BACKGROUND Ca2+ release-activated Ca2+ channel regulator 2A (CRACR2A) has been linked to immunodeficiency attributable to T-cell dysfunction in humans. We discovered that neutrophil CRACR2A promotes neutrophil adhesive and migratory functions by facilitating Ca2+ mobilization and β2 integrin activation. METHODS Myeloid-specific cracr2a conditional knockout mice and intravital microscopy were used to investigate the physiologic role of neutrophil cracr2a in neutrophil recruitment in vascular inflammation. Cracr2a-deficient neutrophils or dHL-60 (differentiated human neutrophil-like) cells and CRACR2A-derived peptides were used in flow cytometry, immunoprecipitation, cytosolic Ca2+ mobilization, and flow chamber assays to elucidate the molecular mechanism. Four-dimensional confocal intravital microscopy of mice after focal brain ischemia and single neutrophil behavioral analysis demonstrated the pathologic role of neutrophil cracr2a in brain damage. RESULTS Compared with wild-type control mice, cracr2a conditional knockout mice exhibited significantly reduced adhesion, crawling, and transmigration of neutrophils on ear and cremaster venules in tumor necrosis factor-α-induced sterile inflammation. Neutrophil cracr2a rapidly interacts with STIM1 (stromal interaction molecule 1) after agonist stimulation and facilitates Ca2+ mobilization, increasing the ligand-binding function of β2 integrin. Our findings in cracr2a-deficient mouse neutrophils are recapitulated in dHL-60 cells, in which CRACR2A is deleted by CRISPR/Cas9. Furthermore, overexpression of CRACR2A in CRACR2A KO dHL-60 cells restores normal function. Using a series of peptides covering the coiled-coil region of CRACR2A, we identified a palmitoylated 20-mer that blocks STIM1-CRACR2A interaction. Treating neutrophils with this 20-mer inhibits Ca2+ mobilization and β2 integrin activation after agonist stimulation, reducing neutrophil recruitment to an activated endothelial cell monolayer under venous shear stress and to ear venules in tumor necrosis factor-α-challenged mice. Cerebral 4-dimensional intravital microscopy of mice after focal brain ischemia revealed that neutrophil cracr2a enhances the emergence of highly migratory neutrophils by increasing the surface level of αMβ2 integrin, thereby facilitating neutrophil infiltration into brain tissue and exacerbating brain injury. CONCLUSIONS Our results demonstrate that neutrophil CRACR2A promotes neutrophil recruitment to sites of sterile inflammation, such as ischemic stroke. Blocking the STIM1-CRACR2A interaction may be a novel therapeutic strategy to mitigate inflammation and consequent tissue injury.

中文翻译：

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中性粒细胞CRACR2A促进无菌性炎症和缺血性中风中的中性粒细胞募集。


背景 Ca2+ 释放激活的 Ca2+ 通道调节因子 2A （CRACR2A） 与人类 T 细胞功能障碍引起的免疫缺陷有关。我们发现中性粒细胞CRACR2A通过促进 Ca2 + 动员和 β 2 整合素激活来促进中性粒细胞粘附和迁移功能。方法 采用髓系特异性 cracr2a 条件性敲除小鼠和活体显微镜检查研究中性粒细胞 cracr2a 在血管炎症中中性粒细胞募集中的生理作用。Cracr2a 缺陷的中性粒细胞或 dHL-60 （分化的人中性粒细胞样）细胞和 CRACR2A 衍生肽用于流式细胞术、免疫沉淀、胞质 Ca2+ 动员和流式室测定，以阐明分子机制。局灶性脑缺血后小鼠的四维共聚焦活体显微镜检查和单个中性粒细胞行为分析表明中性粒细胞 cracr2a 在脑损伤中的病理作用。结果 与野生型对照小鼠相比，在肿瘤坏死因子 α 诱导的无菌炎症中，cracr2a 条件性敲除小鼠在耳和耳突小静脉上的中性粒细胞粘附、爬行和迁移显着减少。中性粒细胞 cracr2a 在激动剂刺激后与 STIM1 （基质相互作用分子 1） 迅速相互作用，并促进 Ca2+ 动员，增加 β2 整合素的配体结合功能。我们在 cracr2a 缺陷小鼠中性粒细胞中的发现在 dHL-60 细胞中进行了概括，其中 CRACR2A 被 CRISPR/Cas9 删除。此外，CRACR2A KO dHL-60 细胞中 CRACR2A 的过表达可恢复正常功能。使用覆盖 CRACR2A 卷曲螺旋区域的一系列肽，我们鉴定了一种阻断 STIM1-CRACR2A 相互作用的棕榈酰化 20 聚体。 用这种 20 聚体处理中性粒细胞可抑制激动剂刺激后的 Ca2 + 动员和 β 2 整合素活化，从而减少中性粒细胞在静脉剪切应激下募集到活化的内皮细胞单层和肿瘤坏死因子α攻击小鼠的耳小静脉。局灶性脑缺血后小鼠脑 4 维活体显微镜检查显示，中性粒细胞 cracr2a 通过增加 αMβ2 整合素的表面水平来增强高度迁移性中性粒细胞的出现，从而促进中性粒细胞浸润脑组织并加剧脑损伤。结论 我们的结果表明，中性粒细胞CRACR2A促进中性粒细胞募集到无菌性炎症部位，例如缺血性中风。阻断 STIM1-CRACR2A 相互作用可能是减轻炎症和随之而来的组织损伤的新型治疗策略。