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Introduction: The Inflammatory response is a fundamental pathophysiological procedure in stroke, and the activation of microglia characterized by either M1 (pro-inflammatory) or M2 (anti-inflammatory) state is crucial in initiating, sustaining and resolving the post-stroke inflammation. IRF5/4 are key determinants in macrophage polarization. Our previous study has also shown IRF5/4 signaling corresponds to microglial M1/M2 activation in the early/late phases of ischemic stroke respectively. In the present study, we tested our hypothesis that manipulation of IRF5/4 regulatory axis impacts on post-stroke inflammation and stroke outcomes.Methods: Microglia specific IRF5/4 conditional knockout (CKO) and lenti-IRF5/4 treated C57BL6 (for IRF5/4 overexpression) mice were subjected to a 60-minute middle cerebral artery occlusion (MCAO). Stroke outcomes including infarct size and behavior deficits were evaluated at 3d and 10d of MCAO. Microglial activation and peripheral immune cell infiltration were determined by flow cytometry (FC). IRF5/4 colocalization with microglia was examined with IHC, and multiplex was performed to measure cytokine levels in both the brain and blood samples.Results: At both time points, IRF4 CKO mice exhibited increased infarct lesions and worse behavior outcomes, and this effect was reversed in IRF4 overexpressed mice. In contrast, IRF5 CKO mice had reduced infarct lesions and improved behavior deficits; whereas IRF5 over expression in microglia worsened stroke outcomes. FC results revealed that microglia exhibited M1 phenotype in IRF4 CKO and IRF5 over-expressed mice that concomitantly had pro-inflammatory profile demonstrated by cytokine data; however, IRF5 CKO and IRF4 over-expressed mice had M2 dominant microglia and anti-inflammatory responses. IHC showed IRF5 expression was upregulated in IRF4 CKO and IRF5 over-expressed mice, and IRF4 expression was augmented in IRF5 CKO and IRF4 over-expressed mice.Conclusion: IRF5/4 regulatory axis regulates the microglial M1/M2 activation and impacts on stroke outcomes. Manipulation of IRF5/4 regulatory axis represents a novel therapeutic strategy for ischemic stroke.