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Infection plays a significant role in the relapse of chronic rhinosinusitis (CRS), however, the role of primary human nasal epithelial cells (HNECs) in this process is largely unknown. Here, we determined the effect of Toll-like receptor (TLR) agonists and inflammatory cytokines on mucosal barrier integrity and immune response of HNECs. TLR 1–9 agonists and inflammatory cytokines were applied to submerged and/or air-liquid interface (ALI) cultures of HNECs from CRS patients and controls for 24 hours. Interleukin-6 (IL-6) protein levels were determined by ELISA. Mucosal barrier integrity was measured via Transepithelial Electrical Resistance and passage of fluorescently-labelled dextrans. IL-1β and IFN- γ significantly increased IL-6 production in HNECs derived from CRS patients and controls, however, a dose-dependent effect was observed in CRS-derived HNECs only. Stimulation with Poly (I:C) LMW induced a 15 to 17 fold increase in IL-6 production by HNEC-ALI control cells (p < 0.05) and HNEC-ALI-CRS cells (p = 0.004) whilst a 2.5 fold increase was observed in CRS HNEC submerged cultures. Priming of cells with Poly (I:C) LMW reduced subsequent IL-6 secretion upon stimulation with TLR 2–4 agonists. Poly (I:C) LMW exerts a potent pro-inflammatory effect on HNECs and reduces a subsequent immune activation by TLR agonists.

Chronic rhinosinusitis (CRS) is a persistent inflammation of the nasal cavity and paranasal sinuses associated with tissue remodelling, dysfunction of the sinuses’ natural defence mechanisms, and induction of different inflammatory clusters. The etiopathogenesis of CRS remains elusive, and both environmental factors, such as bacterial biofilms and the host’s general condition, are thought to play a role. Bacterial biofilms have significant clinical relevance due to their potential to cause resistance to antimicrobial therapy and host defenses. Despite substantial medical advances, some CRS patients suffer from recalcitrant disease that is unresponsive to medical and surgical treatments. Those patients often have nasal polyps with tissue eosinophilia, S. aureus-dominant mucosal biofilm, comorbid asthma, and a severely compromised quality of life. This review aims to summarise the contemporary knowledge of inflammatory cells/pathways in CRS, the role of bacterial biofilm, and their impact on the severity of the disease. Here, an emphasis is placed on S. aureus biofilm and its secreted products. A better understanding of these factors might offer important diagnostic and therapeutic perceptions for recalcitrant disease.

Matrix metalloproteinase (MMP)-9 is thought to be involved in the etiopathogenesis of chronic rhinosinusitis (CRS) with nasal polyps and cleaves collagen IV, causing hyperpermeability of the basement membrane within mucosal tissue. It is known that MMP-9 expression is negatively affected by sirtuin (SIRT)-1 in human monocytotic cells, retinal endothelial cells, and epithelial carcinoma cells. However, it is unknown which factors affect MMP-9 expression and activity in human nasal epithelial cells (HNECs). To examine factors affecting MMP-9 expression and activity in HNECs, HNECs were stimulated with Toll-like receptor (TLR) agonists, followed by quantitative PCR, immunofluorescence, and zymography to examine MMP-9 expression and activity. MMP-9 expression was evaluated in sinonasal tissue of control subjects without CRS, and patients with CRS without nasal polyps and those with CRS with nasal polyps, in relation to the expression of SIRT1 using a tissue microarray. The effect of SIRT1 stimulation/inhibition on MMP-9 expression in HNECs was also tested. TLR3 agonists increased MMP-9 mRNA expression (473 fold, P = 0.0198) and activity (20.4-fold, P < 0.05). SIRT1 activation or inhibition reciprocally affected MMP-9 expression in the presence of TLR3 agonists. MMP-9 and SIRT1 expression within the epithelial layer of sinonasal tissue was inversely correlated only in patients with CRS but not in control subjects. TLR3 agonists increased MMP-9 expression and activity in HNECs, and the effect was abolished in the presence of SIRT1 activation. SIRT1 and MMP-9 expression was inversely correlated in CRS tissue, supporting SIRT1 as a possible therapeutic target for nasal polyp formation.

Normal wound healing is a highly regulated and coordinated process. However, tissue injury often results in inflammation with excessive scar tissue formation after 40–70% of operations. Here, we evaluated the effect of the iron chelator deferiprone on inflammation and the migration of primary nasal fibroblasts and primary human nasal epithelial cells (HNECs) in vitro . The cytotoxicity of deferiprone was examined by the lactate dehydrogenase assay on primary nasal fibroblasts and air-liquid interface (ALI) cultures of HNECs. Wound closure was observed in scratch assays by using time-lapse confocal scanning laser microscopy. Interleukin-6 (IL-6) and type I and III collagen protein levels were determined by ELISA. Intracellular Reactive Oxygen Species (ROS) activity was measured by utilizing the fluorescent probe H2DCFDA. Deferiprone at 10 mM concentration was non-toxic to primary fibroblasts and HNECs for up to 48 hours application. Deferiprone had significant dose-dependent inhibitory effects on the migration, secreted collagen production and ROS release by primary nasal fibroblasts. Deferiprone blocked Poly (I:C)-induced IL-6 production by HNECs but did not alter their migration in scratch assays. Deferiprone has the potential to limit scar tissue formation and should be considered in future clinical applications.

Cytokine mediated changes in paracellular permeability contribute to a multitude of pathological conditions including chronic rhinosinusitis (CRS). The purpose of this study was to investigate the effect of interferons and of Th1, Th2, and Th17 cytokines on respiratory epithelium barrier function. Cytokines and interferons were applied to the basolateral side of air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs) from CRS with nasal polyp patients. Transepithelial electrical resistance (TEER) and permeability of FITC-conjugated dextrans were measured over time. Additionally, the expression of the tight junction protein Zona Occludens-1 (ZO-1) was examined via immunofluorescence. Data was analysed using ANOVA, followed by Tukey HSD post hoc test. Our results showed that application of interferons and of Th1 or Th2 cytokines did not affect the mucosal barrier function. In contrast, the Th17 cytokines IL-17, IL-22, and IL-26 showed a significant disruption of the epithelial barrier, evidenced by a loss of TEER, increased paracellular permeability of FITC-dextrans, and discontinuous ZO-1 immunolocalisation. These results indicate that Th17 cytokines may contribute to the development of CRSwNP by promoting a leaky mucosal barrier.

Antibiotics are often administered to patients perioperatively and have been shown to affect ROS production of nasal cells , but their effect in the setting of active wound healing remains unclear. Reactive oxygen species (ROS) are known to play a significant role in wound healing. This study analyzed a broad array of antibiotics used after sinus surgery to assess their effect on wound healing and ROS production . It was hypothesized that ROS production would be affected by these antibiotics and there would be a negative relationship between ROS activity and cell migration speed. Monolayers of primary human nasal epithelial cells (HNEC) and primary fibroblasts were disrupted with a linear wound, treated with 10 different antibiotics or a ROS inhibitor and observed over 36 h in a controlled environment using confocal microscopy. ROS activity and migration speed of the wound edge were measured at regular intervals. The relationship between the two parameters was analyzed using mixed linear modeling. Performing a linear scratch over the cell monolayers produced an immediate increase in ROS production of ~35% compared to unscratched controls in both cell types. Incubation with mitoquinone and the oxazolidinone antibiotic linezolid inhibited ROS activity in both fibroblasts and HNEC in association with slowed fibroblast cell migration ( < 0.05). Fibroblast cell migration was also reduced in the presence of clarithromycin and mupirocin ( < 0.05). A significant correlation was seen between ROS suppression and cell migration rate in fibroblasts for mitoquinone and all antibiotics except for azithromycin and doxycycline, where no clear relationship was seen. Treatments that slowed fibroblast cell migration compared to untreated controls showed a significant correlation with ROS suppression ( < 0.05). Increased ROS production in freshly wounded HNEC and fibroblast cell monolayers was suppressed in the presence of antibiotics, in correlation with reduced fibroblast cell migration. In contrast, HNEC cell migration was not significantly affected by any of the antibiotics tested. This differential effect of antibiotics on fibroblast and HNEC migration might have clinical relevance by reducing adhesion formation without affecting epithelial healing in the postoperative setting.

Introduction Chronic rhinosinusitis (CRS) pathophysiology and its link to microbiome is an area of ongoing investigation. Certain pathogens, in particular Staphylococcus aureus described to contribute to recalcitrant CRS. In addition, different species of coagulase negative staphylococci (CoNS) are frequently isolated from the sinonasal cavity of CRS patients. However, the influence of Staphylococcal species coexisting in the same niche on the inflammatory process remains unclear. The aim of this study was to explore the impact of exoproteins from various Staphylococcus species isolated from the same patients on the mucosal barrier. Methods Staphylococcal species isolated from CRS and control patients were cultured from sinus swabs in planktonic and biofilm forms, and their exoproteins extracted. Primary human nasal epithelial cells (HNECs) from CRS patients were cultured at an air‐liquid interface (ALI) and exposed to 20 μg/mL exoproteins or control. Barrier disruption and cytotoxicity were assessed by measuring the transepithelial electrical resistance (TEER), passage of fluorescein labeled dextrans and lactate dehydrogenase (LDH) levels. IL‐ 6 concentration was measured employing ELISA. Patient's matched sinonasal tissue samples were analyzed with flow cytometry to detect and quantify immune cells. Results Forty‐four Staphylococcal species were isolated from 22 CRS and control patients including: 22 S. aureus, 12 S. epidermidis, and 10 S. lugdunensis. 15 out of 22 S. aureus exoproteins significantly enhanced cytotoxicity, reduced TEER values and increased paracellular permeability compared to control (p < 0.05). By contrast, S. epidermidis and S. lugdunensis exoproteins caused either mild or negligible effects on the TEER values, cell viability, and paracellular permeability. However, S. lugdunensis exoproteins induced significantly higher IL‐6 compared to control. Correlation analysis indicated S. aureus and S. lugdunensis from the same patient acted in concert to disrupt the nasal epithelial barrier and induce toxicity. Conclusion This study shows the significant and detrimental impact of the presence of S. aureus exoproteins on nasal epithelial cell barrier function. S. aureus and S. lugdunensis isolated from the same patients acted in concert to affect the nasal barrier and inducing toxicity.

Staphylococcus aureus with varying virulence is often isolated from chronic rhinosinusitis (CRS) patients and impacts disease severity. Prophage‐mediated virulence, particularly encoded by φSa3int (NM3) prophages, which often encodes human immune‐evasion cluster genes is well known, but how a new prophage domestication impacts overall expression of core bacterial genes, and the expression of resident prophages is understudied. To understand this, we transduced a φSa3int prophage recovered from hyper‐biofilm forming mucoid S. aureus (SA333) into a high‐biofilm forming non‐mucoid S. aureus (SA222) recovered from same CRS patient but at different time points. Upon φSa3int prophage domestication, we observed a significant upregulation of 21 exoproteins including human immune‐evasion toxins and an intercellular adhesion protein B (IcaB). Further, φSa3int prophage domestication led to reduced phagocytosis implying φSa3int prophage mediates escape of S. aureus from human innate immunity. Our data further show that in addition to adding novel prophage‐encoded virulence, φSa3int prophage domestication also affects the expression of non‐prophage (bacterial) genes and suppresses expression of structural proteins of resident prophages. Since strains without prophage or with specific prophages have varying virulence and pathogenicity, targeted identification virulence factors associated with mobile genetic elements (MGEs) in addition to species identification may lead to better personalized therapy, particularly in chronic infections. Prophages play a significant role in modulating bacterial virulence and pathogenicity. Our results suggest domestication of φSa3int prophage leads to reduced phagocytosis mediating escape of S. aureus from human innate immunity. As such, targeted identification of mobile genetic elements (MGEs) in addition to species and strain identification may lead to better‐personalized therapy as not all bacteria carry the same MGEs .

Dysregulated inflammation is important in the pathogenesis of many diseases including cancer, allergy, and autoimmunity. Macrophage activation and polarisation are commonly involved in the initiation, maintenance and resolution of inflammation. Perhexiline (PHX), an antianginal drug, has been suggested to modulate macrophage function, but the molecular effects of PHX on macrophages are unknown. In this study we investigated the effect of PHX treatment on macrophage activation and polarization and reveal the underlying proteomic changes induced. We used an established protocol to differentiate human THP-1 monocytes into M1 or M2 macrophages involving three distinct, sequential stages (priming, rest, and differentiation). We examined the effect of PHX treatment at each stage on the polarization into either M1 or M2 macrophages using flow cytometry, quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Quantitative changes in the proteome were investigated using data independent acquisition mass spectrometry (DIA MS). PHX treatment promoted M1 macrophage polarization, including increased and expression and IL-1β secretion. This effect occurred when PHX was added at the differentiation stage of the M1 cultures. Proteomic profiling of PHX treated M1 cultures identified changes in metabolic (fatty acid metabolism, cholesterol homeostasis and oxidative phosphorylation) and immune signalling (Receptor Tyrosine Kinase, Rho GTPase and interferon) pathways. This is the first study to report on the action of PHX on THP-1 macrophage polarization and the associated changes in the proteome of these cells.

causes severe chronic respiratory diseases and is associated with recalcitrant chronic rhinosinusitis (CRS). exoproteins contain virulence factors and play important roles in the pathogenicity of , however their role in CRS pathophysiology remains unknown. We isolated clinical isolates (CIs) and obtained clinical information from 21 CRS patients. Elastase activity of the CIs was measured at different phases of growth. Primary human nasal epithelial cells (HNECs) were cultured at air-liquid interface (ALI) and challenged with exoproteins or purified elastase, followed by measuring Transepithelial Electrical Resistance (TEER), permeability of FITC-dextrans, western blot, and immunofluorescence. 14/21 CIs had a significant increase in elastase activity in stationary phase of growth. There was a highly significant strong correlation between the elastase activity of CIs with mucosal barrier disruption evidenced by increased permeability of FITC-dextrans ( = 0.95, = 0.0004) and decreased TEER ( = -0.9333, < 0.01) after 4 h of challenge. Western blot showed a significant degradation of ZO-1, Occludin and β-actin in relation to the elastase activity of the exoproteins. There was a highly significant correlation between the elastase activity of CIs and CRS disease severity (for log phase, = 0.5631, = 0.0097; for stationary phase, = 0.66, = 0.0013) assessed by CT imaging of the paranasal sinuses. Our results implicate exoproteins as playing a major role in the pathophysiology of associated CRS by severely compromising mucosal barrier structure and function.