expression was upregulated in the ischemic penumbra of mouse models and postmortem brain tissues from stroke patients.
Increased NLRC5 levels were found in activated microglia after ischemic stroke.
Deletion of NLRC5 in microglia resulted in significantly improved neurological outcomes and reduced brain damage in mice.
NLRC5 induction by various stimuli led to increased release of pro-inflammatory cytokines and neurotoxicity from microglia.
NLRC5 disrupted lysosomal function, which is crucial for the degradation of proteins and cellular debris.
The interaction between NLRC5 and ISG15 prevented ISG15 from being degraded by , contributing to inflammation.
Simplified
BACKGROUND: Ischemic stroke triggers excessive microglial activation and sustained neuroinflammation, driving secondary neuronal injury. Recent evidence suggests that dysfunction of the - 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 () 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.
Key numbers
17×
Infarct Volume Reduction
Comparison of infarct volumes in mCKO mice vs. control mice post-stroke.
28
Survival Rate Improvement
Survival rate of mCKO mice compared to control mice after tMCAO.
4
Pro-inflammatory Cytokine Reduction
Reduction in TNF-α and IL-6 levels in mCKO mice compared to controls.
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Declarations. Ethics approval and consent to participate: All mice procedures received full approval from the Animal Care and Use Committee of Nanjing University (No. 2023AE01007), and were in compliance with the NIH Guide for the Care and Use of Laboratory Animals. Frozen postmortem human brains from patients who died of cerebral ischemic stroke were obtained from the Chinese Brain Bank Center (approval number: 2021-scuec-034). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.
