Department of Neurology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
Cells. 2021 Jan 30;10(2):276. doi: 10.3390/cells10020276.
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.
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.
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.
IRAK4 signaling is critical for microglial inflammatory responses and a potential therapeutic target for neuroinflammatory diseases including cerebral ischemia.
干扰素调节因子 (IRF) 5 和 4 在调节小胶质细胞对脑缺血的促炎和抗炎反应方面起着决定性作用。小胶质细胞中 IRF5 和 IRF4 信号的激活方式仍不清楚。我们假设白细胞介素-1 受体相关激酶 4 (IRAK4) 在缺血性小胶质细胞中磷酸化并激活 IRF5 和 IRF4。我们旨在探索这两个 IRF 的上游信号，确定 IRAK4-IRF 信号如何调节炎症介质的表达，并影响神经病理学。
培养自发永生化的小鼠（SIM）-A9 小胶质细胞系、原代小胶质细胞和 C57BL/6 WT 小鼠的神经元，并使其暴露于氧葡萄糖剥夺 (OGD)，然后用 LPS 或 IL-4 刺激。使用 IRAK4 抑制剂 (ND2158) 通过共免疫沉淀 (Co-IP)/Western blot、ELISA 和免疫荧光测定来检查 IRAK4 对 IRF5/IRF4 磷酸化的影响及其对神经元形态的影响。
我们证实 IRAK4 与 MyD88/IRF5/IRF4 形成 Myddosome，并磷酸化这两个 IRF，随后它们易位到细胞核中。IRAK4 磷酸化的抑制主要抑制小胶质细胞的促炎反应，并增加缺血后神经元的活力和神经突长度。
IRAK4 信号对于小胶质细胞的炎症反应至关重要，是包括脑缺血在内的神经炎症性疾病的潜在治疗靶点。
