Yutaka Kozu

The Rac1/JNK pathway is critical for EGFR-dependent barrier formation in human airway epithelial cells

The airway epithelial barrier provides defenses against inhaled antigens and pathogens, and alterations of epithelial barrier function have been proposed to play a significant role in the pathogenesis of chronic airway diseases. Although the epidermal growth factor receptor (EGFR) plays roles in various physiological and pathological processes on the airway epithelium, the role of EGFR on barrier function in the airway remains largely unknown. In the present study, we assessed the effects of EGFR activation on paracellular permeability in airway epithelial cells (AECs). EGFR activation induced by the addition of EGF increased transepithelial electrical resistance (TER) in AECs. An EGFR-blocking antibody eradicated the development of TER, paracellular influx of dextran, and spatial organization of tight junction. Moreover, the effects of EGFR activation on paracellular permeability were eradicated by knockdown of occludin. To identify the EGFR signaling pathway that regulates permeability barrier development, we investigated the effects of several MAP kinase inhibitors on permeability barrier function. Pretreatment with a JNK-specific inhibitor, but not an ERK- or p38-specific inhibitor, attenuated the development of TER induced by EGFR activation. Rac1 is one of the upstream activators for JNK in EGFR signaling. Rac1 knockdown attenuated the phosphorylation of JNK activation and EGFR-mediated TER development. These results suggest that EGFR positively regulates permeability barrier development through the Rac1/JNK-dependent pathway.

Glucocorticoids enhance airway epithelial barrier integrity.

Asthma is a chronic inflammatory disorder of the airways, but its pathogenesis is incompletely understood. While asthma is a complex disease caused by multiple factors, epithelial barrier damage is a cardinal feature. Glucocorticoids (GCs) are the most effective anti-inflammatory drugs in the treatment of asthma. However, the effects of GCs on the airway epithelial barrier have not been evaluated. Epithelial barrier functions were evaluated in cultured human airway epithelial cell monolayers, Calu-3 and 16HBE. Then, the cells were treated with dexamethasone (Dex), fulticasone propionate (FP), or budesonide (BD) for 5 days. Permeability measured by transepithelial electrical resistance was increased by treatment with Dex, FP, and BD in a dose-dependent manner. Permeability to fluorescein isothiocyanate-labeled dextran was markedly reduced by these treatments. Immunocytostaining revealed that Dex treatment potentiated tight junction formation in these polarized epithelial cells. Knockdown of epidermal growth factor receptor (EGFR) by small interference RNA blunted the effects of Dex on barrier integrity. Although EGFR expression was not affected by Dex treatment, EGFR phosphorylation was enhanced in Dex-treated cells. This is suggesting that EGFR are important for this phenomenon. These findings suggest that GC inhalation therapy can improve epithelial barrier integrity and might contribute to the therapeutic effects of GCs for treating asthma.

Protocadherin-1 is a glucocorticoid- responsive critical regulator of airway epithelial barrier function

BackgroundImpaired epithelial barrier function renders the airway vulnerable to environmental triggers associated with the pathogenesis of bronchial asthma. We investigated the influence of protocadherin-1 (PCDH1), a susceptibility gene for bronchial hyperresponsiveness, on airway epithelial barrier function.MethodsWe applied transepithelial electric resistance and dextran permeability testing to evaluate the barrier function of cultured airway epithelial cells. We studied PCDH1 function by siRNA-mediated knockdown and analyzed nasal or bronchial tissues from 16 patients with chronic rhinosinusitis (CRS) and nine patients with bronchial asthma for PCDH1 expression.ResultsPCDH1 was upregulated with the development of epithelial barrier function in cultured airway epithelial cells. Immunocytochemical analysis revealed that PCDH localized to cell-cell contact sites and colocalized with E-cadherin at the apical site of airway epithelial cells. PCDH1 gene knockdown disrupted both tight and adhesion junctions. Immunohistochemical analysis revealed strong PCDH1 expression in nasal and bronchial epithelial cells; however, expression decreased in inflamed tissues sampled from patients with CRS or bronchial asthma. Dexamethasone (Dex) increased the barrier function of airway epithelial cells and increased PCDH1 expression. PCDH1 gene knockdown eradicated the effect of Dex on barrier function.ConclusionThese results suggest that PCDH1 is important for airway function as a physical barrier, and its dysfunction is involved in the pathogenesis of allergic airway inflammation. We also suggest that glucocorticoids promotes epithelial barrier integrity by inducing PCDH1.

Heregulin activation of ErbB2/ErbB3 signaling potentiates the integrity of airway epithelial barrier

BACKGROUND Members of the ErbB family of the receptor protein tyrosine kinase superfamily mediate heregulin (HRG)-induced cell responses. Here we investigated HRG activation of ErbB receptors, and the role of this activation in the development of the permeability barrier in airway epithelial cells (AECs). METHODS Two airway epithelial-like cell lines, Calu-3 and 16HBE were exposed to HRG or no stimulus and were evaluated with respect to their paracellular permeability as determined by transepithelial electric resistance (TER) and fluorescein isothiocyanate (FITC)-dextran flux. Tight junctions (TJs) were assessed by immunocytochemical localization of occludin and zonula occludens-1. RESULTS HRG promoted the development of the permeability barrier and TJ formation by monolayers of Calu-3 and 16HBE cells. Calu-3 cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4, on their surface. ErbB3 knockdown by small interference RNA (siRNA) blunted the effects of HRG on the permeability barrier. ErbB3 is known as a kinase-dead receptor and relies on other members of the family for its phosphorylation. To identify its heterodimerization partner, we knocked down the expression of other ErbB family receptors. We found that HRGs effect on the permeability barrier could be significantly attenuated by transfecting cells with ErbB2 siRNA but not with EGFR siRNA. CONCLUSION These results indicate that HRG activation of ErbB2/ErbB3 heterodimers is essential for regulation of the permeability barrier in AECs.

Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates airway epithelial barrier integrity.

BACKGROUND Inhaled corticosteroids enhance airway epithelial barrier integrity. However, the mechanism by which they accomplish this is unclear. Therefore, we investigated steroid-inducible genes and signaling pathways that were involved in enhancing airway epithelial barrier integrity. METHODS A human bronchial epithelial cell line (16HBE cells) was cultured with 10(-6) M dexamethasone (DEX) for 3 days to enhance epithelial barrier integrity. After measuring transepithelial electrical resistance (TER) and paracellular permeability, we extracted total RNA from 16HBE cells and performed microarray and pathway analysis. After we identified candidate genes and a canonical pathway, we measured TER and immunostained for tight junction (TJ) and adherent junction (AJ) proteins in cells that had been transfected with specific small interfering RNAs (siRNAs) for these genes. RESULTS We identified a nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response pathway which was primarily involved in the steroid-induced enhancement of airway epithelial barrier integrity. Transfecting cells with Nrf2 specific siRNA reduced the steroid-induced enhancement of airway epithelial barrier integrity and the accumulation of TJ and AJ proteins at sites of cell-cell contact. Moreover, based on pathway analysis, aldehyde oxidase 1 (AOX1) was identified as a downstream enzyme of Nrf2. Transfecting cells with AOX1-specific siRNA also reduced the steroid-induced enhancement of airway epithelial barrier integrity. CONCLUSIONS Our results indicated that the Nrf2/AOX1 pathway was important for enhancing airway epithelial barrier integrity. Because the airway epithelium of asthmatics is susceptible to reduced barrier integrity, this pathway might be a new therapeutic target for asthma.

DsRNA disrupts airway epithelial barrier integrity through down-regulation of claudin members

Bronchial asthma is a pathological condition characterised by chronic allergic inflammation of the airway.1 In recent years, it has been suggested that there is a close relationship between functional disturbances of airway epithelial cells and the development and exacerbation of asthma.2 The airway is open to the external environment and serves as first line defence of the body against invading environmental factors.3 While immunity is a selfdefence system based on discrimination between self and nonself, the tight junctional barrier, which limits the entry of foreign antigens, can be referred to as the selfenon-self boundary. Environmental factors, such as allergens, viruses and air pollutants cause repeated airway inflammation and direct epithelial injuries, which lead to disrupted barrier functions of the airway epithelium and excessive responsiveness to foreign substances. The E-cadherinbased cell adhesion apparatus of airway epithelial cells is important for the stabilisation of immune cells.4 For example, house dustinduced damage to adherence junctions of the airway epithelium has been reported to promote TSLP production in airway epithelial cells.5 In addition, E-cadherin and tight junction proteins are expressed in CD103-positive dendritic cells and are present between epithelial cells by homo binding to E-cadherin and tight junction proteins of epithelial cells.6 These dendritic cells serve an immune surveillance function in a normal state; however, once epithelial injury is caused by inflammation, they undergo differentiation and migration and induce an immune response. The physical barrier function of the airway epithelium thus serves an immunoregulatory function and is believed to play a critical role in the development and progression of allergic inflammation. Infection with RNA viruses, such as rhinovirus and respiratory syncytial virus, is a major cause of the exacerbation of asthma. At the time of RNA virus infection, double-stranded RNA (dsRNA) is inevitably produced during virus replication within host cells. dsRNA released outside cells is taken up by cells, recognised by Toll-like receptor 3 (TLR3) on endosomes, which induces the production of cytokines and chemokines. TLR3-mediated response of airway epithelial cells to dsRNA is an important element in the pathogenesis of allergic airway inflammation.1,7 In this study, we investigated the mechanism by which dsRNA destroys the airway epithelial cell barrier. Transformed human bronchial epithelial (16HBE140 , abbreviated as 16HBE) cells were kindly donated by Dr. Dieter C. Gruenert (Gene Therapy Center, Cardiovascular Research Institute, Department of Laboratory

Utility of serum YKL-40 levels for identification of patients with asthma and COPD

Eosinophil (no.) 283.2 ± 23.6 309.2 ± 41.4 264.1 ± 26.2 248.1 ± 31.3 0.5119 (%) 4.55 ± 0.30 4.86 ± 0.49 4.57 ± 0.58 3.90 ± 0.39 0.4216 CRP (mg/dL) 0.40 ± 0.15 0.54 ± 0.28 0.18 ± 0.03 0.31 ± 0.09 0.5983 YKL40 (ng/ml) 125.3 ± 7.23 75.8 ± 7.68 157.4 ± 13.6 197.1 ± 13.1 <0.0001* (percentile) 65.4 ± 2.43 49.9 ± 3.26 79.4 ± 4.09 84.1 ± 3.42 <0.0001* log total IgE (IU/ml) 2.25 ± 0.06 2.28 ± 0.08 2.40 ± 0.10 2.04 ± 0.11 0.0680

Selective release of miRNAs via extracellular vesicles is associated with house-dust mite allergen-induced airway inflammation

MicroRNAs (miRNAs) may facilitate cell‐to‐cell communication via extracellular vesicles (EVs). The biological roles of miRNAs in EVs on allergic airway inflammation are unclear.

CpG oligodeoxynucleotides enhance airway epithelial barrier integrity

Asthma is a common chronic disorder of the airways and involves a complex interaction of airflow obstruction, bronchial hyperresponsiveness, and airway remodeling.1 In recent years, functional disturbances in the airway epithelial barrier have been suggested to be involved in the development and exacerbation of asthma.2,3 Environmental factors, such as allergens, viruses, and air pollutants, cause repeated airway inflammation and direct epithelial injuries, which lead to the disruption of the barrier functions of the airway epithelium. The functional disturbance in the airway epithelial cells as a physical barrier plays a critical role in the development and progression of allergic inflammation.2,3 Previously, we reported that the activation of the Toll-like receptor 3 (TLR3)-mediated signaling pathway by double-stranded RNA (dsRNA) impairs the epithelial barrier function.4,5 RNA virus infection is believed to be involved in the pathogenesis of bronchial asthma.1 TLR3 activation has been suggested to be mechanistically related not only to the production of cytokines, such as TSLP, IL-33, and IL-25, but also to the impaired barrier function.4 In addition, TLR9 agonists have demonstrated substantial potential as vaccine adjuvants for treating allergic diseases, including asthma. Commonly referred to as CpG oligodeoxynucleotides (ODN), TLR9 agonists directly induce the activation andmaturation of plasmacytoid dendritic cells and enhance the differentiation of B cells into antibody-secreting plasma cells. Preclinical and early clinical data support the use of TLR9 agonists as vaccine adjuvants, which can enhance the responses to diverse antigens.6 The benefits of CpG ODN have been shown in multiple rodent and primate models of asthma7 and other allergic diseases, with encouraging results obtained in some early clinical trials.7e9 In the present study, we investigated the effect of CpG ODN on the airway epithelial barrier integrity. Transformed human bronchial epithelial (16HBE140 , abbreviated as 16HBE) cells were kindly donated by Dr Dieter C. Gruenert (University of California, San Francisco, USA). Primary cultured normal bronchial epithelial (NBE) cells and 16HBE cells were cultured in a Transwell chamber (Corning Costar, Lowell, MA, USA) for 24 h and for an additional 3 days in the presence of 1.25e5 mM CpG ODN (SigmaeAldrich, St. Louis, MO, USA), a synthetic DNA. The epithelial barrier function was assessed by measuring the transepithelial electrical resistance (TER) using Millicell TER (Millipore, Billerica, MA, USA). Permeability was measured using 4 kDa fluorescein

Serum eosinophil-derived neurotoxin: Correlation with persistent airflow limitation in adults with house-dust mite allergic asthma.

BACKGROUND The serum level of eosinophil-derived neurotoxin (EDN), a protein present in eosinophil granules, correlates with the severity of childhood asthma. However, the relationship between the serum EDN level and the severity of adult asthma has not been sufficiently investigated. OBJECTIVE This study aimed to elucidate the correlation between the serum EDN level and markers of severity in adult asthma. METHODS The subjects comprised 83 adult patients who had asthma and who were undergoing treatment. Of these patients, 40 were positive for house-dust-specific immunoglobulin E (IgE) antibodies; 9 patients with severe adult asthma who were treated with omalizumab were included in the study. We measured the blood eosinophil count, serum EDN, and eosinophil cationic protein levels before investigating the correlations of these parameters with lung function and symptom score. RESULTS There were no significant correlations between the blood eosinophil count or serum EDN or eosinophil cationic protein level with lung function and the symptom score in patients with asthma. However, serum EDN level was inversely correlated with the decrease percentage forced expiratory volume in 1 second (%FEV1) in patients positive for house-dust-specific IgE antibody (R = -0.54; p < 0.05), whereas no such correlation was observed in patients with negative results for house-dust-specific IgE antibody (R = 0.11; p = 0.468). A significant correlation was observed between a decrease in serum EDN level from baseline and lung function improvement after 8 weeks of omalizumab therapy (R = -0.77; p = 0.015). CONCLUSION Serum EDN level may be a useful marker for monitoring persistent airflow limitation in adult patients with asthma who had positive results for house-dust-specific IgE antibodies.