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.

Public Health Relevance

The objective of this proposal is to investigate the hypothesis that the bioactive lipid molecules (hepoxilin A3 and leukotriene B4) operate in tandem to direct neutrophils to breach the mucosal epithelial barrier during bacterial infection. The specific genes and the distinct cell types that express the specific genes responsible for synthesizing these lipid eicosanoid molecules will be identified as will their role in bacterial induced neutrophil trans-epithelial migration. Information gathered from this work will be instrumental in developing a novel therapeutic approach that effectively and exquisitely targets this inflammatory axis thereby alleviating destructive neutrophil driven tissue injury, which 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)
Project #
Application #
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Minnicozzi, Michael
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Massachusetts General Hospital
United States
Zip Code
Levardon, Hannah; Yonker, Lael M; Hurley, Bryan P et al. (2018) Expansion of Airway Basal Cells and Generation of Polarized Epithelium. Bio Protoc 8:
Drenkard, Eliana; Hibbler, Rhianna M; Gutu, D Alina et al. (2018) Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants. J Vis Exp :
Pazos, Michael A; Lanter, Bernard B; Yonker, Lael M et al. (2017) Pseudomonas aeruginosa ExoU augments neutrophil transepithelial migration. PLoS Pathog 13:e1006548
Yonker, Lael M; Pazos, Michael A; Lanter, Bernard B et al. (2017) Neutrophil-Derived Cytosolic PLA2? Contributes to Bacterial-Induced Neutrophil Transepithelial Migration. J Immunol 199:2873-2884
Yonker, Lael M; Mou, Hongmei; Chu, Kengyeh K et al. (2017) Development of a Primary Human Co-Culture Model of Inflamed Airway Mucosa. Sci Rep 7:8182
Chu, Kengyeh K; Kusek, Mark E; Liu, Linbo et al. (2017) Illuminating dynamic neutrophil trans-epithelial migration with micro-optical coherence tomography. Sci Rep 8:45789
Yonker, Lael M; Cigana, Cristina; Hurley, Bryan P et al. (2015) Host-pathogen interplay in the respiratory environment of cystic fibrosis. J Cyst Fibros 14:431-439
Pazos, Michael A; Pirzai, Waheed; Yonker, Lael M et al. (2015) Distinct cellular sources of hepoxilin A3 and leukotriene B4 are used to coordinate bacterial-induced neutrophil transepithelial migration. J Immunol 194:1304-15
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
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

Showing the most recent 10 out of 14 publications