Elucidating the Impact of CC16 on Pulmonary Epithelial-Driven Host Responses

Abstract

Club Cell Secretory Protein (CC16) is a homodimeric pneumoprotein secreted by club cells and other non-ciliated epithelial cells in the distal airways. Although the complete biological functions and mechanisms of CC16 have not been elucidated, several studies have shown that CC16 has anti-inflammatory and antioxidant properties in the lungs. Studies have shown that low circulating levels of CC16 are correlated to accelerated declines in lung function, increased inflammation, and increased disease severity in asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) patients. CC16 has also been shown to directly decrease pathogen burden, pulmonary inflammation, and airway hyperresponsiveness during a variety of bacterial and viral lung infections; thereby, further supporting its role in regulating inflammation and immune responses. The airway epithelium is the first line of defense against inhaled pathogens and environmental toxicants; therefore, making it an important mediator of inflammation and immune responses. Airway epithelial cells are important regulators of normal lung homeostasis, which includes lung fluid balance, metabolism, mucociliary clearance of trapped particulates, activation of immune cells during infection or injury, and regulation of smooth muscle function. The four major epithelial cells observed within the lungs are basal, club, ciliated, and goblet cells, all of which perform different functions within the lung. Dysregulation of epithelial cells contributes to the respiratory conditions manifested in pulmonary diseases including asthma, COPD, or CF. In these studies, we provide data showing that CC16 is important for regulating protective properties within the pulmonary epithelium during naïve conditions and during infection with Mycoplasma pneumoniae (Mp). During early life Mp infection, we show that CC16 protects the lungs by decreasing airway hyperresponsiveness, remodeling, inflammation, and Mp burden within the lung itself, as well as within mouse tracheal epithelial cells (MTECs). Building from this foundational work, we looked further into the protective mechanisms that CC16 elicits within the pulmonary epithelium and discovered that CC16 regulates the expression of multiple antimicrobial proteins. We focused our efforts on one identified antimicrobial protein, Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) since it has been shown to have direct antimicrobial activity against Mp. Mechanistically, we were able to show that CC16 induces SPLUNC1 expression by signaling through Very Late Antigen-2 (VLA-2) and that inhibition of VLA-2 signaling during Mp infection resulted in increased Mp burden within pulmonary epithelial cells. To better understand the protective properties that CC16 elicits within the pulmonary epithelium, we sought to evaluate the therapeutic potential of recombinant CC16 (rCC16) therapy by determining which responses rCC16 elicited within wildtype (WT) MTECs using mass spectrometry and quantitative proteomics. Following rCC16 treatment, we observed that the WT MTECs increase expression of antioxidant proteins and decrease expression of proteins that may be related to airway remodeling. From these results, we hypothesized that rCC16 therapy may be effective in eliciting protective effects within the pulmonary epithelium. Furthermore, based on our in vivo and in vitro data, we next sought to understand if and how CC16 protects the pulmonary epithelium during Mp infection. Using mass spectrometry and quantitative proteomics, we observed that the MTECs deficient in CC16 (CC16-/- MTECs) significantly increased expression of proteins that may be related to airway remodeling and bacterial invasion of epithelial cells. The results presented in this dissertation demonstrate a protective role that CC16 plays with the lungs, and specifically within the pulmonary epithelium, during naïve conditions and during Mp infection. We showed that CC16 increases expression of multiple antimicrobial proteins; thereby, decreasing pulmonary Mp burden, as well as attenuating epithelial-driven antioxidant responses and airway remodeling. Translationally, these results provide insight for the development of pointed epithelial-based therapeutic approaches for individuals with CC16 deficits that suffer from chronic pulmonary infections.