Hematopoietic cell kinase regulates microglial/macrophage activation to drive neuroinflammation after traumatic brain injury.

BACKGROUND: Brain-resident microglia and infiltrating monocyte-derived macrophages are among the main effectors in regulating innate immunity and mediating neuroinflammation after traumatic brain injury (TBI). Our previous work demonstrated that circulating extracellular vesicles from TBI patients (TEVs) aggravate microglial/macrophage activation and associated neuroinflammatory damage. However, the key molecular regulators governing TBI-induced and TEV-aggravated microglial/macrophage responses remain elusive. METHODS: To identify key molecular regulators of microglial/macrophage activation during the acute phase after TBI, we first performed RNA-sequencing on mouse TBI brains at multiple acute time points, and on brains from TBI mice treated with TEVs. We then integrated and analyzed a public single-nucleus RNA-sequencing dataset from human TBI brains (GSE209552) to validate and extend our murine findings. After hub gene identification, we used complementary in vivo (TBI and TBI + TEV mice) and in vitro [oxygen-glucose deprivation/re-oxygenation (OGD/R)-stimulated and TEV-stimulated BV2 cells] models for mechanistic investigation. RESULTS: Multi-dimensional bioinformatics analyses identified six hub genes, including HCK, BLNK, C1QB, CTSC, P2RY6, and VAV1, consistently implicated in microglial/macrophage activation throughout the acute phase after TBI. Subsequent mechanistic investigations focused on hematopoietic cell kinase (HCK), a central node within this hub gene network. Pharmacological inhibition of HCK kinase activity with A-419259 was found to (1) alleviate TBI-induced and TEV-aggravated microglial/macrophage activation and recruitment, (2) promote a phenotypic shift from pro-inflammatory M1 toward anti-inflammatory/reparative M2 polarization, and (3) suppress the activation of microglial/macrophage nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. These effects collectively ameliorated secondary brain damage and improved neurological outcomes after TBI. Complementary in vitro studies confirmed that A-419,259 treatment alleviated OGD/R-stimulated or TEV-stimulated BV2 microglial activation and NLRP3 inflammasome activation, while facilitating the M1-to-M2 transition. CONCLUSIONS: Our findings identify HCK as a pivotal molecular regulator of microglia/macrophage-mediated neuroinflammation, thus providing a potential therapeutic target for ameliorating secondary brain injury after TBI.