Combination of IL-17A/F and TNF-alpha uniquely alters the bronchial epithelial cell proteome to enhance proteins that augment neutrophil migration

Background: The heterodimer interleukin (IL)-17A/F is elevated in the lungs in chronic respiratory disease such as severe asthma, along with the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Although IL-17A/F and TNF-alpha are known to functionally cooperate to exacerbate airway inflammation, proteins altered by their interaction in the lungs are not fully elucidated.Results: We used Slow Off-rate Modified Aptamer-based proteomic array to identify proteins that are uniquely and/or synergistically enhanced by concurrent stimulation with IL-17A/F and TNF-alpha in human bronchial epithelial cells (HBEC). The abundance of 38 proteins was significantly enhanced by the combination of IL-17A/F and TNF-alpha, compared to either cytokine alone. Four out of seven proteins that were increased > 2-fold were those that promote neutrophil migration; host defence peptides (HDP; Lipocalin-2 (LCN-2) and Elafin) and chemokines (IL-8, GRO alpha). We independently confirmed the synergistic increase of these four proteins by western blots and ELISA. We also functionally confirmed that factors secreted by HBEC stimulated with the combination of IL-17A/F and TNF-alpha uniquely enhances neutrophil migration. We further showed that PI3K and PKC pathways selectively control IL-17A/F + TNF-alpha-mediated synergistic production of HDPs LCN-2 and Elafin, but not chemokines IL-8 and GRO alpha. Using a murine model of airway inflammation, we demonstrated enhancement of IL-17A/F, TNF-alpha, LCN-2 and neutrophil chemokine KC in the lungs, thus corroborating our findings in-vivo.Conclusion: This study identifies proteins and signaling mediated by concurrent IL-17A/F and TNF-alpha exposure in the lungs, relevant to respiratory diseases characterized by chronic inflammation, especially neutrophilic airway inflammation such as severe asthma