Investigating the Role of Exosomes in Cell-cell Signaling Contributing to Airway Disease

Exosomes are microvesicles that contain functional proteins, messenger RNAs (mRNAs), and microRNAs. Recently, intercellular communication via exosomes has garnered attention in contributing to both asthma and obesity.;Obesity is often complicated by co-morbid conditions, yet how excess adipose contributes to end-organ dysfunction is poorly understood. Adipose-derived exosomes are a possible direct link contributing to end organ dysfunction in addition to systemic inflammation. To understand the role of exosomes in obesity, the first study of this dissertation describes the development of exosome techniques for exosomes released by adipose tissue from obese subjects. Herein, for aim 1, we developed techniques to quantify, characterize and compare these adipose-derived exosomes between obese and lean donors. Comparison of obese vs. lean visceral adipose donors detected 55 differentially-expressed exosomal microRNAs (p<0.05; fold change≥|1.2|). Quantitative Real Time PCR (qRT-PCR) confirmed downregulation of miR-148b (ratio = 0.2 [95% confidence interval = 0.1, 0.6]) and miR-4269 (0.3 [0.1, 0.8]), and upregulation of miR-23b (6.2 [2.2, 17.8]) and miR-4429 (3.8 [1.1 to 13.4]) in obese compared to lean visceral adipose exosomes. Biological pathway analysis identified transforming growth factor beta (TGF-beta) signaling and the canonical Wnt (Wnt/ beta-catenin) signaling pathway among the top canonical pathways predicted to be altered with visceral adiposity, based on projected mRNA targets for the 55 differentially expressed microRNAs. These data show that visceral adipose tissue sheds exosomal-mediators predicted to regulate key end-organ inflammatory and fibrotic signaling pathways. In aim 2, we demonstrate that obese visceral exosomes regulate mRNA expression of ACVR2B, an important TGF-beta receptor. Interestingly, these exosomes down-regulate ACVR2B in non-asthmatic fibroblasts and up-regulate ACVR2B in asthmatic fibroblasts, suggesting that obese visceral exosomes regulate airway fibroblast gene expression and that these cells respond differently to these exosomes dependent on disease state. This initial study allowed us to utilize exosomal techniques for investigation of exosomal communication in an in vivo animal model for asthma. The development of this model is described in our second study.;For the second study of this dissertation, we introduce a new animal model for asthma. In aim 3, using an immunocompromised murine host, we developed a xenograft model: integrating a proliferating and differentiating human asthmatic airway epithelium with an actively remodeling rodent mesenchyme. In this model, we show that the presence of asthmatic epithelium alone is sufficient to drive aberrant mesenchymal remodeling. Moreover, these xenografts permit direct assessment of exosomal intercellular communication between the epithelial-mesenchymal trophic unit. After developing an in vivo animal model of asthma, we can incorporate techniques developed in the first and second study to understand intercellular communication between the airway epithelium and mesenchyme in asthma.;Therefore for the third study, after establishing exosome techniques in our laboratory using the human obesity model, we extended these techniques in aim 3 to the in vivo xenograft model of asthma. We isolated exosomes shed from non-asthmatic and asthmatic human primary bronchial airway epithelial cells. Global microRNA profiling of these exosomes demonstrated that exosomal microRNAs dysregulated with asthma are predicted to upregulate the TGF-beta signaling pathway, an important contributor to airway remodeling and fibrosis in asthma. We then exposed acellular xenografts once daily for 12 days at a concentration of 3 mug/mL to non-asthmatic and asthmatic bronchial airway epithelial exosomes. We analyzed grafts using Masson's Trichrome for collagen fibers. Asthmatic bronchial airway epithelial exosome exposed grafts were thinner, and had a scarring appearance compared to non-asthmatic bronchial airway epithelial exosome grafts that appeared stiffer and thicker.;Our findings suggest that asthmatic airway epithelial exosomes aberrantly communicate with the underlying mesenchyme. This aberrant intercellular communication between the epithelium and mesenchyme in asthma may lead to improper formation of matrix scaffolding necessary for epithelialization of the airway epithelium.