Innate immunity in bacterial keratitis

铜绿假单胞菌、肺炎链球菌和金黄色葡萄球菌是世界范围内引起微生物角膜炎的重要原因。中性粒细胞在角膜感染、创伤和角膜烧伤中发挥着重要作用，不仅可以杀死微生物和破坏组织，还可以作为促炎细胞因子的主要来源。我们已经证明，IL-1β是调节细菌性角膜炎所需的宿主免疫反应中的关键细胞因子。IL-1β缺乏通过经典分泌途径释放的信号序列，因此需要非常规途径进行输出。GasderminD(GSDMD)是非经典IL-1β释放的关键介质，它是一种胞浆蛋白，在炎症信号转导过程中被Caspase-1裂解，产生片段，形成质膜大孔；这些片段可以直接介导IL-1β的外流，也可以诱导焦链溶解。我们的初步数据显示，中性粒细胞中的GSDMD是分泌IL-1β所必需的，并被Caspase-1所切割。然而，与巨噬细胞不同的是，中性粒细胞不会发生下垂，也不会在质膜上堆积GSDMD孔，相反，裂解的GSDMD似乎与包括自噬在内的细胞内细胞器有关，可能通过分泌自噬促进IL-1β的释放。我们推测，N-GSDMD转运的这种重新定向有助于保护中性粒细胞在角膜炎期间直接杀死细菌的能力，同时仍然允许IL-1β的释放，从而维持局部炎症，直到细菌被清除。虽然中性粒细胞在最初的炎性小体激活时抵抗下垂，但其他初步数据和最近的报道表明，在持续的炎性小体信号传递过程中，GSDMD可以介导中性粒细胞特异性的细胞死亡途径。我们推测，在恢复角膜透明度所需的细菌性角膜炎缓解阶段，这些替代的GSDMD细胞死亡途径有助于降低中性粒细胞的活性，抑制炎症，并增加中性粒细胞的清除。我们还在细菌感染的小鼠角膜中发现了一组IL-1β炎性单核细胞，这可能对感染的结果有重要影响。为了描述和扩展我们的新发现，我们提出了三个目标。目的1验证炎性单核细胞通过产生IL-1β、调节和清除中性粒细胞、恢复角膜透明度来调节细菌性角膜炎的假说。目的2验证中性粒细胞分泌依赖GSDMD的IL-1β是由分泌自噬途径中的特定运输蛋白介导的假说。目的3将验证一种假说，即金黄色葡萄球菌引起的嗜天青颗粒释放的丝氨酸蛋白酶可以裂解GSDMD，从而协同caspase-1非依赖性IL-1β的产生来介导中性粒细胞死亡途径。建议的研究结果将确定自噬和GSDMD在中性粒细胞和单核细胞中在细菌性角膜炎中的作用。我们预计，这些数据还将确定可用于角膜感染的药物干预的靶点。

Pseudomonas aeruginosa, Streptococcus pneumoniae and Staphylococcus aureus are important causes of microbial keratitis worldwide. Neutrophils play an important role in corneal infections and in trauma and corneal burn injuries, not only for microbial killing and tissue damage, but also as a major source of pro-inflammatory cytokines. We have shown that IL-1β is a pivotal cytokine in the host immune response required to regulate bacterial keratitis. IL-1β lacks a signal sequence for release by the classical secretory pathway and thus requires a non-conventional pathway for export. A key mediator of non-canonical IL-1β release is gasdermin D (GSDMD), a cytosolic protein which is cleaved by caspase-1 during inflammasome signaling to generate fragments that form macropores in the plasma membrane; these can directly mediate efflux of IL-1β but also induce pyroptotic lysis. Our preliminary data show that GSDMD in neutrophils is required for IL-1β secretion and is cleaved by caspase-1. However, in contrast to macrophages, neutrophils do not undergo pyroptosis or accumulate GSDMD pores in their plasma membrane instead, cleaved GSDMD appears to associate with intracellular organelles, including autophagosomes, to possibly facilitate IL-1β release by secretory autophagy. We hypothesize that this redirection of N-GSDMD trafficking serves to preserve neutrophil viability for direct bacterial killing during keratitis, while still permitting the IL-1β release which sustains local inflammation until bacteria are cleared. Although neutrophils resist pyroptosis during initial inflammasome activation, other preliminary data and recent reports suggest that GSDMD can mediate neutrophil-specific cell death pathways during sustained inflammasome signaling. We hypothesize that these alternative GSDMD cell death pathways serve to decrease neutrophil viability, dampen inflammation, and increase neutrophil clearance during the resolving phase of bacterial keratitis required for restoration of corneal clarity. We have also identified a population of IL-1β inflammatory monocytes in bacteria-infected corneas of mice that likely contribute significantly to the outcome of infection. To characterize and extend our new findings, we propose three aims. Aim 1 will test the hypothesis that infiltrating inflammatory monocytes regulate bacterial keratitis by production of IL-1β, regulation and clearance of neutrophils, and restoration of corneal clarity. Aim 2 will test the hypothesis that GSDMD-dependent IL-1β secretion in neutrophils is mediated by specific trafficking proteins in the secretory autophagy pathway. Aim 3 will test the hypothesis that serine proteases released from azurophilic granules in response to S. aureus can cleave GSDMD to mediate neutrophil cell death pathways in coordination with caspase-1-independent IL-1β production. Results of the proposed studies will identify the function of autophagy and GSDMD in neutrophils and monocytes during bacterial keratitis. We anticipate that these data will also identify targets that can be used for pharmacological intervention of corneal infections.