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