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: 9927561
• 负责人: BRYAN P HURLEY
• 金额: $ 59.34万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9927561
• 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; Cultured Cells; Cystic Fibrosis; Disease; Eicosanoids; Enzymes; Epithelial; Epithelial Cells; Epithelium; Event; Exhibits; Failure; 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 Inflammation; 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; Resolution; Respiratory System; Role; Signal Transduction; Source; Stroke; Structure; Surface; Synthetic Genes; System; Techniques; Therapeutic; Tissues; Virulence Factors; Work; acute infection; airway epithelium; 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.

项目摘要/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特征是中性粒细胞大量聚集到 空域。感染触发了一个过程，中性粒细胞从循环中迁移到空域，在那里 然而，正如观察到的那样，它们面对的是粘膜入侵者，这可能是过度的，并导致组织损伤 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呼吸道和 控制这一过程的分子机制正在探索中。使用小鼠和人类的初级和 在通透性Transwell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 跨上皮间迁移可被模拟为体外共培养。肺上皮细胞的治疗 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 通过脂肪氧合酶转化为肝素A3(HxA3)。HxA3在肺上皮细胞的顶面释放 引导中性粒细胞通过上皮屏障的单层。中性粒细胞已经越过屏障迁移 随后通过不同的脂氧合酶活性释放白三烯B4(LTB4)。LTB4实质上 增加了这种迁徙过程的规模，导致大量的 中性粒细胞。这项建议旨在建立在目前对HxA3潜在机制的理解基础上 和LTB4在上皮细胞和中性粒细胞中的合成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细胞迁移。基因敲除小鼠和删除的分子技术 上皮细胞、中性粒细胞和细菌中的磷脂酶A2和脂氧合酶基因将被用来精确定位 主导这一过程的酶。通过以下途径触发二十烷类化合物生成的上游信号事件 磷脂酶和脂氧合酶也将被提及。一种差异化的气液界面培养体系 已经建立了从原代呼吸道基底干细胞衍生的细胞，并将其与先进的成像技术配对以建立 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作为重要的中性粒细胞趋化信号协同作用于呼吸道的假说 在活体中驱动中性粒细胞跨上皮迁移的粘膜将通过使用小鼠进行严格评估 铜绿假单胞菌急性肺炎模型的建立。中性粒细胞将被招募到老鼠的空域 在存在和不存在二十烷类合成基因的情况下，以组织特异性的方式以及在 对特定干扰HxA3或LTB4的外源性拮抗剂进入空域的反应。 总的来说，这项建议试图阐明关键基因和表达这些关键基因的细胞，这些关键基因是 参与协调感染诱导的炎症途径，最终导致中性粒细胞破裂 保护性的粘膜屏障。实现这些目标对发展具有巨大的潜力。 一种新型的抗炎疗法，可以减轻黏膜处的破坏性肺部炎症。