Implications for the role of macrophages in a model of myocardial fibrosis: CCR2^-/- mice exhibit an M2 phenotypic shift in resident cardiac macrophages

Background Macrophages (MΦ) are functionally diverse and dynamic. Until recently, cardiac MΦ were assumed to be monocyte derived; however, resident cardiac MΦ (rCMΦ), present at baseline, were identified in myocardia and have been implicated in cardiac healing. Previously, we demonstrated that CCR2^-/- mice are protected from myocardial fibrosis - an observation initially attributed to changes in infiltrating monocytes. Here, we reexplored this observation in the context of our new understanding of rCMΦ. Methods Male CCR2^-/- and C57BL/6 hearts were digested and purified to a single cell suspension, incubated with fluorophore-linked antibodies (CCR2, CX3CR1, CD11b, Ly6C, TNF-α, and IL-10), and assessed by flow cytometry. Differentiated MΦ were cocultured with fibroblasts in order to characterize how MΦ phenotype influences fibroblast activation. Fibroblasts were characterized for their expression of smooth muscle cell actin (SMA). Results A significant decrease in Ly6C expression was observed in the CCR2^-/- cardiac MΦ population relative to WT, which corresponded with significantly lower TNF-α expression and significantly higher IL-10 expression. Using in vitro coculture system, classical MΦ promoted fibroblast activation relative to nonclassical MΦ. Conclusion CCR2^-/- rCMΦ favor a more antiinflammatory phenotype relative to WT controls. Moreover, a shift toward the antiinflammatory promotes proliferation, but not activation in vitro. Together, these observations suggest that antiinflammatory cardiac MΦ populations may inhibit myocardial fibrosis in a pathological setting by preventing the activation of fibroblasts. News and noteworthy Here, we provide novel evidence for baseline differences in rCMΦ phenotypes (i.e. classical vs. nonclassical) and how these differences could modulate cardiac healing. Importantly, we observed differences in how classical vs. nonclassical MΦ influenced fibroblast activation, which could, in turn, affect fibrosis.