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

Abstract

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(hi) 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 Ly6C(hi)CD43(lo)CCR2(+) 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 Ly6C(hi)CD43(lo) 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.

Spatio-temporal profile, phenotypic diversity, and fate of recruited monocytes into the post-ischemic brain

“…Our data are consistent with the observations by Gliem et al [8] of reduced accumulation of differentiated macrophages (identified as Ly6c lo F4/80 hi ) in CCR2 −/− mice 6 days after ischemia. Thus, CCR2 + Ly6C hi Mo/MΦ could be precursors for CX3CR1 + Ly6C lo Mo/MΦ from which they develop through environmental cues provided by the ischemic brain tissue, as has been recently suggested [19]. The signals and their target receptors responsible for this phenotypic shift remain to be determined.…”

“…Low rates of Mo/MΦ proliferation have also been reported in a mouse model of permanent cerebral ischemia [19]. Considering these low proliferation rates, it is more likely that the increased numbers of brain Mo/MΦ are the result of continuous recruitment of hematogenous monocytes than from cell proliferation in situ.…”

Microglia and Monocyte-Derived Macrophages in Stroke

“…In rats, 10 % to 20 % of monocytes that express low CD43 are classified as a proinflammatory subset and the remaining monocytes that expresses higher levels of CD43 are classified as an anti-inflammatory subset [79]. Recently, CD43 has also been used to distinguish monocyte subsets in mice [80]. Classically activated proinflammatory monocytes express CCR2 with low or no expression of CX 3 CR1 across different species.…”

“…Others have suggested the acquisition of the repair M2 phenotype from M1 macrophages in sterile wounds. Early recruited peripheral monocytes/macrophages are predominantly Ly-6C hi (CCR2 + ) cells that become M1 tissue macrophages in the stroked hemisphere [25,80,114,115]. Once recruited, these Fig.…”

“…An additional modulator for tissue repair and remodeling in the CNS injury is arginase-1. Studies indicated that arginase-1 expression is correlated with improved recovery from CNS injury and disease [80,[139][140][141]. A recent study using EAE and spinal cord injury models has shown that infiltrated macrophages mainly express arginase-1 in the inflamed CNS [142], suggesting the importance of infiltrated myeloid cells in the CNS repair after injury.…”