Abstract Rationale Cystic fibrosis (CF) is a genetic disorder that affects multiple organ systems, notably the airways leading to accelerated aging, inflammation, and susceptibility to recurrent opportunistic infections in the lung. Our lab has previously published that increased cellular senescence is evident in the CF bronchial epithelium. Cellular senescence, a product of aging, is characterized by the combination of multiple factors including cell cycle arrest, senescence-associated β-galactosidase (SA-βgal) activity, apoptotic resistance, and secretion of pro-inflammatory cytokines. Pseudomonas aeruginosa (Pa) is a prevalent bacterial colonizer of the CF lung and treatment with the Pa metabolite, pyocyanin (PCN), has been shown to increase the activity of SA-βgal, a lysosomal enzyme. We hypothesized that Pa infection increases cellular senescence and lysosomal stress in the CF bronchial epithelium. Methods A publicly accessible RNA sequencing dataset from primary CF bronchial epithelial cells (P-CFBEs) infected with Pa was mined to assess for evidence of cellular senescence using the published Cell Age comparative gene set. Immortalized CFBEs were treated with PCN or infected with a clinical mucoid Pa strain, PAM57-15. Expression of cellular senescence and lysosomal markers were assessed through protein expression via western blotting and immunofluorescence imaging, mRNA levels via RT-qPCR, and SA-βgal activity was analyzed as positive cells/total cells using brightfield-microscopy. Lysosomal pH was measured using Lysosensor, a ratiometric fluorescent probe, and analyzed with a fluorescence microplate reader. Results Pa-infected P-CFBEs did not exhibit an overall increased senescence score or individual RNA values for cell cycle arrest and apoptotic resistance but did show an increase in GLB1 (β-galactosidase) expression. In-vitro Pa infection and PCN treatment increased SA-βgal activity independently of cellular senescence markers for cell cycle arrest and apoptotic resistance in CFBEs. Pa infected CFBEs also exhibited an increase in pro-inflammatory markers, neutralization of lysosomal pH, and decreased expression of the lysosomal biogenesis markers LAMP1 and TFEB. Rapamycin, an mTOR inhibitor that affects lysosomal biogenesis and function, was found to attenuate the Pa-induced SA-βgal activity and increase lysosomal biogenesis. Conclusions Our study presents a novel link between Pa infection and lysosomal stress via SA-βgal independent of other cellular senescence markers in the CF bronchial epithelium, which is partially mediated by mTOR signaling. Therefore, our results indicate a novel host-directed therapeutic target for Pa-infected CF airways, which involves the restoration of lysosomal homeostasis. This abstract is funded by: R01HL160911 to SK
Matthews et al. (Fri,) studied this question.