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• W2076324249 abstract "Well-differentiated human airway epithelia present formidable barriers to efficient siRNA delivery. We previously reported that treatment of airway epithelia with specific small molecules improves oligonucleotide uptake and facilitates RNAi responses. Here, we exploited the platelet activating factor receptor (PAFR) pathway, utilized by specific bacteria to transcytose into epithelia, as a trigger for internalization of Dicer-substrate siRNAs (DsiRNA). PAFR is a G-protein coupled receptor which can be engaged and activated by phosphorylcholine residues on the lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae and the teichoic acid of Streptococcus pneumoniae as well as by its natural ligand, platelet activating factor (PAF). When well-differentiated airway epithelia were simultaneously treated with either nontypeable Haemophilus influenzae LOS or PAF and transduced with DsiRNA formulated with the peptide transductin, we observed silencing of both endogenous and exogenous targets. PAF receptor antagonists prevented LOS or PAF-assisted DsiRNA silencing, demonstrating that ligand engagement of PAFR is essential for this process. Additionally, PAF-assisted DsiRNA transfection decreased CFTR protein expression and function and reduced exogenous viral protein levels and titer in human airway epithelia. Treatment with spiperone, a small molecule identified using the Connectivity map database to correlate gene expression changes in response to drug treatment with those associated with PAFR stimulation, also induced silencing. These results suggest that the signaling pathway activated by PAFR binding can be manipulated to facilitate siRNA entry and function in difficult to transfect well-differentiated airway epithelial cells. Well-differentiated human airway epithelia present formidable barriers to efficient siRNA delivery. We previously reported that treatment of airway epithelia with specific small molecules improves oligonucleotide uptake and facilitates RNAi responses. Here, we exploited the platelet activating factor receptor (PAFR) pathway, utilized by specific bacteria to transcytose into epithelia, as a trigger for internalization of Dicer-substrate siRNAs (DsiRNA). PAFR is a G-protein coupled receptor which can be engaged and activated by phosphorylcholine residues on the lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae and the teichoic acid of Streptococcus pneumoniae as well as by its natural ligand, platelet activating factor (PAF). When well-differentiated airway epithelia were simultaneously treated with either nontypeable Haemophilus influenzae LOS or PAF and transduced with DsiRNA formulated with the peptide transductin, we observed silencing of both endogenous and exogenous targets. PAF receptor antagonists prevented LOS or PAF-assisted DsiRNA silencing, demonstrating that ligand engagement of PAFR is essential for this process. Additionally, PAF-assisted DsiRNA transfection decreased CFTR protein expression and function and reduced exogenous viral protein levels and titer in human airway epithelia. Treatment with spiperone, a small molecule identified using the Connectivity map database to correlate gene expression changes in response to drug treatment with those associated with PAFR stimulation, also induced silencing. These results suggest that the signaling pathway activated by PAFR binding can be manipulated to facilitate siRNA entry and function in difficult to transfect well-differentiated airway epithelial cells. IntroductionSmall interfering RNA (siRNA) oligonucleotides cannot easily cross cellular membranes because of their size and negative charge. The efficient delivery of siRNA oligonucleotides remains a major challenge for advancing RNA interference (RNAi) technology, particularly to the airway. Although the airways can support direct topical delivery, both extra- and intracellular barriers present obstacles for successful RNAi therapy.1Sanders N Rudolph C Braeckmans K De Smedt SC Demeester J Extracellular barriers in respiratory gene therapy.Adv Drug Deliv Rev. 2009; 61: 115-127Crossref PubMed Scopus (173) Google Scholar,2Zuhorn IS Engberts JB Hoekstra D Gene delivery by cationic lipid vectors: overcoming cellular barriers.Eur Biophys J. 2007; 36: 349-362Crossref PubMed Scopus (204) Google Scholar While several studies reported therapeutic effects following naked siRNA delivery to the lungs3Alvarez R Elbashir S Borland T Toudjarska I Hadwiger P John M et al.RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy.Antimicrob Agents Chemother. 2009; 53: 3952-3962Crossref PubMed Scopus (128) Google Scholar,4Bitko V Musiyenko A Shulyayeva O Barik S Inhibition of respiratory viruses by nasally administered siRNA.Nat Med. 2005; 11: 50-55Crossref PubMed Scopus (627) Google Scholar,5DeVincenzo J Lambkin-Williams R Wilkinson T Cehelsky J Nochur S Walsh E et al.A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus.Proc Natl Acad Sci USA. 2010; 107: 8800-8805Crossref PubMed Scopus (344) Google Scholar,6Kim TH Kim SH Seo JY Chung H Kwak HJ Lee SK et al.Blockade of the Wnt/β-catenin pathway attenuates bleomycin-induced pulmonary fibrosis.Tohoku J Exp Med. 2011; 223: 45-54Crossref PubMed Scopus (100) Google Scholar,7Lomas-Neira JL Chung CS Wesche DE Perl M Ayala A In vivo gene silencing (with siRNA) of pulmonary expression of MIP-2 versus KC results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury.J Leukoc Biol. 2005; 77: 846-853Crossref PubMed Scopus (118) Google Scholar,8Merkel OM Zheng M Debus H Kissel T Pulmonary gene delivery using polymeric nonviral vectors.Bioconjug Chem. 2012; 23: 3-20Crossref PubMed Scopus (55) Google Scholar,9Perl M Chung CS Lomas-Neira J Rachel TM Biffl WL Cioffi WG et al.Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis.Am J Pathol. 2005; 167: 1545-1559Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar,10Rosas-Taraco AG Higgins DM Sánchez-Campillo J Lee EJ Orme IM González-Juarrero M Intrapulmonary delivery of XCL1-targeting small interfering RNA in mice chronically infected with Mycobacterium tuberculosis.Am J Respir Cell Mol Biol. 2009; 41: 136-145Crossref PubMed Scopus (59) Google Scholar,11Senoo T Hattori N Tanimoto T Furonaka M Ishikawa N Fujitaka K et al.Suppression of plasminogen activator inhibitor-1 by RNA interference attenuates pulmonary fibrosis.Thorax. 2010; 65: 334-340Crossref PubMed Scopus (95) Google Scholar,12Zhang X Shan P Jiang D Noble PW Abraham NG Kappas A et al.Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis.J Biol Chem. 2004; 279: 10677-10684Crossref PubMed Scopus (241) Google Scholar others have suggested alternative off target explanations for the results.7Lomas-Neira JL Chung CS Wesche DE Perl M Ayala A In vivo gene silencing (with siRNA) of pulmonary expression of MIP-2 versus KC results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury.J Leukoc Biol. 2005; 77: 846-853Crossref PubMed Scopus (118) Google Scholar,9Perl M Chung CS Lomas-Neira J Rachel TM Biffl WL Cioffi WG et al.Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis.Am J Pathol. 2005; 167: 1545-1559Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar,13Ge Q Filip L Bai A Nguyen T Eisen HN Chen J Inhibition of influenza virus production in virus-infected mice by RNA interference.Proc Natl Acad Sci USA. 2004; 101: 8676-8681Crossref PubMed Scopus (411) Google Scholar,14Robbins M Judge A Ambegia E Choi C Yaworski E Palmer L et al.Misinterpreting the therapeutic effects of small interfering RNA caused by immune stimulation.Hum Gene Ther. 2008; 19: 991-999Crossref PubMed Scopus (206) Google Scholar Additional data support these findings; siRNA oligonucleotides enter cell poorly, and as a result are not well retained in the respiratory epithelium.15Moschos SA Frick M Taylor B Turnpenny P Graves H Spink KG et al.Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense.Mol Ther. 2011; 19: 2163-2168Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,16Platz J Pinkenburg O Beisswenger C Püchner A Damm T Bals R Application of small interfering RNA (siRNA) for modulation of airway epithelial gene expression.Oligonucleotides. 2005; 15: 132-138Crossref PubMed Scopus (19) Google ScholarWe also reported earlier that Dicer-substrate siRNA (DsiRNA) formulations failed to silence genes in well-differentiated airway epithelia, irrespective of the dose or time of transfection, and that this lack of efficiency correlated strongly with limited oligonucleotide entry.17Krishnamurthy S Behlke MA Ramachandran S Salem AK McCray Jr, PB Davidson BL Manipulation of Cell Physiology Enables Gene Silencing in Well-differentiated Airway Epithelia.Mol Ther Nucleic Acids. 2012; 1: e41Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar However, we discovered that pretreatment with certain small molecules, as well as enhancers of macropinocytosis such as epidermal growth factor, improved synthetic oligonucleotide uptake and RNAi responses. This result suggested that mechanisms promoting siRNA oligo internalization into differentiated airway epithelia might facilitate RNAi responses.In this work, we considered mechanisms that microorganisms exploit to enter cells. Following receptor binding, many viruses and bacteria cross cell membranes by one or more endocytic pathway including phagocytosis, clathrin- or caveolae-mediated endocytosis, or macropinocytosis.18Ketterer MR Shao JQ Hornick DB Buscher B Bandi VK Apicella MA Infection of primary human bronchial epithelial cells by Haemophilus influenzae: macropinocytosis as a mechanism of airway epithelial cell entry.Infect Immun. 1999; 67: 4161-4170PubMed Google Scholar,19Mercer J Helenius A Virus entry by macropinocytosis.Nat Cell Biol. 2009; 11: 510-520Crossref PubMed Scopus (582) Google Scholar An elegant study by Ketterer et al.18Ketterer MR Shao JQ Hornick DB Buscher B Bandi VK Apicella MA Infection of primary human bronchial epithelial cells by Haemophilus influenzae: macropinocytosis as a mechanism of airway epithelial cell entry.Infect Immun. 1999; 67: 4161-4170PubMed Google Scholar showed that the opportunistic pathogen of the airway, nontypeable Haemophilus Influenzae (NTHi), entered human bronchial epithelia by macropinocytosis after initiating cytoskeletal rearrangement. Further study by the same group demonstrated that NTHi invaded host cells by first binding to the host receptor platelet activating factor receptor (PAFR) via its lipooligosaccharide (LOS)20Moniot B Declosmenil F Barrionuevo F Scherer G Aritake K Malki S et al.The PGD2 pathway, independently of FGF9, amplifies SOX9 activity in Sertoli cells during male sexual differentiation.Development. 2009; 136: 1813-1821Crossref PubMed Scopus (129) Google Scholar glycoforms containing phosphorylcholine (Chop).21Robbins ML Sekhon RS Meeley R Chopra S A Mutator transposon insertion is associated with ectopic expression of a tandemly repeated multicopy Myb gene pericarp color1 of maize.Genetics. 2008; 178: 1859-1874Crossref PubMed Scopus (18) Google Scholar,22Swords WE Buscher BA Ver Steeg Ii K Preston A Nichols WA Weiser JN et al.Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor.Mol Microbiol. 2000; 37: 13-27Crossref PubMed Scopus (256) Google Scholar The adherence and invasion of another bacterium, Streptococcus pneumoniae (pneumococcus) was also linked to PAFR binding,23Cundell DR Gerard NP Gerard C Idanpaan-Heikkila I Tuomanen EI Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor.Nature. 1995; 377: 435-438Crossref PubMed Scopus (609) Google Scholar possibly through the ChoP moiety present in the bacterial cell wall. Thus, the bacteria's interaction with PAFR provides for cell entry.PAFR is a G-protein coupled receptor whose natural ligand is platelet activating factor (PAF), a potent phospholipid which also consists of the ChoP moiety in NTHi and the pneumococcal cell wall. Since PAFR stimulation is involved in bacterial entry,24Zhang JR Mostov KE Lamm ME Nanno M Shimida S Ohwaki M et al.The polymeric immunoglobulin receptor translocates pneumococci across human nasopharyngeal epithelial cells.Cell. 2000; 102: 827-837Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar we hypothesized that PAFR activation might also facilitate macromolecules entry such as siRNA.We tested this hypothesis on well-differentiated human airway epithelia (HAE) grown at the air–liquid interface (ALI) and assessed the ability of DsiRNAs to silence endogenous or exogenous targets with or without PAFR stimulation. We show that DsriRNA transfection in combination with PAFR engagement by any of its cognate binding partners or by small molecule treatment improves DsiRNA uptake with concomitant reductions in target RNA and protein levels.ResultsTransfection of DsiRNA into HAE along with LOS or PAF treatment results in silencing of mRNA levels of target geneWell-differentiated primary cultures of HAE maintained at the ALI model many aspects of the in vivo morphology of the surface epithelium and are a useful system in which to test the efficacy of inhibitory RNAs in the form of DsiRNAs.25Pezzulo AA Starner TD Scheetz TE Traver GL Tilley AE Harvey BG et al.The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia.Am J Physiol Lung Cell Mol Physiol. 2011; 300: L25-L31Crossref PubMed Scopus (224) Google Scholar We previously reported that DsiRNA transfection in formulation with various transfection agents failed to achieve RNAi in HAE.17Krishnamurthy S Behlke MA Ramachandran S Salem AK McCray Jr, PB Davidson BL Manipulation of Cell Physiology Enables Gene Silencing in Well-differentiated Airway Epithelia.Mol Ther Nucleic Acids. 2012; 1: e41Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar To test if DsiRNA entry and RNAi responses in HAE can be improved, we activated PAFR in airway cells using some of its cognate binding partners.One PAFR ligand is the LOS structure of NTHi. The NTHi LOS contains a highly variable assortment of short polyhexose or lactosamine chains emanating from a tri-heptose-ketodeoxyoctanoate-phosphate-lipid A core region.26Preston A Mandrell RE Gibson BW Apicella MA The lipooligosaccharides of pathogenic gram-negative bacteria.Crit Rev Microbiol. 1996; 22: 139-180Crossref PubMed Scopus (240) Google Scholar In a form of molecular mimicry, NTHi expresses host carbohydrate structures within the oligosaccharide portion of the LOS, including ChoP. NTHi can adhere to and invade human bronchial epithelial cells via interactions between the ChoP+ LOS glycoforms and PAFR.22Swords WE Buscher BA Ver Steeg Ii K Preston A Nichols WA Weiser JN et al.Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor.Mol Microbiol. 2000; 37: 13-27Crossref PubMed Scopus (256) Google Scholar We isolated LOS from the native NTHi strains and the three LOS isoforms tested differed in the number of phosphocholine residues and LOS stability. The details of each LOS isoforms used are provided in Supplementary Table S1. When HAE were transduced with a HPRT DsiRNA-transductin formulation and simultaneously treated with each LOS, HPRT mRNA levels were reduced by up to 35% when analyzed 24 hours after transduction (Figure 1a) compared to negligible transduction in the absence of the LOS.The natural ligand for PAFR is the potent phospholipid activator, PAF. We hypothesized that PAF activation of PAFR during DsiRNA transduction would improve RNAi responses in HAE as seen with LOS. PAF binding to PAFR activates several signaling mechanisms including GTPase activation causing phospholipid turnover (via phospholipases C, D, and A2 pathways) and protein kinase C and tyrosine kinase activation.27Shukla SD Platelet-activating factor receptor and signal transduction mechanisms.FASEB J. 1992; 6: 2296-2301Crossref PubMed Scopus (184) Google Scholar Epithelia were PAF treated for 4 hours in the presence of the DsiRNA mixture, rinsed, and then incubated for a further 24 hours before gene expression was measured by RT-qPCR. Treatment of HAE with DsiRNA-transductin and PAF reduced HPRT mRNA levels between 10 and 40% depending on the donor cell culture (Figure 1b). A dose–response relationship for PAF on DsiRNA silencing was observed (Supplementary Figure S1a). We also investigated the duration of knock down after PAF-assisted transfection. Following delivery, epithelia were studied on days 1, 3, 6, and 10. HPRT mRNA levels were significantly reduced 1 and 3 days post transfection. By 6 and 10 days, the transcript levels were returning to basal levels (Supplementary Figure S1b). Since each mRNA target is likely to have a different rate of turnover, repeated transfection may be necessary to maintain knockdown.A well-studied effect of PAFR stimulation is the activation of mediators that can generate inflammatory responses (38–41). As a mediator of inflammation, PAFR activation can alter tight junction integrity in endothelial and intestinal epithelial cells.28Knezevic II Predescu SA Neamu RF Gorovoy MS Knezevic NM Easington C et al.Tiam1 and Rac1 are required for platelet-activating factor-induced endothelial junctional disassembly and increase in vascular permeability.J Biol Chem. 2009; 284: 5381-5394Crossref PubMed Scopus (72) Google Scholar,29Xu LF Xu C Mao ZQ Teng X Ma L Sun M Disruption of the F-actin cytoskeleton and monolayer barrier integrity induced by PAF and the protective effect of ITF on intestinal epithelium.Arch Pharm Res. 2011; 34: 245-251Crossref PubMed Scopus (14) Google Scholar We examined the effects of PAF on tight junction integrity in airway epithelia. PAF application to the apical and basolateral surfaces of HAE for 8 hours caused no alteration of tight junction morphology as assessed by ZO-1 immunostaining. In contrast, 24 hours of PAF treatment caused significant alterations in tight junction morphology (Supplementary Figure S2a). Our experimental protocol involved PAF treatment of epithelia during DsiRNA transduction for 4 hours; hence its effects on tight junction should be minimized. We also examined epithelial cell morphology after PAF-assisted transfection. Epithelial sheets were fixed, sectioned, and stained with hematoxylin–eosin (H&E) 1 and 10 days after transfection, and these sheets showed no changes in cell morphology when compared with control treated epithelium (Supplementary Figure S2b). Cytotoxicity was also assessed by lactate dehydrogenase release. Both PAF and control treated cells showed minimal evidence of cytotoxicity (Supplementary Figure S2c).The PAFR-dependence of the LOS- and PAF-mediated enhancement of DsiRNA silencing was evaluated using the PAF receptor antagonist (PAFRa) WEB2086. Pre- or post-treatment of epithelia with 100 µmol/l PAFRa completely inhibited HPRT silencing of either LOS- or PAF-assisted transduction samples (Figure 1c). Lower concentrations of PAFRa (1 µmol/l) partially inhibited silencing in cells transduced with LOS or PAF (Figure 1c). These results suggest that the effects of LOS and PAF on siRNA-mediated silencing are a consequence of their interactions with PAFR.PAF treatment enhances DsiRNA entry and uptake into cellsWe next asked whether the improved RNAi responses following PAFR stimulation were due to an increase in uptake and internalization of DsiRNA. When digoxigenin (DIG)-labeled DsiRNA was transduced in formulation with transductin in PAF-treated HAE, we observed a substantial increase in the amount of internalized label 1 hour later (Figure 2b) compared with control HAE (Figure 2a). These data suggests that improved RNAi upon PAFR stimulation is a result of improved DsiRNA internalization.Figure 2PAF treatment enhances DsiRNA entry and uptake into cells. Confocal images (x-y, left panels; x-z stacks, right panels) of epithelia 1 hour after transduction with DIG-labeled DsiRNA complexed with transductin, (a) without or (b) with PAF treatment (200 nmol/l). Blue, nuclei; green, DIG-labeled oligo; red, ZO-1. The images on the right and the left are from different fields.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Spiperone treatment induces RNAi responses in HAEThe Connectivity map (CMAP) database is a repository of transcriptional profiles from a number of human cell lines in response to drug treatments.30Lamb J Crawford ED Peck D Modell JW Blat IC Wrobel MJ et al.The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease.Science. 2006; 313: 1929-1935Crossref PubMed Scopus (3417) Google Scholar We correlated the input signatures of gene expression changes during PAFR activation from a previously published microarray study31Travers JB Edenberg HJ Zhang Q Al-Hassani M Yi Q Baskaran S et al.Augmentation of UVB radiation-mediated early gene expression by the epidermal platelet-activating factor receptor.J Invest Dermatol. 2008; 128: 455-460Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar with those from the CMAP database. The method of analysis is described in more detail in Materials and Methods. The analysis yielded several drugs that correlated positively with PAF-induced changes in gene expression. We reasoned that treatment of HAE with these candidates might induce gene expression changes similar to those associated with PAFR activation. From these data, six candidates were selected and tested. HAE were pretreated with drugs and then subsequently incubated with HPRT targeting DsiRNA-transductin formulations as before. Of these interventions, only spiperone pretreatment was effective. Spiperone, an antipsychotic drug, increases intracellular Ca2+ levels through a protein kinase-coupled phospholipase C-dependent pathway and also stimulates calcium-activated chloride channels in polarized HAE cells.32Liang L MacDonald K Schwiebert EM Zeitlin PL Guggino WB Spiperone, identified through compound screening, activates calcium-dependent chloride secretion in the airway.Am J Physiol Cell Physiol. 2009; 296: C131-C141Crossref PubMed Scopus (12) Google Scholar Of note, PAF also activates the phospholipase C pathway.27Shukla SD Platelet-activating factor receptor and signal transduction mechanisms.FASEB J. 1992; 6: 2296-2301Crossref PubMed Scopus (184) Google Scholar Spiperone treatment of epithelia (10 µmol/l) before DsiRNA transduction resulted in 25% silencing of HPRT mRNA levels (Figure 3) . A spiperone dose–response showed marginal increases in silencing with increasing doses (Supplementary Figure S3).Figure 3Spiperone improves RNAi responses in DsiRNA transduced cells. HAE were pre-treated with various small molecules at the concentration indicated. The drugs were added to the apical surface and the basolateral media for 6 hours. Both surfaces were then rinsed and HPRT or NC1 DsiRNA formulated with transductin added to the apical surface. The cells were rinsed after 4 hours, and RT-qPCR done on cell lysates harvested 24 hours later. Data show HPRT mRNA levels normalized to NC1-treated samples (three biological replicates –in triplicate). ***P < 0.001 (Student's t-test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)PAF or spiperone treatment decreases CFTR protein levels and function in HAE transduced with CFTR DsiRNAWe next asked whether endogenous gene silencing by DsiRNA, facilitated by PAFR activation, reduces target mRNA and protein levels. Cystic fibrosis transmembrane regulator (CFTR) encodes an anion channel in airway epithelia. Loss of CFTR function, caused by mutations in the CFTR gene causes cystic fibrosis, an important chronic disease characterized by progressive pulmonary infection and inflammation. DsiRNA incubation with simultaneous PAF treatment or with spiperone pre-treatment caused ~30% silencing of CFTR mRNA levels (Figure 4a). Immunoprecipitation and then western blot analysis in cells treated with PAF or spiperone and CFTR DsiRNA-transductin showed reduced CFTR protein levels compared to controls (Figure 4b). Fully glycosylated CFTR band C was significantly decreased in PAF and spiperone-treated cells (Figure 4c). Consequently, well-differentiated airway epithelia allow for silencing of target mRNA and protein when PAFR signaling or PAFR-related gene expression changes are activated.Figure 4PAF or spiperone treatment decreases CFTR protein levels in HAE treated with CFTR DsiRNA. (a) PAF and DsiRNA (CFTR-targeting or control NC1 in transductin), were applied to well-differentiated HAE for 4 hours. Alternatively, spiperone was applied apically and basolaterally to well-differentiated HAE for 6 hours, and then removed by rinsing before incubation for 4 hours with CFTR-targeting or NC1 DsiRNAs complexed with transductin. In both cases, 24 hours after transfection, and RNA levels quantified by RT-qPCR. (b) A representative immunoblot from similarly treated wells, but cells were lysed and proteins visualized after immunoprecipitation with anti-CFTR antibody. Alpha-tubulin was used as a loading control. (c) Densitometry readings of immunoblots with results normalized to NC1 treated samples. In all cases, data are from 3 biological replicates and are mean ± SD. ***P < 0.001; **P < 0.01(Student's t-test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next assessed epithelial cell bioelectric properties following PAF-mediated delivery of DsiRNA-transductin targeting CFTR; CFTR is a phosphorylation and nucleotide activated anion channel that helps regulate the volume and composition of airway surface liquid, and loss of function impairs transepithelial anion transport. In control epithelia with or without PAF treatment (without HPRT DsiRNA transfection) or PAF and scrambled control DsiRNA (NC1) treated cells, the addition forskolin and isobutylmethylxantine (IBMX) increased the cAMP-activated transepithelial short-circuit current (Isc and ΔIsc) and conductance (Gt and ΔGt) as expected (Supplementary Figure S4; Figure 5a,b). Subsequent addition of GlyH-101, a CFTR channel inhibitor, decreased both values. In contrast, PAF treated and CFTR DsiRNA-transduced cells showed significantly reduced cAMP-activated changes in transepithelial current and conductance (Supplementary Figure S4; Figure 5a,b). We note that CFTR activity is typically not rate-limiting at normal levels of CFTR expression. Rather, the activity of transporters/channels at the basolateral membrane limits the magnitude of agonist activated current. Therefore, the magnitude of current reduction observed here may reflect a reduction in CFTR protein expression greater than that suggested by current measurements. These results show that PAFR-mediated oligo delivery and CFTR knockdown significantly reduced CFTR channel activity.Figure 5PAF treatment with CFTR DsiRNA reduces CFTR function in HAE. Changes in (a) transepithelial current (ΔIsc) and (b) conductance (ΔGt) induced by sequential addition of amiloride, Forskolin and IBMX (F&I) and GlyH-101 in HAE cultures treated with the indicated agents. Each bar represents nine replicates from three different donors (three replicates from each). Mean ± SD. Statistical significance was determined by repeated measures one-way ANOVA ****P < 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PAF treatment reduces exogenous viral protein with concomitant decrease in titer in DsiRNA transduced HAERespiratory Syncytial Virus (RSV) is an important pathogen in children and elderly and immunocompromised individuals. There is neither a preventive vaccine nor an effective treatment for the disease. Potent RNAi against RSV in airway epithelia has not been rigorously demonstrated. Here, we tested the effect of DsiRNA against the RSV N gene in infected HAE with or without PAF treatment at the time of oligonucleotide application. Epithelia were PAF treated and incubated with DsiRNA-transductin targeting the N gene. Six hours later, the cells were infected with RSV at an MOI of 1. Viral titers calculated from the apical rinses were reduced more than 100-fold in samples treated with PAF (Figure 6a). PAFR activation alone did not reduce viral titers. Immunoblot analysis 48 hours after infection showed reduced RSV N and P protein abundance compared to untreated control cells (Figure 6b,c). These results demonstrate that PAFR activation enables viral load reduction by facilitating RNAi in airway epithelia.Figure 6PAF induced DsiRNA uptake reduces RSV protein levels and titer in HAE. HAE was either treated o" @default.
• W2076324249 created "2016-06-24" @default.
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• W2076324249 date "2014-01-01" @default.
• W2076324249 modified "2023-09-30" @default.
• W2076324249 title "Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia" @default.
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• W2076324249 doi "https://doi.org/10.1038/mtna.2014.26" @default.
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