Multi-omics analyses nominate THBS1 as a regulator of an inflammatory microglial state after intracerebral hemorrhage.

Neuroinflammation is a major determinant of secondary injury after intracerebral hemorrhage (ICH), but the microglial states that drive this process and their upstream regulators remain incompletely resolved. We integrated public single-cell RNA sequencing and bulk RNA sequencing datasets with experimental validation to define inflammatory microglial programs after ICH. Re-clustering of microglia identified an acute MG3 subpopulation enriched for cytokine, NF-κB, and TNF signaling pathways. Integrative analysis with weighted gene co-expression network analysis prioritized thrombospondin-1 (THBS1) as a candidate regulator of this state. In perihematomal tissue, THBS1 increased prominently at day 3 after ICH and localized predominantly to microglia. In oxyhemoglobin-stimulated BV2 cells, Thbs1 knockdown reduced iNOS/CD16/32-associated inflammatory features and the release of TNF-α, IL-6, and IL-1β, while increasing CD206 expression. In CX3CR1Cre/ERT2 mice, microglia-targeted Thbs1 knockdown decreased perihematomal TNF-α, IL-6, and IL-1β levels, attenuated neuronal injury, and improved long-term neurological recovery. Direct RNA sequencing and MeRIP-qPCR further showed dynamic changes in m6A enrichment on Thbs1 transcripts after ICH, supporting an association between RNA methylation and temporal Thbs1 regulation. Together, these data identify a THBS1-high inflammatory microglial state in experimental ICH and support THBS1 as a candidate mediator of inflammatory secondary injury after experimental ICH.