Abstract Body: Background: Microglia-mediated inflammatory response directly affects ischemic stroke outcome. Autophagy is crucial in inflammatory response after cerebral ischemia. NLRC5 can be a positive or negative regulator of inflammatory signaling cascades, but its role after ischemic stroke is unstudied.

Methods: Single-cell RNA sequencing and immunofluorescence staining were used to explore the expression and localization of NLRC5 in mice brain. Conditional (microglia-specific) NLRC5-knockout mice (TMEM119^CreERTNLRC5^f/f) and their littermate control mice (NLRC5^f/f) were subjected to MCAO. Histopathological and behavioral methods were performed to assess ischemic injury in mice. In vitro, oxygen-glucose deprivation (OGD) and OGD neuronal supernatant were used to stimulate microglia to simulate post-stroke inflammatory microenvironment. Primary microglia were extracted from litters of wide type and NLRC5-global-KO mice for proteomic detection after OGD. Mass spectrometry was used to investigate the interaction of NLRC5 with other proteins. A series of cellular and molecular biological techniques were used to evaluate the proinflammatory response, autophagy activity and protein–protein interactions etc.

Results: mRNA and protein expression of NLRC5 were increased after MCAO from 1 to 7 days. NLRC5 was mainly expressed in microglia rather than neurons and astrocytes. After MCAO, the cerebral infarction volume of TMEM119^CreERTNLRC5^f/f mice was smaller than that of NLRC5^f/f mice, and neurological impairment was slighter. In the ischemic penumbra tissue of TMEM119^CreERTNLRC5^f/f mice, autophagy level increased, microglia activation decreased, and inflammation decreased. In vitro experiments, NLRC5 knockdown promoted autophagy level of primary microglia and BV2 in the inflammatory environment, and the inflammatory response was reduced. Proteomic results showed that after OGD, the autophagy levels of NLRC5-global-KO primary microglia were increased compared to that of wide type mice. Additionally, the protein levels of EIF5A, EIF5A hypusination and ATG3 increased in NLRC5-KO primary microglia after OGD. Mass spectrometry revealed that NLRC5 could bind to EIF5A. Specific binding sites and mechanisms need further study.

Conclusions: Down-regulation of microglia NLRC5 can increase hypusination level of EIF5A, increase ATG3 transcription, thus promote autophagy, inhibit neuroinflammation, and protect brain injury after ischemic stroke.