STING hinders XIAP-mediated degradation of NLRP3 to lock the inflammasome in a hyperactivated state during pulmonary ischemia-reperfusion injury.

Abstract

Pulmonary ischemia-reperfusion (I/R) injury activates the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome to drive sterile inflammation. Although stimulator of interferon genes (STING) signaling primes NLRP3 transcription, its post-translational control over NLRP3 stability during lung I/R remains unclear. Murine lung I/R and alveolar epithelial cell hypoxia/reoxygenation (H/R) models were utilized. We combined pharmacological inhibition (H151), genetic manipulations, cycloheximide chase assays, TurboID proximity labeling, and site-directed mutagenesis to investigate the STING-regulated NLRP3 interactome and ubiquitination. STING activation during lung I/R or H/R stabilized NLRP3 protein and enhanced inflammasome assembly. Mechanistically, ischemic stress enhanced STING-NLRP3 spatial proximity, which disrupted the interaction between NLRP3 and X-linked inhibitor of apoptosis protein (XIAP). TurboID-MS and domain mapping revealed that XIAP directly binds NLRP3 via their RING and PYD domains, serving as an E3 ligase to mediate polyubiquitination at four LRR domain lysine residues (K823/K830/K831/K880) for proteasomal degradation. STING activation blocked this XIAP-dependent ubiquitination. Strikingly, NLRP3 overexpression in vitro or total NLRP3 knockout in vivo abolished the protective phenotypic benefits of STING deficiency or H151 treatment. STING signaling exacerbates pulmonary I/R injury by impairing XIAP-mediated NLRP3 ubiquitination and degradation. Targeting the STING-XIAP-NLRP3 axis offers a therapeutic strategy for ischemic acute lung injury.