Abstract 20: Targeted Ablation Of STAT1 Enhances Resolution Of Inflammation By Microglia/macrophages And Promotes Long-term Recovery After Ischemic Stroke

Abstract

Introduction: Identification of key molecules that control conversion of resting microglia/macrophages (Mi/MΦ) to a detrimental or beneficial phenotype may help develop novel therapies to ischemic stroke. The transcription factor STAT1 contributes to acute (< 24h) neuronal death after brain ischemia/reperfusion (I/R), but its role in Mi/MΦ and impact on long term stroke outcome remain unknown. Hypothesis: Activation of STAT1 dictates proinflammatory responses of Mi/MΦ at subacute stage (3-5 d) after I/R. Selective deletion of STAT1 reprograms Mi/MΦ into an inflammation-resolving phenotype and improves long term stroke outcome. Methods: We generated mice with tamoxifen-induced, Mi/MΦ-specific knockout (mKO) of STAT1. Brain injury, inflammation and various behavioral deficits were assessed after 1 h MCAO and 1-35 d reperfusion. Mi/MΦ phenotype was examined in vivo (FACS followed by RNA-seq) and in vitro (primary microglia). Results: STAT1 was activated (phosphorylated) in Mi/MΦ 3 d after I/R in WT but not in STAT1 mKO mice. STAT1 mKO did not alter 24-h infarct size (TTC; p>0.05, n=8), but attenuated Mi/MΦ release of HMGB1 and increased arginase 1-producing Mi/MΦ 3d after I/R (immunostain, n=6), suggesting boosted inflammation-resolving responses of Mi/MΦ. RNA-seq further revealed downregulated proinflammatory genes and a concomitant elevation of phagocytosis-related genes in STAT1 mKO Mi/MΦ 3d after I/R (n=3, FDR<0.05). Consistently, phagocytosis of dead neurons was enhanced in STAT1 mKO mice 3d after I/R (n=7) or in cultured STAT1 KO microglia (n=4). As a result, STAT1 mKO mitigated brain inflammation 5d after I/R (ELISArray; 11 out of 27 markers, p<0.05, n=6), and improved motor/cognitive functions for 35d in mice of both sexes (p<0.01 vs WT, n=10-11). Conclusions: Mi/MΦ-targeted STAT1 KO does not offer acute neuroprotection but primes Mi/MΦ toward an inflammation-resolving phenotype, thereby facilitating long term functional recovery after stroke.