Abstract Body: Objective:
Incomplete clearance of cell debris in stroke lesion contributes to post-stroke cognitive impairment. Microglia and infiltrated macrophages are the major scavenger of debris in stroke lesion. However, as stroke progresses, their phagocytic capacity is impaired, while secretion of pro-inflammatory factors was enhanced. This study aims to identify the mechanisms driving the phenotypic shift after excessive phagocytosis.
Methods:
Brain cells from 8-12 week-old male C57BL/6J mice were collected for single-cell RNA sequencing at 1, 5, and 14 days after transient middle cerebral artery occlusion (tMCAO), as well as from sham-operated control group. Key transcription factors mediating the phenotypic changes of microglia and macrophages were identified through bioinformatics analysis, and further validated in vivo and in vitro using immunofluorescence staining, flow cytometry, and Western blot. Additionally, tMCAO models were treated with the key factor inhibitor to evaluate microglia and macrophage phenotypic shifts and the progression of stroke.
Results:
Through single-cell RNA sequencing analysis, we identified a concurrent activation pattern between the stimulator of interferon genes (STING) mediated Type I interferon signaling and the pro-inflammatory phenotype of microglia/macrophages. In the first 5 days after stroke, microglia and macrophages displayed an inflammatory-resolving phenotype with enhanced phagocytic activity, but from days 5 to 14, they transitioned to a pro-inflammatory phenotype with sustained upregulation of STING-mediated Type I interferon pathway molecules. We validated the phenomenon observed in bioinformatics using tMCAO models. In vitro and in vivo, we found that continuous phagocytosis by macrophages and microglia led to STING activation, triggering Type I interferon signaling and driving the phenotypic shift towards a pro-inflammatory state. Treatment with STING inhibitor H-151 reversed these detrimental shifts..
Conclusions:
These findings suggest that continuous post-stroke phagocytosis by macrophages and microglia led to the activation of STING, thereby triggering type I interferon signaling and driving the phenotypic shift towards a pro-inflammatory state. STING inhibition may serve as a therapeutic strategy for mitigating neuroinflammatory responses following stroke.