Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis.
Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis.
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DOI：
10.1371/journal.pone.0184793
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Vij N
中科院分区：
文献类型：
--
作者：
Brockman SM;Bodas M;Silverberg D;Sharma A;Vij N
囊性纤维化是由囊性纤维化跨膜电导调节基因S突变引起的一种遗传性疾病。最常见的突变是ΔF508，它会导致有缺陷的cftr蛋白在侵袭体中积累。此外，铜绿假单胞菌(PA)是一种常见的CF病原体，可加重阻塞性CF的肺部病理。在本研究中，我们旨在开发和测试一种新的策略，以提高半胱胺的生物利用度，并有可能通过开发基于树枝状大分子(PamAM-DEN)的半胱胺类似物来实现半胱胺的靶向给药。半胱胺是一种具有抗菌特性的有效自噬诱导药物。我们首先评估了树状大分子半胱胺类似物(PAMAM-DENCyS)对IB3-1细胞内在自噬反应的影响，并观察到与普通树状大分子(PamAM-DEN)对照相比，PAMAM-DENCyS处理显著减少了Ub-RFP和LC3-GFP共定位(侵袭体)。接下来，我们观察到，PAMAM-DENCys治疗显示了对C型ΔF508-cftr的适度挽救。免疫荧光显微镜观察显示，PAMAM-DENCyS能将错折叠的ΔF508-CFTR从侵袭体中解救出来，并诱导其转运到细胞膜上。我们通过流式细胞术进一步验证了这些结果，并观察到PAMAM-DEN与PAMAM-DENCyS处理的非通透性Δ-1细胞的CFTRF508-CFTR膜的拯救显著(P<；0.05；PAMAM-DEN与PAMAM-DENCyS)。最后，我们通过处理感染PA01-GFP的IB3-1细胞，评估了PAMAM-DENCyS自噬介导的细菌清除能力，并通过免疫荧光显微镜和流式细胞仪观察到细胞内细菌数量显著减少(P<；0.01；PamAM-DEN与PamAM-DENCyS相比)。此外，PAMAM-DENCyS处理显著抑制PA01-GFP细菌的生长，并显示出强大的粘液溶解特性。我们在这里展示了基于树枝状大分子的自噬诱导在防止ΔF508-cftr对侵袭体的隔离，同时促进其向质膜运输的有效性。此外，PAMAM-DENCys可减少PA的感染和生长，同时具有粘液溶解特性，提示其在抢救PA诱导的ΔF508-CF肺部疾病方面的潜力值得在CF小鼠模型上进一步研究。
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. 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 (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. 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-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.