Microglial Vitamin D Receptor Modulates Acute Brain Injury After Ischemic Stroke

Background and objective:Vitamin D deficiency is a largely underestimated epidemic and frequently coexists with cardiovascular diseases.Mounting evidence suggests that vitamin D deficiency is associated with poor recovery after acute ischemic stroke.Nevertheless,the potential impact of vitamin D and VDR signaling on stroke progression and the underlying mechanisms are not well-defined owing to a paucity of mechanistic studies.In light of the well-documented immunomodulatory effects of VDR,we aimed to determine how microglial VDR affects ischemia-elicited neuroinflammation and the progression of ischemic brain injury during the acute phase.Methods:We first employed transient middle cerebral artery occlusion（MCAO）procedure in C57BL/6 background mice to explore the expression of VDR in the ischemic brain and the individual cell subsets within the central nervous system.Next,we constructed a Cre-loxp genetic system that conditionally eliminates VDR gene in microglia.We evaluated the neurological function,infarct volume,neural cell apoptosis,blood-brain barrier permeability,cytokine levels,the number of immune cells within the ischemic brain and spleen,and chemokine levels of VDR^f/f and VDR-c KO mice after MCAO.The method we applied in this study included:immunofluorescence,q RT-PCR,Western blot analysis,flow cytometric analysis,Annexin V-propidium iodide（PI）apoptosis assay,microglia sorting from adult mouse brain,RNA sequencing（RNA-seq）,isolation and culture of mouse primary microglia and endothelial cells（ECs）,in vitro drug administration,cell culture and lentiviral transfection,oxygen glucose deprivation/reperfusion（OGD/R）,Transwell coculture system,T cell isolation and in vitro migration assay,ELISA,measurement of reactive oxygen species（ROS）generation in vitro.Results:Here,we found that microglia-specific VDR knockout markedly expanded infarct volume and aggravated neurological deficits after experimental ischemic stroke.Mechanistically,microglial VDR elimination exacerbated postischemic neuroinflammation,including increased production of proinflammatory cytokines and leukocyte chemokines.Specifically,microglia within the ischemic penumbra of VDR-c KO mice exhibited a more primed phenotype,and importantly,facilitated CXCL10 release from endothelial cells via increased TNF-αsecretion,which culminated in massive recruitment of peripheral T lymphocytes.In vitro OGD models revealed that VDR knockdown augmented ROS production,NF-κB activation,and the downstream release of TNF-αin microglia,whereby VDR activation ameliorated this proinflammatory response.Furthermore,microglial VDR upregulation dampened the generation of endothelial CXCL10,the process of which was at least partially driven by microglia-secreted TNF-α.Conclusion:This study revealed for the first time that microglial VDR plays essential roles in regulating the inflammatory responses evoked by cerebral ischemia through a coordinated network of cytokines and chemokines.Following AIS,microglia lacking VDR not only induces a more primed proinflammatory phenotype in itself,but also leaves ECs involved in postischemic inflammatory cascades,thereby causing CXCL10 upregulation and ultimately augmenting the infiltration of peripheral T lymphocytes.Our findings at least in part provide a mechanistic explanation for the adverse effect of vitamin D deficiency on disease progression and recovery in AIS patients.Individuals at high risk of stroke are encouraged to maintain vitamin D sufficiency to at least partially circumvent such devastating pathological events that impede stroke recovery.