Microglial NLRC5 drives lysosomal dysfunction to disrupt autophagic flux and promote post-stroke neuroinflammation

Ischemic stroke triggers excessive microglial activation and sustained neuroinflammation, driving secondary neuronal injury. Recent evidence suggests that dysfunction of the autophagy-lysosome system may be a crucial factor sustaining microglial pro-inflammatory responses, yet the underlying regulatory mechanisms remain unclear. NOD-like receptor family caspase recruitment domain-containing protein 5 (NLRC5) has been widely studied in various immune and inflammatory diseases and exhibits functional heterogeneity under different pathological conditions. However, the role of NLRC5 in modulating post-stroke neuroinflammation remains unclear.

NLRC5 expression and localization was examined in a mouse transient middle cerebral artery occlusion (tMCAO) model and postmortem brain tissue from stroke patients. A microglia-specific Nlrc5 knockout (mCKO) mice line was generated to evaluate the effects of Nlrc5 deletion on neurological function, infarct volume, neuronal apoptosis, and inflammatory response after ischemic stroke. Proteomics, mass spectrometry, and molecular biology assays were conducted to elucidate the mechanisms.

NLRC5 expression was upregulated in the ischemic penumbra of mouse models and appeared higher in postmortem brain tissues from stroke patients, specifically in activated microglia. Strikingly, mCKO mice exhibited significantly improved neurological outcomes, reduced infarct volumes, and attenuated neuronal apoptosis post-stroke. In vitro studies demonstrated that NLRC5 induction by various stimuli, including oxygen-glucose deprivation/reperfusion (OGD/R), lipopolysaccharide (LPS), as well as neuronal debris and supernatant, promoted pro-inflammatory cytokine release and microglia-mediated neurotoxicity, whereas Nlrc5 deletion exerted protective effects. Mechanistically, NLRC5 did not influence autophagosome formation but profoundly disrupted autophagic flux by impairing lysosomal function. Proteomic and biochemical analyses revealed that NLRC5 binds interferon-stimulated gene 15 (ISG15) via its CARD domain, shielding ISG15 from autophagy-lysosomal degradation. Furthermore, NLRC5-induced lysosomal defects and inflammatory responses were abolished in the absence of Isg15.

NLRC5 promotes microglial inflammation and exacerbates post-stroke brain injury by stabilizing ISG15 and disrupting lysosomal function and autophagic flux. NLRC5-ISG15 axis is a therapeutic target for immune modulation in ischemic stroke.