Brain Immune Cells Use NLRC5 to Block Self-Cleaning and Fuel Stroke Damage

Ischemic stroke kills neurons not only during the initial blood-flow interruption but through a prolonged inflammatory wave driven by over-activated microglia. Suppressing this secondary injury without crippling needed immune surveillance has proven difficult, partly because the molecular switches governing microglial inflammation remain incompletely understood. This study zeroes in on NLRC5—the largest known NOD-like receptor protein—as a previously unrecognized driver of post-stroke neuroinflammation.

The team used a mouse transient middle cerebral artery occlusion (tMCAO) model alongside postmortem human brain tissue from stroke patients. NLRC5 protein was markedly elevated in the ischemic penumbra, and immunostaining confirmed this upregulation was concentrated in activated microglia rather than neurons or astrocytes. To test causation, the researchers bred microglia-specific conditional knockout (mCKO) mice using a Tmem119-CreERT2 system crossed with floxed Nlrc5 alleles, ensuring tamoxifen-inducible, cell-type-selective deletion.

Strikingly, mCKO mice showed significantly reduced infarct volumes, less neuronal apoptosis, and improved neurological scores compared to controls at multiple post-stroke time points. In vitro, primary microglia exposed to oxygen-glucose deprivation/reperfusion (OGD/R), LPS, or neuronal debris all upregulated NLRC5 and released more TNF-α, IL-1β, and IL-6; Nlrc5 deletion blunted these responses and reduced neurotoxicity in co-culture assays.

Proteomic profiling and biochemical assays provided the mechanistic explanation: NLRC5 did not alter the formation of autophagosomes but profoundly impaired their clearance by disrupting lysosomal acidification and hydrolase activity. Mass spectrometry identified ISG15 as a direct binding partner of NLRC5's CARD domain. By physically associating with ISG15, NLRC5 shields it from autophagy-lysosomal degradation, allowing ISG15 to accumulate and further impair lysosomal function in a feed-forward loop. Critically, knocking out Isg15 in microglia abolished both the lysosomal defects and the inflammatory amplification caused by NLRC5, confirming ISG15 as the essential downstream effector.

The findings position the NLRC5-ISG15 axis as a druggable checkpoint where lysosomal biology and neuroinflammation intersect. Because restoring autophagic flux in microglia appears sufficient to dampen the sustained pro-inflammatory state that drives secondary brain injury, pharmacological disruption of the NLRC5-ISG15 interaction could extend the therapeutic window beyond current recanalization strategies.