Nasal Epithelial Cell Line Research Articles

Anticancer Effects & Comparative Study of Thymoquinone, Cordyceps, Spirulina, Ganoderma Lucidium, Poria Cocos, and Lion’s Mane on Human Nasal Cancer Cells (RPMI-2650)

Recently, many studies have been conducted to discover or improve cancers treatment. The current study aims to investigate the anticancer effect of thymoquinone, cordyceps, spirulina, ganoderma lucidium, poria cocos, and lion’s mane in four different concentrations 4, 8, 16, and 32 ug (equivalent to 1 mg/mL) in two different time treatments (48 and 96 hours) on human nasal epithelial cell line RPMI 2650. By using cell culture cytotoxicity techniques and assay, the highest anticancer effect on RPMI 2650 was obtained by thymoquinone. The lowest anticancer effect was demonstrated by poria cocos and cordyceps. However, these two medications showed higher anticancer effect when given in short-term treatment (48 hours) compared to long-term treatment (96 hours). Ganoderma lucidium and spirulina showed better impact than poria cocos, cordyceps, and lion’s mane in term of cells cytotoxicity. Mild to moderate antineoplastic effect was seen by utilizing lion’s mane treatment compared other drugs. Therefore, adopting a long-term treatment of high concentrations and doses of thymoquinone, cordyceps, spirulina, ganoderma lucidium, poria cocos, and lion’s mane can be more effective in the treatment of nasal cancer. In conclusion, these drugs were found to be a promising cancer remedy; therefore, they can be utilized as alternative treatment for nasal cancer or any other type of cancer therapy.

Suitability and functional characterization of two Calu-3 cell models for prediction of drug permeability across the airway epithelial barrier.

The Calu-3 cell line has been largely investigated as a physiological and pharmacological model of the airway epithelial barrier. Its suitability for prediction of drug permeability across the airway epithelia, however, has not been yet evaluated by using large enough set of model drugs. We evaluated two Calu-3 cell models (air-liquid and liquid-liquid) for drug permeability prediction based on the recent regulatory guidelines on showing suitability of in vitro permeability methods for drug permeability classification. Bidirectional permeability assays using 22 model drugs and several zero permeability markers, as well as using ABC transporter substrates were conducted. Functional activity of P-gp, but not of BCRP was revealed. The potential of the Calu-3 cells to be used as a model of the nasal epithelial barrier, despite their different anatomical origin, has been demonstrated by the obtained excellent correlation with the fully differentiated 3D human nasal epithelial model (MucilAir™) for 11 model drugs, as well as by the good correlation obtained with the human nasal epithelial cell line RPMI 2650. In addition, the permeability values determined in the two Calu-3 models correlated well with the intestinal permeability model Caco-2.

Epithelial cells expressed IL-33 to promote degranulation of mast cells through inhibition on ST2/PI3K/mTOR-mediated autophagy in allergic rhinitis

ABSTRACT Nasal epithelial cells are the first barrier against allergen infiltration in allergic rhinitis (AR), and the relationship between nasal epithelial cells and mast cell-mediated hypersensitivity remains unclear. This study aimed to investigate the possible association between allergen-challenged nasal epithelial cells (AR-HNEpC) and mast cell degranulation in AR. Our data revealed that calcium influx and degranulation were increased in AR-HNEpC-co-cultured mast cells. Expression of IL-33, a factor that binds to ST2 receptors on mast cells and regulates their degranulation, was elevated in AR-HNEpC. Blocking IL-33/ST2 pathway activated autophagy and inhibited degranulation and inflammatory factor release in mast cells. Furthermore, PI3K/mTOR was increased in IL-33-treated mast cells. Inhibition on PI3K/mTOR pathway enhanced autophagy and inhibited degranulation. Analysis using an in vivo AR model supported the above findings. In conclusion, IL-33 from epithelial cells promotes degranulation of mast cells in AR through inhibition on ST2/PI3K/mTOR-mediated autophagy, which provides a potential therapeutic target for the disease. Abbreviations: AR: allergic rhinitis; IL: interleukin; TNF-α: tumor necrosis factor-alpha; INF-γ: interferon-gamma; HNEpC: human nasal epithelial cell line; ATCC: American Type Culture Collection; C48/80: compound 48/80; 3-MA: 3-methyladenine; qPCR: quantitative PCR; AR-HNEpC: dust mite allergen-treated nasal epithelial cells; IgE: immunoglobulin E; Atg7: autophagy-related gene 7

A 3D "In Vitro" Model to Study Hyaluronan Effect in Nasal Epithelial Cell Line Exposed to Double-Stranded RNA Poly(I:C).

Environmental agents, including viral and bacterial infectious agents, are involved in the alteration of physicochemical and biological parameters in the nasal epithelium. Hyaluronan (HA) has an important role in the regulation of tissue healing properties. High molecular weight HA (HMW-HA) shows greater anti-inflammatory responses than medium molecular weight HA (MMW-HA) and low molecular weight HA (LMW-HA). We investigated the effect of HMW-HA, MMW-HA and LMW-HA on the regulation of physicochemical and biological parameters in an “in vitro” model that might mimic viral infections of the nasal epithelium. Human nasal epithelial cell line RPMI2650 was stimulated with double-stranded RNA (dsRNA) Poly(I:C) for 5 days in air-liquid-interface (ALI) culture (3D model of airway tissue). dsRNA Poly(I:C) treatment significantly decreased transepithelial electrical resistance (TEER) in the stratified nasal epithelium of RPMI2650 and increased pH values, rheological parameters (elastic G’ and viscous G’’), and Muc5AC and Muc5B production in the apical wash of ALI culture of RPMI2650 in comparison to untreated cells. RPMI2650 treated with dsRNA Poly(I:C) in the presence of HMW-HA showed lower pH values, Muc5AC and Muc5B production, and rheological parameters, as well as increased TEER values in ALI culture, compared to cells treated with Poly(I:C) alone or pretreated with LMW-HA and MMW-HA. Our 3D “in vitro” model of epithelium suggests that HMW-HA might be a coadjuvant in the pharmacological treatment of viral infections, allowing for the control of some physicochemical and biological properties affecting the epithelial barrier of the nose during infection.

Capsaicin and Piperine as Functional Excipients for Improved Drug Delivery across Nasal Epithelial Models.

The fruit from various pepper plants has been employed for the seasoning of food, as perfuming agents, and also as traditional medicines. Phytochemicals isolated from different pepper species have been found to modulate the pharmacokinetics of orally administered drugs. This study investigated the possibility to apply capsaicin and piperine (extracted alkaloids) as modulators for drug delivery across the nasal epithelium. Both a nasal epithelial cell line (RPMI 2650) and excised sheep nasal tissue were used as models to investigate the effects of the selected pepper compounds on drug permeation. FITC-dextran 4400 (MW 4400 Da) was used as a large molecular weight marker compound for paracellular transport, while rhodamine 123 was used as a marker compound that is a substrate for P-glycoprotein-mediated efflux. From the permeation results, it was clear that capsaicin inhibited P-glycoprotein efflux to a larger extent, while piperine showed drug permeation enhancement via other mechanisms. The cell cytotoxicity studies indicated that capsaicin was noncytotoxic up to a concentration of 200 µM and piperine up to a concentration of 500 µM as indicated by cell viability above 80%. The histological analysis of the excised nasal tissue and cultured RPMI 2650 cell layers indicated that some damage occurred after treatment with 200 µM capsaicin, but no changes were observed for piperine up to a concentration of 50 µM.

Mixed Mucoadhesive Amphiphilic Polymeric Nanoparticles Cross a Model of Nasal Septum Epithelium in Vitro.

Intranasal administration of nano-drug-delivery systems emerged as an appealing strategy to surpass the blood-brain barrier and thus increase drug bioavailability in the central nervous system. However, a systematic study of the effect of the structural properties of the nanoparticles on the nose-to-brain transport is missing. In this work, we synthesized and characterized mixed amphiphilic polymeric nanoparticles combining two mucoadhesive graft copolymers, namely, chitosan- g-poly(methyl methacrylate) and poly(vinyl alcohol)- g-poly(methyl methacrylate), for the first time. Chitosan enables the physical stabilization of the nanoparticles by ionotropic cross-linking with tripolyphosphate and confers mucoadhesiveness, while poly(vinyl alcohol) is also mucoadhesive and, owing to its nonionic nature, it improves nanoparticle compatibility in nasal epithelial cells by reducing the surface charge of the nanoparticles. After a thorough characterization of the mixed nanoparticles by dynamic light scattering and nanoparticle tracking analysis, we investigated the cell uptake by fluorescence light and confocal microscopy and imaging flow cytometry. Mixed nanoparticles were readily internalized at 37 °C, while the uptake was inhibited almost completely at 4 °C, indicating the involvement of energy-dependent mechanisms. Finally, we assessed the nanoparticle permeability across liquid-liquid and air-liquid monolayers of a nasal septum epithelial cell line and studied the effect of nanoparticle concentration and temperature on the apparent permeability. Overall, our findings demonstrate that these novel amphiphilic nanoparticles cross this in vitro model of intranasal epithelium mainly by a passive (paracellular) pathway involving the opening of epithelial tight junctions.

The role of IL-36γ and its regulation in eosinophilic inflammation in allergic rhinitis

BackgroundAllergic rhinitis (AR) is characterized by eosinophilic inflammation. However, the function and regulation of eosinophils in AR are largely unknown. This study aimed to explore the expression and role of interleukin-36 (IL-36) cytokines in AR. MethodsSixty AR patients and 20 control subjects were recruited in this study. The mRNA and protein expression of serum IL-36 family cytokines and IL-36R in AR were detected by quantitative RT-PCR and enzyme-linked immunosorbent assay ELISA, respectively. IL-36R expression and regulation by eosinophils and the role of IL-36γ in the survival, adhesion, migration and activation of eosinophils were performed in purified eosinophils. Human nasal epithelial cell line was cultured and treated with different stimulators and IL-36γ was measured. ResultsThe mRNA and protein expression of serum IL-36 cytokines and IL-36R were significantly higher in AR compared with control, especially in asthmatic patients. Among the IL-36 cytokines, the expression of IL-36γ was the highest. The expression of IL-36R by eosinophils were significantly increased compared with normal controls and was up-regulated by recombinant IL-17, IL-25, IL-33 and Dermatophagoides pteronyssinus group 1. The IL-36γ promote the survival, adhesion, migration and activation of eosinophils. Human nasal epithelial cells can secrete IL-36γ after treated with recombinant IL-17, IL-25, IL-33. ConclusionsHigh expression of IL-36γ exaggerates eosinophilic inflammation in AR by promoting the survival, adhesion, and activation of eosinophils.

Tc17/IL-17A Up-Regulated the Expression of MMP-9 via NF-κB Pathway in Nasal Epithelial Cells of Patients With Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is a common chronic inflammatory disease of the upper airways involving nasal cavity and sinus. Deriving both from its clinical complexity with protean clinical manifestations as well its pathogenetic heterogeneity, the molecular mechanisms contributing to the pathogenesis of CRS remain unclear, and attract a wide interest in the field. Current evidences indicate that IL-17A is highly expressed in chronic rhinosinusitis with nasal polyps (CRSwNP). However, its pathogenetic role in regulation of tissue remodeling of CRSwNP remains unknown. The present study aimed to investigate the cellular origins and functions of IL-17A cytokine in CRSwNP, and further determined whether IL-17A could affect the expression of metalloproteinases (MMPs), the remodeling factors of CRSwNP. The results showed that the expression of IL-17A was upregulated in nasal tissues of patients with CRSwNP compared to those with chronic rhinosinusitis without nasal polyps (CRSsNP) and controls. CD8+ cytotoxic T lymphocytes (Tc) were major IL-17A producers in nasal tissues of CRSwNP. Interleukin (IL)-17-producing CD8+ T cells (Tc17) was significantly higher in nasal tissues of CRSwNP than CRSsNP and controls. Nonetheless, no difference was observed among the IL-17A in peripheral blood lymphocytes of these three groups. Moreover, in the same patients, IL-17A expression was negligible in lymphocytes of peripheral blood when compared with nasal tissues. Increased gene and protein expression of MMP-7 and MMP-9 in patients with CRSwNP compared with controls were observed. In CRSwNP samples, IL-17A receptor (IL-17AR) co-localized with MMP-9 and they were mainly expressed in the epithelial cells. MMP-9 expression was up-regulated both in Primary human nasal epithelial cells (PHNECs) and a nasal epithelial cell line (RPMI 2650) by IL-17A treatment, and diminished by anti-IL-17AR treatment. Furthermore, IL-17A promoted the expression of MMP-9 by activating the NF-κB signal pathway. Thus, our results have revealed a crucial role of IL-17A and Tc cells on pathogenesis and tissue remodeling of CRSwNP.

Human cystatin SN is an endogenous protease inhibitor that prevents allergic rhinitis

Protease allergens disrupt epithelial barriers to exert their allergenicity. Cystatin SN (encoded by CST1) is an endogenous cysteine protease inhibitor upregulated in nasal epithelia in patients with allergic rhinitis (AR). We sought to investigate the protective effect of human cystatin SN on AR symptoms using pollen-induced AR mouse models. We performed an invitro protease activity assay to evaluate the effect of recombinant human cystatin SN (rhCystatin SN) on Japanese cedar (JC) or ragweed proteases. Ahuman nasal epithelial cell line, RPMI 2650, was used to examine tight junction (TJ) disruption invitro. Mice were sensitized and nasally challenged with JC or ragweed pollens with or without rhCystatin SN to examine the effect of rhCystatin SN on AR symptoms and the epithelial barrier invivo. Because mice lack CST1, we generated transgenic (Tg) mice expressing human CST1 under control of its genomic control region (hCST1-Tg mice) to examine the role of cystatin SN in physiologically expressed conditions. rhCystatin SN inhibited JC but not ragweed protease activities and prevented JC-induced but not ragweed-induced TJ disruption invitro. Exogenous administration of rhCystatin SN ameliorated JC-induced but not ragweed-induced sneezing and nasal TJ disruption invivo. Furthermore, hCST1-Tg mice showed decreased JC-induced but not ragweed-induced sneezing symptoms and nasal TJ disruption compared with wild-type mice. Human cystatin SN suppresses AR symptoms through inhibiting allergen protease activities and protecting the nasal TJ barrier in an allergen-specific manner. We propose that upregulation of nasal endogenous protease inhibitors, including cystatin SN, is a novel therapeutic strategy for protease allergen-induced AR.

Silenced DMBT1 promotes nasal mucosa epithelial cell growth.

The aim of this study was to investigate the role of the deleted in malignant brain tumors 1 (DMBT1) gene in the development of nasal polyps, as well as related mechanisms. A stable human nasal mucosa epithelial cell (HNEpC) line with low expression of DMBT1 was generated. Three groups were established: a control group (HNEpCs without any treatment), a control short interference RNA (shRNA) group (HNEpCs transfected with an empty vector), and a DMBT1 shRNA group (HNEpCs with silenced DMBT1). Cell viability, apoptosis, and cell cycle distribution were measured after incubation. Expression of p53, signal transducer and activator of transcription 3 (STAT3), protein kinase B (AKT) and extracellular signal-regulated kinase1/2 (ERK1/2) was detected by western blotting. Compared with the control cell line, HNEpCs with silenced DMBT1 had increased viability and decreased apoptosis. Moreover, after DMBT1 silence, cell numbers were decreased significantly in the G1 phase and increased in the G2 and S phases. DMBT1 silence was associated with increased AKT expression and decreased p53 expression, but it did not alter expression of ERK1/2 or STAT3 (P > 0.05). Compared with the control cell line, HNEpCs transfected with an empty vector did not have altered cell viability, apoptosis, cell cycle distribution or expression of AKT, p53, ERK1/2, or STAT3 (P > 0.05). DMBT1 plays an important role in the growth and division of nasal epithelial cells. The possible mechanism might involve DMBT1 regulating the AKT-p53 pathway to promote cell viability and reduce apoptosis of nasal epithelial cells.

The application of silver nano-particles on developing potential treatment for chronic rhinosinusitis: Antibacterial action and cytotoxicity effect on human nasal epithelial cell model

Chronic rhinosinusitis (CRS) has raised attentions both in many countries due to its high relapsing rate and the resistance of certain treatments especially antibiotics application on both acute and chronic bacterial rhinosinusitis. The aim of this research is stressing on developing an effective alternative treatment for treating CRS and reducing the use of antibiotics to avoid further resistance forming. The antibacterial functions of silver nano-particles (AgNPs) are well known according to previous reports and studies. However, for developing a suitable treatment for further clinical application, a variety of AgNPs cell cytotoxicity experiments and AgNPs antibacterial properties experiments were examined in vitro in this study. For imitating the clinical condition of CRS, the human nasal epithelial cell line (RPMI2650) has chosen as experimental model. Moreover, Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) were selected for antibacterial function experiments. The analytical results demonstrated that 5ppm of AgNPs not only maintains >80% of cell activity to RPMI2650, but also possesses >80% of antibacterial function to S. aureus and 100% of antibacterial function to E. coli. Therefore, 5ppm of AgNPs might be considered as a promising antibacterial agent for treating CRS.

Effects of benzalkonium chloride on histamine H1 receptor mRNA expression in nasal epithelial cells.

To better understand the causes of the exacerbation of rhinitis medicamentosa (RM) induced by oxymetazoline (OMZ) or benzalkonium chloride (BKC), we examined the impact of pretreatment with OMZ or BKC on cultured human nasal epithelial cells. We also examined the effect of mometasone furoate (MF) on the cultured human nasal epithelial cells treated with OMZ or BKC. Cells of the human nasal epithelial cell line HNEpC were treated with OMZ or BKC, and the OMZ- and BKC-induced expression of histamine H1 receptor (H1R) mRNA was assayed using real-time polymerase chain reaction. In some experiments, 1.0×10(-5)M MF was added to the HNEpC cells for 24h before treatment with OMZ or BKC. Treatment with OMZ slightly increased the expression level of H1R mRNA in HNEpC cells. This enhanced expression was not significantly reduced by pretreatment with MF. In contrast, treatment with BKC remarkably increased the expression level of H1R mRNA in HNEpC cells. In addition, this enhanced expression was significantly reduced by pretreatment with MF. These results suggest that the increased expression of H1R mRNA due to treatment with OMZ or BKC might be one of the factors underlying the exacerbation of symptoms in patients with RM and those complicated with allergic rhinitis. The concomitant use of a nasal steroid might reduce the exacerbation of symptoms caused by BKC, although there remains a risk of developing histamine hypersensitivity from the long-term use of a topical steroid-containing BKC.

Transfer of the Cystic Fibrosis Transmembrane Conductance Regulator to Human Cystic Fibrosis Cells Mediated by Extracellular Vesicles.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in a deficiency in chloride channel activity. In this study, extracellular vesicles (EVs), microvesicles, and exosomes were used as vehicles to deliver exogenous CFTR glycoprotein and its encoding mRNA (mRNA(GFP-CFTR)) to CF cells to correct the CFTR chloride channel function. We isolated microvesicles and exosomes from the culture medium of CFTR-positive Calu-3 cells, or from A549 cells transduced with an adenoviral vector overexpressing a GFP-tagged CFTR (GFP-CFTR). Both microvesicles and exosomes had the capacity to package and deliver the GFP-CFTR glycoprotein and mRNA(GFP-CFTR) to target cells in a dose-dependent manner. Homologous versus heterologous EV-to-cell transfer was studied, and it appeared that the cellular uptake of EVs was significantly more efficient in homologous transfer. The incubation of CF15 cells, a nasal epithelial cell line homozygous for the ΔF508 CFTR mutation, with microvesicles or exosomes loaded with GFP-CFTR resulted in the correction of the CFTR function in CF cells in a dose-dependent manner. A time-course analysis of EV-transduced CF cells suggested that CFTR transferred as mature glycoprotein was responsible for the CFTR-associated channel activity detected at early times posttransduction, whereas GFP-CFTR translated from exogenous mRNA(GFP-CFTR) was responsible for the CFTR function at later times. Collectively, this study showed the potential application of microvesicles and exosomes as vectors for CFTR transfer and functional correction of the genetic defect in human CF cells.

Proprotein convertase 5/6A is associated with bone morphogenetic protein‐2‐induced squamous cell differentiation

Results Using a retinoic acid deficiency-induced squamous metaplasia model of HNECs, we observed a significant increase in the expression of PC5/6A, a PC member, and BMP-2, a candidate substrate for PC5/6A. Specific lentiviral shRNA-mediated PC5/6A knockdown decreased BMP-2 expression and maturation, decreased expression of squamous cell markers, and increased expression of ciliated cell markers. Dec-RVKR-CMK, a PC inhibitor, and LDN-193189, a BMP receptor inhibitor, suppressed squamous differentiation, promoted mucociliary differentiation, and down-regulated the BMP-2/Smad1/5/8/p38 signalling pathways. Dec-RVKRCMK also decreased expression of PC5/6A, but not furin, another PC member, suggesting the involvement of PC5/6A in squamous differentiation of HNECs. Overexpression of PC5/6A and BMP-2 in the human nasal epithelial cell line RPMI-2650 demonstrated that PC5/6A can activate BMP-2. Under retinoic acid-sufficient culture conditions for mucociliary differentiation of HNECs, short-term expression of PC5/6A by the adenovirus system and addition of exogenous BMP-2 induced squamous differentiation. Furthermore, PC5/6A and BMP-2 were highly expressed in metaplastic squamous epithelium of human nasal polyps.

Human parainfluenza virus type 3 (HPIV3) induces production of IFNγ and RANTES in human nasal epithelial cells (HNECs).

BackgroundHuman parainfluenza virus type 3 (HPIV3), while infecting lower airway epithelial cells induces pneumonia and bronchiolitis in infants and children, and may lead to asthma exacerbations in children and adults. Respiratory viruses invading the airway epithelium activate innate immune response and induce inflammatory cytokine release contributing to the pathophysiology of upper and lower airway disorders. However, the effects of HPIV3 infection on nasal epithelial cells have not been well defined.The aim of this study was to evaluate the effect of the HPIV3 infection on cultured human nasal epithelial cells (HNECs) and the release of interferon gamma and other cytokines.MethodsRPMI 2650, a human nasal epithelial cell line was cultured into confluence and was infected with HPIV3 (MOI of 0.1, 0.01 and 0.001). The protein release into supernatants and mRNA expression of selected cytokines were assessed 24, 48 and 72 h after infection. Cytokine concentrations in supernatants were measured by ELISA and expression of cytokine mRNA in RPMI 2650 cells confirmed by real time RT-PCR analysis.ResultsHNECs infection with HPIV3 did not induce cytotoxicity for at least 48 hours, but significantly increased IFN-γ protein concentration in the cell supernatants at 24 h and 48 h post infection (by 387% and 485% respectively as compared to mock infected cells). At 24 h a significant increase in expression of mRNA for IFNγ was observed. RANTES protein concentration and mRNA expression were significantly increased at 72 h after infection (mean protein concentration: 3.5 ± 1.4 pg/mL for 0.001 MOI, 10.8 ± 4.6 pg/mL for 0.01 MOI and 61.5 ± 18.4 pg/mL for 0.1 MOI as compared to 2.4 ± 1.3 pg/mL for uninfected cells). No measurable concentrations of TNF-α, IL-10, TSLP, IL-8, GM-CSF or eotaxin, were detected in virus infected cells supernatants.ConclusionsHPIV3 effectively infects upper airway epithelial cells and the infection is associated with induction of IFN-γ and generation of RANTES.

Proprotein convertase 5/6a is associated with bone morphogenetic protein-2-induced squamous cell differentiation.

Squamous metaplasia in airway epithelium is a pathological process arising from abnormal remodeling/repair responses to injury. Proteolytic maturation of many growth and differentiation factors involved in tissue remodeling is controlled by proprotein convertases (PCs). However, the role of these convertases in airway remodeling remains poorly understood. Using a retinoic acid deficiency-induced squamous metaplasia model of cultured human nasal epithelial cells (HNECs), we observed a significant increase in the expression of PC5/6A, a PC member, and bone morphogenetic protein-2 (BMP-2), a candidate substrate for PC5/6A. Specific lentiviral short hairpin RNA-mediated PC5/6A knockdown decreased BMP-2 expression and maturation, decreased expression of squamous cell markers, and increased expression of ciliated cell markers. Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK), a PC inhibitor, and LDN-193189, a BMP receptor inhibitor, suppressed squamous differentiation, promoted mucociliary differentiation, and down-regulated the BMP-2/Smad1/5/8/p38 signaling pathways. Dec-RVKR-CMK also decreased expression of PC5/6A, but not furin, another PC member, suggesting the involvement of PC5/6A in squamous differentiation of HNECs. Overexpression of PC5/6A and BMP-2 in the human nasal epithelial cell line RPMI-2650 demonstrated that PC5/6A can activate BMP-2. Under retinoic acid-sufficient culture conditions for mucociliary differentiation of HNECs, short-term expression of PC5/6A by the adenovirus system and addition of exogenous BMP-2 induced squamous differentiation. Furthermore, PC5/6A and BMP-2 were highly expressed in metaplastic squamous epithelium of human nasal polyps. Taken together, PC5/6A is involved in squamous differentiation of HNECs, possibly through up-regulation of the BMP-2/pSmad1/5/8/p38 signaling pathway, pointing to a potential therapeutic target for the prevention of chronic airway diseases that exhibit squamous metaplasia.

The characterization of the human nasal epithelial cell line RPMI 2650 under different culture conditions and their optimization for an appropriate in vitro nasal model.

The further characterization of the cell line RPMI 2650 and the evaluation of different culture conditions for an in vitro model for nasal mucosa. Cells were cultured in media MEM or A-MEM at air-liquid (A-L) or liquid-liquid (L-L) interfaces for 1 or 3 weeks. Different cryopreservation methods and cell culture techniques were evaluated with immunolabelling of junctional proteins, ultrastructural analysis using electron microscopy, transepithelial electrical resistance (TEER) measurements, permeation studies with dextran and jacalin, and gene expression profiling of 84 drug transporters. Cell proliferation and differentiation depended on the used medium. The established epithelia expressed occludin, claudin-1, and E-cadherin under all conditions. Cells grown at the A-L interface formed more layers and exhibited a higher TEER and lower dextran and jacalin permeability than at the L-L interface, where cells morphologically exhibited a more differentiated phenotype. The expression of ABC and SLC transporters depended on culture duration and interface. The RPMI 2650 cells form a polarized epithelium resembling nasal mucosa. However, different culture conditions have a significant effect on cell ultrastructure, barrier integrity, and gene expression, and should be considered when using this cell line as an in vitro model for drug permeability studies and screening of nasal drug candidates.

Fluticasone furoate maintains epithelial homeostasis via leptin/leptin receptor pathway in nasal cells.

Leptin is involved in the lung epithelial homeostasis. Its role in the nasal tract is largely unknown. Allergic rhinitis (AR) is induced by the allergen exposure leading to consequential structural abnormalities in the nasal epithelium. Topical corticosteroids are recommended as first-line therapy in AR. Parietaria pollen is one of the most important allergenic sources in the southern Europe. In vitro, in human nasal epithelial cell line RPMI 2650, we aimed to determine whether allergen stimulation acts on leptin/leptin receptor pathway and how fluticasone furoate (FF) influences this pathway. The effects of the major allergen recombinant Par j 1 (rPar j 1), of FF, of leptin, and of TGF-β1 on cell proliferation, on leptin/leptin receptor expression and modulation (by clonogenic test, by RT-q-RT-PCR, by immunocytochemistry and by flow-cytometry), and on STAT-3 activation (assessing nuclear translocation by western blot analysis) were assessed. We found that rPar j 1 and TGF-β1 significantly decreased cell proliferation and down-regulated the leptin/leptin receptor pathway, whereas FF and leptin reverted them, both alone and in combination. Furthermore, rPar j 1 reduced, while leptin and FF increased STAT-3 activation. In conclusion, FF and leptin itself are able to preserve nasal epithelial homeostasis restoring the leptin/leptin receptor pathway altered by rPar j 1 exposure.

Hypoxia-mediated mechanism of MUC5AC production in human nasal epithelia and its implication in rhinosinusitis.

BackgroundExcessive mucus production is typical in various upper airway diseases. In sinusitis, the expression of MUC5AC, a major respiratory mucin gene, increases. However, the mechanisms leading to mucus hypersecretion in sinusitis have not been characterized. Hypoxia due to occlusion of the sinus ostium is one of the major pathologic mechanisms of sinusitis, but there have been no reports regarding the mechanism of hypoxia-induced mucus hypersecretion.Methods and FindingsThis study aims to identify whether hypoxia may induce mucus hypersecretion and elucidate its mechanism. Normal human nasal epithelial (NHNE) cells and human lung mucoepidermoid carcinoma cell line (NCI-H292) were used. Sinus mucosa from patients was also tested. Anoxic condition was in an anaerobic chamber with a 95% N2/5% CO2 atmosphere. The regulatory mechanism of MUC5AC by anoxia was investigated using RT-PCR, real-time PCR, western blot, ChIP, electrophoretic mobility shift, and luciferase assay. We show that levels of MUC5AC mRNA and the corresponding secreted protein increase in anoxic cultured NHNE cells. The major transcription factor for hypoxia-related signaling, HIF-1α, is induced during hypoxia, and transfection of a mammalian expression vector encoding HIF-1α results in increased MUC5AC mRNA levels under normoxic conditions. Moreover, hypoxia-induced expression of MUC5AC mRNA is down-regulated by transfected HIF-1α siRNA. We found increased MUC5AC promoter activity under anoxic conditions, as indicated by a luciferase reporter assay, and mutation of the putative hypoxia-response element in MUC5AC promoter attenuated this activity. Binding of over-expressed HIF-1α to the hypoxia-response element in the MUC5AC promoter was confirmed. In human sinusitis mucosa, which is supposed to be hypoxic, expression of MUC5AC and HIF-1α is higher than in control mucosa.ConclusionThe results indicate that anoxia up-regulates MUC5AC by the HIF-1α signaling pathway in human nasal epithelia and suggest that hypoxia might be a pathogenic mechanism of mucus hypersecretion in sinusitis.

Formulation and Characterization of Nanoemulsion Intranasal Adjuvants: Effects of Surfactant Composition on Mucoadhesion and Immunogenicity

The development of effective intranasal vaccines is of great interest due to their potential to induce both mucosal and systemic immunity. Here we produced oil-in-water nanoemulsion (NE) formulations containing various cationic and nonionic surfactants for use as adjuvants for the intranasal delivery of vaccine antigens. NE induced immunogenicity and antigen delivery are believed to be facilitated through initial contact interactions between the NE droplet and mucosal surfaces which promote prolonged residence of the vaccine at the site of application, and thus cellular uptake. However, the details of this mechanism have yet to be fully characterized experimentally. We have studied the physicochemical properties of the NE droplet surfactant components and demonstrate that properties such as charge and polar headgroup geometry influence the association of the adjuvant with the mucus protein, mucin. Association of NE droplets with mucin in vitro was characterized by various biophysical and imaging methods including dynamic light scattering (DLS), zeta potential (ZP), and surface plasmon resonance (SPR) measurements as well as transmission electron microscopy (TEM). Emulsion surfactant compositions were varied in a systematic manner to evaluate the effects of hydrophobicity and polar group charge/size on the NE-mucin interaction. Several cationic NE formulations were found to facilitate cellular uptake of the model antigen, ovalbumin (OVA), in a nasal epithelial cell line. Furthermore, fluorescent images of tissue sections from mice intranasally immunized with the same NEs containing green fluorescent protein (GFP) antigen demonstrated that these NEs also enhanced mucosal layer penetration and cellular uptake of antigen in vivo. NE-mucin interactions observed through biophysical measurements corresponded with the ability of the NE to enhance cellular uptake. Formulations that enhanced antigen uptake in vitro and in vivo also led to the induction of a more consistent antigen specific immune response in mice immunized with NEs containing OVA, linking NE-facilitated mucosal layer penetration and cellular uptake to enhancement of the immune response. These findings suggest that biophysical measurement of the mucoadhesive properties of emulsion based vaccines constitutes an effective in vitro strategy for selecting NE candidates for further evaluation in vivo as mucosal adjuvants.