BTK promotes neuroinflammation after intracerebral hemorrhage involving hub genes and alterations in microglial functions

Bruton's tyrosine kinase (Btk) plays a key role in the inflammatory response in many diseases. However, its specific function and underlying mechanisms in intracerebral hemorrhage (ICH) remain unclear. Here, we used a mouse ICH model and transcriptomic datasets to investigate the role and mechanism of Btk in neuroinflammation after ICH. Inhibiting Btk with ibrutinib alleviated ICH-induced neurological deficits and reduced the expression of inflammatory markers in mice. Weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) analysis of RNA-sequencing data from ICH and control mice identified Btk as a hub gene in the green module (ranked 10th by kWithin). Also, 12 hub genes that strongly interacted with Btk were identified within the key gene module; all of them are critically involved in inflammatory processes. Single cell RNA-sequencing analysis revealed that microglia were the predominant BTK-expressing immune cells in the mouse brain, expressing 76.4% of total Btk in sham mice and 83.7% in ICH mice. Microglia from ICH mice were further stratified into BTK_high and BTK_low subpopulations. Enrichment analysis of differentially expressed genes (DEGs) between these groups showed that a substantial proportion of the top 30 enriched pathways (12 for GO and 20 for KEGG) were immune-related. Gene set enrichment analysis (GSEA) demonstrated that four anti-inflammatory and phagocytosis-related pathways were significantly downregulated in BTK_high microglia compared with BTK_low microglia (all P < 0.001). Gene set variation analysis (GSVA) further found differential activation of multiple immune pathways between the two subpopulations. In addition, six microglia polarization scores revealed that BTK_high microglia preferentially polarized toward M1 and M2b phenotypes, whereas BTK_low microglia favored M2 (M2a, M2c) states (all P < 0.0001). Finally, intercellular communication analysis indicated that Btk promoted signaling between microglia and Other immune cells, both globally and within specific inflammatory pathways. In conclusion, our findings show that Btk is a critical driver of post-ICH neuroinflammation. This effect is mediated, at least in part, through Btk-associated hub genes and through modulation of microglial immune pathway activation, polarization state, and intercellular communication.