Interferon regulatory factor 4/5 signaling impacts on microglial activation after ischemic stroke in mice

Microglial activation is a key element in initiating and perpetuating inflammatory responses to stroke. Interferon regulatory factor 5 (IRF5) and IRF4 signaling have been found critical in mediating macrophage pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, respectively, in peripheral inflammation. We hypothesize that the IRF5/4 regulatory axis also mediates microglial activation after stroke. C57BL6 mice of 8-12weeks were subject to a 90-min middle cerebral artery occlusion, and the brains evaluated at 24h, 3, 10 and 30days after reperfusion. Flow cytometry was utilized to examine microglial activation and cytokine expression. RT-PCR was performed for mRNA levels of IRF5/4 in sorted microglia. Microglial expression of IRF5/4 was examined by immunohistochemistry, and brain cytokine levels were determined by ELISA. Our results revealed that the IRF5 mRNA level in sorted microglia increased at 3days of stroke; whereas IRF4 mRNA level exhibited biphasic increases, with a transient rise at 24h and a peak at 10days. The same pattern was seen in IRF5/4 protein colocalization with Iba-1(+) cells by IHC. Intracellular levels of TNF-alpha and IL-1 beta in microglia peaked at 3days of stroke, and IL-4(+)IL-10(+) double-positive microglia significantly increased at day 10. Brain levels of these cytokines were consistent with microglial cytokine changes. Worse behavior test results were seen at 3days vs. 10days of stroke. We conclude that microglia phenotypes are dynamic to ischemic stroke, and IRF5/4 signaling may regulate microglial M1/M2 activation and impact on stroke outcomes.