Cholesterol Drives IFITM3⁺ Microglia Activation and Induces STING Mediated Neuroinflammation After Ischemic Stroke

Cerebral ischemic stroke triggers extensive neuronal membrane breakdown, releasing a massive load of cholesterol that overwhelms resident microglia. Dysregulated microglial cholesterol metabolism has been implicated in post-stroke neuroinflammation, yet the specific pathogenic microglial subpopulations, their molecular signatures, and the downstream inflammatory cascades remain poorly defined.

We employed a permanent distal middle cerebral artery occlusion (dMCAO) model combined with single-cell RNA sequencing (scRNA-seq) to profile immune cell transcriptomes and identify cholesterol-associated microglial markers. Cholesterol dynamics, lipid droplet accumulation, and inflammatory marker expression were quantified via immunofluorescence and transmission electron microscopy. Therapeutic interventions included pharmacological cholesterol mobilization with 2-hydroxypropyl-β-cyclodextrin (HβCD), pharmacological STING inhibition with C-176, and microglia-targeted STING knockdown using AAV9 vectors. Cerebral injury and neurological function were assessed through infarct volume measurement, white matter integrity analysis, and behavioral assays (rotarod and grip strength) in dMCAO, tMCAO, and perioperative stroke (PIS) models.

Using scRNA-seq, we identified interferon-induced transmembrane protein 3 (IFITM3) as a specific marker for a microglial subpopulation that was characterized by upregulated ACAT1, enhanced cholesterol esterification, and accumulation of cholesterol crystals and lipid droplets. This IFITM3⁺ microglia population peaked at 7 days post-stroke and correlated with NLRP3 inflammasome activation and STING signaling. Pharmacological reduction of cholesterol burden with HβCD attenuated lipid droplet formation, suppressed mitochondrial DNA leakage, and inhibited STING pathway activation. Correspondingly, HβCD and C-176 administration significantly reduced cerebral infarct size, mitigated white matter demyelination, and improved motor function in dMCAO and tMCAO models. We further found that AAV-mediated STING knockdown recapitulated the above protective effects in HβCD and C-176 treated stroke mice. Furthermore, HβCD treatment ameliorated microglial inflammation and improved functional outcomes in a PIS model.

IFITM3⁺ microglia is a pro-inflammatory and cholesterol-laden subpopulation that exacerbates post-stroke cerebral ischemic brain injury. Targeting the microglial cholesterol axis by HβCD or inhibiting the STING pathway represents a promising therapeutic strategy to mitigate ischemic brain injury and improve neurological function.