The mucosal epithelial surface serves as a protective barrier to external threats both in the physical sense and as a vital contributor to immune surveillance and orchestration. One of the hallmarks of infectious inflammatory mucosal disease in the respiratory tract is massive mobilization of neutrophils across the mucosal barrier into the airway lumen. Encounter with a pathogenic organism triggers a process whereby neutrophils migrate from the bloodstream to airspace in order to directly confront mucosal invaders through engulfment and release of noxious substances. Neutrophil accumulation can become over-exuberant resulting in host lung tissue damage as with the infectious process involving Pseudomonas aeruginosa in the diseases pneumonia and cystic fibrosis. The final step in neutrophil recruitment from the bloodstream to the airway involves migration across the epithelial barrier of the mucosal surface. Our studies have revealed a molecular mechanism that underlies this phenomenon. Using lung epithelial cells grown on permeable Transwell filters, we have modeled neutrophil trans-epithelial migration in response to infection. Our studies have demonstrated that treatment of lung epithelial monolayers with P. aeruginosa results in activation of phospholipase A2, which causes the release of arachidonic acid. Arachidonic acid is then converted to the neutrophil chemo-attractant hepoxilin A3 by the actions of 12-lipoxygenases / hepoxilin synthases. Hepoxilin A3 is released at the apical surface of lung epithelial monolayers and guides neutrophils across the epithelial barrier. In the current application, we aim to further explore the mechanism underlying hepoxilin A3 production in mouse and human lung epithelial cells by identifying both the specific phospholipase A2 and 12-lipoxygenase / hepoxilin synthase enzyme(s) responsible for generating hepoxilin A3 upon infection with pathogen. Furthermore, we propose to establish air-liquid interface cultures of primary mouse and human lung epithelial cells as well as lung epithelial cells with defective cystic fibrosis transmembrane conductance regulator. The ability of P. aeruginosa to instigate hepoxilin A3 production and neutrophil trans-epithelial migration will be assessed in these air-liquid models. Finally, we will evaluate hepoxilin A3 as a neutrophil chemo-attractant operating at the airway mucosal surface in vivo employing a mouse model of P. aeruginosa pneumonia. Multiple interventions to interfere with hepoxilin A3 synthesis, stability, and action will be utilized and correlated with outcomes that include severity of pathological tissue damage, expression of 12-lipoxygenases, and neutrophil accumulation in the infected airspace. These studies aim to further elucidate the role of this newly appreciated eicosanoid neutrophil chemo-attractant termed hepoxilin A3 that is produced at various mucosal surfaces. A better understanding of this novel innate inflammatory pathway holds tremendous promise towards unveiling a distinct class of unexplored targets to exploit therapeutically in order to alleviate destructive lung inflammation.

Public Health Relevance

The objective of this application is to further investigate the hypothesis that hepoxilin A3 represents an important molecule that is critical for directing neutrophils to breach the mucosal epithelial barrier. Information gathered from this work will be instrumental in the development of a novel class of anti- inflammatory therapeutics that targets the destructive neutrophil driven tissue injury that occurs in a wide array of lung inflammatory diseases, both infectious and otherwise.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Host Interactions with Bacterial Pathogens Study Section (HIBP)
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Minnicozzi, Michael
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Massachusetts General Hospital
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Kusek, Mark E; Pazos, Michael A; Pirzai, Waheed et al. (2014) In vitro coculture assay to assess pathogen induced neutrophil trans-epithelial migration. J Vis Exp :e50823
Kubala, S A; Patil, S U; Shreffler, W G et al. (2014) Pathogen induced chemo-attractant hepoxilin A3 drives neutrophils, but not eosinophils across epithelial barriers. Prostaglandins Other Lipid Mediat 108:1-8
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