Abstract Body: Introduction: Leukocyte immunoglobulin-like receptor B4 (LILRB4) is a crucial regulator of immune responses, particularly within microglia, where it may influence the infiltration of peripheral T cells into the brain. However, the specific role of LILRB4 in modulating brain damage after acute ischemic stroke remains unclear. This study aims to investigate the role of LILRB4 in modulating the immune response and its potential protective effects against ischemic brain injury in mice. Methods: Microglia-specific LILRB4 conditional knockout (LILRB4-KO) and overexpression transgenic (LILRB4-TG) mice were constructed using a Cre-loxP system. A transient MCAO model was employed to induce ischemic stroke. Single-cell RNA sequencing (scRNA-seq), RT-PCR, Flow cytometry (FACS), and immunofluorescence, TTC staining, laser speckle imaging, and neurological behavioral tests were employed. Results: Spatial transcriptomics analysis revealed increased LILRB4 expression in the ischemic hemisphere. ScRNA-seq identified microglia-cluster3, an ischemia-associated microglia subcluster with elevated LILRB4 expression in the ischemic brain. FACS and immunofluorescence staining showed increased CD8⁺ T cell infiltration into the brain in LILRB4-KO-tMCAO mice. Behavioral tests, cortical perfusion maps, and infarct size measurements indicated that LILRB4-KO-tMCAO mice had more severe functional deficits and larger infarct sizes compared to Control-tMCAO and LILRB4-TG-tMCAO mice. LILRB4-KD microglia promoted CD8⁺ T cell recruitment and activation in vitro and in vivo, which was mitigated by CCL2 inhibition and recombinant Arginase-1 addition. ScRNA-seq and spatial transcriptomics identified that CCL2 was predominantly secreted from activated microglia/macrophage and that increased CCL2 expression in LILRB4-KD microglia. Conclusion: LILRB4 in microglia plays a crucial role in modulating the post-stroke immune response by regulating CD8⁺ T cell infiltration and activation. Knockout of LILRB4 exacerbates ischemic brain injury by promoting CD8⁺ T cell recruitment. Overexpression of LILRB4, conversely, offers neuroprotection. These findings highlight the therapeutic potential of targeting LILRB4 and its downstream pathways to mitigate immune-mediated damage in ischemic stroke.
Keywords: Ischemic stroke, LILRB4, Microglia, Neuroinflammation, CD8⁺ T cells