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A3814 - Regulation of IL-17C Expression by Proinflammatory Cytokines and Th2 Cytokines in Airway Epithelial Cells
Author Block: K. Yamanaka1, T. Fujisawa2, H. Kusagaya2, K. Mori2, M. Niwa2, K. Furuhashi2, M. Kono2, E. Hamada1, T. Suda2, M. Masato1; 1Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan, 2Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Rationale IL-17C is one of the IL-17 cytokine family members predominantly produced by epithelial cells in gastrointestinal tract, skin, and lung. IL-17C binds to the IL-17RE/IL-17RA receptor complex, located primarily on epithelial cells to activate downstream signalling pathways and promote innate immune responses. We have previously shown that polyI:C, a ligand of Toll-like receptor 3, induced IL-17C expression in normal human bronchial epithelial (NHBE) cells, and IL-17C enhanced the expression of human β-defensin 2, colony-stimulating factor 3, and S100A12 in an autocrine/paracrine manner (Kusagaya, AJRCMB 2014), indicating that IL-17C play an important role in airway host defense. In the pathogenesis of chronic airway diseases (e.g. asthma, COPD), a variety of cytokines influences cellular function of airway epithelium, however, regulation of airway IL-17C expression by cytokines remains poorly understood. The purpose of the present study is to investigate molecular mechanism of cytokine-mediated IL-17C expression in human airway epithelial cells. Methods NHBE cells were stimulated with proinflammatory cytokine, IL-1β , and/or Th2 cytokines, IL-4 or IL-13. We detected the expression of IL-17C mRNA and protein using real-time PCR and ELISA. To demonstrate the signalling pathway in the expression, inhibitor study and small interfering RNA (siRNA) knockdown were performed. Western blotting was performed to investigate intracellular signalling. Chromatin immunoprecipitation (ChIP) assay was performed to analyse the transcriptional mechanisms. Results IL-1β strongly induced both IL-17C mRNA and protein expression in NHBE cells. In contrast, treatment with IL-4 or IL-13 resulted in significant reduction of IL-1β-induced IL-17C expression. Attenuation of NF-κB signalling pathway, using either the IκB-α inhibitor BAY11-7082 or an NF-κB subunit p65-specific siRNA, decreased IL-1β-induced IL-17C expression. The inhibitory effects of IL-13 on IL-17C expression were abolished when the JAK/STAT6 signalling pathway was impaired using either the JAK inhibitor ruxolitinib or a STAT6-specific siRNA. Western blotting analysis revealed that IL-1β promoted phosphorylation and degradation of IκB-α and p65 nuclear translocation. While IL-13 induced STAT6 phosphorylation and nuclear translocation, IL-13 had no effect on IL-1β-mediated IκB-α phosphorylation and p65 translocation. ChIP assay confirmed enhanced binding of p65 to two proximal regions of IL-17C promoters (-163/-142, -135/-114) following IL-1β stimulation. Interestingly, addition of IL-13 resulted in significant reduction of the binding of p65 to these regions. Conclusions IL-1β increases IL-17C expression via the NF-κB-based transcriptional mechanism in airway epithelium, whereas the Th2 cytokines attenuate this induction via STAT6-dependent mechanism by suppressing NF-κB binding to the IL-17C promoter regions.