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A7186 - Defect in Translational Control Leads to Hyperresponsive Bronchial Epithelial Cells in Asthma Patients
Author Block: A. Ravi1, S. Chowdhury2, A. Dijkhuis3, P. J. Sterk4, R. Lutter2; 1Respiratory Medicine and Experimental Immunology, Acamedic Medical Center, Amsterdam, Netherlands, 2Respiratory Medicine, Academic Medical Center, Amsterdam, Netherlands, 3Experimental Immunology, Acamedic Medical Center, Amsterdam, Netherlands, 4Academic Medical Center, Amsterdam NL-1105 AZ, Netherlands.
Rationale: We have shown that primary bronchial epithelial cells (PBECs) from mild and severe asthmatics, in comparison to PBECs from healthy individuals, can release significantly higher levels of interleukin-8 (CXCL-8), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF) and macrophage inhibitory factor (MIF) when stimulated with tumor necrosis factor-α (TNF-α) and interleukin-17A (IL-17A). PBECs producing exaggerated amounts of mediators are referred to as hyperresponsive PBECs, as opposed to normoresponsive PBECs with levels comparable to those by PBECs from most healthy controls. In addition, hyperresponsive PBECs are unresponsive to corticosteriods while normoresponsive PBECs are responsive. We aimed to reveal the underlying molecular defect that leads to hyperresponsive PBECs.
Methods: PBECs were cultured from bronchial brushes of mild and severe asthma patients and healthy controls and in parallel bronchoalveolar lavage fluid (BALF) was collected to determine inflammatory parameters. When confluent, PBECs were stimulated with TNF-α and IL-17A, in the presence or absence of dexamethasone for 16 hours. Cytokines were measured by luminex and microRNA16 (miR16), CXCL-8 and IL-6 mRNA were measured by qPCR. TiAR was visualised in PBECs by immunohistochemistry using confocal microscopy and also knocked down with lentiviral constructs.
Results: Previously, we have shown that in human lung mucoepidermoid carcinoma cell lines, CXCL-8 mRNA degradation is inhibited in the presence of IL-17A by AU-binding factor-1 (AUF-1), particularly by attenuating miR16. Interestingly, in hyperresponsive PBECs from asthmatics, had elevated levels of miR16 with TNF-α and IL-17A stimulation, but no reduction in CXCL-8 and IL-6 mRNA and its half-life. Hence, we investigated AUBps involved in translational control like T-cell internal antigen receptor (TiAR) in PBECs. Notably, TiAR failed to translocate to the cytoplasm in hyperresponsive PBECs, however, in normoresponsive PBECs TiAR co-localised with eukaryotic initiation factor-3η, which actively involves in translational arrest. Immunoprecipitation showed that TiAR interacts with CXCL-8 and IL-6 mRNA after TNF-α and IL-17A stimulations. Knocking down TiAR in hyperresponsive PBECs did not have an impact on CXCL-8 levels, however, in normoresponsive PBECs CXCL-8 levels increased after TiAR knockdown. Moreover, CXCL-8 released from PBECs of asthma patients in vitro, strongly correlated with CXCL-8 levels and neutrophil percentage in BALF, but not with eosinophil percentage.
Conclusion: We show that PBECs from most asthma patients are hyperresponsive to IL-17A and are corticosteroid unresponsive. Translational repressor TiAR, can halt translation of CXCL-8 mRNA in normoresponsive PBECs, but not in hyperresponsive PBECs due to a defective translocation to cytoplasm. Hyperresponsive PBECs relate to neutrophilic inflammation.