Kerstin Wanke

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

BACKGROUND Chronic rhinosinusitis (CRS) is a common disease with still unclear pathophysiologic mechanisms. Epithelial tight junctions (TJs) have been shown to be involved in different chronic disorders, including bronchial asthma, inflammatory bowel diseases, and skin disorders. The regulation of epithelial barrier function and TJ expression has not been extensively studied in patients with CRS and in the paranasal sinus epithelium thus far. OBJECTIVE We sought to elucidate the TJ expression pattern in the epithelium of the sinonasal mucosa and its regulation in patients with CRS. METHODS Trans-tissue resistance was measured in biopsy specimens from healthy control subjects and patients with CRS with and without nasal polyps. TJ protein expression was determined by using immunofluorescence, Western blotting, and real-time PCR. Primary epithelial cell cultures from patients with CRS and control subjects were used in air-liquid interface (ALI) cultures for the measurement of transepithelial resistance (TER) and TJ expression. The effect of IFN-γ, IL-4, and IL-17 on ALI cultures was assessed. RESULTS A decreased trans-tissue resistance was found in biopsy specimens from patients with CRS with nasal polyps along with an irregular, patchy, and decreased expression of the TJ molecules occludin and zonula occludens 1. TER was reduced in ALI cultures from patients with CRS with nasal polyps. The cytokines IFN-γ and IL-4 decreased TER, whereas IL-17 did not have any influence on epithelial integrity. CONCLUSION A defective epithelial barrier was found in patients with CRS with nasal polyps along with a decreased expression of TJ proteins. The disruption of epithelial integrity by IFN-γ and IL-4 in vitro indicates a possible role for these proinflammatory cytokines in the pathogenesis of patients with CRS.

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

Background: Tight junctions (TJs) form a barrier on the apical side of neighboring epithelial cells in the bronchial mucosa. Changes in their integrity might play a role in asthma pathogenesis by enabling the paracellular influx of allergens, toxins, and microbes to the submucosal tissue. Objective: The regulation of bronchial epithelial TJs by TH2 cells and their cytokines and their involvement in epigenetic regulation of barrier function were investigated. Methods: The expression, regulation, and function of TJs were determined in air‐liquid interface (ALI) cultures of control and asthmatic primary human bronchial epithelial cells (HBECs) by means of analysis of transepithelial electrical resistance, paracellular flux, mRNA expression, Western blotting, and immunofluorescence staining. Results: HBECs from asthmatic patients showed a significantly low TJ integrity in ALI cultures compared with HBECs from healthy subjects. TH2 cell numbers and levels of their cytokines, IL‐4 and IL‐13, decreased barrier integrity in ALI cultures of HBECs from control subjects but not in HBECs from asthmatic patients. They induced a physical separation of the TJs of adjacent cells in immunofluorescence staining of the TJ molecules occludin and zonula occludens‐1. We observed that expression of histone deacetylases (HDACs) 1 and 9, and Silent information regulator genes (sirtuins [SIRTs]) 6 and 7 were significantly high in HBECs from asthmatic patients. IL‐4 and IL‐13 significantly increased the expression of HDACs and SIRTs. The role of HDAC activation on epithelial barrier leakiness was confirmed by HDAC inhibition, which improved barrier integrity through increased synthesis of TJ molecules in epithelium from asthmatic patients to the level seen in HBECs from control subjects. Conclusion: Our data demonstrate that barrier leakiness in asthmatic patients is induced by TH2 cells, IL‐4, and IL‐13 and HDAC activity. The inhibition of endogenous HDAC activity reconstitutes defective barrier by increasing TJ expression.

The broad spectrum of interepithelial junctions in skin and lung

and serum 25(OH)D concentration correlates positively with Foxp3 Treg cells in the peripheral blood. A, Representative dot plots demonstrating the gating strategy to define Treg cells. Values represent % of gated live CD4CD3 lymphocyte population. B, Frequency of Foxp3 Treg cells in SS and SR asthmatic patients. Data shown asmean, 5%-95% CI, assessed by t test. C, Correlation of Foxp3 Treg cells with serum 25(OH)D in all the patients with moderate to severe asthma. Assessed by Pearson correlation test. J ALLERGY CLIN IMMUNOL AUGUST 2012 544 LETTERS TO THE EDITOR

Histamine Receptor 2 Modifies iNKT Cell Activity within the Inflamed Lung

Histamine is a key immunoregulatory mediator and can dampen proinflammatory responses via activation of histamine receptor 2 (H2R). The aim of this study was to determine the role of H2R in modulating lung inflammatory responses.

Respiratory syncytial virus infection influences tight junction integrity

Respiratory syncytial virus (RSV) is an important risk factor of asthma development and is responsible for severe respiratory tract infections. However, the influence of RSV infection on barrier function of bronchial epithelial cells in vitro and in vivo is still unclear. The aim of this study was to analyse the role of RSV in tight junction (TJ) regulation and to compare epithelial integrity between asthmatic and healthy individuals upon RSV infection. Healthy and asthmatic human bronchial epithelial cells (HBECs) were differentiated at air–liquid interface (ALI) and infected with RSV and ultraviolet (UV)‐irradiated RSV. TJ expression and their integrity were analysed by quantitative polymerase chain reaction (qPCR), transepithelial resistance (TER) and paracellular flux. To determine the effect in vivo, BALB/c mice were infected intranasally with RSV or UV‐irradiated RSV A2. Bronchoalveolar lavage and TJ integrity were analysed on days 1, 2, 4 and 6 post‐infection by qPCR, bioplex and confocal microscopy. RSV increased barrier integrity in ALI cultures of HBEC from healthy subjects, but no effect was found in HBECs from asthmatics. This was not associated with an increase in TJ mRNA expression. In vivo, RSV induced lung inflammation in mice and down‐regulated claudin‐1 and occludin mRNA expression in whole lungs. Surprisingly, RSV infection was not observed in bronchial epithelial cells, but was found in the lung parenchyma. Decreased expression of occludin upon RSV infection was visible in mouse bronchial epithelial cells in confocal microscopy. However, there was no regulation of claudin‐1 and claudin‐7 at protein level.

Platelet-activating factor decreases skin keratinocyte tight junction barrier integrity

To the Editor: Keratinocytes (KCs) build the epidermis, an efficient barrier that prevents microbial pathogens and environmental agents including allergens from entering into the skin. Pathological changes in skin KCs are commonly associated with many skin diseases, such as atopic dermatitis (AD), urticaria, and psoriasis. It was recently reported in the Journal of Allergy and Clinical Immunology that there are tight junction (TJ) defects that lead to a disruption of epithelial barrier function of KCs in the skin of patients with AD and in the meantime evidence for the genetic and immune regulatory mechanisms that control TJ defects has been reported. Barrier-related molecules in the epidermis are regulated by inflammatory responses derived from both the epidermal (ie, KCs and Langerhans cells) and immune cells (ie, T cells, dendritic cells, and other cells). Platelet-activating factor (PAF) is an important proinflammatory factor produced and secreted by several types of cells, including mast cells, monocytes, tissue macrophages, platelets, eosinophils, endothelial cells, and neutrophils, and is involved in allergic inflammation. In this letter, we emphasize the influence of PAF on skin barrier and KC TJ integrity in AD and further analyze TJ disruption in diseased skin for better understanding the molecular and cellular mechanisms in the regulation of TJ barrier integrity. First, we investigated the role of PAF in the regulation of TJ function of human KCs. Normal human skin KCs from healthy subjects and skin KCs from patients with AD were cultured at air-liquid interface (ALI) and transepithelial resistance (TER) was measured as a readout for barrier integrity. Two days after reaching maximal TER, ALI cultures were stimulated with different concentrations of PAF and TER was measured before and 24, 48, 72, and 96 hours after stimulation. In resting conditions without any stimulation, TER levels in AD KCs were significantly lower than in healthy KCs (Fig 1, A). PAF decreased the TER starting from 48 hours and after stimulation of ALI cultures of human KCs. Although AD KCs seem to show a relatively late response to PAF compared with healthy KCs in the early time points such as 24 and 48 hours, there was no difference in percent increase after 72 and 96 hours (Fig 1, B). Notably, changes in TER are taking place relatively late, becoming significant after 48 hours. A part of this effect can be due to secondary mediators, such as cytokines that modify TJ integrity. It has been reported that PAF activates epithelial cells to release several cytokines and several cytokines have been reported to regulate TJs. In accordance with this, we observed an increase in paracellular flux of fluorescein isothiocyanate– labeled dextran 4 kDa in response to PAF treatment, indicating a comparable decrease in TJ barrier integrity in both groups (Fig 1, C). Upon binding to the PAF receptor, PAF activates downstream signaling pathways leading to increased intracellular Ca flux. To investigate the downstream regulation of PAF on TJ, we stimulated KCs of healthy individuals and KCs of individuals with AD with ionomycin, an agonist inducing intracellular Ca flux, and found that ionomycin rapidly decreased TER (Fig 1, D). The present study strongly suggests that the influence of PAF on barrier function is controlled by the induction of intracellular Ca influx, because induction of Ca flux alone with Ca ionophore in the epithelium disrupts TJ barrier integrity. This finding does not exclude other signaling events via the PAF receptor, and has implications on other molecules working on the same family of G-protein coupled receptors that are inducing Ca flux. As repeatedly reported in other cells, we also observed that the leakiness of AD KCs stays stable after many passages. We agree that there might be many reasons for this finding including prolonged exposure to TH2 environment starting early in life. It is practically not possible that these epigenetic changes are the same in every individual, so variation between donors can be observed. To investigate TJ barrier directly in the context of human skin diseases, we analyzed mRNA expression and TJ protein integrity in skin biopsies. Immunofluorescence staining of occludin and claudin-7 showed an intact TJ layer in healthy skin (Fig 2, A and B). TJ integrity was disrupted in AD skin, more severely in lesional AD than in nonlesional AD skin, manifesting with a patchy, disrupted, and less dense arrangement of protein expression of TJs (Fig 2, B). A full disruption of TJs expression was observed in some parts of AD lesions. Skin prick tests induce immediate allergic reaction and urticaria is another PAF-associated skin disease. As an important finding that may open a new window for future research, the TJ layer integrity was decreased in skin prick tests and urticaria in comparison to healthy skin (Fig 2, A). We then further analyzed whether the defects in protein expression of TJs were due to a transcriptional regulation that affects mRNA levels. We used a low-density array microfluidic card (Applied Biosystems, Carlsbad, Calif), which contains all known junctional and junction-associated proteins that are expressed in the epidermis and epithelia. In addition to TJ proteins, desmosomes, gap junctions, adherens junctions, and adaptor protein genes were included in the analysis. We observed decreased mRNA expression of claudin-7 in skin prick test biopsies, and decreased mRNA expressions of claudin-3, claudin-7, claudin-8, claudin-14, and occludin in urticaria biopsies compared with healthy skin biopsies (see Fig E1 in this article’s Online Repository at www.jacionline.org). Similarly, the mRNA expressions of occludin, claudin-7, claudin-2, claudin-8, claudin-12, claudin-23, as well as several other junctional proteins such as multi-PDZ domain protein-1, plakophillin-2, and connexin-43 were downregulated in both nonlesional and lesional AD skin. In accordance with the confocal staining data, the decrease in mRNAs expression was stronger in lesional AD than in nonlesional AD skin (see Fig E2 in this article’s Online Repository at www.jacionline.org). Epithelial barrier disturbance is now recognized as a common feature in many inflammatory diseases including food allergy, chronic rhinosinusitis, asthma, and AD. In the study by De Benedetto et al, the expressions of claudin-1 and claudin-23 were reduced in nonlesional AD skin, which was also verified in the present study. Silencing of claudin-1 led to a reduction in TER, suggesting that the reduced expression of claudin-1 in AD epidermis is an important factor disturbing barrier integrity. Here, we demonstrate that barrier mechanisms are more complex

Soluble mediators derived from bronchial epithelium are able to drive Th2 differentiation in the context of rhinovirus infection

The majority of acute asthma exacerbations follow upper respiratory infections, and most are rhinovirus induced. The pathways by which a rhinovirus infection may lead to asthma development are still under scrutiny, but the role of bronchial epithelium in driving this mechanism has been considered of great importance. It has been shown that the epithelial derived cytokines IL25, IL33 and TSLP are upregulated in bronchial asthma and could be further induced by virus infection. We hypothesise that such cytokines might influence Th2 cell differentiation in the context of rhinovirus infection.