Abstract WP312: Microglial Tmem119 Inhibits IRAK4 Activation and Confers Neuroprotection After Ischemic Stroke

Abstract

Introduction: Ischemic microglia trigger post-stroke inflammation leading to secondary neuronal death. Microglial responses are regulated through membrane protein signals that in turn activate inflammatory transcription factors. Transmembrane 119 (Tmem119) is a type 1A membrane protein that is exclusively expressed by microglia. It has been suggested that Tmem119 interferes with myeloid differentiation primary response 88 (MyD88)-interleukin-1 receptor-associated kinase 4 (IRAK4) axis that is involved in microglial activation. However, the exact role of Tmem119 in microglial activation has been elusive. Hypothesis: Tmem119 inhibits IRAK4 activation to suppress microglial pro-inflammatory responses and is protective against ischemic injury. Methods: Young (6-8-week-old) C57BL/6 WT mice were injected stereotaxically Lenti-Tmem119 to induce overexpression in microglia. Six weeks later, the mice were subjected to a 60-min middle cerebral artery occlusion (MCAO) model. Oxygen-glucose deprivation (OGD) was also performed in primary microglia cultures treated with Lenti-Tmem119 or Tmem119 siRNA. Western blotting was performed on brain homogenates or microglia cells respectively for expression of p-IRAK4. ELISA was conducted to measure cytokine levels in the plasma and cell culture supernatant. Results: p-IRAK4 protein levels were significantly lower in ischemic brains from Lenti-Tmem119 vs. Lenti-GFP injected mice (p=0.0035) at 3d after MCAO (n=4-6), accompanied by smaller infarct size in Lenti-Tmem119 injected mice. Cultured microglia treated with Lenti-Tmem119 or Tmem119 siRNA had decreased and increased expression of p-IRAK4 respectively than controls after OGD (p=0.0090 and p=0.0152 respectively, n=3-4). Pro-inflammatory cytokine (TNF-α, IL-1β, IL-6) levels were significantly lower in the plasma of Lenti-Tmem119 treated mice compared to controls. Levels of these cytokines were decreased or increased in microglia culture medium treated with Lenti-Tmem119 or Tmem119 siRNA respectively than in control treated medium. Conclusion: Tmem119 signaling regulates microglial inflammatory responses through interaction with IRAK4 and is beneficial to stroke. Keywords: Stroke; microglia; Tmem119; IRAK4; inflammation