Human Sinonasal Epithelial Cells Research Articles

Retraction: The expression of ephrinA1/ephA2 receptor increases in chronic rhinosinusitis and ephrinA1/ephA2 signaling affects rhinovirus-induced innate immunity in human sinonasal epithelial cells

Retraction: The expression of ephrinA1/ephA2 receptor increases in chronic rhinosinusitis and ephrinA1/ephA2 signaling affects rhinovirus-induced innate immunity in human sinonasal epithelial cells

House dust mite allergens induce Ca2+ signalling and alarmin responses in asthma airway epithelial cells

Type 2 inflammation in asthma develops with exposure to stimuli to include inhaled allergens from house dust mites (HDM). Features include mucus hypersecretion and the formation of pro-secretory ion transport characterised by elevated basal Cl− current. Studies using human sinonasal epithelial cells treated with HDM extract report a higher protease activated receptor-2 (PAR-2) agonist-induced calcium mobilisation that may be related to airway sensitisation by allergen-associated proteases. Herein, this study aimed to investigate the effect of HDM on Ca2+ signalling and inflammatory responses in asthmatic airway epithelial cells. Primary bronchial epithelial cells (hPBECs) from asthma donors cultured at air-liquid interface were used to assess electrophysiological, Ca2+ signalling and inflammatory responses. Differences were observed regarding Ca2+ signalling in response to PAR-2 agonist 2-Furoyl-LIGRLO-amide (2-FLI), and equivalent short-circuit current (Ieq) in response to trypsin and 2-FLI, in ALI-asthma and healthy hPBECs. HDM treatment led to increased levels of intracellular cations (Ca2+, Na+) and significantly reduced the 2-FLI-induced change of Ieq in asthma cells. Apical HDM-induced Ca2+ mobilisation was found to mainly involve the activation of PAR-2 and PAR-4-associated store-operated Ca2+ influx and TRPV1. In contrast, PAR-2, PAR-4 antagonists and TRPV1 antagonist only showed slight impact on basolateral HDM-induced Ca2+ mobilisation. HDM trypsin-like serine proteases were the main components leading to non-amiloride sensitive Ieq and also increased interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP) from asthma hPBECs. These studies add further insight into the complex mechanisms associated with HDM-induced alterations in cell signalling and their relevance to pathological changes within asthma.

Hypoxia-induced cystic fibrosis transmembrane conductance regulator dysfunction is a universal mechanism underlying reduced mucociliary transport in sinusitis.

Hypoxia due to sinus obstruction is a major pathogenic mechanism leading to sinusitis. The objective of the current study is to define the electrophysiologic characteristics of hypoxia in vitro and in vivo. Cystic fibrosis bronchoepithelial cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and human sinonasal epithelial cells were exposed to 1% or atmospheric O2 for 24h. Time-dependent production of cytoplasmic free radicals was measured. Cells were subjected to Ussing chamber and patch clamp technique where CFTR currents were recorded in whole-cell and cell-attached mode for single channel studies. Indices of mucociliary transport (MCT) were measured using micro-optical coherence tomography. In a rabbit hypoxic maxillary sinus model, tissue oxygenation, relative mRNA expression of HIF-1α, pH, sinus potential difference (SPD), and MCT were determined. Ussing chamber (p<0.05), whole-cell (p<0.001), and single channel patch-clamp (p<0.0001) showed significant inhibition of Cl- currents in hypoxic cells. Cytoplasmic free radicals showed time-dependent elevation peaking at 4h (p<0.0001). Airway surface liquid (p<0.0001), periciliary liquid (p<0.001), and MCT (p<0.01) were diminished. Co-incubation with the free radical scavenger glutathione negated the impact of hypoxia on single channel currents and MCT markers. In sinusitis rabbits, mucosa exhibited low tissue oxygenation (p<0.0001), increased HIF1α mRNA (p<0.05), reduced pH (p<0.01), and decreased MCT (p<0.001). SPD measurements demonstrated markedly diminished transepithelial Cl- transport (p<0.0001). Hypoxia induces severe CFTR dysfunction via free radical production causing reduced MCT in vitro and in vivo. Improved oxygenation is critical to reducing the impact of persistent mucociliary dysfunction.

The role of TAS2R38 genotype in surgical outcomes and culturable bacteria in chronic rhinosinusitis with or without nasal polyps.

Recent studies reported the relationship between genetic variations and TAS2R38, which is a bitter taste receptor expressed in the cilia of human sinonasal epithelial cells, among the predisposing factors playing role in immune response to upper respiratory tract bacterial infection. The present study aims to examine the relationship of TAS2R38 genotype with the active microorganism and the effect of genotype on the surgical outcomes among chronic rhinosinusitis patients. 34 patients undergoing endoscopic sinus surgery (ESS) for chronic rhinosinusitis with or without polyps (23 CRSwNP, 11 CRSsNP) and 30 patients undergoing septoplasty surgery for isolated nasal septum deviation were included. All the patients were genotyped for TAS2R38. Scoring was made using endoscopic Modified Lund-Kennedy and radiological Lund-Mackay systems preoperatively. Sino-Nasal Outcome Test with 22 items (SNOT-22) was implemented preoperatively and postoperatively. Nasal swab culture samples were taken intraoperatively from CRS patients and the active microorganism were isolated. In the TAS2R38 genotyping of the study group, PAV/PAV was found in 32.4% of patients, PAV/AVI in 47.1%, and AVI/AVI in 20.6%. In the control group, PAV/PAV was found in 26.7%, PAV/AVI in 36.7%, and AVI/AVI in 36.7%. In the study group, there was no statistically significant difference between the CRS and CRS subgroups in terms of TAS2R38 genotype distributions. The changes in patients' preoperative and postoperative SNOT-22 scores were similar between the genotypes. Proliferation was detected in culture in the whole AVI-AVI group, 81.8% of PAV-PAV group, and 56.3% of PAV-AVI group but the difference was not found to be statistically significant. The proliferation level of Staphylococcus epidermidis by TAS2R38 genotype was found to be statistically significantly higher among patients, who had AVI-AVI genotype, in CRSwNP. We did not find a statistically significant relationship between the TAS2R38 genotype and CRS subtype, sinonasal bacterial infection risk increase and surgical success rate in CRS patients. Long-term and large-scale studies are needed, which are to be carried out by individual genotyping and sequencing to provide more information on the effects of these genetic variants.

The Expression of ephrinA1/ephA2 Receptor Increases in Chronic Rhinosinusitis and ephrinA1/ephA2 Signaling Affects Rhinovirus-Induced Innate Immunity in Human Sinonasal Epithelial Cells.

EphA2 receptor and its ephrin ligands are involved in virus infection, epithelial permeability, and chemokine secretion. We hypothesized that ephrinA1/ephA2 signaling participates in rhinovirus (RV)-induced antiviral immune response in sinonasal mucosa of patients with chronic rhinosinusitis (CRS). Therefore, we investigated the expression of ephrinA1/ephA2 in normal and inflamed sinonasal mucosa and evaluated whether they regulate chemokine secretion and the production of antiviral immune mediators including interferons (IFNs) in RV-infected human primary sinonasal epithelial cells. For this purpose, the expression and distribution of ephrinA1/ephA2 in sinonasal mucosa were evaluated with RT-qPCR, immunofluorescence, and western blot. Their roles in chemokine secretion and the production of antiviral immune mediators such as type I and III IFNs, and interferon stimulated genes were evaluated by stimulating ephA2 with ephrinA1 and inactivating ephA2 with ephA2 siRNA or inhibitor in cells exposed to RV and poly(I:C). We found that ephrinA1/ephA2 were expressed in normal mucosa and their levels increased in inflamed sinonasal mucosa of CRS patients. RV infection or poly(I:C) treatment induced chemokine secretion which were attenuated by blocking the action of ephA2 with ephA2 siRNA or inhibitor. The production of antiviral immune mediators enhanced by rhinovirus or poly (I:C) is increased by blocking ephA2 compared with that of cells stimulated by either rhinovirus or poly(I:C) alone. In addition, blocking ephA2 attenuated RV replication in cultured cells. Taken together, these results describe a novel role of ephrinA1/ephA2 signaling in antiviral innate immune response in sinonasal epithelium, suggesting their participation in RV-induced development and exacerbations of CRS.

PM2.5 Upregulates the Expression of MUC5AC via the EGFR-PI3K Pathway in Human Sinonasal Epithelial Cells

Background: Fine particulate matter (PM) (PM with an aerodynamic diameter <2.5 μm, PM2.5) exposure contributes to respiratory disease development and exacerbation. Objective: We sought to investigate the effect of PM2.5 exposure on mucociliary function in primary human nasal epithelial cells (HNECs) and the underlying mechanism. Methods: HNECs derived from control subjects and patients with chronic rhinosinusitis with nasal polyps were established as air-liquid interface cultures. Confluent cultures were exposed to 100 or 200 μg/mL PM2.5 for 24 h and assessed for expression of specific mucociliary-associated factors, the percentage of β-tubulin IV-positive and MUC5AC-positive cells, expression of epidermal growth factor receptor (EGFR) ligand and activation of phosphoinositide 3-kinase (PI3K)-AKT/ERK. In addition, cultures pretreated for 30 min with AG1478 (an EGFR inhibitor) or LY294002 (a PI3K inhibitor) following PM2.5 exposure were assessed for MUC5AC mRNA and protein expression. Results: PM2.5 exposure at 100 or 200 μg/mL for 24 h did not affect geminin coiled-coil domain containing, multiciliate differentiation and DNA synthesis associated cell cycle protein, FOXJ1, or DNAI2 mRNA expression or the percentage of β-tubulin IV-positive cells. However, 200 μg/mL PM2.5 exposure significantly increased mRNA expression of SAM-pointed domain-containing ETS transcription factor and MUC5AC and the percentage of MUC5AC-positive cells. PM2.5 also increased expression of EGFR ligands, including heparin-binding EGF-like growth factor and amphiregulin. Furthermore, PM2.5induced activation of PI3K, AKT, and ERK, and pretreatment of HNECs with AG1478 or LY294002 attenuated PM2.5-induced MUC5AC mRNA and protein expression. Conclusions and Clinical Relevance: This study demonstrates that short-term PM2.5 exposure increases MUC5AC expression in HNECs. Furthermore, this study shows that PM2.5-induced MUC5AC expression is likely mediated through the EGFR-PI3K pathway.

S100A7 Co-localization and Up-regulation of Filaggrin in Human Sinonasal Epithelial Cells.

Filaggrin (FLG) is a protein expressed in the epidermis and involved in the maintenance of the epidermal barrier. However, the expression and localization of FLG in the upper airway remain controversial. The present study aimed to determine the significance of FLG and the effect of S100A7 on FLG expression in the upper respiratory mucosa. Human nasal epithelial cells (HNECs) were cultured and examined for FLG expression and S100A7 effects by real-time polymerase chain reaction and Western blotting. The localization and distribution of FLG were assessed using sinonasal mucosa. A significant expression of FLG was detected at the mRNA and protein levels in HNECs. A moderate FLG immunoreactivity was observed in the epithelial cells, but no staining was seen in epithelial goblet cells. S100A7 increased the FLG mRNA level in HNECs in a dose-dependent manner and also up-regulated the FLG protein in a dose-dependent manner. This study significantly contributes to a better understanding of the role of FLG in the pathogenesis of airway inflammation from the viewpoint of the epithelial barrier function. FLG-related events in response to S100A7 protein may represent novel therapeutic targets for the treatment of upper airway inflammation.

In vitro evaluation of a novel oxygen-generating biomaterial for chronic rhinosinusitis therapy.

Hypoxia due to closure at the ostiomeatal complex is widely considered one of the major pathogenic mechanisms leading to chronic inflammation in chronic rhinosinusitis (CRS). The objective of this study was to develop and characterize an oxygen-generating biomaterial (OGB) as an innovative treatment strategy for CRS. An OGB was fabricated by coating hydrophobic beeswax (BW, 15mg or 30mg) on the surface of calcium peroxide - catalase complex (CPO-CA, 30mg) and characterized using scanning electron microscopy (SEM). In vitro releases of both oxygen and hydrogen peroxide (H2 O2 ) were spectrophotometrically quantified, and cytotoxicity in human sinonasal epithelial cells (HSNECs) was evaluated. The influence of OGB on transepithelial Cl- secretion was also determined by pharmacologically manipulating HSNECs, cultured under hypoxic conditions, in Ussing chambers. Three groups of OGBs: (1) CPO only; (2) CPO coated with CA and BW (1:1 ratio, CPO-CA(1)-BW(1)); and (3) CPO coated with CA and BW (1:0.5 ratio, CPO-CA(1)-BW(0.5) were analyzed for accumulated oxygen release over 7 days: highest release (mmol/mg) was observed in CPO-CA(1)-BW(1)=0.11±0.003, followed by CPO-CA(1)-BW(0.5)=0.08 ± 0.010, and CPO=0.05 ± 0.004 (p<0.0001). H2 O2 production (mM) was significantly higher in CPO (1.87±0.50) compared to CPO-CA (1)-BW(1) (0.00±0.00) (p<0.001) after 24h. CPO-CA(1)-BW(1) showed significantly reduced cytotoxicity and increased Cl- transport compared to the CPO group. A novel OGB (CPO-CA-BW complex) exhibited sustained oxygen release over 7 days without significant cytotoxicity after 24h in vitro. Preclinical studies evaluating the efficacy of OGB in CRS are warranted, especially for potential therapy in an obstruction-based CRS model.

LPS decreases CFTR open probability and mucociliary transport through generation of reactive oxygen species

Lipopolysaccharide (LPS) serves as the interface between gram-negative bacteria (GNB) and the innate immune response in respiratory epithelial cells (REC). Herein, we describe a novel biological role of LPS that permits GNB to persist in the respiratory tract through inducing CFTR and mucociliary dysfunction. LPS reduced cystic fibrosis transmembrane conductance regulater (CFTR)-mediated short-circuit current in mammalian REC in Ussing chambers and nearly abrogated CFTR single channel activity (defined as forskolin-activated Cl- currents) in patch clamp studies, effects of which were blocked with toll-like receptor (TLR)-4 inhibitor. Unitary conductance and single-channel amplitude of CFTR were unaffected, but open probability and number of active channels were markedly decreased. LPS increased cytoplasmic and mitochondrial reactive oxygen species resulting in CFTR carbonylation. All effects of exposure were eliminated when reduced glutathione was added in the medium along with LPS. Functional microanatomy parameters, including mucociliary transport, in human sinonasal epithelial cells in vitro were also decreased, but restored with co-incubation with glutathione or TLR-4 inhibitor. In vivo measurements, following application of LPS in the nasal cavities showed significant decreases in transepithelial Cl- secretion as measured by nasal potential difference (NPD) – an effect that was nullified with glutathione and TLR-4 inhibitor. These data provide definitive evidence that LPS-generated reactive intermediates downregulate CFTR function in vitro and in vivo which results in cystic fibrosis-type disease. Findings have implications for therapeutic approaches intent on stimulating Cl- secretion and/or reducing oxidative stress to decrease the sequelae of GNB airway colonization and infection.

WDPCP Modulates Cilia Beating Through the MAPK/ERK Pathway in Chronic Rhinosinusitis With Nasal Polyps.

Cilia loss and dysfunction is one of the typical pathological features of chronic rhinosinusitis with nasal polyps (CRSwNP). Tryptophan-aspartic acid (W-D) repeat containing planar cell polarity effector (WDPCP) has been proven to be an essential element for ciliogenesis in human nasal epithelium, but its role in the beating of cilia remains unclear. In this study, we sought to investigate the role of WDPCP and its underlying mechanism behind the dysfunction in the beating of cilia in nasal polyp tissue. We demonstrated WDPCP expression in the epithelium of nasal polyps. We also investigated the MAPK/ERK pathway in primary human sinonasal epithelial cells to explore the function of WDPCP. The air–liquid interface culture system was used as a model to verify the role of WDPCP and the MAPK/ERK pathway in the beating of cilia. With the dysfunction of cilia beating, we observed a low expression of WDPCP in the epithelium of nasal polyp tissues. Within the in vitro study, we found that WDPCP was critical for mitochondrial biogenesis and mitochondrial function in human sinonasal epithelial cells, possibly due to the activation of the MAPK/ERK pathway. The mitochondrial dysfunction caused by U0126 or lacking WDPCP could be partially recovered by dexamethasone. The low expression of WDPCP in nasal epithelium could affect mitochondria via the MAPK/ERK pathway, which may contribute to the dysfunction in the beating of cilia in CRSwNP.

Azithromycin and ciprofloxacin inhibit interleukin-8 secretion without disrupting human sinonasal epithelial integrity in vitro.

We recently developed a ciprofloxacin and azithromycin sinus stent (CASS) to target recalcitrant infections in chronic rhinosinusitis (CRS). The objective of this study was to evaluate the anti-inflammatory activity of azithromycin released from the CASS and assess the impact on the integrity and function of primary human sinonasal epithelial cells (HSNECs). Pseudomonas aeruginosa lipopolysaccharide (LPS)-stimulated HSNECs were treated with azithromycin and/or ciprofloxacin at concentrations attainable from CASS release. Interleukin-8 (IL-8) secretion was quantified by enzyme-linked immunosorbent assay (ELISA). Epithelial integrity (transepithelial resistance [TEER], paracellular permeability [fluorescein isothiocyanate-labeled dextran], lactate dehydrogenase [LDH] assays) and function (ciliary beat frequency [CBF]) were also evaluated. Azithromycin significantly reduced secreted IL-8 from P. aeruginosa LPS-stimulated HSNECs at all concentrations tested (mean ± standard deviation; control = 5.77 ± 0.39 ng/mL, azithromycin [6 μg/mL] = 4.58 ± 0.40 ng/mL, azithromycin [60 µg/mL] = 4.31 ± 0.06, azithromycin [180 µg/mL] = 4.27 ± 0.26 ng/mL, p < 0.05). Co-incubation with azithromycin (6 µg/mL) and ciprofloxacin (2.4 µg/mL) in LPS-stimulated HSNECs also displayed a significant reduction in secreted IL-8 when compared to P. aeruginosa LPS alone (co-treatment = 4.61 ± 0.29 ng/mL, P. aeruginosa LPS = 7.35 ± 0.89 ng/mL, p < 0.01). The drugs did not negatively impact TEER, paracellular permeability, LDH release, or CBF, indicating retention of cell integrity and function. Azithromycin decreased P. aeruginosa LPS IL-8 production in HSNECs at drug concentrations attainable with sustained release of azithromycin from the CASS. In addition to antibacterial activity, anti-inflammatory properties of the CASS should provide further benefit for patients with recalcitrant CRS.

Topical Vitamin D May Modulate Human Sinonasal Mucosal Responses to House Dust Mite Antigen.

Respiratory epithelium is a key defense against inhaled pathogens. Vitamin D3 (VD) has been suggested to modulate airway inflammation; however, its effect on innate airway defenses, the physical barrier, mucociliary apparatus, and cytokine release remains unclear. To investigate the outcomes of VD application prior to challenge in an in vitro model of human sinonasal epithelium, through assessment of epithelial transepithelial resistance (TER), cilia beat frequency (CBF), and interleukin (IL)-6 release, and secondarily to determine whether topical VD is beneficial to patients with inflammatory sinonasal pathology. Primary human sinonasal epithelial cells from patients with eosinophilic chronic rhinosinusitis (eCRS) and healthy controls were cultured in air-liquid interface (ALI). Well-differentiated cultures from each patient were pretreated for 24 hours with 4 different VD doses. Toxicity was quantified at 24 hours in unchallenged ALI by lactate dehydrogenase (LDH) assay. Innate responses were assessed by measuring TER and CBF before and up to 24 hours after house dust mite Dermatophagoides pteronyssinus challenge. IL-6 release was evaluated 24-hour postchallenge. Fifteen patients (53 ± 13.5 years, 60% females, 53% eCRS) representing 120 ALI wells were assessed. VD (0, 25, 50, 150 IU/mL) released less LDH than vehicle, indicating noncytotoxicity (0.15 ± 0.02; 0.15 ± 0.00; 0.14 ± 0.02; 0.11 ± 0.01 vs 0.17 ± 0.03, P = .004). VD increased TER for eCRS wells at 5 minutes (50 IU/mL: Δ6.76 ± 3.93 vs Δ3.87 ± 2.46, P = .04) and 24 hours (50 IU/mL: Δ0.88 ± 0.49 vs Δ0.40 ± 0.42, P = .02; 150 IU/mL: Δ1.06 ± 0.58 vs Δ0.47 ± 0.46, P = .01). CBF increased at 1 hour for eCRS wells (50 IU/mL: Δ0.62 ± 0.14 vs Δ0.41 ± 0.13, P = .001; 150 IU/ml: Δ0.60 ± 0.13 vs Δ0.38 ± 0.11, P < .001). IL-6 release was similar between normal and eCRS wells. Topical VD supplementation in eCRS patients may be beneficial for innate epithelial defenses. VD is noncytotoxic and does not adversely affect the physical barrier, mucociliary apparatus, or IL-6 release. Further studies should clarify its potential as a therapeutic agent.

In vitro safety of ketotifen as a topical nasal rinse.

Ketotifen is a second-generation noncompetitive H1-antihistamine and mast-cell stabilizer. It is commonly used to treat or prevent allergic conjunctivitis, asthma, chronic urticaria, anaphylaxis, mast-cell, and other allergic-type disorders. However, it has never been studied in aspirin-exacerbated respiratory disease (AERD), an aggressive phenotype of chronic rhinosinusitis with nasal polyps, where the mast cell plays a prominent role its pathogenesis. Human sinonasal epithelial cells were grown at an air-liquid interface (ALI). Ketotifen powder was dissolved in saline to make 4 test solutions at 1.04, 2.08, 10.4, and 20.8 µg/mL. Control (saline) or ketotifen solution was added apically to ALI cultures from tissue of 5 unique patients, and ciliary beat frequency (CBF) changes were recorded. Lactate dehydrogenase was measured at 24 and 48 hours to estimate long-term cellular toxicity. Apical application of ketotifen at all concentrations was neither ciliotoxic nor ciliostimulatory, with no change in CBF over a period of 15 minutes after application. Cellular toxicity for all concentrations at 24 and 48 hours after application was <3% and <7%, respectively, that of lysed cultures. Topical application of ketotifen to an in vitro model of sinonasal epithelium is safe, as evaluated by CBF and lactate dehydrogenase. Ketotifen is neither ciliotoxic nor ciliostimulatory, and no long-term cellular toxicity was observed. Ketotifen may have promise as a topical nasal rinse in the treatment of AERD.

Contribution of Epithelial Cell Dysfunction to the Pathogenesis of Chronic Rhinosinusitis with Nasal Polyps

BackgroundIn the past, the airway epithelium was thought to be primarily an inert physical barrier. We now know that the upper airway epithelium plays a critical role in both innate and adaptive immunity, and that epithelial dysfunction is strongly associated with inflammatory airway disease. The pathogenesis of chronic rhinosinusitis is poorly understood, but growing evidence supports a key role for the airway epithelium in the pathophysiology of the disease.ObjectiveThe purpose of this study is to explore our current understanding of how dysfunction in human sinonasal epithelial cells (HSNECs) contributes to the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) and to examine how current and developing therapies affect epithelial cell functions.MethodsA literature review of papers published in English pertaining to epithelial cell dysfunction in patients with CRSwNP was performed using the PubMed database. The search utilized combinations of the following key words: sinusitis, polyps, epithelium, pathophysiology, barrier function, dendritic cells, eosinophils, T cells, complement, mucociliary clearance, vitamin D, cytokines, chemokines, taste receptors, steroids, saline, and therapy.ResultsHSNEC mucociliary clearance, barrier function, secretion of cytokines, influence on dendritic cells, influence on T-cells, regulation of eosinophils, vitamin D metabolism, complement production, and taste receptor function are altered in patients with CRSwNP and contribute to the pathogenesis of the disease. Current therapies utilized to manage CRSwNP counteract the effects of HSNEC dysfunction and relieve key symptoms of the disease.ConclusionHSNECs are key players in both innate and adaptive immunity, and altered epithelial functions are closely intertwined with the pathogenesis of CRSwNP. Our review supports further investigation of altered HSNEC function in patients with CRSwNP and supports development of novel epithelial-targeted therapies for its management.

Responses of Well-differentiated Human Sinonasal Epithelial Cells to Allergen Exposure and Environmental Pollution in Chronic Rhinosinusitis.

Background Evidence suggests that intrinsic cell dysfunction leads to dysregulated immune responses to environmental triggers in chronic rhinosinusitis (CRS). Although epidemiological and in vivo studies support this theory, in vitro studies are lacking. Methods Epithelial cells from human sinonasal mucosa were cultured using an air–liquid interface culture model producing a well-differentiated phenotype. Specimens were characterized as chronic rhinosinusitis with (CRSwNP) or without (CRSsNP) nasal polyps and healthy control mucosa. Culture wells were exposed to house dust mite (HDM), diesel exhaust particles (DPM), or a combination (HDM + DPM) over 24 hours and responses in the 3 groups compared. Ciliary beat frequency (CBF) and transepithelial electrical resistance (TEER) were measured to assess mucociliary and barrier function, respectively. Interleukin-6 (IL-6) and 33 (IL-33) were measured after 24 hours. Results following challenge testing are expressed as fold change from baseline. Results Baseline CBF was lower in CRSsNP compared with control (5.27 ± 0.51 Hz vs 5.88 ± 1.22 Hz, P = .003). HDM significantly reduced CBF and TEER in the CRSwNP group compared with its vehicle (CBF: 0.55 ± 0.25 vs 1.03 ± 0.22, P &lt; .001; TEER: 0.54 [0.13] Ω cm2 vs 0.93 [0.5] Ω cm2, P = .001). In CRSwNP and CRSsNP, HDM induced an increase in IL-6 compared with its vehicle (CRSwNP: 81.11 [67.19] pg/mL vs 3.15 [44.64] pg/mL, P = .016; CRSsNP: 321.46 [182.04] pg/mL vs 21.54 [53.93] pg/mL, P = .004). Results are expressed as median (interquartile range) and in IL-33 in CRswNP (84.04 [69.96] pg/mL vs 16.62 [20.19] pg/mL, P = .025). Exposure to DPM did not affect CBF, TEER, and cytokine release in all groups. Conclusion CRSwNP and CRSsNP cells exhibit altered responses particularly to HDM even after they have been removed from their host and cultured in vitro, suggesting an intrinsic cell dysfunction of the upper airway epithelium.

Aspergillus fumigatus induction of IL-33 expression in chronic rhinosinusitis is PAR2-dependent.

In the pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP), Aspergillus fumigatus (A. fumigatus) can upregulate IL-33 from human sinonasal epithelial cells (SNECs), which then activates innate lymphoid cells causing release of IL-13, an important driver of allergic inflammation. However, the mechanism by which A. fumigatus mediates the induction of IL-33 expression remains to be elucidated. The objectives of this study were to determine the specific fungal component(s) and the receptor responsible for mediating the A. fumigatus induced increase in IL-33 expression in SNECs from patients with CRSwNP. SNECs from CRSwNP patients were cultured and stimulated with various fungal components in the absence or presence of 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride, an irreversible serine protease inhibitor, or GB83, a reversible protease activated receptor 2 (PAR2) inhibitor. IL-33 expression was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). PAR2 expression was examined in inflamed mucosa from nonatopic control and CRSwNP patients. Elevation of IL-33 expression in primary SNECs was found in response to fungal protease but not fungal cell wall components. PAR2 expression was elevated in inflamed mucosa from CRSwNP patients in comparison to controls. The A. fumigatus fungal protease-mediated elevation in IL-33 expression by human SNECs was serine protease- and PAR2-dependent. These data suggest that serine protease activity of A. fumigatus is capable of inducing IL-33 expression in CRSwNP SNECs via PAR2, a potential therapeutic target in the treatment of CRSwNP. NA Laryngoscope, 129:2230-2235, 2019.

Broncho-Vaxom® (OM-85 BV) soluble components stimulate sinonasal innate immunity.

Broncho-Vaxom® (OM-85 BV) is an extract of infectious respiratory bacteria that is used as an immunostimulant outside of the United States for the prevention and treatment of bronchitis and rhinosinusitis. Prior studies have shown that use of OM-85 BV is associated with reduction in frequency of respiratory infection and decreased duration of antibiotic usage. However, the effects of OM-85 BV on respiratory mucosal innate immunity are unknown. Human sinonasal epithelial cells were grown at an air-liquid interface (ALI). Ciliary beat frequency (CBF) and nitric oxide (NO) production in response to stimulation with OM-85 BV was measured in vitro. Pharmacologic inhibitors of bitter taste receptor (T2R) signaling were used to determine if this pathway was taste-receptor-mediated. Apical application of OM-85 BV resulted in an NO-mediated increase in CBF (p<0.05) and increased NO production (p<0.0001) when compared to saline-stimulated control cultures. ALI pretreatment with taste receptor pathway inhibitors blocked OM-85 BV-induced increases in NO. OM-85 BV has ciliostimulatory and immunogenic properties that may be partially responsible for its observed efficacy as a respiratory therapeutic. These responses were NO-dependent and consistent with T2R activation. Further work is necessary to elucidate specific component-receptor signaling relationships.

The ratio of 11β-hydroxysteroid dehydrogenase 1/11β-hydroxysteroid dehydrogenase 2 predicts glucocorticoid response in nasal polyps.

Glucocorticoids are the first-line medical treatment for chronic rhinosinusitis with nasal polyps (CRSwNP), whose local metabolism is catalyzed by 11β-HSD1 and 11β-HSD2. This study investigates the role of 11β-HSD1 and 11β-HSD2 on the glucocorticoid response of CRSwNP patients and the pathogenic mechanism of these polyps. Forty-three adult CRSwNP patients were enrolled in this study. We evaluated the endoscopic scores by a nasal polyp grading system before and after treatment. We estimated the response to glucocorticoids by the total endoscopic scores. The logistic regression models and inflammatory characteristic curves were conducted to explore the prediction of the response to glucocorticoid in CRSwNP. The expression of 11β-HSD1 and 11β-HSD2 on human sinonasal epithelial cells (HSECS) was measured under the stimulation of toll-like receptor agonists and dexamethasone. The endoscopic scores in the CRSwNP group declined, the expression of 11β-HSD1/11β-HSD2 increased (r = 0.5276, P = 0.0011), and the cutoff value of the ratio of 11β-HSD1/11β-HSD2 was 0.4654 (sensitivity 79.17%, specificity 88.89%). Dexamethasone induced a decrease in the ratio of 11β-HSD1/11β-HSD2 (P = 0.049) by the stimulation of PGN-BS. We found a strong correlation between the response to glucocorticoids and the ratio of 11β-HSD1/11β-HSD2, which could be used as a marker in predicting the level of tissue response to glucocorticoid therapy in CRSwNP. In addition, PGN-BS could also be a therapeutic target, as it is the negative factor that will decrease the sensitivity of glucocorticoids by reducing the ratio of 11β-HSD1/11β-HSD2.

Resveratrol and ivacaftor are additive G551D CFTR-channel potentiators: therapeutic implications for cystic fibrosis sinus disease.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in defective Cl- transport and cause chronic bacterial infections in the upper and lower airways of cystic fibrosis (CF) patients. Ivacaftor is a CFTR potentiator that improves Cl- transport in CF patients with at least 1 copy of the G551D mutation. Resveratrol is also a potent CFTR potentiator that increases determinants of mucociliary transport. The objective of this study is to determine whether resveratrol and ivacaftor improve Cl- secretion in G551D CFTR over either agent alone. Fisher rat thyroid cells (FRT) transfected with G551D CFTR and human sinonasal epithelial cells (HSNE) containing the CFTR G551D mutation were subjected to pharmacologic manipulation of transepithelial ion transport in Ussing chambers. Activity was further evaluated using whole-cell patch clamp methods in G551D FRT cells. In G551D FRT cells, resveratrol (100 μM) and ivacaftor (10 μM) significantly increased Cl- transport (change in short-circuit current, δISC = μA/cm2 ) compared with single-agent and dimethylsulfoxide vehicle controls (resveratrol + ivacaftor 4.97 ± 0.57 vs ivacaftor 0.74 ± 0.12 vs resveratrol 2.96 ± 0.52 vs control 0.74 ± 0.12; p < 0.001). Maximal Cl- secretion (20 μM forskolin) was also significantly enhanced (p < 0.0001). Activity was confirmed in G551D HSNE (resveratrol + ivacaftor 4.48 ± 0.39 vs ivacaftor 1.05 ± 0.11 vs. resveratrol 0.84 ± 0.3 vs control, 0.0 ± 0.02; p < 0.001), and whole-cell patch clamp analysis in G551D FRT cells (resveratrol + ivacaftor -2535 ± 179.3 pA vs ivacaftor -1408.9 ± 101.3 pA vs resveratrol; -766.2 ± 71.2 pA; p < 0.0001). Additive improvement in G551D CFTR-mediated Cl- secretion suggests that resveratrol could enhance ivacaftor therapy in these patients and improve CF-related rhinosinusitis.

C3a receptor antagonism as a novel therapeutic target for chronic rhinosinusitis.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory disease with an unknown etiology. Recent studies have implicated the complement system as a potential modulator of disease immunopathology. We preformed proteomic pathway enrichment analysis of differentially increased proteins, and found an enrichment of complement cascade pathways in the nasal mucus of individuals with CRSwNP as compared to control subjects. Sinonasal mucus levels of C3 correlated with worse subjective disease severity, whereas no significant difference in systemic C3 levels could be determined in plasma samples. Given that human sinonasal epithelial cells were the predominate sinonasal source of C3 and C3a staining, we focused on their role in in-vitro studies. Baseline intracellular C3 levels were higher in CRSwNP cells, and following exposure to Aspergillus fumigatus (Af) extract they released significantly more C3 and C3a. Inhibition of C3aR signaling led to a decrease in Af-induced C3 and C3a release, both in-vitro and in-vivo. Finally, we found in-vivo that C3aR deficiency or inhibition significantly reduced inflammation and CRS development in a mouse model of Af induced CRS. These findings demonstrate that local sinonasal complement activation correlates with subjective disease severity, and that local C3aR antagonism significantly ameliorates Af induced CRS in a rodent model.