Mechanisms of host protection against pathogen-associated proteases in acute lung injury

项目摘要/摘要：广泛的，长期的目标是确定主机保护机制， 对抗病原体引发的肺部炎症和损伤。许多病原体分泌蛋白酶， 但细菌、真菌和病毒也可以吸收宿主蛋白酶，增加致病性 并促进肺损伤。我们研究了铜绿假单胞菌（PA）作为感染诱导的 损伤，以探测病原体编码的蛋白酶称为假单胞菌弹性蛋白酶LasB，金属蛋白酶 通过PA II型分泌系统释放到细胞外环境中，诱导肺组织损伤， 宿主来源的丝氨酸蛋白酶如中性粒细胞弹性蛋白酶（NE）的次级触发物。我们之前发现 血小板反应蛋白-1（TSP-1）是一种由多种细胞在损伤后分泌的基质细胞蛋白， 病原体编码的LasB和宿主蛋白酶NE，以限制肺损伤和炎症。关键问题 从这项工作中产生的是不受调节的蛋白水解环境如何驱动过度的炎症反应。 损伤后的反应和失调的修复，以及校准这种反应的宿主因素是什么？ 肺我们的初步研究结果表明，前馈嗜酸性炎症反应发生在 PA感染的蛋白水解环境通过IL-36γ的N末端加工，其在PA感染中被夸大。 缺乏TSP-1。此外，血小板TSP-1似乎对PA诱导的肺损伤有保护作用，但精确的 与TSP-1在肺泡-毛细血管界面的作用相关的机制仍然未知。此外，我们显示 临床菌株中PA弹性蛋白酶活性导致小鼠过度炎症和损伤， 与非弹性蛋白酶生产者相比，临床结果更差。基于这些发现，我们 利用遗传缺陷小鼠、细胞特异性条件性敲除系统提出以下目的，以及 从ICU获得的PA临床呼吸道分离株（1）阐明TSP-1 对抗由促炎细胞因子的蛋白水解加工介导的炎症反应 IL-36γ;（2）检查TSP-1和血小板TSP-1在保护肺泡屏障中的贡献 肺损伤后早期临时基质的破坏和稳定;（3）确定PA ICU是否 具有弹性蛋白溶解特性的呼吸道分离株会导致不必要的炎症和持续的组织损伤， 易感宿主更好地了解严重呼吸道感染期间的宿主生物学可能是有用的 在针对病原体衍生的蛋白酶和失调的宿主的靶向治疗的合理设计中， 炎症作为当前抗菌药物的替代品和支持性肺部和重症监护 管理

Project Summary/Abstract: The broad, long term objective is to identify host-protective mechanisms that counter pathogen-initiated lung inflammation and injury. Many pathogens secrete proteases to cause direct damage to the lung, but bacteria, fungi and viruses can also co-opt host proteases to increase pathogenicity and promote lung injury. We have studied Pseudomonas aeruginosa (PA) as a model of infection-induced injury to probe how a pathogen-encoded protease called Pseudomonas elastase LasB, a metalloprotease released into the extracellular milieu by the PA type II secretion system, induces lung tissue damage and secondary trigger of host-derived serine proteases such as neutrophil elastase (NE). We previously identified thrombospondin-1 (TSP-1), a matricellular protein secreted by a variety of cells following injury that disarms both pathogen-encoded LasB and host protease NE, to limit lung injury and inflammation. Key questions that have arisen from this work is how an unregulated proteolytic environment drives excessive inflammatory response and dysregulated repair following injury, and what are the host factors that calibrate this response in the lung. Our preliminary findings suggest that a feed-forward neutrophilic inflammatory response occurs in the proteolytic environment of PA infection through N-terminal processing of IL-36γ that is exaggerated in the absence of TSP-1. Moreover, platelet TSP-1 appears protective against PA-induced lung injury, but the precise mechanism related to TSP-1's role at the alveolar-capillary interface remains unknown. Furthermore, we show that PA elastase activity in clinical strains confer excessive inflammation and injury in mice and is associated with worse clinical outcomes when compared with non-elastase producers. Based upon these findings, we propose the following aims utilizing genetically deficient mice, cell-specific conditional knockout systems, and PA clinical respiratory isolates obtained from the ICU to (1) elucidate the mechanisms by which TSP-1 counters the hyperinflammatory response mediated by proteolytic processing of the pro-inflammatory cytokine IL-36γ; (2) examine the contribution of TSP-1 and platelet TSP-1 in protection against alveolar barrier disruption and stabilization of the early provisional matrix following lung injury; (3) determine whether PA ICU respiratory isolates with elastolytic properties drive unwarranted inflammation and persistent tissue injury in the susceptible host. A better understanding of host biology during severe respiratory infection could prove useful in the rational design of targeted therapeutics against pathogen-derived proteases and deregulated host inflammation as adjuncts to current antimicrobial agents and supportive pulmonary and critical care management.