Abstract Body: Objective: Microglia, the central nervous system's immune cells, are crucial in neuroinflammation after ischemic brain injury. Previous single-cell and spatial transcriptomics showed that LILRB4 was upregulated in microglia after MCAO in C57BL/B6J mice, but its role is unclear. This study investigates LILRB4's function and mechanism in ischemic injury.
Methods: In C57BL/B6J mice, LILRB4 expression was analyzed at various time points post-MCAO using Q-PCR, Western blot, and flow cytometry. A microglia-specific LILRB4 knockout mouse model (LILRB4-cKO) and control mice (LILRB4^fl/fl) were established, and MCAO was induced. Infarct volume and behavioral changes were assessed by MAP2 staining, mNSS scoring, grip strength, gait, and rotarod tests. qPCR and Western blot were used to detect brain inflammatory factors and DAMPs, while microglial morphology, cell counts, and RIPK3 levels were analyzed by immunofluorescence and flow cytometry. In vitro, LILRB4 expression changes post-OGD were validated, and LILRB4 knockdown BV2 cells (sh-LILRB4 BV2) were constructed. Necroptosis was induced using RIPK3 inhibitor and OGD, and necroptosis markers were detected. The JASPAR database was used to identify ATF2 as a potential RIPK3 transcription factor, and its role in gene regulation was validated by transfecting ATF2 plasmid into sh-LILRB4 BV2 cells.
Results：LILRB4 expression in microglia was upregulated after MCAO, peaking at day 3. LILRB4-cKO mice showed larger infarct volumes, greater neurological deficits, and higher pro-inflammatory cytokines (TNF, IL-6, IL-1β) than LILRB4^fl/fl mice. Microglia from LILRB4-cKO mice had larger cell bodies, shorter processes, reduced numbers, and more RIPK3⁺ necroptotic cells. In primary microglia and sh-LILRB4 BV2 cells, OGD increased LILRB4, RIPK3, MLKL, and cytokines, which were suppressed by GSK-872. The JASPAR database identified ATF-2 as a potential RIPK3 transcription factor, and its role in promoting RIPK3 transcription was confirmed. The JASPAR database identified ATF-2 as a potential RIPK3 transcription factor, and its role in promoting RIPK3 transcription was confirmed. Knockdown of ATF-2 reduced the elevated levels of inflammatory cytokines, DAMPs, and necroptosis markers induced by LILRB4 deletion.
Conclusion：LILRB4 inhibits microglial necroptosis through the ATF2/RIPK3 pathway, reducing neuroinflammation and offering neuroprotection. Therefore, LILRB4 may serve as a potential therapeutic target for ischemic brain injury