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

赫泊西林 A3 在中性粒细胞破坏受感染气道粘膜中的作用。

• 批准号: 10404506
• 负责人: BRYAN P HURLEY
• 金额: $ 59.24万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-08 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10404506
• 关键词: AIDS/HIV problem; Achievement; Acute; Acute Pneumonia; Address; Age; Air; Anti-Inflammatory Agents; Apical; Arachidonic Acids; Automobile Driving; Bacteria; Bacterial Infections; Bacterial Model; Blood Circulation; Bone Marrow; CRISPR/Cas technology; CXC Chemokines; Cells; Chemotactic Factors; Child; Chronic; Coculture Techniques; Complement 5a; Complex; Consequentialism; Cystic Fibrosis; Disease; Eicosanoids; Enzymes; Epithelial; Epithelial Cells; Event; Exhibits; Generations; Genes; Hereditary Disease; Human; Image; Immunologics; In Vitro; Individual; Infection; Inflammasome; Inflammation; Inflammatory; Investigation; Knockout Mice; Leukotriene B4; Lipids; Lipoxygenase; Liquid substance; Lung; Lung infections; Malaria; Malignant Neoplasms; Mediating; Mediator of activation protein; Microbe; Modeling; Molecular; Mucous Membrane; Mus; Myocardial Infarction; Neutrophil Infiltration; Pathologic; Pathway interactions; Permeability; Phospholipase; Phospholipase A2; Play; Pneumonia; Process; Protein Isoforms; Pseudomonas aeruginosa; Pulmonary Inflammation; Resolution; Respiratory Failure; Respiratory Mucosa; Respiratory System; Role; Signal Transduction; Source; Stroke; Surface; Synthetic Genes; System; Techniques; Therapeutic; Tissues; Virulence Factors; Work; acute infection; airway epithelium; antagonist; cell type; design; driving force; global health; in vivo; in vivo Model; inflammatory lung disease; inflammatory modulation; member; migration; monolayer; mouse model; neutrophil; novel; novel therapeutic intervention; pathogenic bacteria; pneumonia model; response; stem cells; targeted treatment; tissue injury

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.

项目概要/摘要 粘膜上皮表面是抵御外部威胁的物理和免疫屏障。一个 呼吸道感染性炎症性疾病的标志是中性粒细胞大量积聚 空域。感染触发一个过程，中性粒细胞从循环系统迁移到空气空间，其中 它们面对粘膜入侵者，然而，这可能过度并导致观察到的组织损伤 肺炎和囊性纤维化期间。中性粒细胞突破粘膜上皮屏障到达气道并 正在探索控制这一过程的分子机制。使用小鼠和人类初级和 在可渗透的 Transwell 过滤器上培养的转化极化肺上皮细胞，细菌诱导的中性粒细胞 跨上皮迁移可以建模为体外共培养。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。花生四烯酸是 由脂氧合酶转化为Hepoxilin A3 (HxA3)。 HxA3 在肺上皮顶端表面释放 单层引导中性粒细胞穿过上皮屏障。已迁移穿过屏障的中性粒细胞 随后通过独特的脂氧合酶活性释放白三烯 B4 (LTB4)。 LTB4 实质上 增加了这种迁移过程的规模，导致大量的气道屏障突破 中性粒细胞。本文的这项提议旨在建立在当前对 HxA3 基础机制的理解之上 和 LTB4 分别在上皮细胞和中性粒细胞中合成，以及这些事件如何协调中性粒细胞 响应铜绿假单胞菌的跨上皮迁移。基因敲除小鼠和分子删除技术 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 驱动这一过程的主要酶。通过上游信号事件触发类二十烷酸生成 磷脂酶和脂氧合酶也将得到讨论。差异化的气液界面培养系统 源自初级气道基底干细胞的细胞已经建立，并与先进的成像技术相结合进行建模 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， HxA3 与 LTB4 协同作为气道中关键的中性粒细胞趋化信号的假设 将通过使用小鼠对粘膜驱动中性粒细胞跨上皮迁移的体内进行严格评估 铜绿假单胞菌诱发的急性肺炎模型。中性粒细胞募集到小鼠空域将 以组织特异性方式以及在是否存在类二十烷酸合成基因的情况下进行分析 对外源递送到空域的拮抗剂的反应，这些拮抗剂专门干扰 HxA3 或 LTB4。 总的来说，该提案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 参与协调感染诱导的炎症途径，最终导致中性粒细胞破坏 保护性粘膜屏障。这些目标的实现为发展提供了巨大的潜力 一类新型抗炎疗法，可以减轻粘膜处的破坏性肺部炎症。

项目成果

Host-pathogen interplay in the respiratory environment of cystic fibrosis.

• DOI: 10.1016/j.jcf.2015.02.008
• 发表时间: 2015-07
• 期刊: Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society
• 作者: Yonker LM;Cigana C;Hurley BP;Bragonzi A
• 通讯作者: Bragonzi A