Sinonasal Epithelial Cells Research Articles

Prostaglandin E Receptor 2 (EP2) Dysregulation in Allergic Fungal Rhinosinusitis Nasal Polyp Epithelium.

Allergic fungal rhinosinusitis (AFRS) is an eosinophilic subtype of chronic rhinosinusitis with nasal polyposis (CRSwNP). This study aimed to investigate the transcriptome of AFRS nasal polyp epithelium. Sinonasal epithelial cells were harvested from healthy nasal mucosa and polyp tissue collected from participants undergoing elective sinonasal surgery. Primary epithelial cells were subsequently grown in air/liquid interface and subjected to RNA-seq analysis, RT-qPCR, immunoblotting, and immunostaining. A total of 19 genes were differentially expressed between healthy and AFRS sample epithelium. The second top candidate gene, ranked by adjusted p-value, was prostaglandin E receptor 2 (PTGER2). The upregulation of PTGER2 was confirmed by RT-qPCR and immunoblot. The presence of the EP2 receptor, encoded by the PTGER2 gene, was confirmed by immunocytochemistry. PTGER2 is a potential novel therapeutic target for AFRS. EP2 dysregulation is associated with aspirin-exacerbated respiratory disease, potentially giving insight into common mechanisms of disease in severe CRSwNP. NA Laryngoscope, 2024.

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.

[This retracts the article DOI: 10.3389/fimmu.2021.793517.].

Chemokine CCL19 and Its Receptors CCR7 and CCRL1 in Chronic Rhinosinusitis.

CCL19 has been shown to predict disease severity in COVID-19 and treatment response in rheumatoid arthritis. CCL19 can exert both pro- and anti-inflammatory effects and is elevated in chronic rhinosinusitis (CRS). However, its role in CRS remains unknown. This study sought to determine the transcriptional changes in CCL19, its receptors, and associated cytokines and their association with disease severity in CRS. A clinical database of control subjects and patients with CRS was examined. Lund-Kennedy, Lund-Mackay, Sinonasal Outcomes Test 22 (SNOT-22), and rhinosinusitis disability index (RSDI) scores were collected at enrollment. mRNA was extracted from sinonasal tissues and subjected to multiplex gene expression analysis. Gene transcript differences between patients with CRS and controls were compared and correlated with disease severity metrics. Immunohistochemical analyses of CCL19, CCR7, and CCRL1 were conducted to compare differences in protein expression between cohorts. A subgroup analysis was performed to compare transcriptional and protein expression difference between patients with (CRSwNP) and without (CRSsNP) nasal polyps and controls. Thirty-eight subjects (control group, n=7; CRS group, n=31) were included in this study. CCRL1 (p=0.0093) and CCR7 (p=0.017) levels were significantly elevated in CRS compared to those in controls. CCL19 (p=0.038) and CCR7 (p=0.0097) levels were elevated in CRSwNP and CCRL1 was elevated in CRSsNP (p=0.0004). CCR7 expression was significantly elevated in sinonasal epithelial cells in CRSwNP (p=0.04). CCL19 expression was positively correlated with TNFA expression (p<0.0002). CCL19 and CCR7 expression was positively correlated with SNOT-22 and RSDI scores (p<0.05). CCL19 and CCR7 may modulate TNF-α-driven pro-inflammatory signaling and contribute to increased disease severity in CRS. Mechanistic studies are required to further elucidate the role of CCRL1 in CRS.

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.

Matrix-Mediated Delivery of Silver Nanoparticles for Prevention of Staphylococcus aureus and Pseudomonas aeruginosa Biofilm Formation in Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is a chronic health condition affecting the sinonasal cavity. CRS-associated mucosal inflammation leads to sinonasal epithelial cell death and epithelial cell barrier disruption, which may result in recurrent bacterial infections and biofilm formation. For patients who fail medical management and elect endoscopic sinus surgery for disease control, bacterial biofilm formation is particularly detrimental, as it reduces the efficacy of surgical intervention. Effective treatments that prevent biofilm formation in post-operative patients in CRS are currently limited. To address this unmet need, we report the controlled release of silver nanoparticles (AgNps) with silk-elastinlike protein-based polymers (SELPs) to prevent bacterial biofilm formation in CRS. This polymeric network is liquid at room temperature and forms a hydrogel at body temperature, and is hence, capable of conforming to the sinonasal cavity upon administration. SELP hydrogels demonstrated sustained AgNp and silver ion release for the studied period of three days, potent in vitro antibacterial activity against Pseudomonas aeruginosa (**** p < 0.0001) and Staphylococcus aureus (**** p < 0.0001), two of the most commonly virulent bacterial strains observed in patients with post-operative CRS, and high cytocompatibility with human nasal epithelial cells. Antibacterial controlled release platform shows promise for treating patients suffering from prolonged sinonasal cavity infections due to biofilms.

Epigenetic responses to rhinovirus exposure in airway epithelial cells are correlated with key transcriptional pathways in chronic rhinosinusitis.

Viruses may drive immune mechanisms responsible for chronic rhinosinusitis with nasal polyposis (CRSwNP), but little is known about the underlying molecular mechanisms. To identify epigenetic and transcriptional responses to a common upper respiratory pathogen, rhinovirus (RV), that are specific to patients with CRSwNP using a primary sinonasal epithelial cell culture model. Airway epithelial cells were collected at surgery from patients with CRSwNP (cases) and from controls without sinus disease, cultured, and then exposed to RV or vehicle for 48 h. Differential gene expression and DNA methylation (DNAm) between cases and controls in response to RV were determined using linear mixed models. Weighted gene co-expression analysis (WGCNA) was used to identify (a) co-regulated gene expression and DNAm signatures, and (b) genes, pathways, and regulatory mechanisms specific to CRSwNP. We identified 5585 differential transcriptional and 261 DNAm responses (FDR <0.10) to RV between CRSwNP cases and controls. These differential responses formed three co-expression/co-methylation modules that were related to CRSwNP and three that were related to RV (Bonferroni corrected p < .01). Most (95%) of the differentially methylated CpGs (DMCs) were in modules related to CRSwNP, whereas the differentially expressed genes (DEGs) were more equally distributed between the CRSwNP- and RV-related modules. Genes in the CRSwNP-related modules were enriched in known CRS and/or viral response immune pathways. RV activates specific epigenetic programs and correlated transcriptional networks in the sinonasal epithelium of individuals with CRSwNP. These novel observations suggest epigenetic signatures specific to patients with CRSwNP modulate response to viral pathogens at the mucosal environmental interface. Determining how viral response pathways are involved in epithelial inflammation in CRSwNP could lead to therapeutic targets for this burdensome airway disorder.

Epithelial innate immune response to Pseudomonas aeruginosa-derived flagellin in chronic rhinosinusitis.

Pseudomonas aeruginosa is a common colonizing pathogen in the upper respiratory tract and is associated with recalcitrant chronic rhinosinusitis (CRS). Herein we sought to characterize the effect of P. aeruginosa-derived flagellin on human sinonasal epithelial cell (HSNEC) immune responses and determine whether these pathways are disrupted in CRS. Air-liquid interface cultures were established from CRS and healthy control donors. Cells were incubated with P. aeruginosa-derived flagellin for 24hours and transcriptional changes were assessed using whole transcriptome RNA sequencing. Apical and basolateral secretion of the pro-inflammatory cytokines in interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 were measured after stimulation by lipopolysaccharide or flagellin and responses were compared between CRS and healthy control patients. HSNECs were weakly responsive to lipopolysaccharide, whereas flagellin stimulated a profound innate immune response dominated by TNF-α, IL-1β, and IL-17 signaling and activation of the IL-17C/IL-23 axis. CRS-derived HNSECs showed an altered innate immune response to flagellin, characterized by a profound increase in TNF-α secretion coupled with reduced IL-6 secretion. Flagellin activates a potent innate immune response in HSNECs characterized by pro-inflammatory mediators and cytokines/chemokines associated with neutrophilic inflammation. HSNECs from CRS patients have a dysregulated innate immune response to flagellin characterized by an imbalance between IL-6 and TNF-α secretion.

Hydrogen peroxide attenuates rhinovirus-induced anti-viral interferon secretion in sinonasal epithelial cells.

Altered innate defense mechanisms, including an imbalance between oxidants and antioxidants release, have been implicated in the pathogenesis of chronic rhinosinusitis (CRS). The aim of this study is to investigate whether oxidative stress may attenuate the secretion of anti-viral interferons in human sinonasal mucosa. The levels of H2O2 in nasal secretion were increased in patients with CRS with nasal polyps, compared with that of CRS patients without nasal polyps and control subjects. Normal sinonasal epithelial cells derived from healthy subjects were cultured under an air-liquid interface. The cultured cells were infected with rhinovirus 16 (RV 16) or treated with poly (I: C), TLR3 agonist, after being pretreated with an oxidative stressor, H2O2 or antioxidant, N-acetylcysteine (NAC). Thereafter, the expression levels of type I (IFN-β) and type III (IFN-λ1 and λ2) interferons and interferon-stimulated genes (ISGs) were evaluated with RT-qPCR, ELISA, and western blot. The data showed that the production of type I (IFN-β) and type III (IFN-λ1 and λ2) interferons and ISGs was upregulated in cells infected with RV 16 or treated with poly (I: C). However, their up-regulated expression was attenuated in cells pretreated with H2O2, but not inhibited in cells pretreated with NAC. In line with these data, the up-regulated expression of TLR3, RIG-1, MDA5, and IRF3 was reduced in cells pretreated with H2O2, but not attenuated in cells treated with NAC. Furthermore, cells transfected with Nrf2 siRNA showed decreased secretion of anti-viral interferons whereas sulforaphane treatment enhanced the secretory capacity of antiviral interferons. These results suggest that the production of RV16-induced antiviral interferons may be attenuated by oxidative stress.

S-Nitrosoglutathione Reduces the Density of Staphylococcus aureus Biofilms Established on Human Airway Epithelial Cells.

Patients with chronic rhinosinusitis (CRS) often show persistent colonization by bacteria in the form of biofilms which are resistant to antibiotic treatment. One of the most commonly isolated bacteria in CRS is Staphylococcus aureus (S. aureus). Nitric oxide (NO) is a potent antimicrobial agent and disperses biofilms efficiently. We hypothesized that S-nitrosoglutathione (GSNO), an endogenous NO carrier/donor, synergizes with gentamicin to disperse and reduce the bacterial biofilm density. We prepared GSNO formulations which are stable up to 12 months at room temperature and show the maximum amount of NO release within 1 h. We examined the effects of this GSNO formulation on the S. aureus biofilm established on the apical surface of the mucociliary-differentiated airway epithelial cell cultures regenerated from airway basal (stem) cells from cystic fibrosis (CF) and CRS patients. We demonstrate that for CF cells, which are defective in producing NO, treatment with GSNO at 100 μM increased the NO levels on the apical surface and reduced the biofilm bacterial density by 2 log units without stimulating pro-inflammatory effects or inducing epithelial cell death. In combination with gentamicin, GSNO further enhanced the killing of biofilm bacteria. Compared to placebo, GSNO significantly increased the ciliary beat frequency (CBF) in both infected and uninfected CF cell cultures. The combination of GSNO and gentamicin also reduced the bacterial density of biofilms grown on sinonasal epithelial cells from CRS patients and improved the CBF. These findings demonstrate that GSNO in combination with gentamicin may effectively reduce the density of biofilm bacteria in CRS patients. GSNO treatment may also enhance the mucociliary clearance by improving the CBF.

Nitric oxide-generating microparticles: An in vitro evaluation of anti-biofilm efficacy and sinonasal epithelial cell cytotoxicity.

International Forum of Allergy & RhinologyAccepted Articles RESEARCH NOTE Nitric Oxide Generating Microparticles: An In Vitro Evaluation of Anti-Biofilm Efficacy and Sinonasal Epithelial Cell Cytotoxicity Kevin L. Li MD, Kevin L. Li MD Department of Otorhinolaryngology – Head and Neck Surgery, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorDaniel Z. Miranda PhD, Daniel Z. Miranda PhD Department of Medicine (Division of Infectious Diseases) and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorLevi G. Cleare BA, Levi G. Cleare BA Department of Medicine (Division of Infectious Diseases) and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorNadeem A. Akbar MD, Nadeem A. Akbar MD Department of Otorhinolaryngology – Head and Neck Surgery, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorJoel M. Friedman MDPhD, Joel M. Friedman MDPhD Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorAndrew Draganski PhD, Andrew Draganski PhD Zylö Therapeutics, Greenville, SC, USASearch for more papers by this authorJoshua D. Nosanchuk MD, Joshua D. Nosanchuk MD Department of Medicine (Division of Infectious Diseases) and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorWaleed M. Abuzeid MD, Corresponding Author Waleed M. Abuzeid MD wabuzeid@uw.edu Department of Otolaryngology: Head and Neck Surgery, University of Washington, Seattle, WA, USA Correspondence Waleed M. Abuzeid, MD, Associate Professor, Director of Rhinology Research, Director of Equity, Diversity, and Inclusion, Department of Otolaryngology: Head and Neck Surgery, University of Washington. Email: wabuzeid@uw.eduSearch for more papers by this author Kevin L. Li MD, Kevin L. Li MD Department of Otorhinolaryngology – Head and Neck Surgery, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorDaniel Z. Miranda PhD, Daniel Z. Miranda PhD Department of Medicine (Division of Infectious Diseases) and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorLevi G. Cleare BA, Levi G. Cleare BA Department of Medicine (Division of Infectious Diseases) and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorNadeem A. Akbar MD, Nadeem A. Akbar MD Department of Otorhinolaryngology – Head and Neck Surgery, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorJoel M. Friedman MDPhD, Joel M. Friedman MDPhD Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorAndrew Draganski PhD, Andrew Draganski PhD Zylö Therapeutics, Greenville, SC, USASearch for more papers by this authorJoshua D. Nosanchuk MD, Joshua D. Nosanchuk MD Department of Medicine (Division of Infectious Diseases) and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USASearch for more papers by this authorWaleed M. Abuzeid MD, Corresponding Author Waleed M. Abuzeid MD wabuzeid@uw.edu Department of Otolaryngology: Head and Neck Surgery, University of Washington, Seattle, WA, USA Correspondence Waleed M. Abuzeid, MD, Associate Professor, Director of Rhinology Research, Director of Equity, Diversity, and Inclusion, Department of Otolaryngology: Head and Neck Surgery, University of Washington. Email: wabuzeid@uw.eduSearch for more papers by this author First published: 08 October 2022 https://doi.org/10.1002/alr.23096 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as https://doi.org/10.1002/alr.23096 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Accepted ArticlesAccepted, unedited articles published online and citable. The final edited and typeset version of record will appear in the future. RelatedInformation

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.

Rhinovirus-induced anti-viral interferon secretion is not deficient and not delayed in sinonasal epithelial cells of patients with chronic rhinosinusitis with nasal polyp.

Dysregulated innate and adaptive immune response to rhinoviral infection plays an important role in the exacerbation or progressive course of chronic rhinosinusitis (CRS). However, few studies have evaluated whether rhinovirus-induced production of anti-viral interferon is deficient or delayed in inflammatory epithelial cells of patients with CRS with nasal polyps. The aim of the present study is to investigate the replication rates of rhinovirus 16 (RV 16), RV16-induced antiviral interferon secretion, and the expression levels of pattern recognition receptors after RV 16 infection or TLR3 stimulation with poly (I: C) in normal and inflammatory epithelial cells. Inflammatory epithelial cells were obtained from CRS patients with nasal polyps and normal epithelial cells were derived from ethmoid sinus mucosa during endoscopic reduction of blowout fracture or uncinate process mucosa of patients with septal deviation. Cultured cells were infected with RV 16 or treated with poly (I: C) for 24, 48, and 72h. Cells and media were harvested at each time point and used to evaluate RV16 replication rates, the secretion of IFN-β, -λ1, -λ2, viperin, Mx, and OAS, and the expression levels of TRL3, RIG-I, MDA5, phospho-NFκB, and phospho-IRF3. RV replication rates reached peak levels 48h after inoculation in both normal and inflammatory epithelial cells and showed no difference between both groups of epithelial cells at any time point. The release of IFN-β, -λ1, and -λ2 in normal and inflammatory epithelial cells was also strongly induced 48h after RV16 inoculation but reached peak levels 24h after poly (I: C) treatment. The expression levels of viperin, Mx, OAS, TLR3, RIG-I, MDA5, phospho-NFκB, and phospho-IRF3 showed similar patterns in both groups of epithelial cells. These results suggest that the production of RV16-induced antiviral interferons is not deficient or delayed in inflammatory epithelial cells from CRS patients with nasal polyps.

Role of C3a as a Novel Regulator of 25(OH)D3 to 1α,25-Dihydroxyvitamin D3 Metabolism in Upper Airway Epithelial Cells.

In patients with chronic rhinosinusitis with nasal polyps, primary human sinonasal epithelial cell (HSNEC) 1α-hydroxylase levels are reduced, as is their ability to metabolize 25-hydroxycholecalciferol [25(OH)D3] to its active metabolite, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In this study, we sought to identify the factor responsible for the regulation of HSNEC metabolism of 25(OH)D3, focusing on C3 and C3a. Multiple inhaled irritants trigger the release of complement components, C3 and C3a, leading to suppression of 1α-hydroxylase levels in HSNECs. Recombinant C3a was able to decrease 1α-hydroxylase and impair 25(OH)D3 to 1,25(OH)2D3 metabolism, while addition of a C3a receptor antagonist restored conversion. Conversely, 1,25(OH)2D3 suppressed Aspergillus fumigatus-induced C3 and C3a levels in HSNEC supernatant. Given the ability of 1,25(OH)2D3 to modulate LL37 in other cell types, we examined its regulation in HSNECs and relationship to C3a. 1,25(OH)2D3 stimulated the secretion of LL37, whereas A. fumigatus and C3a suppressed it. Conversely, LL37 reduced the release of C3/C3a by HSNECs. Lastly, oral steroid use and in vitro dexamethasone application both failed to increase 1α-hydroxylase or reduce C3a levels. In summary, in this article, we describe for the first time a novel relationship between complement activation and local vitamin D metabolism in airway epithelial cells. The presence of elevated C3/C3a in patients with asthma and/or chronic rhinosinusitis with nasal polyps may account for their impaired HSNEC 25(OH)D3 to 1,25(OH)2D3 metabolism and explain why they receive limited therapeutic benefit from oral vitamin D3 supplementation.

In Vitro Effects of Permethrin on Sinonasal Epithelia.

Inhalant toxicants are postulated to contribute to the pathogenesis of chronic rhinosinusitis. Permethrin is a pesticide widely used in agricultural, industrial, and residential settings. The objective of this pilot study is to investigate the in vitro effects of permethrin on sinonasal epithelial cells (SNECs). Sinus mucosa was collected from 4 patients undergoing transsphenoidal pituitary surgery without a history of chronic rhinosinusitis. Cultured SNECs were exposed to varied concentrations of permethrin (0-156 µM) for 6 days. Cell viability and proliferation were determined via the MTT colorimetric assay and the Incucyte Live Cell Imaging System. Cellular reactive oxygen species (ROS) activity was measured by the DCFDA ROS detection assay. A statistically significant reduction in cell viability and proliferation was observed between the exposure and control groups at certain concentrations, and a dose-dependent increase in ROS activity was also observed. These findings indicate that permethrin may have deleterious effects on SNECs in a dose-dependent manner.

Temporal Changes in Treatment Patterns for Rural and Urban Patients With Early-Stage Non-Small Cell Lung Cancer

BACKGROUND Our objectives were to evaluate geographic access to lung cancer treatment modalities in North Carolina and to characterize how practice patterns are changing over time. We hypothesized that rural patients would be less likely to undergo treatment compared to urban patients, with widening disparities over time.METHODS We identified patients with Stage I non-small cell lung cancer (NSCLC) from 2006 to 2015 using the North Carolina Central Cancer Registry linked with Medicaid, Medicare, and private insurance claims. The primary outcome was first-course treatment: surgery, radiation, or no treatment. Calendar years were split into earlier (2006-2010) and later (2011-2015) periods. We estimated the adjusted odds ratio (OR) of rural/urban status and time period with 1) surgery and 2) any treatment (surgery or radiation) using multivariable logistic regression.RESULTS Among 5504 patients, 3206 (58%) underwent surgery as initial therapy, 1309 (24%) received radiation as initial therapy, and 989 (18%) had no therapy. There were no rural-urban disparities in treatment patterns. For rural and urban patients, the odds of surgery decreased over time and the odds of radiation increased. We also found that only 48% of those receiving no treatment ever reached a surgeon or radiation oncologist.LIMITATIONS This was an insured, single-state population. Treatment preferences are unknown.CONCLUSIONS Among all treated patients, whether urban or rural, there was increasing use of radiation and decreasing use of surgery over time. Many patients without treatment never had a consultation with a surgeon/radiation oncologist, and this is an actionable target for improving treatment access for early-stage NSCLC.

The correlation between second-hand tobacco smoke exposure and biofilm formation in chronic rhinosinusitis

Introduction and purpose&#x0D; Tobacco smoke is a major health concern globally. Due to tobacco epidemic, approximately 8 million people died as a result of cigarette smoking in 2020 alone, where 1.2 million were caused by non-smokers inhaling second-hand smoke. Chronic rhinosinusitis (CRS) is an inflammatory condition that has a significant health and economic impact worldwide. Despite its great burden on the health-care system and patients' quality of life, the variety of therapy options for CRS is currently limited. Tobacco-induced biofilm formation may contribute to the refractory nature of many respiratory diseases reported in smokers and second-hand smokers, due to increased resistance to antibiotics. The aim of this study is to present that exposure to household passive smoking may induce the formation of nasal biofilms.&#x0D; Brief description of the state of knowledge&#x0D; Chronic bacterial infections involving biofilms have recently been recognised as a factor in CRS pathogenesis. The presence of biofilms on the mucosa of CRS patients is associated with more severe pre-operative disease, persistent postoperative symptoms, ongoing mucosal inflammation, and infections following endoscopic sinus surgery. Tobacco smoke and CRS have been associated because of the immunosuppressive and irritating effects of tobacco smoke on sinonasal epithelial cells. Smoking uptake and cessation have been shown to affect microbial communities, with smokers having not just distinct microbial communities, but also a higher frequency of possible pathogens in those communities.&#x0D; Summary&#x0D; Biofilms may play a significant role in the development of chronic rhinosinusitis. The impact of tobacco smoke on biofilm development could have major implications not only for CRS but also for other respiratory infections.&#x0D;

HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages.

Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two innate immune cell types are activated by bitter products, including those secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells. NO also increases phagocytosis by macrophages. Using biochemistry and live-cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). Immunofluorescence showed that H441 cells express eNOS and T2Rs and that the bitter agonist denatonium benzoate activates NO production in a Ca2+- and HSP90-dependent manner in cells grown either as submerged cultures or at the air–liquid interface. In primary sinonasal epithelial cells, we determined that HSP90 inhibition reduces T2R-stimulated NO production and ciliary beating, which likely limits pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves as an innate immune modulator by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream Ca2+ signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases such as chronic rhinosinusitis, asthma, or cystic fibrosis.

CDHR3 rs6967330 risk allele influences CRS disease severity

Cadherin Related Family Member 3 (CDHR3) is the receptor for Rhinovirus-C and expressed in sinonasal epithelial cells. We have previously determined that the CDHR3 (rs6967330) base change from G to A causes a two-fold increase in the odds of adults with chronic rhinosinusitis (CRS) and that RV-C infections result in increased CRS severity. However, the influence of the rs6967330 SNP on CRS disease is not known.

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.