Autophagy regulates DUOX1 localization and superoxide production in airway epithelial cells during chronic IL-13 stimulation

Abstract

The airway epithelium is a broad interface with the environment, mandating well-orchestrated responses to properly modulate inflammation. Classically, autophagy is a homeostatic pathway triggered in response to external cellular stresses, and is elevated in chronic airway diseases. Recent findings highlight the additional role of autophagy in vesicle trafficking and protein secretion, implicating autophagy pathways in complex cellular responses in disease. Th2 cytokines, IL-13 and IL-4, are increased in asthma and other airway diseases contributing to chronic inflammation. Previously, we observed that IL-13 increases reactive oxygen species (ROS) in airway epithelial cells in an autophagy-dependent fashion. Here, we tested our hypothesis that autophagy is required for IL-13-mediated superoxide production via the NADPH oxidase DUOX1. Using a mouse model of Th2-mediated inflammation induced by OVA-allergen, we observed elevated lung amounts of IL-13 and IL-4 accompanied by increased autophagosome levels, determined by LC3BII protein levels and immunostaining. ROS levels were elevated and DUOX1 expression was increased 70-fold in OVA-challenged lungs. To address the role of autophagy and ROS in the airway epithelium, we treated primary human tracheobronchial epithelial cells with IL-13 or IL-4. Prolonged, 7-day treatment increased autophagosome formation and degradation, while brief activation had no effect. Under parallel culture conditions, IL-13 and IL-4 increased intracellular superoxide levels as determined by electron paramagnetic resonance (EPR) spectroscopy. Prolonged IL-13 activation increased DUOX1, localized at the apical membrane. Silencing DUOX1 by siRNA attenuated IL-13-mediated increases in superoxide, but did not reduce autophagy activities. Notably, depletion of autophagy regulatory protein ATG5 significantly reduced superoxide without diminishing total DUOX1 levels. Depletion of ATG5, however, diminished DUOX1 localization at the apical membrane. The findings suggest non-canonical autophagy activity regulates DUOX1-dependent localization required for intracellular superoxide production during Th2 inflammation. Thus, in chronic Th2 inflammatory airway disease, autophagy proteins may be responsible for persistent intracellular superoxide production.

Mucin granules are degraded in the autophagosome-lysosome pathway as a means of resolving airway mucous cell metaplasia

“…We previously showed that in human AECs cultured with IL-13, there was increased cytoplasmic positive MUC5AC granules when autophagy protein, ATG5 was depleted by shRNA (7). In addition, we found that that LC3 positive autophagosome levels were increased as measured by LC3 II accumulation during flux assay in human AECs challenged with type 2 cytokines IL-13 and IL-4 (7,8). ATG5-12 and ATG16L1 form a complex that facilitates autophagosome maturation.…”

“…Finally, cells can utilize selective autophagy by targeting proteins, protein aggregates, and organelles including mitochondria(4, 5) and even cilia components(6) to the autophagosome for degradation in the lysosome. We (7,8), and others(9-13), have observed that autophagy markers are increased in models of human airway disease, and in asthma and COPD airways. This led us to infer that autophagy played a key role in the response to airway inflammation in the epithelium.Mucociliary clearance is a vital feature of innate immunity (14,15).…”

“…Mice given OVA sensitization and challenge had substantial increase in Muc5ac production by qPCR that decreased by day 10 and returned to baseline by day 17 (Figure 5A). We previously reported that levels of autophagosome marker, LC3B II, remain elevated in AECs even after withdrawal of IL-13 or IL-4 from culture media suggesting persistent autophagy activity during resolution of mucous cell metaplasia (8). We therefore examined isolated lysosomes derived from mouse lung homogenates following OVA stimulation.…”

“…In the mouse, secretory cells marked by cytoplasmic Secretoglobin 1a1, Muc5b, along with proteins involved in the regulated exocytic machinery transition from a club cell appearance to goblet cell morphology with cytoplasm packed with Muc5ac granules (21). Depending on the model, 50-300-fold inductions of Muc5ac have been observed (8,20,29,30).…”

“…The sheer magnitude of Muc5ac production leads to ER stress responses (31) and activated unfold protein responses (32) along with epithelial ROS (8). A number of inflammatory and allergen factors have been identified that initiate mucous cell metaplasia of the airway epithelium.…”