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

本提案的主要目标是：（一）确定由下列物质产生的挥发性有机化合物（VOC）： 铜绿假单胞菌（PA）可引起囊性病变中杯状细胞化生（GCM）和粘液分泌过多 纤维化（CF）气道，（ii）表征诱导机制，（iii）确定其重要性CF 发病机制PA是急性和慢性肺部感染的常见原因，包括CF和非CF 支气管扩张、呼吸机相关性肺炎（VAP）和慢性阻塞性肺病（COPD）。 PA感染与这些患者的发病率和死亡率显著增加相关。之一 这些病变肺共有的主要病理特征是粘液分泌失调 以及清除失败，导致气道阻塞，从而减少气体交换并恶化肺功能。 已知PA分泌多种次级代谢物，包括VOC。近年来，微生物VOCs 在患者的呼吸中检测到的蛋白质已被仔细检查为疾病诊断的潜在生物标志物，包括 CF、VAP和COPD。不幸的是，VOCs对肺发病机制的生物学效应，包括GCM和 粘液分泌过多，知之甚少。在初步研究中，我们证明，几个占主导地位的 先前在患有CF的人的呼吸中鉴定的PA VOC种类（例如，2-氨基苯乙酮，2-AA） 在人气道上皮细胞和小鼠中诱导GCM和粘液分泌过多。在这个应用程序中，我们将 测试VOC是患病气道中GCM和粘液分泌过多的主要贡献者的假设。 目标1将单独表征能够诱导GCM的主要PA VOC物质，并将其混合在一起 正常人原代支气管上皮细胞（NHBEC）与CF患病的原代 将支气管上皮细胞（CF-DHBECs）在空气-液体界面（ALI）中培养， 条件，分别模拟正常和CF气道。将对排名靠前的挥发性有机化合物进行单独或 在混合物中，它们在野生型与b-ENAC过表达中诱导GCM和粘液分泌过多的能力 （Scnn 1b-Tg）CF小鼠。最后，野生型PA菌株PA 14及其2-AA缺陷的同基因DpqsE突变体， 将比较它们在野生型与b-ENAC小鼠中诱导GCM和粘液分泌过多的能力。目的 2将研究GCM和PA VOCs诱导粘液生物合成的机制。首先，我们将确定 VOC是否激活前GCM通路，包括AhR和EGFR-AKT/ERK 1/2，以抑制 FOXA 2，气道粘液稳态的关键调节因子。然后，我们将研究VOCs是否也会诱导GCM， 粘液生物合成通过调节巨噬细胞极化、中性粒细胞流入和Th 1/Th 2/Th 17/ILC 2免疫 这些反应是已知的GCM和患病气道中粘液分泌过多的驱动因素。完成 该提案将揭示CF和其他病变肺部中VOC和过量粘液之间的机械联系， 以及针对细菌性肺炎的潜在新治疗方法。

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.