Abstract Introduction Airway diseases such as COPD and asthma are characterized by epithelial mucous metaplasia leading to hypersecretion of mucin granules and subsequent airway obstruction. We previously identified that autophagy proteins regulate the degradation of excess mucin granules in airway secretory cells. While we previously assumed this was a passive response to excess mucin granules, we now hypothesize that recycling of mucin granule glycoproteins is at the epicenter of a comprehensive cellular response to stress. Methods A secretory cell specific, tamoxifen (TAM) inducible mouse model was generated by crossing Scgb1a1-creERTM;tdT+ with Atg5flox/flox mice (Atg5 deletant). tdT+;Atg5flox/flox mice without Cre (Atg5flox/flox) were utilized as controls. C57bl6j wildtype (WT;Atg16L1+/+) or Atg16L1 hypomorph (Atg16L1-/-) mice were also used. Mice were challenged with OVA sensitization and nebulized challenge, or intra-nasal IL-1b. Human or mouse primary airway epithelial cells (AEC) were grown under air-liquid interface (ALI) conditions until fully differentiated. Results Atg5-deficient mice had modest increases in Muc5b levels under homeostatic conditions. However, following either OVA or IL-1b-mediated mucous cell metaplasia, Atg5 deletant mice had significantly increased lung Muc5ac and Muc5b. Bulk RNA seq data from mouse AEC revealed significant decreases in gene pathways regulating the integrated stress response (ISR) and mitochondrial function/oxidative phosphorylation from the Atg16L1-/- mice. This was confirmed by significant decreases in immunostaining of nuclear Atf4 and mitochondrial membrane marker Tom20 from Atg16L1-/- mouse airway epithelial cells. Atg16L1-/- cells also had functional deficits with reduced cilia beat frequency. To establish a relationship between ISR activation and degradation of mucin granules, we challenged human airway epithelial cells (hAEC) with either amino acid starvation with HBSS or oligomycin, which inhibits oxidative phosphorylation. Both conditions led to increases in nuclear ATF4 and an increase in LAMP1-labeled lysosomes that clustered around mucin granules in secretory cells (Fig.1). Conclusion Deletion of autophagy proteins leads to accumulation of mucin granules in airway secretory cells. These data link autophagy and energy homeostasis through the integrated stress response in airway epithelial cells and suggest that the secretory cell may serve as a nutrient-sensing cell. Figure 1: A) ISR pathway sensors, GCN2 and PERK, phosphorylate EIF2 and activate transcription factor ATF4. B) Representative ATF4 immunoblot for hAEC nuclear and cytoplasmic fractions under media-fed, HBSS, or oligomycin conditions for 6 hours. C) Representative Super-Resolution Microscopy-SIM images for LAMP1, MUC5AC, with nuclear counterstain by DAPI. Scale bar 5 microns or 1 micron in inset images. This abstract is funded by: R01 HL157269
Dickinson et al. (Fri,) studied this question.