Phosphorylation of Microglial IRF5 and IRF4 by IRAK4 Regulates Inflammatory Responses to Ischemia

Background: Interferon Regulatory Factor (IRF) 5 and 4 play a determinant role in regulating microglial pro- and anti-inflammatory responses to cerebral ischemia. How microglial IRF5 and IRF4 signaling are activated has been elusive. We hypothesized that interleukin-1 receptor associated kinase 4 (IRAK4) phosphorylates and activates IRF5 and IRF4 in ischemic microglia. We aimed to explore the upstream signals of the two IRFs, and to determine how the IRAK4-IRF signaling regulates the expression of inflammatory mediators, and impacts neuropathology. Methods: Spontaneously Immortalized Murine (SIM)-A9 microglial cell line, primary microglia and neurons from C57BL/6 WT mice were cultured and exposed to oxygen-glucose deprivation (OGD), followed by stimulation with LPS or IL-4. An IRAK4 inhibitor (ND2158) was used to examine IRAK4′s effects on the phosphorylation of IRF5/IRF4 and the impacts on neuronal morphology by co-immunoprecipitation (Co-IP)/Western blot, ELISA, and immunofluorescence assays. Results: We confirmed that IRAK4 formed a Myddosome with MyD88/IRF5/IRF4, and phosphorylated both IRFs, which subsequently translocated into the nucleus. Inhibition of IRAK4 phosphorylation quenched microglial pro-inflammatory response primarily, and increased neuronal viability and neurite lengths after ischemia. Conclusions: IRAK4 signaling is critical for microglial inflammatory responses and a potential therapeutic target for neuroinflammatory diseases including cerebral ischemia.