Immature monocytes recruited to the ischemic mouse brain differentiate into macrophages with features of alternative activation

Acute stroke induces a local inflammatory reaction causing leukocyte infiltration. Circulating monocytes are recruited to the ischemic brain and become tissue macrophages morphologically indistinguishable from reactive microglia. However, monocytes are a heterogeneous population of cells with different functions. Herein, we investigated the infiltration and fate of the monocyte subsets in a mouse model of focal brain ischemia by permanent occlusion of the distal portion of the middle cerebral artery. We separated two main subtypes of CD11b monocytes according to their expression of the surface markers Ly6C and CD43. Using adoptive transfer of reporter monocytes and monocyte depletion, we identified the pro-inflammatory Ly6CCD43CCR2 subset as the predominant monocytes recruited to the ischemic tissue. Monocytes were seen in the leptomeninges from where they entered the cortex along the penetrating arterioles. Four days post-ischemia, they had invaded the infarcted core, where they were often located adjacent to blood vessels. At this time, Iba-1 and Iba-1 cells in the ischemic tissue incorporated BrdU, but BrdU incorporation was rare in the reporter monocytes. The monocyte phenotype progressively changed by down-regulating Ly6C, up-regulating F4/80, expressing low or intermediate levels of Iba-1, and developing macrophage morphology. Moreover, monocytes progressively acquired the expression of typical markers of alternatively activated macrophages, like arginase-1 and YM-1. Collectively, the results show that stroke mobilized immature pro-inflammatory Ly6CCD43 monocytes that acutely infiltrated the ischemic tissue reaching the core of the lesion. Monocytes differentiated to macrophages with features of alternative activation suggesting possible roles in tissue repair during the sub-acute phase of stroke.
Financed by the Spanish Ministries of Economy MINECO (SAF2011-30492, SAF2014-56279) and Health (FIS PI12/01437), and the European Community FP7 (InMiND project n°278850). We acknowledge the Image and Cytometry platforms of IDIBAPS, and the Confocal Microscopy Unit of the Serveis Cientifico-Tècnics of the University of Barcelona for technical help. F.M.M. was supported by the ‘Red Invictus’ of the Instituto de Salud Carlos III (RD12/0014/0011).