Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke.

Neutrophil infiltration has been linked to worse clinical outcomes after ischemic stroke. Microglia, a key type of immune-competent cell, engage in cross-talk with the infiltrating immune cells in the inflamed brain area, yet the molecular mechanisms involved remain largely unexplored. In this study, we investigated the mechanisms of how canonical transient receptor potential 1 (TRPC1) modulated neutrophil infiltration in male mouse cerebral ischemia and reperfusion injury (CIRI) models. Our findings revealed a notable upregulation of TRPC1 in microglia within both middle cerebral artery occlusion reperfusion (MCAO/R) and in vitro oxygen-glucose deprivation/regeneration (OGD/R) model. Conditional Trpc1 knockdown in microglia markedly reduced infarct volumes and alleviated neurological deficits. Microglia conditional Trpc1 knockdown mice displayed less neutrophil infiltration in peri-infarct area. Trpc1 knockdown microglia exhibited a reduced primed proinflammatory phenotype with less secretion of CC-Chemokines ligand (CCL) 5 and CCL2 after MCAO/R. Blocking CCL5/2 significantly mitigated neutrophil infiltration in microglia/neutrophil transwell co-culture system upon OGD/R condition. Trpc1 knockdown markedly reduced store-operated calcium entry and nuclear factor of activated T-cells c1 (NFATc1) level in OGD/R treated microglia. Overexpression of Nfatc1 reversed the CCL5/2 reducing effect of Trpc1 knockdown, which is mediated by small interfering RNA in BV2 cells upon OGD/R. Our data indicate that upregulation of TRPC1 in microglia stimulates the production of CCL5/2 through the Ca2+/NFATc1 pathway. Upregulated CCL5/2 leads to an increase in neutrophil infiltration into the brain, thereby aggravating reperfusion injury. Our results demonstrate the importance of TRPC1 in microglia-mediated neuroinflammation and suggest a potential means for reducing CIRI induced neurological injury.

Astrocyte KDM4A mediates chemokines and drives neutrophil infiltration to aggravate cerebral ischemia and reperfusion injury.

Neutrophils plays a crucial role in acute ischemic brain injury and have emerged as potential treatment targets to mitigate such injuries. Lysine-specific demethylase 4 A (KDM4A), a member of the histone lysine demethylase family of enzymes involved in transcriptional regulation of gene expression, is upregulated during hypoxic events. However, the exact role of KDM4A in the pathological process of ischemic stroke remains largely unexplored. Our findings reveal that there was an upregulation of KDM4A levels in reactive astrocytes within both stroke mouse models and in vitro oxygen-glucose deprivation/regeneration (OGD/R) models. Using a conditional knockout mouse, we observed that astrocytic Kdm4a knockout regulates neutrophil infiltration and alleviates brain injury following middle cerebral artery occlusion reperfusion. Furthermore, Kdm4a deficiency astrocytes displayed lower chemokine C-X-C motif ligand 1 (CXCL1) level upon OGD/R and decreased neutrophil infiltration in a transwell system. Mechanistically, KDM4A, in cooperation with nuclear factor-kappa B (NF-κB), activates Cxcl1 gene expression by demethylating histone H3 lysine 9 trimethylation at Cxcl1 gene promoters in astrocytes upon OGD/R injury. Our findings suggest that astrocyte KDM4A-mediated Cxcl1 activation contributes to neutrophil infiltration via cooperation with NF-κB, and KDM4A in astrocytes may serve as a potential therapeutic target to modulate neutrophil infiltration after stroke.