Mesenchymal stem cells drive M2 microglia polarization, associated with protection after acute brain injury : in vitro and in vivo evidence

Objectives: We have previously shown that microglia activation is associated with a protective phenotype after ischemic and traumatic brain injury and that infused stem cells induce a microglial switch toward a nonphagocytic phenotype. Here we investigate if mesenchymal stem cells (MSC) can drive protective microglia M2 polarization in vitro and in vivo. Material and methods: Primary murine microglial cultures were exposed for 72h to: 1) TNF/INFγ for M1 classical activation; 2) IL4 for M2 alternative activation; 3) human MSC. Co-cultures were then analyzed for IL1 β, iNOS and Ym1 by real time RT-PCR and immunohistochemistry. C57Bl/6 mice were subjected to sham or traumatic (TBI) brain injury. PBS or MSC (150,000/5ul) were infused intracerebroventricularly 24h later. The activation of microglia/macrophage and the expression of M1 and M2 markers was determined in the pericontusional tissue by real time RT-PCR 72h post injury and by immunohistochemistry 7 days post injury. Results: in vitro experiments on primary murine microglial cultures showed that 1) M1 activation induced an up-regulation of the pro-inflammatory genes IL1β and iNOS, 2) M2 activation induced an up-regulation of the protective marker Ym1, 3) exposure to MSC increased Ym1 mRNA and protein expression, thus polarizing microglia towards M2 phenotype. In vivo experiments showed that TBI at 72h post injury induced a comparable increase in CD11b expression (a marker of microglia activation) in MSC treated (345%) and untreated (342%) mice. Notably MSC infusion in TBI mice induced a significant up-regulation of the microglia M2 markers Ym1 (200%) and Arginase1 (182%) compared to TBI-PBS mice. At 7 days post injury in TBI-MSC mice positivity of CD11b cells was increased (260%) and associated with a significant decrease of CD68 positivity (58%) and an increase of Ym1 positivity (346%) thus showing a protective M2 microglial commitment. These early events anticipated sensorimotor and cognitive protection observable starting from 3 weeks post injury in TBI-MSC compared to TBI-PBS mice. Conclusions: This study shows that, both in vitro and in vivo, MSC activate a M2 microglial polarization associated with tissue repair program.