3 to establish the VMC model, and blood samples from the mice were examined for targeting analysis of folate metabolism-related compounds. The myeloid cell-specific MTHFD2 knockout mice Mthfd2fl/flLyz2-Cre+ (MTHFD2-KO-Mφ) and littermate mice underwent peripheral blood proteomic analysis. We observed activation of the one-carbon metabolism folate cycle and upregulation of MTHFD2 in macrophages during myocarditis. Further, CVB3-infected MTHFD2-KO-Mφ mice exhibited higher cardiac immunocyte infiltration, especially pro-inflammatory macrophages, aggravated myocardial injury, and cardiac dysfunction. MTHFD2 knockdown also enhanced the migration of bone marrow-derived macrophages and increased their polarization toward a pro-inflammatory phenotype. Proteomic analysis identified Rap1 as a direct downstream target of MTHFD2 in VMC. Specifically, MTHFD2 modulated integrin-regulated monocyte-macrophage migration via Rap1a and reduced cellular pro-inflammatory differentiation in VMC by inhibiting Rap1/p38 MAPK signaling. Both MTHFD2 administration and high-folate diet feeding reduced cardiac inflammation and fibrosis and improved cardiac function in mice with VMC. Conclusions We identified MTHFD2 as an immune regulator of monocyte-macrophage homeostasis to protect against CVB3-induced VMC. Targeted regulation of MTHFD2 is a potential therapeutic option for VMC clinically.