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A3682 - LTA4 Hydrolase Directs Bacterial-Induced Neutrophil Transepithelial Migration
Author Block: L. Yonker, R. Hibbler, H. Mou, H. Leung, K. Gipson, B. Lanter, A. Eaton, G. J. Tearney, B. P. Hurley; Massachusetts General Hospital, Boston, MA, United States.
Rationale: Infected airway epithelial cells apically release hepoxilin A3 (HxA3), a bioactive lipid and neutrophil chemoattractant, initiating neutrophil transepithelial migration. Migrated neutrophils release leukotriene B4 (LTB4) synthesized via cPLA2α and 5-lipoxygenase activity, augmenting neutrophil chemotactic responses. Certain diseases, such as cystic fibrosis or acute respiratory distress syndrome (ARDS) exhibit excessive, detrimental neutrophil inflammatory responses, exacerbating airway disease. Reducing airway inflammation by targeting LTB4 synthesis is a promising therapeutic strategy but effective approaches remains elusive. LTA4 hydrolase (LTA4H) confers the final enzymatic step in LTB4 production via its epoxide hydrolase activity. However, LTA4H is bi-functional exhibiting aminopeptidase activity responsible for degrading a distinct neutrophil chemoattractant, proline-glycine-proline. Further, although epithelial cells cannot independently make LTB4, they do express LTA4H; the role of epithelial LTA4H in airway infection is not well defined. We assessed the balance of anti-inflammatory and pro-inflammatory properties of both neutrophil and epithelial LTA4H in the context of bacterial-induced neutrophil transepithelial migration.
Methods: Polarized airway epithelial cells were grown on the underside of permeable transwells. Neutrophils isolated from healthy volunteers were placed in the basolateral compartment and migrated towards apical signals released by Pseudomonas aeruginosa (PAO1)-infected epithelial cells or imposed chemotactic gradients (IL8). Neutrophils and epithelial cells were pre-treated with LTA4H inhibitors prior to migration. Total neutrophil migration was quantified using myeloperoxidase assays. LTB4 production was quantified by ELISA. Micro-Optical Coherence Tomography (µOCT) imaging allowed real-time visualization of PAO1-induced neutrophil transepithelial migration at 1µm resolution, without fixation or staining.
Results: Pre-treatment of neutrophils or epithelium with LTA4H epoxide hydrolase inhibitor, ARM1, reduced PAO1-induced neutrophil transepithelial migration in a dose dependent manner, and this effect was additive following combined neutrophil/epithelial treatment. In contrast, pre-treatment of neutrophils or epithelium with LTA4H aminopeptidase inhibitor, bestatin, resulted in no observable decrease in PAO1-induced neutrophil trans-epithelial migration. µOCT imaging of PAO1-induced transmigration following LTA4H epoxide hydrolase inhibition reveals diminished neutrophilic breach of the epithelium.
Conclusions: Our findings suggest that both neutrophil- and epithelial-derived LTA4H play a key role in bacterial-induced neutrophil transepithelial migration through its targeting of LTA4 to generate LTB4. Our findings suggest that the epoxide activity of LTA4H, but not the aminopeptidase activity, contributes to bacterial-induced neutrophil transepithelial migration. Further understanding the role of LTA4H as it pertains to airway inflammation is critical for understanding how to optimally exploit LTA4H as a therapeutic target and will guide future anti-inflammatory therapeutic development.