Phagocytosis converts infiltrated monocytes to microglia-like phenotype in experimental brain ischemia

초록

Background Monocyte-derived macrophages (MDMs) and microglia elicit neural inflammation and clear debris for subsequent tissue repair and remodeling. The role of infiltrating MDMs in the injured brain, however, has been controversial due to overlapping antigen expression with microglia. In this study, we define the origin and function of MDMs in cerebral ischemia. Methods Using adoptive transfer of GFP(+) splenocytes into adult asplenic mice subjected to transient middle cerebral artery occlusion, we compared the role of CD11b(+)/CD45(+)/NK1.1(-)/Ly6G(-) MDMs and microglia in the ischemic brain. The phagocytic activities of MDMs and microglia were measured by the uptake of fluorescent beads both in vivo with mice infused with GFP(+) splenocytes and ex vivo with cultures of isolated brain immune cells. Results Stroke induced an infiltration of MDMs [GFP+] into the ipsilateral hemisphere at acute (3 days) and sub-acute phases (7 days) of post-stroke. At 7 days, the infiltrating MDMs contained both CD45(High) and CD45(Low) subsets. The CD45(High) MDMs in the injured hemisphere exhibited a significantly higher proliferation capacity (Ki-67 expression levels) as well as higher expression levels of CD11c when compared to CD45(Low) MDMs. The CD45(High) and CD45(Low) MDM subsets in the injured hemisphere were approximately equal populations, indicating that CD45(High) MDMs infiltrating the ischemic brain changes their phenotype to CD45(Low) microglia-like phenotype. Studies with fluorescent beads reveal high levels of MDM phagocytic activity in the post-stroke brain, but this phagocytic activity was exclusive to post-ischemic brain tissue and was not detected in circulating monocytes. By contrast, CD45(Low) microglia-like cells had low levels of phagocytic activity when compared to CD45(High) cells. Both in vivo and ex vivo studies also show that the phagocytic activity in CD45(High) MDMs is associated with an increase in the CD45(Low)/CD45(High) ratio, indicating that phagocytosis promotes MDM phenotype conversion. Conclusions This study demonstrates that MDMs are the predominant phagocytes in the post-ischemic brain, with the CD45(High) subset having the highest phagocytic activity levels. Upon phagocytosis, CD45(High) MDMs in the post-ischemic brain adopt a CD45(Low) phenotype that is microglia-like. Together, these studies reveal key roles for MDMs and their phagocytic function in tissue repair and remodeling following cerebral ischemia.

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