Defective epithelial barrier in chronic rhinosinusitis: The regulation of tight junctions by IFN-γ and IL-4

LIGHT Signals Directly to Intestinal Epithelia to Cause Barrier Dysfunction via Cytoskeletal and Endocytic Mechanisms

Studies have independently indicated that eosinophils and histone deacetylases (HDACs) may compromise the integrity of the epithelial barrier in nasal polyps; however, the underlying mechanisms are not clear. In this study, we aimed to investigate the role of eosinophilia and HDACs in regulation of tight junctions (TJs) and nasal epithelial barrier integrity in chronic rhinosinusitis with nasal polyps (CRSwNP) patients. Expression of mRNAs and proteins of TJs and HDACs of biopsy specimens and air-liquid interface (ALI) human nasal epithelial cell cultures (HNECs) from eosinophilic and noneosinophilic CRSwNP patients and healthy controls was assessed. The ALI HNECs were also assessed for changes in transepithelial electrical resistance (TER) and paracellular flux of fluorescein isothiocyanate (FITC)-labelled dextran. Meanwhile, the assessments for the effect of HDAC inhibitor in eosinophilic nasal polyps were also conducted. Decreased TER and increased paracellular flux of FITC-labelled dextran in the ALI cultures were found in both eosinophilic and noneosinophilic CRSwNP, along with irregular, patchy and reduced expression of claudin-1, 4, 7, occludin, zonula occludens (ZO)-1 and ZO-2 and increased expression of HDAC1, 9 and SIRT7 for both ALI culture cells and biopsy specimens, especially for the eosinophilic CRSwNP group. Treatment of eosinophilic CRSwNP ALI-HNECs with an HDAC inhibitor improved the TJs expression and epithelial barrier integrity. Our data suggest that eosinophilia and HDACs influence epithelial barrier function in CRSwNP patients by regulating TJ protein expression. Targeting HDACs with specific inhibitors may be a potential treatment option for patients with eosinophilic CRSwNP.

Impaired barrier function in patients with house dust mite–induced allergic rhinitis is accompanied by decreased occludin and zonula occludens-1 expression

Regulation of bronchial epithelial barrier integrity by type 2 cytokines and histone deacetylases in asthmatic patients

Interleukin-13 Is the Key Effector Th2 Cytokine in Ulcerative Colitis That Affects Epithelial Tight Junctions, Apoptosis, and Cell Restitution

Sealing the ducts

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Staphylococcus aureus colonization and release of enterotoxin B (SEB) has been associated with severe chronic rhinosinusitis with nasal polyps (CRSwNP). The pathogenic mechanism of SEB on epithelial barriers, however, is largely unexplored. We investigated the effect of SEB on nasal epithelial barrier function. SEB was apically administered to air-liquid interface (ALI) cultures of primary polyp and nasal epithelial cells of CRSwNP patients and healthy controls, respectively. Epithelial cell integrity and tight junction expression were evaluated. The involvement of Toll-like receptor 2 (TLR2) activation was studied in vitro with TLR2 monoclonal antibodies and in vivo in tlr2-/- knockout mice. SEB applied to ALI cultures of polyp epithelial cells decreased epithelial cell integrity by diminishing occludin and zonula occludens (ZO)-1 protein expression. Antagonizing TLR2 prevented SEB-induced barrier disruption. SEB applied in the nose of control mice increased mucosal permeability and decreased mRNA expression of occludin and ZO-1, whereas mucosal integrity and tight junction expression remained unaltered in tlr2-/- mice. Furthermore, in vitro SEB stimulation resulted in epithelial production of IL-6 and IL-8, which was prevented by TLR2 antagonization. SEB damages nasal polyp epithelial cell integrity by triggering TLR2 in CRSwNP. Our results suggest that SEB might represent a driving factor of disease exacerbation, rather than a causal factor for epithelial defects in CRSwNP. Interfering with TLR2 triggering might provide a way to avoid the pathophysiological consequences of S.aureus on inflammation in CRSwNP.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a typical type-2 inflammation involving several cytokines and is associated with epithelial cell dysfunction. Oncostatin M (OSM) (belonging to the interleukin(IL)-6 family) could be a key driver of epithelial barrier dysfunction. Therefore, we investigated the presence of OSM and IL-6 and the expression pattern of tight junctions (TJs) in the nasal tissue of CRSwNP patients and controls using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Then, their potential role in the epithelial barrier was evaluated in vitro in 27 different primary cultures of human nasal epithelial cells (HNECs) by measuring TJ expression and transepithelial electric resistance (TEER) with or without OSM or IL-6 (1, 10, and 100 ng/mL). The effect on ciliary beating efficiency was evaluated by high-speed videomicroscopy and on repair mechanisms with a wound healing model with or without OSM. OSM and IL-6 were both overexpressed, and TJ (ZO-1 and occludin) expression was decreased in the nasal polyps compared to the control mucosa. OSM (100 ng/mL) but not IL-6 induced a significant decrease in TJ expression, TEER, and ciliary beating efficiency in HNECs. After 24 h, the wound repair rate was significantly higher in OSM-stimulated HNECs at 100 ng/mL. These results suggest that OSM could become a new target for monoclonal antibodies.

Mycophenolic acid (MPA) is a potent inhibitor of inosine monophosphate dehydrogenase (IMPDH), a key regulator of purine biosynthesis. MPA is frequently used as an immunosuppressant drug to prevent acute graft rejection for kidney, liver and lung transplantation. The use of MPA is associated with GI toxicity which is a problem to the patients, and a challenge for clinicians. The present study was undertaken to identify novel molecular targets of MPA using a proteomics approach. Two dimensional gel electrophoresis (2-DE) and mass spectrometry were used to identify proteome alterations in human embryonic cells (HEK-293) following exposure to therapeutic concentrations of MPA. Cells were treated for 72 hours, and total cell lysate was resolved by 2-DE followed by QTOF-MS/MS analysis of all identified differentially regulated proteins. A total of 12 proteins were differentially regulated in HEK-293 cells following exposure to MPA. Among these, 7 proteins were up-regulated (complement component 1 Q subcomponent-binding protein, electron transfer flavoprotein subunit beta, cytochrome b-c1 complex subunit, peroxiredoxin 1, thioredoxin domain-containing protein 12, myosin regulatory light chain 2, and profilin 1), while 5 proteins were down-regulated (protein SET, stathmin, 40S ribosomal protein S12, histone H2B type 1 A, and histone H2B type 1-C/E/F/G/I). Functional annotation tool analysis showed that MPA modulated proteins were mainly involved in the cytoskeleton (26%), chromatin structure/dynamics (17%), and energy production/conversion (17%). Considering both putative functions and their clinical significance, peroxiredoxin-1 (Prdx-1) and myosin light chain 2 (MLC2) were selected for Western blot and real time PCR analysis. Both proteins showed up-regulation at mRNA as well as at protein level following MPA exposure.MLC2 is known to be involved in several functions including tight junctions (TJ) regulation. Epithelial barrier disruption by phosphorylation of MLC2 has been implicated in several bowel diseases. Since MPA treatment often causes diarrhea when used clinically, we hypothesized that MPA regulated epithelial TJ by modulation of MLC2. To test this hypothesis, we investigated the effect of MPA on the expression of MLC2 in two colonic cell lines, HT-29 and Caco-2. Increased MLC2 expression was observed in both cell lines following MPA exposure. These findings suggest that the increase in MLC2 expression after exposure to MPA is not a cell specific effect. Moreover, we observed similar up-regulation of MLC2 expression in whole cell lysates prepared from MMF treated rats, which implies that MPA has similar effects both in vitro and in vivo. We then used Caco-2 cells grown for 21 days post confluence to develop polarize monolayers to conduct physiological, expressional and microscopic analysis to establish the possible role of MPA in disruption of TJ. MPA exposure caused a time and dose dependent decreases in transepithelial resistance (TER), and increases in the FITC-dextran 4 KDa (FD4) paracellular influx in these Caco-2 monolayers. In addition, we found that AcMPAG (a pharmacologically active metabolite of MPA) was also able to cause decreases in TER and increases in FD4 influx. These MPA and AcMPAG mediated increases in permeability were not due to cellular toxicity, as shown by the fact that no significant apoptosis or cell death was observed. In MPA and AcMPAG treated cells, we also found altered expression and distribution of TJ proteins (ZO-1 and occludin).Since MLC phosphorylation is a key modulator of TJ disruption; we investigated whether MPA also increased MLC2 phosphorylation. Using immunoblot analysis we found that MPA significantly increased MLC2 phosphorylation. We then investigated whether MPA mediated increases in MLC2 phosphorylation was through effects on MLCK. Immunoblot analysis revealed that MPA increased MLCK expression both at mRNA and protein levels. To further confirm that MLCK was the key player in MPA mediated MLC2 phosphorylation and its associated TJ disruption, we pre-incubated cells with ML-7 (a specific MLCK inhibitor), and observed that ML-7 was able to partially prevent the MPA mediated increase in MLC2 phosphorylation. Furthermore, we found that ML-7 partially reversed MPA mediated decreases in TER, and increases in FD4 paracellular influx. ML-7 also prevented the MPA associated disruption of the distribution and expression of TJ proteins. These findings suggest that MPA may regulate TJ function via MLCK-driven MLC2 phosphorylation. However, these results do not exclude the possibility that other pathways may also be involved in MPA induced regulation of TJ function. Taking together findings of the present studies showed that therapeutic concentrations of MPA can modulate the expression of important proteins which are crucial for various cellular functions. MPA may modulate epithelial TJ integrity via MLC2 phosphorylation. These findings will be helpful to understand the molecular mechanisms of MPA-induced proteome alterations, including proteins that are involved in disruption of TJ. Further studies are needed to clarify the mechanism(s) and consequences of MPA mediated disruption of TJs, especially in in vivo models, to know whether these TJ barrier changes are responsible for the GI adverse events associated with MPA treatment.

National burden of antibiotic use for adult rhinosinusitis

Bronchial epithelial tight junctions (TJ) have been extensively assessed in healthy airway epithelium. However, no studies have yet assessed the effect of human rhinovirus (HRV) infection on the expression and resultant barrier function in epithelial tight junctions (TJ) in childhood asthma. To investigate the impact of HRV infection on airway epithelial TJ expression and barrier function in airway epithelial cells (AECs) of children with and without asthma. Furthermore, to test the hypothesis that barrier integrity and function is compromised to a greater extent by HRV in AECs from asthmatic children. Primary AECs were obtained from children with and without asthma, differentiated into air-liquid interface (ALI) cultures and infected with rhinovirus. Expression of claudin-1, occludin and zonula occluden-1 (ZO-1) was assessed via qPCR, immunocytochemistry (ICC), in-cell western (ICW) and confocal microscopy. Barrier function was assessed by transepithelial electrical resistance (TER; RT ) and permeability to fluorescent dextran. Basal TJ gene expression of claudin-1 and occludin was significantly upregulated in asthmatic children compared to non-asthmatics; however, no difference was seen with ZO-1. Interestingly, claudin-1, occludin and ZO-1 protein expression was significantly reduced in AEC of asthmatic children compared to non-asthmatic controls suggesting possible post-transcriptional inherent differences. HRV infection resulted in a transient dissociation of TJ and airway barrier integrity in non-asthmatic children. Although similar dissociation of TJ was observed in asthmatic children, a significant and sustained reduction in TJ expression concurrent with both a significant decrease in TER and an increase in permeability in asthmatic children was observed. This study demonstrates novel intrinsic differences in TJ gene and protein expression between AEC of children with and without asthma. Furthermore, it correlates directly the relationship between HRV infection and the resultant dissociation of epithelial TJ that causes a continued altered barrier function in children with asthma.

The epithelial barrier of the upper respiratory tract, such as that of the nasal mucosa, plays a crucial role in host defense. The epithelial barrier is regulated in large part by the apical-most intercellular junctions, referred to as tight junctions. However, the mechanisms regulating of tight junction barrier in human nasal epithelial cells remain unclear because the proliferation and storage of epithelial cells in primary cultures are limited. In the present study, we introduced the catalytic component of telomerase, the hTERT gene, into primary cultured human nasal epithelial cells and examined the properties of the transfectants, including their expression of tight junctions, compared with primary cultures. The ectopic expression of hTERT in the epithelial cells resulted in adequate growth potential and a longer lifespan of the cells. The properties of the passaged hTERT-transfected cells including tight junctions were similar to those of the cells in primary cultures. The barrier function in the transfectants after treatment with 10% FBS was significantly enhanced with increases of integral tight junction proteins claudin-1 and -4. When the transfectants were treated with TGF-beta, which is assosciated with nasal polyposis and chronic rhinosinusitis, upregulation of only claudin-4 was observed, without a change of barrier function. In human nasal epithelial cells, the claudins may be important for barrier function and a novel target for a drug-delivery system. Our results indicate that hTERT-transfected human nasal epithelial cells with an extended lifespan can be used as an indispensable and stable model for studying the regulation of claudins in human nasal epithelium.

Odontogenic sinusitis (OS) presents more satisfactory therapeutic effect after endoscopic surgery compared with chronic rhinosinusitis (CRS) of other origin. The aim of the present study was to investigate the clinical characteristics, morphological features, and epithelial barrier function of sinus mucosa of OS and discuss the possible relationship with good prognosis. A total of 25 subjects with OS, 7 CRS without nasal polyps (CRSsNP), 10 CRS with nasal polyps (CRSwNP), and 9 control subjects were recruited. The biopsy specimens were stained with hematoxylin and eosin for general observation of cytomorphologic features. Epithelial tight junctions (TJs) protein claudin-4 expression was determined to evaluate the epithelial barrier integrity by using immunofluorescence and Image-Pro Plus software analysis. The representative cytokine profiles regarding T helper 1 (Th1) (interferon [IFN]-γ), Th2 (interleukin [IL]-5), and Th17 (IL-17) were examined by reverse transcription-polymerase chain reaction (RT-PCR). Extensively small papillary protrusions could be seen in the maxillary sinus mucosa of OS patients under nasal endoscopy, similar to the morphological behavior, which also presented as papillary folds in the surface of the epithelium. The epithelium in OS kept an increased claudin-4 expression compared with that seen in CRSsNP, CRSwNP, and control subjects. The inflammatory pattern analysis demonstrated that OS belonged to the lymphocyte and plasma cell-dominant cellular phenotypes, whereas IL-17 was dominant compared with IFN-γ as well as IL-5. The odontogenic infections might induce the formation of papillary mucosa folds and enhance the epithelial TJ barrier function. OS exhibited as lymphocyte and plasma cell-dominant cellular phenotypes and Th17 cytokine profiles.

Intestinal barrier function is reduced in inflammatory bowel disease (IBD). Tumor necrosis factor (TNF) and interleukin (IL)-13, which are up-regulated in IBD, induce barrier defects that are associated with myosin light chain kinase (MLCK) activation and increased claudin-2 expression, respectively, in cultured intestinal epithelial monolayers. Here we report that these independent signaling pathways have distinct effects on tight junction barrier properties and interact in vivo. MLCK activation alters size selectivity to enhance paracellular flux of uncharged macromolecules without affecting charge selectivity and can be rapidly reversed by MLCK inhibition. In contrast, IL-13-dependent claudin-2 expression increases paracellular cation flux in vitro and in vivo without altering tight junction size selectivity but is unaffected by MLCK inhibition in vitro. In vivo, MLCK activation increases paracellular flux of uncharged macromolecules and also triggers IL-13 expression, claudin-2 synthesis, and increased paracellular cation flux. We conclude that reversible, MLCK-dependent permeability increases cause mucosal immune activation that, in turn, feeds back on the tight junction to establish long-lasting barrier defects. Interactions between these otherwise distinct tight junction regulatory pathways may contribute to IBD pathogenesis.