Cargando…

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 pathoge...

Descripción completa

Detalles Bibliográficos
Autores principales: Brockman, Scott Mackenzie, Bodas, Manish, Silverberg, David, Sharma, Ajit, Vij, Neeraj
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597233/
https://www.ncbi.nlm.nih.gov/pubmed/28902888
http://dx.doi.org/10.1371/journal.pone.0184793
_version_ 1783263676223979520
author Brockman, Scott Mackenzie
Bodas, Manish
Silverberg, David
Sharma, Ajit
Vij, Neeraj
author_facet Brockman, Scott Mackenzie
Bodas, Manish
Silverberg, David
Sharma, Ajit
Vij, Neeraj
author_sort Brockman, Scott Mackenzie
collection PubMed
description 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.
format Online
Article
Text
id pubmed-5597233
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-55972332017-09-15 Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis Brockman, Scott Mackenzie Bodas, Manish Silverberg, David Sharma, Ajit Vij, Neeraj PLoS One Research Article 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. Public Library of Science 2017-09-13 /pmc/articles/PMC5597233/ /pubmed/28902888 http://dx.doi.org/10.1371/journal.pone.0184793 Text en © 2017 Brockman et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Brockman, Scott Mackenzie
Bodas, Manish
Silverberg, David
Sharma, Ajit
Vij, Neeraj
Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis
title Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis
title_full Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis
title_fullStr Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis
title_full_unstemmed Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis
title_short Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis
title_sort dendrimer-based selective autophagy-induction rescues δf508-cftr and inhibits pseudomonas aeruginosa infection in cystic fibrosis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597233/
https://www.ncbi.nlm.nih.gov/pubmed/28902888
http://dx.doi.org/10.1371/journal.pone.0184793
work_keys_str_mv AT brockmanscottmackenzie dendrimerbasedselectiveautophagyinductionrescuesdf508cftrandinhibitspseudomonasaeruginosainfectionincysticfibrosis
AT bodasmanish dendrimerbasedselectiveautophagyinductionrescuesdf508cftrandinhibitspseudomonasaeruginosainfectionincysticfibrosis
AT silverbergdavid dendrimerbasedselectiveautophagyinductionrescuesdf508cftrandinhibitspseudomonasaeruginosainfectionincysticfibrosis
AT sharmaajit dendrimerbasedselectiveautophagyinductionrescuesdf508cftrandinhibitspseudomonasaeruginosainfectionincysticfibrosis
AT vijneeraj dendrimerbasedselectiveautophagyinductionrescuesdf508cftrandinhibitspseudomonasaeruginosainfectionincysticfibrosis