The role of hepoxilin A3 in neutrophil breach of the Infected airway mucosa.

项目概要/摘要 粘液上皮表面是保护免受外部威胁的物理和免疫屏障。一 呼吸道中感染性炎性疾病的标志是嗜中性粒细胞大量积聚到 领空。感染触发了一个过程，中性粒细胞从循环迁移到空气中， 然而，它们面对粘膜入侵者，这可能是过度的，并有助于组织损伤，如观察到的 肺炎和囊性纤维化神经纤维素突破粘膜上皮屏障到达气道， 控制这一过程的分子机制正在探索中。使用小鼠和人的主要和 转化极化肺上皮细胞在可渗透的Transwell过滤器上培养，细菌诱导的中性粒细胞 跨上皮迁移可以模拟为体外共培养。用本发明的化合物治疗肺上皮细胞 细菌病原体铜绿假单胞菌激活磷脂酶A2，释放花生四烯酸。花生四烯酸 通过脂氧合酶转化为肝氧素A3（HxA 3）。HxA 3在肺上皮细胞的顶面释放 引导中性粒细胞穿过上皮屏障的单层。穿过屏障的中性粒细胞 随后通过不同脂氧合酶活性释放白三烯B4（LTB 4）。LTB 4基本上 增加了这种迁移过程的程度，导致大量的 中性粒细胞本文的这一提议旨在建立在目前对HxA 3潜在机制的理解基础上 和LTB 4合成分别在上皮细胞和中性粒细胞，以及这些事件如何协调中性粒细胞 铜绿假单胞菌引起的跨上皮迁移。基因敲除小鼠和分子技术删除 上皮细胞、中性粒细胞和细菌中的磷脂酶A2和脂氧合酶基因，将用于精确定位 主要的酶驱动这个过程。上游信号传导事件，其通过以下途径触发类花生酸生成： 磷脂酶和脂氧合酶也将被提及。一种分化型气液界面培养系统， 来源于原代气道基底干细胞的干细胞，并与先进的成像技术相结合， 细菌诱导的中性粒细胞跨上皮迁移，并评估分子和细胞机制。最后， 假设HxA 3与LTB 4协同作用作为在气道中起作用的关键中性粒细胞趋化信号， 将通过使用小鼠来严格评估粘膜在体内驱动中性粒细胞跨上皮迁移的能力。 铜绿假单胞菌诱导的急性肺炎模型。在老鼠领空招募神经元 在类花生酸合成基因存在和不存在的情况下以组织特异性方式分析， 对特异性干扰HxA 3或LTB 4的外源递送拮抗剂的反应。 总的来说，这项建议旨在阐明关键基因和表达这些关键基因的细胞，这些基因是 参与策划感染诱导的炎症途径，最终导致嗜中性粒细胞破坏 保护粘膜屏障。这些目标的实现对发展具有巨大潜力 一种新型抗炎治疗剂，可减轻粘膜处的破坏性肺部炎症。

PROJECT SUMMARY / ABSTRACT Mucosal epithelial surfaces are physical and immunological barriers that protect against external threats. A hallmark of infectious inflammatory disease in the respiratory tract is massive accumulation of neutrophils into the airspace. Infection triggers a process whereby neutrophils emigrate from circulation to the airspace where they confront mucosal invaders, however, this can be excessive and contributes to tissue damage as observed during pneumonia and cystic fibrosis. Neutrophil breach mucosal epithelial barriers to reach the airway and the molecular mechanisms that control this process are being explored. Using mouse and human primary and transformed polarized lung epithelial cells cultured on permeable Transwell filters, bacterial-induced neutrophil trans-epithelial migration can be modeled as an in vitro co-culture. Treatment of lung epithelia with the bacterial pathogen P. aeruginosa activates phospholipase A2, releasing arachidonic acid. Arachidonic acid is converted by a lipoxygenase to hepoxilin A3 (HxA3). HxA3 is released at the apical surface of lung epithelial monolayers guiding neutrophils across the epithelial barrier. Neutrophils that have migrated across the barrier subsequently release leukotriene B4 (LTB4) through a distinct lipoxygenase activity. LTB4 substantially augments the magnitude of this migratory process causing breach of the airway barrier by large numbers of neutrophils. This proposal herein aims to build upon current understanding of mechanisms underlying HxA3 and LTB4 synthesis in epithelial cells and neutrophils respectively and how these events orchestrate neutrophil trans-epithelial migration in response to P. aeruginosa. Knockout mice and molecular techniques to delete phospholipase A2 and lipoxygenase genes in epithelial cells, neutrophils, and bacteria, will be used to pinpoint dominant enzymes driving this process. Upstream signaling events that trigger eicosanoid generation through phospholipases and lipoxygenases will also be addressed. A differentiated air-liquid interface culture system derived from primary airway basal stem cells has been established and paired with advanced imaging to model bacterial-induced neutrophil trans-epithelial migration and assess molecular and cellular mechanisms. Finally, the hypothesis that HxA3 collaborates with LTB4 as key neutrophil chemotactic signals operative at airway mucosa to drive neutrophil trans-epithelial migration in vivo will be critically evaluated by employing a mouse model of P. aeruginosa-induced acute pneumonia. Neutrophil recruitment into mouse airspace will be analyzed in the presence and absence of eicosanoid synthetic genes in a tissue specific manner as well as in response to exogenously delivered antagonists into the airspace that specifically interfere with HxA3 or LTB4. Collectively, this proposal seeks to elucidate key genes and the cells that express these key genes, which are involved in orchestrating an infection-induced inflammatory pathway culminating in neutrophilic breach of protective mucosal barriers. Achievement of these objectives holds tremendous potential towards developing a novel class of anti-inflammatory therapeutics that can alleviate destructive lung inflammation at the mucosa.