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A3681 - Ahr Promotes Pseudomonas Aeruginosa-Induced Intracellular Infection in Bronchial Epithelial Cells
Author Block: P. Bortolotti1, E. Faure1, J. Berube2, C. Baglole3, S. Rousseau3; 1Medicine, McGill University Research Institute, Montreal, QC, Canada, 2McGill University, Montreal, QC, Canada, 3Meakins-Christie Laboratories, McGill University, Montreal, QC, Canada.
Rationale. Pseudomonas aeruginosa (Pa) is the main pathogen involved in CF lung disease. Pa persists in the airways despite the host immune response and concomitant antibiotic treatments. Pa persistence mechanisms in CF lung is mainly attributed to biofilm formation, but the question of an intracellular epithelial reservoir has recently risen. Persistent Pa infection triggers a dysregulated proinflammatory host response with enhanced recruitment of polymorphonuclear cells which is responsible for tissue damage and airways remodeling, leading to the impairment of lung function and mortality. Previous studies showed a critical role for the arylhydrocarbon receptor (AhR) in regulating the immune response to pathogens at epithelial barriers. AhR is a ligand-dependent transcription factor involved in the regulation of innate immunity related genes. AhR also supports non-genomic functions, including the regulation Src kinase activity. Understanding the role of the AhR in the Pa-induced intracellular epithelial infection and its subsequent immune response could set the stage for the development of adjuvant therapeutic strategies aiming to decrease inflammation while leaving intact innate immune signaling. Methods. CFBE∆F508, CFBE and Beas2B cells were exposed to Pa (PaO1 or clinical CF strain CHA) at a multiplicity of infection of 2. To study the involvement of Ahr, cells were treated with synthetic AhR agonists or an AhR inhibitor CH223191. AhR knock-down cells via CRISPR technique were also used. Involvement of Src kinase was evaluated using Src kinase family inhibitors. After 4 hours, cells were treated with tobramycin to kill extracellular bacteria while preserving internalized bacteria. Intracellular bacterial count was performed at 4 hours and 24 hours by serial dilution of cell lysates on agar plates. In the same time, bacteria-induced cytotoxicity was measured by LDH release. Epithelial response was assessed by IL-8 and ICAM-1 gene expression. Additionally, IL-8 and ICAM-1 proteins production was measured by respectively ELISA and flow cytometry. Results. AhR activation enhances Pa internalization at 4 hours in each cell type. The AhR-mediated increase in Pa internalization involves Src kinase-like activity. AhR activation also promotes intracellular persistence at 24 hours. Intracellular Pa infection is associated with a pro-inflammatory immune response characterized by increased IL-8 and ICAM-1 production at both transcriptional and post-transcriptional levels. Pa intracellular infection by itself enhanced AhR activity, suggesting a possible self-sustained mechanism. Conclusion. AhR is critically involved in the pathogenesis of Pa intracellular epithelial cell infection through enhancement of Pa internalization and Pa intracellular persistence, promoting a pro-neutrophilic inflammatory immune response.