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Background: ARF nucleotide-binding site opener (ARNO) is a guanine nucleotide-exchange factor for ADP-ribosylation factor proteins. ARF nucleotide-binding site opener also binds MyD88, and small-molecule inhibition of ARNO reduces inflammation in animal models of inflammatory arthritis and acute inflammation. However, whether genetic deletion of Arno in mice reduces pathologic inflammation has not yet been reported. Furthermore, its role in the nasal cavity has yet to be investigated. Objective: To generate Arno knockout mice and to determine whether genetic loss of ARNO reduces eosinophilic inflammation in the ovalbumin (OVA) murine model of rhinitis. Methods: Arno knockout mice were generated and wild type and knockout littermates were subjected to the OVA-induced mouse model of rhinosinutitis. Eosinophilic inflammation was assessed through immunofluorescent quantification of EMBP+ eosinophils in the septal mucosa and cytokine expression was assessed by quantitative polymerase chain reaction. Results: Arno knockout mice are viable and fertile without any noted deficits. Arno wild type and knockout mice subjected to the OVA-induced model of rhinitis demonstrated an average of 314.5 and 153.8 EMBP+ cells per mm2 septal tissue, respectively ( P < .05). Goblet cells per mm of basal lamina were assessed via Alcian blue and there was no statistically significant difference between Arno wild type and knockout mice. Ovalbumin-induced expression of interleukin-5 (IL-5) was significantly reduced in Arno knockout mice ( P < .05). There was no statistically significant reduction in IL-4, IL-13, or eotaxin-1 expression. Conclusions: These data demonstrate that deletion of Arno reduces eosinophilic inflammation and IL-5 expression in an OVA-induced model of rhinitis.

Allergic asthma is a pulmonary disease characterized by airway hyperresponsiveness (AhR) in response to inhaled allergens. AhR is largely mediated by allergen-specific (Ag-specific) IgE induced mast cell (MC) degranulation which drives inflammation of the airways following allergen exposure. Ag-specific IgE production requires T follicular helper 13 cells (Tfh13s) which must first be polarized by dendritic cells (DCs). The mechanisms by which DCs polarize Tfh13s, however, are unclear. We hypothesized that the Tfh13 inducing allergen Alternaria would induce a unique metabolic response compared to non-Tfh13 inducing stimuli. We found, by untargeted metabolomic analysis, that Alternaria induced accumulation of the glutamine-derived TCA cycle metabolite α-ketoglutarate compared to LPS stimulated or unstimulated DCs. Glutamine is converted to α-ketoglutarate by glutaminase (Gls), so we predicted that inhibition of Gls may suppress Tfh13 polarization by DCs. Indeed, we found that treatment with the Gls inhibitor, CB-839, reduced Tfh13 polarization and increased T follicular regulatory cells following Alternaria exposure. This finding was recapitulated in a DC adoptive transfer model, indicating that this mechanism is DC intrinsic. We next tested if CB-839 causes allergen desensitization in a model of allergic asthma. Mice were intranasally sensitized and challenged with Alternaria and intranasally treated with CB-839 or vehicle control prior to challenge. CB-839 treated mice exhibited reduced AhR, Ag-specific IgE, and airway MC degranulation. These data demonstrate that DC glutaminolysis is required for Tfh13 polarization, and that Gls inhibitors may promote allergen desensitization in allergic asthma. This work was funded by VCU's National Center for Advancing Translational Sciences (UL1TR002649) and NIAID (R56AI139658).

ARF nucleotide-binding site opener (ARNO) is a guanine nucleotide-exchange factor for ADP-ribosylation factor proteins. ARF nucleotide-binding site opener also binds MyD88, and small-molecule inhibition of ARNO reduces inflammation in animal models of inflammatory arthritis and acute inflammation. However, whether genetic deletion ofTo generateThese data demonstrate that deletion of

OBJECTIVES/HYPOTHESIS Oxidative stress has been postulated to play an important role in chronic rhinosinusitis. Nrf2 is a transcription factor that is involved in the regulation of multiple antioxidant genes, and its function has been previously shown to be important in sinonasal inflammation. Although the sinonasal implications of whole body Nrf2-/- has been reported, the function of sinonasal epithelial expression of Nrf2 has not been studied. The primary aim of this study was to generate a mouse model that is genetically deficient in epithelial-specific Nrf2 and to understand its role in regulating sinonasal inflammation. STUDY DESIGN Basic science. METHODS An epithelial-specific Nrf2 knockout mouse was generated by crossing Krt5-cre(K5) with Nrf2flox/flox . A papain-induced model of rhinosinusitis was performed in the resulting K5 Nrf2-/- mouse. Immunohistochemistry was performed to quantify goblet cell hyperplasia. Mucosal cellular infiltrates were quantified using flow cytometry, and tissue cytokines were measured using an enzyme-linked immunosorbent assay. Lastly, the cellular source of type 2 cytokines was determined using intracellular cytokine staining. RESULTS Papain-sensitized mice lacking epithelial-specific Nrf2 demonstrate increased goblet cell hyperplasia, significant tissue eosinophilia, and statistically significant increase in mucosal IL-13 when compared to Nrf2 wild-type mice. Lastly, mucosal T cells were identified as the cellular source of IL-13. CONCLUSIONS We demonstrate enhanced severity of eosinophilic sinonasal inflammation from disruption of the epithelial-specific Nrf2 pathway. The responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to eosinophilic inflammation and may have potential as a therapeutic target for chronic rhinosinusitis. LEVEL OF EVIDENCE NA Laryngoscope, 131:713-719, 2021.

Background Oxidative stress exacerbates lower airway diseases including asthma and chronic obstructive pulmonary disease (COPD); however, its role in upper airway (sinonasal) chronic inflammatory disorders is less clear. Nuclear erythroid 2 p45-related factor (Nrf2) is an endogenous mechanism that upon activation invokes an antioxidant response pathway via nuclear translocation and upregulation of cytoprotective genes. We sought to determine whether deletion of Nrf2 enhances susceptibility to allergic sinonasal inflammation in vivo. Methods Nrf2-/- mice were subjected to the ovalbumin (Ova)-induced murine model of rhinosinusitis and indices of sinonasal inflammation and epithelial barrier dysfunction were assessed. Results We show that deletion of Nrf2 results in enhances indices of allergen-induced sinonasal inflammation including aggravated eosinophil accumulation and goblet cell hyperplasia. An exaggerated increase in epithelial derived inflammatory cytokines including interleukin 33 (IL-33) and thymic stromal lymphopoietin (TSLP) was observed in the nasal lavage fluid and sinonasal mucosal tissue of Nrf2-/- mice. Furthermore, Nrf2-/- mice showed heightened Ova-induced barrier dysfunction as measured by serum albumin accumulation in nasal lavage fluid of mice. Conclusion These data show that the endogenous Nrf2 pathway limits Ova-induced sinonasal inflammation, epithelial derived inflammatory cytokine production, and epithelial barrier dysfunction in vivo and identify a potential therapeutic target in the management of allergic sinonasal inflammatory disorders. This is the first study to our knowledge which shows that Nrf2 regulates allergic inflammation in the sinonasal cavity in vivo.

Background Eosinophilic chronic rhinosinusitis (ECRS) is a disease characterized by eosinophilic inflammatory infiltrate and a local type 2 cytokine milieu. Current animal models fail to recapitulate many of the innate and adaptive immunologic hallmarks of the disease, thus hindering the development of effective therapeutics. In the present study, mice were exposed intranasally to the cysteine protease papain, which shares functional similarities with parasitic proteases and aeroallergens, to generate a rapidly inducible murine model of eosinophilic rhinosinusitis. Methods C57BL/6 mice were intranasally instilled with 20 μg papain or heat-inactivated papain (HP) on days 0-2 and days 7-10, and then euthanized on day 11. Nasal lavage fluid (NALF) was analyzed to quantify eosinophils and inflammatory cytokine secretion. Sinonasal tissue was sectioned and stained for goblet cells or homogenized to analyze cytokine levels. Serum samples were assayed for immunoglobulin E (IgE) by enzyme-linked immunoassay. Sinonasal mucosal tissue was dissociated and analyzed by flow cytometry. Results Compared with HP treatment, papain induced significant eosinophilia in NALF, goblet cell hyperplasia, innate and adaptive immune cell infiltration, type 2 cytokine production, and IgE responses. Flow cytometric analysis of sinonasal tissues revealed significant inflammatory cell infiltration and interleukin-13-producing cell populations. Conclusion In this study, we demonstrated that the cysteine protease papain induces allergic sinonasal eosinophilic rhinosinusitis and resembles T-helper 2 cell inflammation and innate immune characteristics of ECRS. This model permits further study into the molecular mechanisms underlying ECRS pathology and provides a model system for the evaluation of potential pharmacologic interventions.

Exposure to airborne particulate matter (PM) has been linked to aggravation of respiratory symptoms, increased risk of cardiovascular disease, and all-cause mortality. Although the health effects of PM on the lower pulmonary airway have been extensively studied, little is known regarding the impact of chronic PM exposure on the upper sinonasal airway. We sought to test the impact of chronic airborne PM exposure on the upper respiratory system in vivo. Mice were subjected, by inhalation, to concentrated fine (2.5 μm) PM 6 h/d, 5 d/wk, for 16 weeks. Mean airborne fine PM concentration was 60.92 μm/m3, a concentration of fine PM lower than that reported in some major global cities. Mice were then killed and analyzed for evidence of inflammation and barrier breakdown compared with control mice. Evidence of the destructive effects of chronic airborne PM on sinonasal health in vivo, including proinflammatory cytokine release, and macrophage and neutrophil inflammatory cell accumulation was observed. A significant increase in epithelial barrier dysfunction was observed, as assessed by serum albumin accumulation in nasal airway lavage fluid, as well as decreased expression of adhesion molecules, including claudin-1 and epithelial cadherin. A significant increase in eosinophilic inflammation, including increased IL-13, eotaxin-1, and eosinophil accumulation, was also observed. Collectively, although largely observational, these studies demonstrate the destructive effects of chronic airborne PM exposure on the sinonasal airway barrier disruption and nonallergic eosinophilic inflammation in mice.

BACKGROUND Dysregulated sinonasal epithelial cell (SNEC) barrier function has been proposed to contribute to the pathogenesis of sinonasal inflammatory conditions such as allergic rhinitis (AR) and chronic rhinosinusitis (CRS). Allergens such as house dust mite (HDM) have been reported to disrupt SNEC barrier integrity. We have recently identified nuclear erythroid 2-related factor 2 (Nrf2) activation via sulforaphane (SFN) stimulation to stabilize SNEC barrier function. The purpose of this study was to explore whether Nrf2 activation could ameliorate HDM-induced SNEC barrier dysfunction. METHODS Human SNECs (HSNECs) were grown from patients at the air-liquid interface (ALI). HSNECs were stimulated with HDM with or without pharmacologic activation of Nrf2 with SFN. HSNECs were then stained for the epithelial cell junction protein zonula occludens-1 (ZO-1) and cell surface localization was evaluated by confocal microscopy. Transepithelial electrical resistance (TER) and paracellular fluorescein isothiocyanate (FITC)-dextran permeability was measured in response to stimulation with HDM and SFN. RESULTS HDM stimulation caused a global disruption of the protein ZO-1 along with an associated decrease in TER (p < 0.001) and increased FITC-dextran paracellular permeability (p < 0.0001). Enhancing Nrf2 activation through treatment with SFN prior to stimulation with HDM was associated with increased localization of ZO-1 at the cell surface and statistically significant increases in TER (p < 0.05) and decrease in paracellular FITC-dextran permeability (p < 0.001). CONCLUSION This is the first study to demonstrate that HDM-induced SNEC barrier dysfunction may be preventable by Nrf2 activation. The Nrf2 antioxidant pathway may represent a potential therapeutic target for allergen-induced sinonasal inflammation.

Allergic asthma is a respiratory disease characterized by airway inflammation driven by T-helper 2 (Th2) cytokines and antigen-specific IgE (Ag-specific IgE). Ag-specific IgE production requires T follicular helper 13 cells (Tfh13s), which are a population of IL-4/13+ Tfh cells. Tfh13 polarization is induced by type 2 conventional dendritic cells (cDC2s), however, the mechanisms by which cDC2s regulate Tfh13 polarization are unclear. We hypothesized that cDC2s would exhibit distinct transcriptional responses following in vivo exposure to the Tfh13 inducing stimuli, Alternaria, compared to non-Tfh13 polarizing stimuli, such as LPS or the protease papain. Thus, we treated mice intranasally with LPS, papain, or Alternaria plus fluorescently tagged OVA and sorted OVA+ migratory cDCs from the mediastinal lymph node (mLN) for RNA-seq analysis. Principal component analysis revealed that these stimuli induced unique transcriptional signatures in migratory cDCs. Gene set enrichment analysis demonstrated significant downregulation of oxidative phosphorylation (OXPHOS) genes in Alternaria stimulated cDCs compared to LPS or papain. We therefore hypothesized that Alternaria induces metabolic perturbations in cDC2s, whereby they upregulate glycolytic metabolism and decrease OXPHOS, and this metabolic profile was involved in Tfh13 polarization. To test this hypothesis, mice were either fed ad libitum or fasted for 12 hours prior to Alternaria exposure. Fasted mice exhibited a significant reduction in mLN Tfh13s and Th2 cells. Thus, we demonstrate that glucose metabolism may regulate allergic sensitization in asthma. Future experiments will aim to elucidate the role of cDC2-intrinsic metabolism in Tfh13 polarization.

Background Antibiotics are widely and heavily used in the treatment of chronic sinusitis. Bactericidal antibiotics can stimulate reactive oxygen species (ROS) formation, a proinflammatory response, and cell death in cultured human sinonasal epithelial cells (SNECs). Sulforaphane (SFN) is a potent stimulator of the antioxidant nuclear factor erythroid 2-related factor 2 (Nrf-2) system and a suppressor of inflammation. In this study we utilized SFN to further explore the relationship between levofloxacin treatment, ROS formation, and the cell death response. Methods SNECs were collected from patients during endoscopic sinus surgery and grown in culture at the air-liquid interface. Differentiated SNECs were stimulated with levofloxacin with or without SFN pretreatment. ROS were quantified. Apoptosis markers of caspase-3 activity and DNA fragmentation were quantified. Results Cultured SNECs treated with levofloxacin resulted in a significant increase in activity of the proapoptotic caspase-3 protease (5.9-fold, p = 0.01). The increase in activity was suppressed by pretreatment with SFN (1.9-fold). ROS levels increased with levofloxacin treatment (range, 1.2-fold to 1.8-fold), but were not significantly suppressed by pretreatment with SFN (range, 1.0-fold to 1.3-fold). Conclusion In this study, we demonstrate that treatment of cultured SNECs with levofloxacin leads to an increase in caspase-3 activity. SFN pretreatment suppresses the increased apoptotic response possibly through its antioxidant stimulating properties. Our results suggest that levofloxacin treatment stimulates a potent proapoptotic possibly through an ROS-dependent mechanism. Future studies will explore if this antibiotic-induced response is harmful to recovery of function in those with sinusitis.

Background Environmental factors such as inhaled pollutants like cigarette smoke may play a significant role in diseases of the upper airway including chronic rhinosinusitis (CRS). Recent studies have shown that cigarette smoke causes impaired airway epithelial cell barrier function likely through environmental oxidative stress related pathways. The purpose of this study is to explore whether enhancing nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2], the body's master antioxidant system, can ameliorate cigarette smoke–induced sinonasal epithelial cell (SNEC) barrier dysfunction. Methods Human SNECs (HSNECs) were grown from control patients at the air-liquid interface (ALI). HSNECs were stimulated with cigarette smoke extract (CSE) with and without pharmacologic activation of Nrf2. HSNECs were then stained for the epithelial cell junctional proteins zonula occludens 1 (ZO-1) and junctional adhesion molecule A (JAM-A) using confocal microscopy. In addition, transepithelial electrical resistance (TER) was measured in cultures before and after stimulation with CSE. Results CSE stimulation caused a global disruption of the epithelial junctional proteins ZO-1 and JAM-A along with an associated decrease in TER levels. Enhancing Nrf2 levels prior to stimulation with CSE was associated with increased localization of ZO-1 and JAM-A levels at the cell surface and statistically significant increases in TER levels. Conclusion This is the first study to demonstrate that cigarette smoke induced SNEC barrier dysfunction is reversible by Nrf2 activation. The Nrf2 antioxidant pathway may represent a potential therapeutic target for cigarette smoke–associated sinonasal inflammation.

Background Few efficacious topical therapies exist for chronic rhinosinusitis (CRS). The lack of a reproducible mouse model of CRS limits the pilot testing of potential novel anti-inflammatory therapies. Although the ovalbumin-induced mouse model of sinonasal inflammation is commonly used, it is difficult to reproduce and can generate variable histologic results. In this study, we explore a variation of this model in different strains of mice and explore various inflammatory cytokines as reproducible molecular markers of inflammation. Methods Allergic sinonasal inflammation was generated in BALB/c and C57BL/6 mice using intraperitoneal high-dose injections of ovalbumin (Ova; Sigma Chemical Co.) followed by 10 days of high-dose intranasal sensitization. Real-time polymerase chain reaction (RT-PCR) for eotaxin, interleukin 4 (IL-4), and IL-13 were measured from sinonasal mucosa. We also pilot tested a known topical budesonide to characterize the anti-inflammatory response. Histological sections were analyzed for epithelial thickness and eosinophilia. Results Both BALB/c and C57BL/6 mice consistently showed increases in T helper 2 (Th2) cytokines after sensitization with high-dose Ova (p < 0.0001) when compared to controls. There were also significant increases in epithelial thickening in Ova-sensitized mice and eosinophilia in both BALB/c and C57BL/6 strains. In addition, topical budesonide significantly reduced anti-inflammatory cytokines, eosinophilia, and epithelial thickness. Conclusion Our variation of the ovalbumin-induced mouse model of sinonasal inflammation in both BALB/c and C57BL/6 mice provides an efficacious model for testing potential topical anti-inflammatory therapies for CRS. The utilization of sinonasal mucosal Th2 cytokines along with histologic markers provides a consistent and quantifiable marker of inflammation in assessing the efficacy of candidate drugs.

Anaphylaxis is a type of severe allergic reaction that is initiated by cross-linking of high-affinity IgE on mast cells leading to mast cell degranulation. Recent evidence demonstrates that T follicular helper cells (Tfh) producing both IL-4 and IL-13, termed Tfh13s, are necessary for the generation of anaphylactic IgE. The mechanisms regulating the polarization of these T cells, however, remain unclear. Parasite infection, which induces strong IgE responses, fails to induce Tfh13 polarization. We therefore hypothesized that papain, a cysteine protease that resembles parasitic CA family peptidases, would similarly fail to induce Tfh13 polarization compared to the aeroallergen Alternaria due to differential action on dendritic cells (DCs). We found that intranasal immunization with LPS+OVA and Alternaria+OVA induced a 2-fold Tfh expansion in the draining lymph node compared to papain+OVA. Additionally, Alternaria stimulated Tfh13 polarization and high-affinity IgE production while papain only induced Th2 responses and non-specific IgE. Analysis of DC migration by immunization with fluorescently tagged OVA alongside these adjuvants revealed markedly different migration kinetics following each stimulus. However, numbers of Ag+ DCs, amount of antigen uptake, and proportions of Ag+ migratory vs. resident DCs in the mediastinal lymph nodes were similar between all groups. Thus, we demonstrate that papain, in contrast to Alternaria, is unable to induce Tfh13 polarization and anaphylactic IgE. We propose that the differential effects of these adjuvants are likely mediated by alternative mechanisms of DC licensing and distinct DC transcriptional profiles.

Background Bactericidal antibiotics have been shown to stimulate reactive oxygen species (ROS) formation in mammalian cells through mitochondrial dysfunction. This results in oxidative tissue damage that may have negative consequences for long-term antibiotic use. Antibiotics are widely and heavily used in the treatment of acute and chronic sinusitis; however, the relationship between antibiotics and ROS formation in sinonasal epithelial cells (SNECs) has not yet been demonstrated. Methods Human SNECs were collected from patients during endoscopic sinus surgery and grown in culture at the air-liquid interface. Differentiated SNECs were stimulated with the bactericidal antibiotics amoxicillin and levofloxacin and the bacteriostatic antibiotic clarithromycin for 24 hours. ROS were quantified via fluorescence. Cell death was quantified by lactate dehydrogenase (LDH) secretion. Expression of inflammatory markers such as tumor necrosis factor α (TNF-α) and nuclear factor erythroid 2–related factor 2 (Nrf2)-mediated antioxidant genes were measured by real-time polymerase chain reaction (RT-PCR). Results Cultured SNECs treated with the bactericidal antibiotics amoxicillin and levofloxacin resulted in a significant increase in production of ROS (p < 0.05) and secretion of LDH (p < 0.05). The increase in ROS formation correlated with an increase in expression of Nrf2-mediated antioxidant genes as well as the expression and production of proinflammatory cytokine TNF-α, and interleukin 1 β (IL-1β) (p < 0.05). SNECs treated with clarithromycin did not demonstrate statistically significant increases in ROS or proinflammatory cytokine production. Conclusion In this study, we show that treatment of cultured human SNECs with bactericidal antibiotics leads to formation of ROS with an associated increase in inflammatory and antioxidant gene expression and cell death. This suggests that long-term or inappropriate antibiotic use in the treatment of sinusitis may result in oxidative tissue damage to the sinonasal epithelium. Future studies will explore the clinical implications of such damage to the sinonasal epithelium.

Dysregulated sinonasal epithelial cell (SNEC) barrier function has been proposed to contribute to the pathogenesis of sinonasal inflammatory conditions such as allergic rhinitis (AR) and chronic rhinosinusitis (CRS). Allergens such as house dust mite (HDM) have been reported to disrupt SNEC barrier integrity. We have recently identified nuclear erythroid 2-related factor 2 (Nrf2) activation via sulforaphane (SFN) stimulation to stabilize SNEC barrier function. The purpose of this study was to explore whether Nrf2 activation could ameliorate HDM-induced SNEC barrier dysfunction.Human SNECs (HSNECs) were grown from patients at the air-liquid interface (ALI). HSNECs were stimulated with HDM with or without pharmacologic activation of Nrf2 with SFN. HSNECs were then stained for the epithelial cell junction protein zonula occludens-1 (ZO-1) and cell surface localization was evaluated by confocal microscopy. Transepithelial electrical resistance (TER) and paracellular fluorescein isothiocyanate (FITC)-dextran permeability was measured in response to stimulation with HDM and SFN.HDM stimulation caused a global disruption of the protein ZO-1 along with an associated decrease in TER (p0.001) and increased FITC-dextran paracellular permeability (p0.0001). Enhancing Nrf2 activation through treatment with SFN prior to stimulation with HDM was associated with increased localization of ZO-1 at the cell surface and statistically significant increases in TER (p0.05) and decrease in paracellular FITC-dextran permeability (p0.001).This is the first study to demonstrate that HDM-induced SNEC barrier dysfunction may be preventable by Nrf2 activation. The Nrf2 antioxidant pathway may represent a potential therapeutic target for allergen-induced sinonasal inflammation.

Antibiotics are widely and heavily used in the treatment of chronic sinusitis. Bactericidal antibiotics can stimulate reactive oxygen species (ROS) formation, a proinflammatory response, and cell death in cultured human sinonasal epithelial cells (SNECs). Sulforaphane (SFN) is a potent stimulator of the antioxidant nuclear factor erythroid 2-related factor 2 (Nrf-2) system and a suppressor of inflammation. In this study we utilized SFN to further explore the relationship between levofloxacin treatment, ROS formation, and the cell death response.SNECs were collected from patients during endoscopic sinus surgery and grown in culture at the air-liquid interface. Differentiated SNECs were stimulated with levofloxacin with or without SFN pretreatment. ROS were quantified. Apoptosis markers of caspase-3 activity and DNA fragmentation were quantified.Cultured SNECs treated with levofloxacin resulted in a significant increase in activity of the proapoptotic caspase-3 protease (5.9-fold, p = 0.01). The increase in activity was suppressed by pretreatment with SFN (1.9-fold). ROS levels increased with levofloxacin treatment (range, 1.2-fold to 1.8-fold), but were not significantly suppressed by pretreatment with SFN (range, 1.0-fold to 1.3-fold).In this study, we demonstrate that treatment of cultured SNECs with levofloxacin leads to an increase in caspase-3 activity. SFN pretreatment suppresses the increased apoptotic response possibly through its antioxidant stimulating properties. Our results suggest that levofloxacin treatment stimulates a potent proapoptotic possibly through an ROS-dependent mechanism. Future studies will explore if this antibiotic-induced response is harmful to recovery of function in those with sinusitis.

Allergy has been inferred to contribute to the pathophysiology of chronic rhinosinusitis (CRS) although this role is controversial and the mechanism is debated. Furthermore, the role of aeroallergens in CRS is poorly defined and has been postulated to contribute to CRS through direct penetration in the sinuses or downstream systemic consequences. Common aeroallergens implicated in chronic rhinosinusitis include air pollution/second hand smoke, dust mite and pollen [citeref rid="ref1"1/citeref,citeref rid="ref2"2/citeref,citeref rid="ref3"3/citeref]. One emerging potential mechanism whereby aeroallergens contribute to CRS is through sinonasal epithelial barrier disruption (fig.figref rid="fig01"1/figref). Characterization of cytokine disruption of sinonasal epithelial cell barrier has been described including interleukin (IL)-4 and IL-13, as well as aeroallergens such as house dust mite and cigarette smoke. Recent results have demonstrated severe barrier disruption in response to direct application of either particulate matter (PM) or house dust mite (HDM) to sinonasal epithelial cells. Sinonasal epithelial barrier disruption may contribute to CRS by enabling the perpetual and chronic exposure of inflammatory allergens and stimuli. The sinonasal epithelial barrier plays a significant role in innate immune host defense. Mechanisms of innate immune defense include pattern recognition receptors (PRRs), secreted endogenous antimicrobials and inflammatory cytokines that aid in repair mechanisms including IL-33. Here we discuss recent evidence implicating aeroallergens and dysregulated host innate immune responses in the development of CRS.figure id="fig01"no1/nofigcapFig. 1. Aeroallergens and inflammatory stimuli disrupt sinonasal epithelial barrier function. These agents act to destabilize the barrier through stimulating endocytosis and destruction of cell junction proteins via oxidative stress and MyD88-dependent mechanisms. Furthermore, aeroallergens and inflammatory stimuli induce secretion of IL-25, IL-33, and TSLP from sinonasal epithelial cells./figcapfigbody filename="F01" filetype="JPG"F01/figbody/figure.

Environmental factors such as inhaled pollutants like cigarette smoke may play a significant role in diseases of the upper airway including chronic rhinosinusitis (CRS). Recent studies have shown that cigarette smoke causes impaired airway epithelial cell barrier function likely through environmental oxidative stress related pathways. The purpose of this study is to explore whether enhancing nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2], the body's master antioxidant system, can ameliorate cigarette smoke-induced sinonasal epithelial cell (SNEC) barrier dysfunction.Human SNECs (HSNECs) were grown from control patients at the air-liquid interface (ALI). HSNECs were stimulated with cigarette smoke extract (CSE) with and without pharmacologic activation of Nrf2. HSNECs were then stained for the epithelial cell junctional proteins zonula occludens 1 (ZO-1) and junctional adhesion molecule A (JAM-A) using confocal microscopy. In addition, transepithelial electrical resistance (TER) was measured in cultures before and after stimulation with CSE.CSE stimulation caused a global disruption of the epithelial junctional proteins ZO-1 and JAM-A along with an associated decrease in TER levels. Enhancing Nrf2 levels prior to stimulation with CSE was associated with increased localization of ZO-1 and JAM-A levels at the cell surface and statistically significant increases in TER levels.This is the first study to demonstrate that cigarette smoke induced SNEC barrier dysfunction is reversible by Nrf2 activation. The Nrf2 antioxidant pathway may represent a potential therapeutic target for cigarette smoke-associated sinonasal inflammation.

Allergy has been inferred to contribute to the pathophysiology of chronic rhinosinusitis (CRS) although this role is controversial and the mechanism is debated. Furthermore, the role of aeroallergens in CRS is poorly defined and has been postulated to contribute to CRS through direct penetration in the sinuses or downstream systemic consequences. Common aeroallergens implicated in chronic rhinosinusitis include air pollution/second hand smoke, dust mite and pollen [ 1 , 2 , 3 ]. One emerging potential mechanism whereby aeroallergens contribute to CRS is through sinonasal epithelial barrier disruption (fig. 1 ). Characterization of cytokine disruption of sinonasal epithelial cell barrier has been described including interleukin (IL)-4 and IL-13, as well as aeroallergens such as house dust mite and cigarette smoke. Recent results have demonstrated severe barrier disruption in response to direct application of either particulate matter (PM) or house dust mite (HDM) to sinonasal epithelial cells. Sinonasal epithelial barrier disruption may contribute to CRS by enabling the perpetual and chronic exposure of inflammatory allergens and stimuli. The sinonasal epithelial barrier plays a significant role in innate immune host defense. Mechanisms of innate immune defense include pattern recognition receptors (PRRs), secreted endogenous antimicrobials and inflammatory cytokines that aid in repair mechanisms including IL-33. Here we discuss recent evidence implicating aeroallergens and dysregulated host innate immune responses in the development of CRS. 1 Fig. 1. Aeroallergens and inflammatory stimuli disrupt sinonasal epithelial barrier function. These agents act to destabilize the barrier through stimulating endocytosis and destruction of cell junction proteins via oxidative stress and MyD88-dependent mechanisms. Furthermore, aeroallergens and inflammatory stimuli induce secretion of IL-25, IL-33, and TSLP from sinonasal epithelial cells. F01 .

Bactericidal antibiotics have been shown to stimulate reactive oxygen species (ROS) formation in mammalian cells through mitochondrial dysfunction. This results in oxidative tissue damage that may have negative consequences for long-term antibiotic use. Antibiotics are widely and heavily used in the treatment of acute and chronic sinusitis; however, the relationship between antibiotics and ROS formation in sinonasal epithelial cells (SNECs) has not yet been demonstrated.Human SNECs were collected from patients during endoscopic sinus surgery and grown in culture at the air-liquid interface. Differentiated SNECs were stimulated with the bactericidal antibiotics amoxicillin and levofloxacin and the bacteriostatic antibiotic clarithromycin for 24 hours. ROS were quantified via fluorescence. Cell death was quantified by lactate dehydrogenase (LDH) secretion. Expression of inflammatory markers such as tumor necrosis factor α (TNF-α) and nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant genes were measured by real-time polymerase chain reaction (RT-PCR).Cultured SNECs treated with the bactericidal antibiotics amoxicillin and levofloxacin resulted in a significant increase in production of ROS (p0.05) and secretion of LDH (p0.05). The increase in ROS formation correlated with an increase in expression of Nrf2-mediated antioxidant genes as well as the expression and production of proinflammatory cytokine TNF-α, and interleukin 1 β (IL-1β) (p0.05). SNECs treated with clarithromycin did not demonstrate statistically significant increases in ROS or proinflammatory cytokine production.In this study, we show that treatment of cultured human SNECs with bactericidal antibiotics leads to formation of ROS with an associated increase in inflammatory and antioxidant gene expression and cell death. This suggests that long-term or inappropriate antibiotic use in the treatment of sinusitis may result in oxidative tissue damage to the sinonasal epithelium. Future studies will explore the clinical implications of such damage to the sinonasal epithelium.