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性角膜炎的重要原因。中性粒细胞在角膜感染和创伤及角膜烧伤中起重要作用，不仅用于杀死微生物和组织损伤，而且作为促炎细胞因子的主要来源。 我们已经表明，IL-1β是调节细菌性角膜炎所需的宿主免疫应答中的关键细胞因子。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输出。非典型IL-1β释放的关键介质是gasdermin D（GSDMD），GSDMD是一种胞质蛋白，在炎性小体信号传导期间被半胱天冬酶-1切割，产生在质膜中形成大孔的片段;这些片段可以直接介导IL-1β的流出，但也诱导热解裂解。 我们的初步数据表明，中性粒细胞中的GSDMD是IL-1β分泌所必需的，并被caspase-1裂解。然而，与巨噬细胞相反，中性粒细胞不经历焦亡或在其质膜中积累GSDMD孔，相反，裂解的GSDMD似乎与细胞内细胞器（包括自噬体）缔合，以可能通过分泌性自噬促进IL-1β释放。我们假设，N-GSDMD运输的这种重定向用于在角膜炎期间保持中性粒细胞活力以直接杀死细菌，同时仍然允许IL-1β释放，其维持局部炎症直到细菌被清除。尽管中性粒细胞在初始炎性小体激活期间抵抗焦亡，但其他初步数据和最近的报告表明，GSDMD可以在持续的炎性小体信号传导期间介导嗜中性粒细胞特异性细胞死亡途径。我们假设，这些替代GSDMD细胞死亡途径有助于降低中性粒细胞活力，抑制炎症，并在恢复角膜透明度所需的细菌性角膜炎消退期增加中性粒细胞清除。我们还在细菌感染的小鼠角膜中鉴定了一群IL-1β炎性单核细胞，它们可能对感染的结果有显著影响。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将验证浸润的炎性单核细胞通过产生IL-1β、调节和清除中性粒细胞以及恢复角膜透明度来调节细菌性角膜炎的假设。目的2将检验中性粒细胞中GSDMD依赖性IL-1β分泌由分泌性自噬途径中的特异性运输蛋白介导的假设。目的3将验证嗜天青颗粒在S.金黄色葡萄球菌可以切割GSDMD，介导中性粒细胞死亡途径，与非半胱天冬酶-1依赖性IL-1β的产生相协调。拟议的研究结果将确定自噬和GSDMD在中性粒细胞和单核细胞在细菌性角膜炎的功能。我们预计，这些数据也将确定可用于角膜感染的药物干预的目标。

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.