The role of Gasdermin E (DFNA5) in bacterial keratitis

项目摘要 Gasdermin(GSDM)A-E包括一个与感染相关的成孔蛋白家族， 炎症和癌症。RNAseq分析显示，唯一表达的GSDM家族成员 中性粒细胞有Gsdmd和Gsdme/DFNA5，初步数据表明，中性粒细胞可以激活caspase-1。 3，它将Pro-GSDME裂解到成孔的N-GSDME上，以驱动质膜孔的形成 导致前炎性嗜酸性细胞死亡。这与我们自然通讯的结果形成了鲜明对比 2020年的一篇论文表明，中性粒细胞中由caspase-1和中性粒细胞弹性蛋白酶激活的GSDMD发生在 无嗜酸细胞死亡。GSDMD和GSDME均介导中性粒细胞产生生物活性IL-2 1β，我们报道了它是宿主免疫反应中的关键细胞因子，需要 调节肺炎链球菌和铜绿假单胞菌的细菌生长和角膜混浊。 我们建议表征中性粒细胞对肺炎链球菌的GSDME调节和功能。 和铜绿假单胞菌，并确定GSDME在这些致病菌引起的角膜炎中的体内作用(S)。 细菌。目标1将描述GSDME在中性粒细胞中的调节和功能，并确定其在 协调中性粒细胞嗜酸细胞死亡和中性粒细胞胞外陷阱的形成 肺炎或铜绿假单胞菌。目标2将描述感染的角膜中GSDME的处理过程，并评估 GSDME-/-和GSDME-//GSDMD-/-小鼠中性粒细胞GSDME在肺炎链球菌或铜绿假单胞菌中的作用 生存能力、细菌杀灭和角膜疾病的严重程度，并将使用过继转移模型 检测中性粒细胞GSDME的作用。拟议研究的结果将使我们深入了解 这些中性粒细胞中的孔形成蛋白对这些眼部病原体的反应，并将识别潜在的 细菌性角膜炎免疫干预的靶点。

PROJECT ABSTRACT Gasdermin (GSDM) A-E comprise a family of pore-forming proteins that are associated with infection, inflammation and cancer. RNAseq analysis revealed that the only GSDM family members expressed by neutrophils are Gsdmd and Gsdme/DFNA5, and preliminary data show that neutrophils can activate caspase- 3, which cleaves pro- GSDME to the pore-forming N-GSDME to drive formation of plasma membrane pores leading to pro-inflammatory pyroptotic cell death. This is in contrast to results from our Nature Communications 2020 paper showing that that GSDMD activated by caspase-1 and neutrophil elastase in neutrophils occurs in the absence of pyroptotic cell death. Both GSDMD and GSDME mediate neutrophil production of bioactive IL- 1β, which we reported is a pivotal cytokine in the host immune response that is required to regulate Streptococcus pneumoniae and Pseudomonas aeruginosa bacterial growth and corneal opacification. We propose to characterize GSDME regulation and function in neutrophils in response to S. pneumoniae and P. aeruginosa in vitro, and to define the vivo role(s) of GSDME in keratitis caused by these pathogenic bacteria. Aim 1 will characterize GSDME regulation and function in neutrophils and define its role in coordinating neutrophil pyroptotic cell death and formation of neutrophil extracellular traps in response to S. pneumoniae or P. aeruginosa. Aim 2 will characterize GSDME processing in infected corneas, and assess the role of GSDME in S. pneumoniae or P. aeruginosa using GSDME-/- and GSDME-/-/GSDMD-/- mice in neutrophil viability, bacterial killing and the severity of corneal disease, and will use an adoptive transfer model to examine the role of neutrophil GSDME. Results of the proposed studies will provide insight into the role of these pore forming proteins in neutrophil responses to these ocular pathogens, and will identify potential targets for immune intervention in bacterial keratitis.