Abstract 105: Neutrophil Cracr2a Is Crucial For Calcium Signaling And Enhances Neutrophil Adhesive Function Under Thromboinflammatory Conditions

Ca 2+ release-activated channel regulator 2A (CRACR2A) is a Ca 2+ -binding protein that facilitates store-operated Ca 2+ entry during T cell activation. However, it is unknown whether neutrophil CRACR2A regulates neutrophil functions in inflammation. Our confocal intravital microscopy (IVM) revealed that compared to wild-type (WT) mice, myeloid-specific cracr2a conditional knockout (CKO, cracr2a fl/fl;Lyz-cre ) mice exhibited a significant increase in neutrophil rolling and a decrease in neutrophil adhesion, crawling, and transmigration on ear and cremaster venules in TNF-α-induced inflammation. Deletion of neutrophil cracr2a impaired β2 integrin function without affecting the expression of L-selectin, PECAM-1, and JAM-A. Neutrophil cracr2a increased Ca 2+ mobilization after stimulation with fMLP, A23187, and thapsigargin by its rapid interaction with STIM1. Due to the lack of CRACR2A inhibitors, we generated CRACR2A-deleted HL60 cells using CRISPR/Cas9. CRACR2A deletion in differentiated HL60 cells recapitulated the defects seen in cracr2a-null neutrophils, including reduced β2 integrin function and Ca 2+ mobilization after agonist stimulation. Since excessively recruited neutrophils cause brain damage in ischemic stroke, we examined whether neutrophil cracr2a contributes to stroke pathology. Using a transient middle cerebral artery occlusion (tMCAO)-induced ischemic stroke model, we found that compared to WT mice, cracr2a CKO mice exhibited improved neurological deficits and reduced infarct volume. Longitudinal 4D IVM of mouse brain revealed that neutrophil cracr2a is crucial for neutrophil adhesion and transmigration on cerebral vessels 16-24 hours after tMCAO and upregulated αMβ2 integrin on adherent neutrophils. Further, single-cell behavior analysis of 4D IVM showed that a proinflammatory subset of neutrophils arise in WT mice, but not cracr2a CKO mice, after tMCAO. We observed that the number of circulating pro-inflammatory neutrophils was markedly decreased 24 hours after tMCAO in cracr2a CKO mice compared with WT mice. Our findings indicate that neutrophil CRACR2A is a key regulator for Ca 2+ signaling and enhances neutrophil pro-inflammatory and adhesive activities in vascular inflammation and ischemic stroke.