Abstract TP283: Phosphorylation of IRF5/4 by IRAK4/MyD88 Regulates Microglial Inflammatory Responses to Ischemia

Abstract

Introduction: Interferon Regulatory Factors 5 and 4 (IRF5/4) have been suggested to play a determinant role in regulating pro-inflammatory (M1) or anti-inflammatory (M2) microglia/macrophage response. However, how IRF5/4 signaling are activated in these phagocytes has not been well understood. Our previous study has shown an increased level of phosphorylated-IRF4 in both the cytosolic and nuclear fraction of cultured microglial homogenates after ischemia, suggesting phosphorylation is important for IRF4 activation. Interleukin-1 receptor associated kinase 4 (IRAK4) and the adaptor protein MyD88 are the upstream signals of IRF5. In the present study we hypothesized that IRAK4/MyD88 phosphorylate IRF5/4 in ischemic microglia and trigger the translocation of IRF5/4 from the cytoplasm to the nucleus, to promote expression of inflammatory mediators. Methods: Spontaneously Immortalized (SIM)-A9 or primary microglia from C57BL/6 mice were cultured and exposed to oxygen-glucose deprivation (OGD) for 4 hours; after OGD the cells were reperfused overnight with glucose-containing medium in room air. Phosphorylation of IRF5/4 by IRAK4 was determined with IRAK4 inhibitor (ND-2158). The interaction between IRAK4/MyD88 with IRF5/4 and the nuclear translocation of phosphorylated-IRF5/4 were examined by immunocytochemistry (ICC) and co-immunoprecipitation (Co-IP)/Western Blots (WB). Production of inflammatory cytokines was measured by ELISA in the culture medium. Results: WB showed more robust nuclear p-IRF5 signals; ELISA found increased level of TNFα, IL-1β, and IL6 in OGD vs. normoxic microglia. A similar pattern was observed for p-IRF4 nuclear translocation but with moderately increased anti-inflammatory cytokine (IL-4 and IL-10) levels with OGD. Co-IP revealed a higher ratio of p-IRF5/4 over total IRF5/4 in the nuclear vs. cytosol fraction, and interactions of IRAK4/MyD88 with both IRFs. ND-2158 treatment led to decreased phosphorylation of IRF5/4 after OGD compared to the vehicle group. Conclusion: IRAK4/MyD88 complex phosphorylates microglial IRF5/4 which subsequently mediates the production of inflammatory cytokines after ischemia. Manipulation of IRF5/4 signaling potentially represents a novel therapeutic strategy for stroke.