The origin of wound repair macrophages is incompletely defined and was examined here in sterile wounds using the subcutaneous polyvinyl alcohol sponge implantation model in mice. Phenotypic analysis identified F4/80+Ly6ChiCD64+MerTK– monocytes and F4/80+Ly6ClowCD64+MerTK+ macrophages in the wound. Circulating monocytes were the precursors of inflammatory Ly6Chi wound monocytes. Ly6ClowMerTK+ macrophages appeared later, expressed CD206, CD11c, and MHC class II, produced cytokines consistent with repair function, and lacked a gene expression profile compatible with mesenchymal transition or fibroblastic transdifferentiation. Data also demonstrated that Ly6Chi wound cells were precursors of Ly6Clow macrophages, although monocytes did not undergo rapid maturation but rather persisted in the wound as Ly6ChiMerTK– cells. MerTK-deficient mice were examined to determine whether MerTK-dependent signals from apoptotic cells regulated the maturation of wound macrophages. MerTK-deficient mice had day 14 cell compositions that resembled more immature wounds, with a smaller proportion of F4/80+ cells and higher frequencies of Ly6G+ neutrophils and Ly6Chi monocytes. The cytokine profile and number of apoptotic cells in day 14 wounds of MerTK-deficient mice was unaffected despite the alterations in cell composition. Overall, these studies identified a differentiation pathway in response to sterile inflammation in which monocytes recruited from the circulation acquire proinflammatory function, persist in the wound, and mature into repair macrophages.

Monocytes/macrophages are critical early innate immune responders during murine CMV (MCMV) infection. It has been established that inflammatory monocyte/macrophages are released from the bone marrow and into the peripheral blood before entry into infected tissue sites. We previously reported a role for IFN-α/β in promotion of CCR2-mediated recruitment of monocyte/macrophages into the liver in response to MCMV infection. However, the mechanisms that support the migration of monocyte/macrophages from the bone marrow and into the peripheral blood under conditions of MCMV infection have not been elucidated. Herein, we demonstrate an accumulation of monocyte/macrophages in the bone marrow of MCMV-infected CCR2-deficient mice, whereas circulating monocyte/macrophages are profoundly diminished. The CCR2 ligands MCP-1, MCP-3, and MCP-5 are detected in bone marrow and in serum from MCMV-infected mice. Furthermore, bone marrow leukocytes from naive mice produce high levels of MCP-1 and MCP-5, and moderate levels of MCP-3, when stimulated with recombinant IFN-α in culture. We identify bone marrow F4/80+ cells as major producers of MCP-1, MCP-3, and MCP-5. Moreover, induction of CCR2 ligands is dependent on IFN-α/β-mediated signals and MCMV infection. Taken together, the results reveal a critical role for inflammatory cytokines in stimulating production of CCR2-binding chemokines from F4/80+ cells in the bone marrow, and they suggest that local production of chemokines supports monocyte/macrophage egress from the bone marrow into the blood during a virus infection.

Chemokine responses critical for inflammation and promotion of effective innate control of murine CMV (MCMV) in liver have been shown to be dependent on immunoregulatory functions elicited by IFN-αβ. However, it remains to be determined whether upstream factors that promote viral sensing resulting in the rapid secretion of IFN-αβ in liver differ from those described in other tissues. Because plasmacytoid dendritic cells (pDCs) are known producers of high levels of systemic IFN-α in response to MCMV, this study examines the in vivo contribution of pDCs to IFN-α production in the liver, and whether production of the cytokine and ensuing inflammatory events are dependent on TLR9, MyD88, or both. We demonstrate that whereas MyD88 deficiency markedly impaired secretion of IFN-α, production of the cytokine was largely independent of TLR9 signaling, in the liver. MyD88 and TLR9 were needed for IFN-α production in the spleen. Moreover, hepatic but not splenic pDCs produced significant amounts of intracellular IFN-α in the absence of TLR9 function during infection. Furthermore, production of CCL2, CCL3, and IFN-γ, as well as the accumulation of macrophages and NK cells, was not affected in the absence of functional TLR9 in the liver. In contrast, these responses were dramatically reduced in MyD88−/− mice. Additionally, MyD88−/− but not TLR9−/− mice exhibited increased sensitivity to virus infection in liver. Collectively, our results define contrasting compartmental functions for TLR9 and MyD88, and suggest that the infected tissue site uniquely contributes to the process of virus sensing and regulation of localized antiviral responses.