Progesterone Attenuates Stress-Induced NLRP3 Inflammasome Activation and Enhances Autophagy Following Ischemic Brain Injury

Abstract

NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome inhibition and autophagy induction attenuate inflammation and improve outcome in rodent models of cerebral ischemia. However, the impact of chronic stress on NLRP3 inflammasome and autophagic response to ischemia remains unknown. Progesterone (PROG), a neuroprotective steroid, shows promise in reducing excessive inflammation associated with poor outcome in ischemic brain injury patients with comorbid conditions, including elevated stress. Stress primes microglia, mainly by the release of alarmins such as high-mobility group box-1 (HMGB1). HMGB1 activates the NLRP3 inflammasome, resulting in pro-inflammatory interleukin (IL)-1β production. In experiment 1, adult male Sprague-Dawley rats were exposed to social defeat stress for 8 days and then subjected to global ischemia by the 4-vessel occlusion model, a clinically relevant brain injury associated with cardiac arrest. PROG was administered 2 and 6 h after occlusion and then daily for 7 days. Animals were killed at 7 or 14 days post-ischemia. Here, we show that stress and global ischemia exert a synergistic effect in HMGB1 release, resulting in exacerbation of NLRP3 inflammasome activation and autophagy impairment in the hippocampus of ischemic animals. In experiment 2, an in vitro inflammasome assay, primary microglia isolated from neonatal brain tissue, were primed with lipopolysaccharide (LPS) and stimulated with adenosine triphosphate (ATP), displaying impaired autophagy and increased IL-1β production. In experiment 3, hippocampal microglia isolated from stressed and unstressed animals, were stimulated ex vivo with LPS, exhibiting similar changes than primary microglia. Treatment with PROG reduced HMGB1 release and NLRP3 inflammasome activation, and enhanced autophagy in stressed and unstressed ischemic animals. Pre-treatment with an autophagy inhibitor blocked Progesterone’s (PROG’s) beneficial effects in microglia. Our data suggest that modulation of microglial priming is one of the molecular mechanisms by which PROG ameliorates ischemic brain injury under stressful conditions.