Abstract
BackgroundCystic 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.ResultsWe first evaluated the effect of dendrimer-based cysteamine analogue (PAMAM-DENCYS) 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-DENCYS treatment as compared to plain dendrimer (PAMAM-DEN) control. Next, we observed that PAMAM-DENCYS 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-DENCYS 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-DENCYS) rescue of membrane-ΔF508-CFTR with PAMAM-DENCYS treatment using non-permeabilized IB3-1 cells immunostained for CFTR. Finally, we assessed the autophagy-mediated bacterial clearance potential of PAMAM-DENCYS by treating IB3-1 cells infected with PA01-GFP, and observed a significant (p<0.01; PAMAM-DEN vs. PAMAM-DENCYS) decrease in intracellular bacterial counts by immunofluorescence microscopy and flow cytometry. Also, PAMAM-DENCYS treatment significantly inhibits the growth of PA01-GFP bacteria and demonstrates potent mucolytic properties.ConclusionsWe 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-DENCYS 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.
Highlights
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel found on epithelial cell membranes [1,2,3,4,5,6,7,8,9,10], and its dysfunction is associated with cystic fibrosis (CF) that involves dysregulation of epithelial fluid transport in the lungs, pancreas, and other organs of the body [7, 8, 11, 12]
We first evaluated the effect of dendrimer-based cysteamine analogue (PAMAM-DENCYS) on the intrinsic autophagy response in IB3-1 cells and observed a significant reduction in Ub-RFP and LC3-GFP co-localization by PAMAM-DENCYS treatment as compared to plain dendrimer (PAMAM-DEN) control
Immunofluorescence microscopy of HEK-293 cells transfected with ΔF508-CFTR-GFP showed that PAMAM-DENCYS is able to rescue the misfolded-ΔF508-CFTR from aggresome-bodies by inducing its trafficking to the plasma membrane
Summary
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel found on epithelial cell membranes [1,2,3,4,5,6,7,8,9,10], and its dysfunction is associated with cystic fibrosis (CF) that involves dysregulation of epithelial fluid transport in the lungs, pancreas, and other organs of the body [7, 8, 11, 12]. Mutations in the Cftr gene (most common being the ΔF508) is characterized by build-up of thick mucus, frequent respiratory infections (such as Pseudomonas aeruginosa, Pa) and inflammation leading to severe lung damage [7,8,9, 13,14,15,16,17] These pathological manifestations caused by dysfunctional CFTR potentially involve autophagyimpairment as an important cellular mechanism associated with pathogenesis of CF lung disease [8, 18, 19]. We anticipate that ROS activation and resulting autophagy-impairment is induced by accumulation of misfolded ΔF508-CFTR protein in perinuclear aggresome-bodies that leads to membrane CFTR-dysfunction and chronic inflammation, initiating the pathogenesis of chronic obstructive CF lung disease. 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.
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