Mechanistic triggers of excessive mucus during lung infections by bacterial volatile organic compounds

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The main objectives of this proposal are to (i) identify volatile organic compounds (VOCs) produced by Pseudomonas aeruginosa (PA) that cause goblet cell metaplasia (GCM) and mucus hypersecretion in Cystic Fibrosis (CF) airways, (ii) characterize the mechanism of induction, and (iii) determine its importance to CF pathogenesis. PA is a frequent cause of acute and chronical lung infections, including CF and non-CF bronchiectasis, ventilator-associated pneumonia (VAP), and chronic obstructive pulmonary disease (COPD). Infections by PA are associated with a significant increase in morbidity and mortality in these patients. One of the major pathological features shared by these diseased lungs is in their dysregulated, hypersecretion of mucus and failure in clearance, resulting in clogged airways that reduces gas exchanges and deteriorates lung function. PA is known to secrete a variety of secondary metabolites, including the VOCs. In recent years, microbial VOCs detected in the breath of patients have been scrutinized as potential biomarkers for disease diagnosis, including CF, VAP and COPD. Unfortunately, the biological effects of VOCs on lung pathogenesis, including GCM and mucus hypersecretion, are poorly understood. In preliminary studies, we demonstrate that several dominant species of PA VOCs previously identified in the breath of people with CF (e.g., 2-aminoacetophenone, 2-AA) induce GCM and mucus hypersecretion in human airway epithelial cells, and in mice. In this application, we will test the hypothesis that VOCs are major contributors of GCM and mucus hypersecretion in the diseased airways. Aim 1 will characterize major PA VOC species individually and in a cocktail that are capable of inducing GCM and mucus secretion in normal human primary bronchial epithelial cells (NHBECs) vs. CF diseased primary bronchial epithelial cells (CF-DHBECs) cultured in the air-liquid interface (ALI), under normoxic and hypoxic conditions, mimicking normal and CF airways, respectively. Top VOCs will be further examined individually or in cocktail for their ability to induce GCM and mucus hypersecretion in wild-type vs. b-ENAC overexpressing (Scnn1b-Tg) CF mice. Finally, both wild-type PA strain PA14 and its isogenic DpqsE mutant deficient in 2-AA will be compared for their ability to induce GCM and mucus hypersecretion in wild-type vs. b-ENAC mice. Aim 2 will examine mechanisms of GCM and mucus biosynthesis induction by PA VOCs. First, we will determine whether VOCs activate pro-GCM pathways, including AhR and EGFR-AKT/ERK1/2, to inhibit the expression of FOXA2, a key regulator of airway mucus homeostasis. Then, we will examine if VOCs also induce GCM and mucus biosynthesis by modulating macrophage polarization, neutrophil influx and Th1/Th2/Th17/ILC2s immune responses, which are known drivers of GCM and mucus hypersecretion in diseased airways. Completion of the proposal will reveal the mechanistic link between VOCs and excessive mucus in CF and other diseased lungs, and, potentially novel therapeutic approaches against bacterial pneumonias.