Sinonasal Epithelial Cells Research Articles

Human sinonasal epithelial cells direct dendritic function and T‐cell T helper 1/T helper 2 skewing following Aspergillus exposure

In lower airway disease such as asthma, epithelial cells have been shown to be potent regulators of dendritic cell (DC) functions. However, it is unclear how human sinonasal epithelial cells (HSNECs) from patients with sinusitis regulate DC functions. Therefore, in these studies we investigated the ability of Aspergillus fumigatus exposed HSNECs to regulate DC antigen uptake, maturation, and direction of T-cell T helper 1 (Th1)/Th2 skewing. Primary HSNECs were cultured from control (n = 8), chronic sinusitis without nasal polyps (CRSsNP) (n = 9), and chronic sinusitis with nasal polyps (CRSwNP) (n = 7) patients and exposed to Aspergillus. Conditioned media was placed upon monocyte-derived DCs from healthy controls. DC antigen uptake was assessed by dextran-fluorescein isothiocyanate (FITC) uptake. DC differentiation and maturation was assessed by immunostaining for CD209, CD80, and CD86 followed by flow cytometric analysis. DC direction of T-cell Th1/Th2 skewing was evaluated by immunostaining followed by intracellular flow cytometric analysis for interferon (IFN)-γ and interleukin (IL)-5. Control and CRSsNP HSNECs have the capacity to stimulate DC antigen uptake, differentiation, and maturation following Aspergillus exposure. CRSwNP HSNECs stimulate DC activation independent of Aspergillus exposure. Furthermore, Aspergillus-exposed CRSwNP HSNECs skew T-cells toward a Th2 phenotype. CRSwNP-derived HSNECs stimulate DC maturation and Th2 skewing independent of Aspergillus exposure. However, control and CRSsNP HSNECs induce DC maturation and Th2 skewing after Aspergillus exposure. These in vitro studies demonstrate that HSNECs are key regulators of DC functions in the sinus microenvironment.

Allergic rhinitis is associated with decreased expression of Toll‐like receptor 9 by sinonasal epithelial cells

Toll-like receptors (TLRs) are important in sinonasal mucosal innate immunity. Previous studies demonstrate that sinonasal epithelial cell (SNEC) TLR9 expression is reduced in T-helper 2 (Th2) cytokine-predominant chronic rhinosinusitis with polyps, and with the in vitro application of Th2 cytokines. To further investigate in vivo modulation of TLR9 by the local cytokine environment, this study examines TLR9 expression in freshly isolated SNECs from subjects with and without active allergic rhinitis (AR). SNECs were gathered via endoscopic-guided middle meatal brushings from 9 AR subjects who were skin-prick test (SPT)-positive to environmental allergens in season at the time of study, and 8 controls. Flow cytometry was utilized to compare SNEC TLR9 expression in the 2 groups. TLR9 expression by SNEC in the AR group was significantly reduced compared to normals (35% ± 26% vs 76% ± 10%, p = 0.002). Similar to observations in eosinophilic chronic rhinosinusitis, this study shows that active AR is associated with decreased SNEC TLR9 expression. These findings are consistent with the concept that Th2 cytokines suppress expression of TLR9 and other innate immune genes. Multiple endogenous and microbial factors likely modulate sinonasal innate immunity to maintain homeostasis and prevent infection in AR.

The Role of Hepatocyte Growth Factor/c-Met in Chronic Rhinosinusitis with Nasal Polyps

Hepatocyte growth factor (HGF) is a growth factor thought to attenuate Th2-driven eosinophilic airway inflammatory responses. Increased expression of HGF and its receptor c-Met in nasal polyps suggests a role in disease pathogenesis. The effect of HGF on human sinonasal epithelial cell (SNEC) responses to Th2 inflammatory cytokines in chronic rhinosinusitis with nasal polyps (CRSwNP) has not been explored. SNECs isolated from patients with CRSwNP and control subjects were grown in cell culture at the air-liquid interface. The Th2 cytokine IL-13 was applied for 24 hours in the presence or absence of HGF. Eotaxin-3 and c-Met expression was assessed using real-time PCR, immunohistochemistry, and flow cytometry. SNECs obtained from both CRSwNP and control subjects showed markedly increased expression of eotaxin-3 after exposure to IL-13. HGF significantly blocked IL-13-induced expression of eotaxin-3 in control SNECs, but not in SNECs derived from CRSwNP subjects. SNECs are active participants in sinonasal mucosal immunity, expressing inflammatory mediators in response to potential pathogens and endogenous cytokines. Although Th2 cytokines can elicit expression of proeosinophilic mediators by SNECs, HGF appears to have a down-regulating effect on this response. In patients with CRSwNP, SNECs are resistant to this attenuation, showing continued IL-13-induced eotaxin-3 expression despite HGF treatment. Abnormalities in the regulation of epithelial cell responses to endogenous cytokines and growth factors may contribute to the persistent eosinophilic inflammatory state in CRSwNP.

Allergic rhinitis patients with recurrent acute sinusitis have increased sinonasal epithelial cell TLR9 expression

Objective Pilot study examining toll-like receptor 9 (TLR9) expression by sinonasal epithelial cells in allergic rhinitis (AR) subjects with and without a history of recurrent acute rhinosinusitis (RARS). Study Design Cohort study. Setting Outpatient clinic. Subjects and Methods Adult subjects were eligible for study if skin tested positive for inhalant allergens, and positive allergens were in season at time of study. Subjects were considered to have AR+RARS if they had four symptomatic episodes with major/minor factors in 12 months and CT evidence of sinusitis. Eight AR-only subjects and 13 AR+RARS subjects underwent endoscopic-guided cell brushing from the middle meatus. Flow cytometry for TLR9 expression was performed on collected fresh sinonasal epithelial cells. Results The AR+RARS group was found to have a significant increase in TLR9 expression in the sinonasal epithelium (66% ± 30%) compared with that of AR-only patients (32% ± 21%; P = 0.011). Conclusion The significant difference in expression of TLR9 in allergic sinusitis patients compared with allergy-only patients in this study may indicate a difference in the role of innate immunity in these groups. The results suggest that expression of innate immune markers such as TLR9 may be upregulated in response to repeated microbial insults in AR+RARS. Further research is necessary to determine whether an initial impairment of innate immune gene expression may predispose some AR patients to subsequent development of RARS.

Treatment-Recalcitrant Chronic Rhinosinusitis with Polyps is Associated with Altered Epithelial Cell Expression of Interleukin-33

Abnormalities in host mucosal immunity exist in chronic rhinosinusitis with nasal polyps (CRSwNPs), but it is unclear whether this is a cause or an effect of the eosinophilic inflammation and frequent microbial colonization that characterizes the disease. Sinonasal epithelial cells (SNECs) are critical participants in healthy antimicrobial innate immune defense. They also can promote Th2 inflammation with various mediators, including interleukin (IL)-33, which induces T helper cells to produce Th2 cytokines. CRSwNP SNECs were obtained during sinus surgery and stored. Patients were subsequently classified as either treatment responsive or treatment recalcitrant, based on long-term outcomes of medical and surgical therapy. Epithelial cells from these patients were grown in air-liquid interface (ALI) culture and treated with IL-13, as well as the bacteria-associated molecule, CpG. Expression of IL-33 mRNA was determined by real-time polymerase chain reaction. Recalcitrant CRSwNP epithelial cells had increased baseline expression of IL-33 compared with responsive CRSwNPs, which was further increased by 24-hour exposure to CpG. Treatment-responsive epithelial cells were not induced by CpG to express IL-33. Prolonged treatment with IL-13 during differentiation at the ALI diminished the baseline expression of IL-33 and prevented the subsequent induction of IL-33 by CpG. Mucosal innate immunity likely plays an important role in CRSwNP pathogenesis. A definitive link between infectious triggers and the development of Th2 inflammation has been elusive. We have found constitutive IL-33 expression by SNECs in recalcitrant CRSwNPs, which can be further induced by a bacteria-associated molecular pattern. Dysregulated epithelial cell immune interactions between host and environment may contribute to Th2 inflammation in CRSwNPs.

Expression of B7-H1/PD-1 in mucosa of chronic rhinosinusitis

To explore the expression of B7-H1/PD-1 and the localization of these proteins in human sinus mucosa of chronic rhinosinusitis (CRS). Twenty-eight patients with CRS (17 with nasal polyps (CRSwNP) and 11 without (CRSsNP)) and 11 control patients were recruited. B7-H1/PD-1 expressions in sinus mucosa among these 3 groups were detected by immunofluorescent staining, real-time quantitative PCR and Western blot. Flow cytometry was also employed to identify the type of B7-H1 or PD-1 positive cells in nasal polyps. Compared with normal mucosa, B7-H1/PD-1 mRNA was highly expressed in sinonasal mucosa of CRSwNP and CRSsNP (P < 0.01). There was no difference between CRSwNP and CRSsNP (P > 0.05). The protein level of B7-H1/PD-1 was similar to mRNA level. As indicated by immunofluorescent assay, B7-H1 was mainly expressed in sinonasal epithelial cells, submucosal gland cells and inflammatory cells within the subepithelial layer while PD-1 was expressed in inflammatory cells within the subepithelial layer. The B7-H1 or PD-1 positive cells in nasal polyps were partially CD19+ B cells and CD3+ T cells respectively. B7-H1/PD-1 is up-regulated in sinus mucosa of CRS. B7-H1/PD-1 may play an important role in the pathogenesis of CRS.

Cigarette Smoke Extract Stimulates Interleukin-8 Production in Human Airway Epithelium and is Attenuated by Superoxide Dismutase in Vitro

Cigarette smoke exposure (CSE) results in extensive inflammation in the upper and lower airways. Reactive oxygen species, such as superoxide, have been shown to be potent mediators of this inflammation. Mucosal biopsy specimens were collected from patients undergoing sinonasal surgery and were used as a source of primary epithelial cells. Human sinonasal epithelial (HSNE) cells and were isolated from sinus tissue, maintained in culture, and ultimately treated with varying concentrations of CSE with or without free superoxide dismutase (SOD). Supernatants and cell lysates were examined for the proinflammatory cytokine interleukin (IL)-8. Similar experiments were performed using normal human bronchial epithelial (NHBE) cell lines. CSE induces both secretion and intracellular production of the proinflammatory cytokine IL-8 by HSNE cells in a dose-dependent manner. Furthermore, this up-regulation can be suppressed by SOD. CSE induces secretion of IL-8 in NHBEs that is also suppressed by SOD. Inflammation in the airway after CSE can be blocked by SOD in this in vitro model. The ability to attenuate CSE-induced inflammation with SOD could provide a therapeutic/preventative approach for individuals with cigarette smoke exposure.

Nasal Challenge with LPS Stimulates the Release of Macrophage Inflammatory Protein 1α

Background: Bacterial infections can cause a variety of airway diseases. Toll-like receptors (TLRs) directly respond to the presence of microbes and partake in the innate immune defense. TLR4 is activated by lipopolysaccharide (LPS), and has been detected in sinonasal tissue, epithelial cells and various inflammatory cells. Macrophage inflammatory protein 1α (MIP-1α) is a chemokine released during the inflammatory process. The present study investigated the potential role and regulation of MIP-1α in LPS-induced nasal inflammation. Methods: Thirty-two healthy individuals were intranasally challenged with LPS or vehicle. Nasal lavage was performed, followed by a nasal biopsy. Inflammatory cells were counted, MIP-1α levels analyzed and expression of MIP-1α mRNA in biopsies quantified. Neutrophils isolated from peripheral blood were treated with LPS and effects on MIP-1α release, cell survival, and the involved signal pathways, were investigated. Results: LPS challenge caused an increase of MIP-1α in nasal lavage. No corresponding change in mRNA expression was seen in nasal biopsies, suggesting the increase was not due to epithelial synthesis. Neutrophil numbers increased after LPS provocation. Treatment of isolated neutrophils with LPS delayed neutrophil apoptosis and resulted in a time- and concentration-dependent release of MIP-1α, which was reduced by inhibitors of transcription and of nuclear factor (NF)-κB, protein kinase C (PKC) and p38 MAPK pathways. Conclusions: Nasal LPS challenge results in release of MIP-1α. The release most likely originates from recruited neutrophils, via NF-κB-, PKC- and p38 MAPK-dependent pathways. LPS stimulation delayed neutrophil apoptosis. MIP-1α may constitute an important mediator in neutrophilic airway disease.

Chitin Stimulates Expression of Acidic Mammalian Chitinase and Eotaxin-3 by Human Sinonasal Epithelial Cells in Vitro

Sinonasal epithelial cells participate in host defense by initiating innate immune mechanisms against potential pathogens. Antimicrobial innate mechanisms have been shown to involve Th1-like inflammatory responses. Although epithelial cells can also be induced by Th2 cytokines to express proeosinophilic mediators, no environmental agents have been identified that promote this effect. Human sinonasal epithelial cells from patients with chronic rhinosinusitis with nasal polyps (CRSwNPs) and controls were harvested and grown in primary culture. Cell cultures were exposed to a range of concentrations of chitin for 24 hours, and mRNA for acidic mammalian chitinase (AMCase), eotaxin-3, and thymic stromal-derived lymphopoietin (TSLP) were assessed. Other cultures were exposed to interleukin 4 (IL- 4) alone and in combination with dust-mite antigen (DMA) for 36 hours. Extracted mRNA and cell culture supernatant were analyzed for expression of AMCase and eotaxin-3. Chitin induced a dose-dependent expression of AMCase and eotaxin-3 mRNA but not TSLP. Patients with recalcitrant CRSwNPs showed lower baseline expression of AMCase when compared with treatment-responsive CRSwNP and less induction of AMCase expression by chitin. DMA did not directly induce expression of AMCase or eotaxin-3. Expression of eotaxin-3 was stimulated by IL-4 and further enhanced with the addition of DMA. Levels of AMCase were not significantly affected by either IL-4 or DMA exposure. In some cases, the combination of IL-4 and DMA was able to induce AMCase expression in cell cultures not producing AMCase at baseline. The abundant biopolymer chitin appears to be recognized by a yet uncharacterized receptor on sinonasal epithelial cells. Chitin stimulates production of AMCase and eotaxin-3, two pro-Th2 effector proteins. This finding suggests the existence of a novel innate immune pathway for local defense against chitin-containing organisms in the sinonasal tract. Dysregulation of this function could precipitate or exacerbate Th2 inflammation, potentially acting as an underlying factor in recalcitrant CRSwNP.

Th2 Cytokines associated with Chronic Rhinosinusitis with Polyps Down-Regulate the Antimicrobial Immune Function of Human Sinonasal Epithelial Cells

Chronic rhinosinusitis with nasal polyps (CRSwNPs) is a disorder characterized by persistent eosinophilic Th2 inflammation and frequent sinonasal microbial colonization. It has been postulated that an abnormal mucosal immune response underlies disease pathogenesis. The relationship between Th2 inflammatory cytokines and the innate immune function of sinonasal epithelial cells (SNECs) has not been explored. Human SNECs (HSNECs) isolated from control subjects and patients with CRS were assessed for expression of antimicrobial innate immune genes and proinflammatory cytokine genes by real-time polymerase chain reaction, ELISA, and flow cytometry. A model of the Th2 inflammatory environment was created by exposure of primary HSNEC to the Th2 cytokine interleukin (IL)-4 or IL-13 for 36 hours, with subsequent assessment of innate immune gene expression. HSNEC obtained from CRSwNP patients displayed decreased expression of multiple antimicrobial innate immune markers, including toll-like receptor 9, human beta-defensin 2, and surfactant protein A. Baseline expression of these genes by normal and CRS HSNEC in culture is significantly down-regulated after incubation with IL-4 or IL-13. Expression of multiple innate immune genes by HSNEC is reduced in CRSwNP. One mechanism appears to be a direct effect of the leukocyte-derived Th2 cytokines present in the sinonasal mucosa in CRSwNP. Impaired mucosal innate immunity may contribute to microbial colonization and abnormal immune responses associated with CRSwNP.

The Cigarette Smoke Component Acrolein Inhibits Expression of the Innate Immune Components IL-8 and Human Beta-Defensin 2 by Sinonasal Epithelial Cells

Tobacco use is associated with poorer outcomes of medical and surgical therapy for chronic rhinosinusitis (CRS), although the underlying mechanism is unknown. Acrolein (AC) is a major component of cigarette smoke that has been shown to suppress innate immune gene expression by human bronchial epithelial cells and murine macrophages. In this study, we explore whether exposure of human sinonasal epithelial cells (HSNECs) to AC similarly reduces their innate immune gene expression. Primary HSNECs from CRS patients were grown in culture, either differentiated or submerged. HSNECs were treated for 30 minutes with 0-50 microM of AC and were subsequently analyzed by real-time polymerase chain reaction and ELISA to determine IL-8 and human beta-defensin (HBD) 2 expression. Total glutathione was measured to see the oxidative stress within the treatment range. In primary HSNEC, IL-8 mRNA levels decreased dose dependently in the range of 10-50 microM of AC with an eightfold decrease at 50 microM. In addition, a 125-fold decrease at 50 microM for IL-8 protein was observed. HBD-2 mRNA decreased twofold and HBD-2 protein decreased fourfold at 50 microM of AC in primary HSNEC. However, differentiated HSNEC showed a marginal decrease in a dose-dependent manner for both IL-8 and HBD-2 within the range of 10-50 microM of AC. There was no oxidative stress observed over this range of AC concentration. The tobacco smoke component AC has the capacity to suppress the inflammatory and innate immune function of sinonasal epithelial cells. Whether this effect contributes to the negative clinical impact of smoking on CRS outcomes merits additional investigation.

Reversal of Chronic Rhinosinusitis-Associated Sinonasal Ciliary Dysfunction

Although multiple etiologies contribute to the development of chronic rhinosinusitis (CRS), a common pathophysiological sequelae is ineffective sinonasal mucociliary clearance, leading to stasis of sinonasal secretions, with subsequent infection and/or persistent inflammation. Proper therapeutic intervention typically restores mucociliary activity, suggesting that the pathophysiological process(es) responsible for CRS-associated mucostasis may be reversible. We previously demonstrated a blunted response of CRS sinonasal cilia after purinergic stimulation. This study investigated whether the blunted ciliary response is unique to purinergic stimulation and addressed whether the blunted effect is primarily caused by local CRS-associated mediators or inherent genetic defects in ciliary function. A dual temperature-controlled perfusion chamber, differential interference contrast microscopy, and high-speed digital video were used to analyze both basal as well as cholinergic, adrenergic, and purinergic stimulation of cilia in human sinonasal mucosal explants. Additionally, enzymically dissociated sinonasal ciliated cells were maintained ex vivo in submersion, on glass coverslips, and assessed daily for purinergic ciliary beat frequency stimulation. Cholinergic and adrenergic stimulation generally were blunted in mucosal explants obtained from CRS patients. Ex vivo maintenance of samples demonstrated that the majority of CRS samples developed a stimulatory phenotype within 36 hours of culturing. CRS is a common debilitating disease principally affecting sinonasal epithelial function with a resultant diminution of mucociliary transport. Presently, little is known about how this disease process affects the sinonasal epithelial ciliated cells. Our data suggest that ciliary response to environmental insults is blunted in a reversible manner in CRS patients.

Sinonasal Epithelial Cell Expression of Toll-Like Receptor 9 is Decreased in Chronic Rhinosinusitis with Polyps

Innate immune recognition of pathogens by sinonasal epithelial cells may play an important role in the pathogenesis of chronic rhinosinusitis (CRS). Previous studies have indicated that toll-like receptor (TLR) mRNA is present in sinonasal mucosa, and levels of TLR9 expression are decreased in recalcitrant CRS with nasal polyps (CRSwNP). However, the cellular source and function of TLR9 in the sinonasal epithelium is not known. In this study, primary epithelial cell cultures were analyzed from control subjects and CRSwNP patients to determine the presence and function of TLR9 protein. Primary epithelial cell cultures were established from 5 controls and 10 CRSwNP patients undergoing sinus surgery. Flow cytometry was used to confirm purity of epithelial cells and to assess expression of TLR9 protein. Epithelial cells were stimulated with TLR9 agonist, and mRNA was analyzed by real-time PCR for expression of human beta-defensin (HBD) 2 and interleukin (IL)-8. Flow cytometry showed TLR9 protein in 100% of epithelial cells from controls and CRSwNP patients. The level of expression was 50% lower in CRS patients than in controls. Stimulation of epithelial cells with TLR9 agonist produced a 1.5- to 9-fold increase in HBD-2 and IL-8 mRNA expression. Functional TLR9 protein is expressed by normal and diseased sinonasal epithelial cells. The level of TLR9 expression is decreased in CRSwNP patients, consistent with the previous finding of decreased TLR9 mRNA in whole sinonasal tissue. These findings suggest that impaired innate immune responses to pathogens via TLR9 on sinonasal epithelial cells may represent a critical mechanism in chronic inflammatory sinus disease.

Quantification of Ciliary Beat Frequency in Sinonasal Epithelial Cells Using Differential Interference Contrast Microscopy and High-Speed Digital Video Imaging

Mucociliary clearance is a critical upper airway host defense mechanism. Ciliated epithelium in the mammalian airway continually beat at a baseline frequency. Importantly, during times of stress such as exercise or infection, the cilia beat faster to increase clearance. Nasal epithelial ciliary beat frequency (CBF) has been analyzed previously in ex vivo specimens using a variety of methods including photodiode detectors and conventional video recording. Recent studies performed using lower airway ciliated mucosa have shown poor correlation between CBF quantified by photodiode/conventional video and those using high-speed digital video capture at temperatures close to physiological temperatures. Thus, to more rigorously interrogate sinonasal CBF at physiological conditions, we have incorporated a high-speed digital video camera to our CBF analysis system. This is the first report of sinonasal epithelial CBF analysis performed using high-speed video digital analysis. Ex vivo samples of sinonasal epithelium were placed in lactated Ringer's in a temperature-controlled microscope stage chamber. An edge of tissue containing beating cilia was observed at a magnification of 630x using differential interference contrast microscopy. The images were captured using a high-speed digital camera with a sampling rate of 250 frames per second. CBF was determined using computerized data analysis. The mean nasal CBF was calculated from a minimum of five regions for each sample. Temperature curves were generated from tissue obtained from chronically infected subjects. Analysis of high-speed digital video capture of sinonasal CBF observed under differential interference contrast microscopy is a powerful method to investigate environmental as well as host influences on mucociliary clearance within the upper airways.

MRNA for genes associated with antigen presentation are expressed by human middle meatal epithelial cells in culture.

Although the mechanisms underlying the initiation and maintenance of inflammation in chronic rhinosinusitis are poorly understood, the activation of memory T cells within the nasal mucosa is thought to play an important role. T-cell activation requires specialized antigen processing and presentation of antigen by immunocompetent cells in the context of cell surface immune molecules. The purpose of this study was to investigate the expression of such molecules by human sinonasal epithelial cells grown in culture at the air-liquid interface (ALI). Middle meatal epithelium was obtained from six patients undergoing endoscopic sinus surgery. Dissociated epithelial cells were grown to confluence in serum-free, defined medium and transferred to filter inserts for culture at the ALI. Cells were harvested at 2 and 21 days of growth at the ALI and processed for real-time polymerase chain reaction (PCR). The presence and relative abundance of constitutively expressed mRNA for human leukocyte antigen (HLA)-B, HLA-DR, B7 to 1, B7 to 2, B7-H2, B7-H3, and cathepsin D were assessed. After 2 days at the ALI, middle meatal epithelial cells demonstrated expression of genes for each of the antigen processing associated genes tested. The expression of HLA-B and HLA-DR increased significantly with cellular maturation at the ALI. Expression of HLA-DR and B7 to 1 increased with cytokine stimulation. Primary human epithelial cells obtained from the middle meatus express genes associated with antigen presentation function. The pattern of gene expression is modulated by cytokine stimulation and changes as the cells differentiate at the ALI. These findings suggest that mature middle meatal epithelial cells have the cellular machinery to interact with T cells and therefore may be direct participants in the modulation of T-cell activity in chronic sinusitis.