Dendrimer-based selective autophagy-induction rescues δF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis

Background Cystic Fibrosis (CF) is a genetic disorder caused by mutation(s) in the CF-transmembrane conductance regulator (Cftr) gene. The most common mutation, δF508, leads to accumulation of defective-CFTR protein in aggresome-bodies. Additionally, Pseudomonas aeruginosa (Pa), a common CF pathogen, exacerbates obstructive CF lung pathology. In the present study, we aimed to develop and test a novel strategy to improve the bioavailability and potentially achieve targeted drug delivery of cysteamine, a potent autophagy-inducing drug with anti-bacterial properties, by developing a dendrimer (PAMAM-DEN)-based cysteamine analogue. Results We first evaluated the effect of dendrimer-based cysteamine analogue (PAMAM-DEN^CYS) on the intrinsic autophagy response in IB3-1 cells and observed a significant reduction in Ub-RFP and LC3-GFP co-localization (aggresome-bodies) by PAMAM-DEN^CYS treatment as compared to plain dendrimer (PAMAM-DEN) control. Next, we observed that PAMAM-DEN^CYS treatment shows a modest rescue of δF508-CFTR as the C-form. Moreover, immunofluorescence microscopy of HEK-293 cells transfected with δF508-CFTR-GFP showed that PAMAM-DEN^CYS is able to rescue the misfolded-δF508-CFTR from aggresome-bodies by inducing its trafficking to the plasma membrane. We further verified these results by flow cytometry and observed significant (p<0.05; PAMAM-DEN vs. PAMAM-DEN^CYS) rescue of membrane-δF508-CFTR with PAMAM-DEN^CYS treatment using non-permeabilized IB3-1 cells immunostained for CFTR. Finally, we assessed the autophagy-mediated bacterial clearance potential of PAMAM-DEN^CYS by treating IB3-1 cells infected with PA01-GFP, and observed a significant (p<0.01; PAMAM-DEN vs. PAMAM-DEN^CYS) decrease in intracellular bacterial counts by immunofluorescence microscopy and flow cytometry. Also, PAMAM-DEN^CYS treatment significantly inhibits the growth of PA01-GFP bacteria and demonstrates potent mucolytic properties. Conclusions We demonstrate here the efficacy of dendrimer-based autophagy-induction in preventing sequestration of δF508-CFTR to aggresome-bodies while promoting its trafficking to the plasma membrane. Moreover, PAMAM-DEN^CYS decreases Pa infection and growth, while showing mucolytic properties, suggesting its potential in rescuing Pa-induced δF508-CF lung disease that warrants further investigation in CF murine model.