Concordance of ALK fusion gene-rearrangement between immunohistochemistry and next-generation sequencing.

BACKGROUND: Although various companion diagnostic tests of ALK fusion gene-rearrangement are approved, few reports have assessed the concordance of ALK fusion gene-rearrangement in two companion diagnostic tests: next-generation sequencing (NGS) testing and immunohistochemistry (IHC). METHODS: The samples evaluated for gene alterations using NGS testing between May 2019 and November 2021 were included in this study. The inclusion criteria were as follows: samples were diagnosed with non-small cell lung cancer; the results of the NGS analysis were informative; and samples had residual specimens for IHC. We performed IHC on the residual specimens and retrospectively collected sample characteristics from medical records. RESULTS: A total of 185 samples were analyzed using NGS. Twenty-six samples were excluded because of failure to analyze gene alterations using NGS, no residual samples, and inadequate IHC. We analyzed 159 samples. The major histological type was adenocarcinoma (115 samples). The number of surgical and transbronchial lung biopsy specimens was 59 and 56, respectively. ALK fusion gene-rearrangement was detected in four samples using NGS, and five were detected using IHC. The sensitivity and specificity of IHC referred to by NGS were 75.0% and 98.7%, respectively. The concordance rate between IHC and NGS was 98.1%. ALK rearrangement was detected in two patients using IHC but not using NGS. In addition, ALK rearrangement was detected in one patient using NGS but not using IHC. CONCLUSION: Our results suggest that IHC and NGS might be complementary tests. In patients suspected of harboring ALK fusion gene-rearrangement, it should be analyzed using another diagnostic method.

Fusobacterium nucleatum putatively affects the alveoli by disrupting the alveolar epithelial cell tight junction, enlarging the alveolar space, and increasing paracellular permeability.

Fusobacterium nucleatum (Fn) is abundant in the human oral cavity and has been associated with periodontal disease, which in-turn has been linked to respiratory disease development. Tight junctions (TJs) line the airway and alveoli surfaces serving as a first line of defense against multiple pathogens. Fn has already been linked to respiratory diseases, however, how Fn affects the alveolar TJ was not fully elucidated. Here, we designed and analyzed a TJ network, grew Fn cells and inoculated it in vitro (16HBE and primary cells) and in vivo (mice lung), measured transepithelial electrical resistance, performed RT-PCR, checked for in vitro cell and mice lung permeability, and determined air space size through morphometric measurements. We found that Fn can potentially affect TJs proteins that are directly exposed to the alveolar surface. Additionally, Fn could possibly cause neutrophil accumulation and an increase in alveolar space. Moreover, Fn putatively may cause an increase in paracellular permeability in the alveoli.

Anti-synthetase syndrome-associated interstitial lung disease possibly caused by atezolizumab in a patient with lung adenocarcinoma: a case report.

BACKGROUND: Anti-aminoacyl-tRNA synthetase (ARS) antibody-positive patients present with a variety of symptoms, including interstitial lung disease (ILD), which is termed anti-synthetase syndrome (ASS). But it is rare that ASS-ILD is considered an immune-related adverse event after the use of immune checkpoint inhibitors (ICIs). CASE PRESENTATION: A 47-year-old male with advanced lung adenocarcinoma was treated with platinum and ICI combination immunotherapy and was followed up as an outpatient. Nine months after the start of treatment, he developed a fever and cough, and imaging findings showed lung consolidations in the bilateral lower lung fields. The patient was positive for anti- ARS antibodies and was considered to have developed ASS-ILD due to ICIs remitted with steroid therapy. The patient was found to be positive for anti-ARS antibodies before ICI administration, and the antibody titer was elevated compared to that before ICI administration. CONCLUSIONS: The examination of anti-ARS antibodies pior to the administration of ICIs may be useful in predicting the development of ASS-ILD.

Once-daily, single-inhaler indacaterol/mometasone versus twice-daily salmeterol/fluticasone in Asian patients with inadequately controlled asthma: post hoc pooled analysis from PALLADIUM and IRIDIUM studies.

OBJECTIVE: PALLADIUM and IRIDIUM studies demonstrated efficacy and safety of indacaterol/mometasone (IND/MF) versus salmeterol/fluticasone (SAL/FLU). This post hoc analysis of pooled data from PALLADIUM and IRIDIUM studies evaluated efficacy and safety of IND/MF versus SAL/FLU in Asian patients with inadequately controlled asthma. METHODS: Both studies were Phase III, 52-week, randomized, double-blind, active-controlled that included patients with predicted pre-bronchodilator FEV1 (PALLADIUM, ≥50%-<85%; IRIDIUM, <80%), ACQ-7 score ≥1.5. Patients treated with IND/MF high- (150/320 µg) or medium-dose (150/160 µg) or SAL/FLU high-dose (50/500 µg) were included. Lung function, asthma control, and asthma exacerbations were evaluated. RESULTS: In total, 323 patients (IND/MF high-dose, n = 107; IND/MF medium-dose, n = 106, SAL/FLU high-dose, n = 110) were included. IND/MF high-dose showed improvement in trough FEV1 versus SAL/FLU high-dose at Weeks 26 (Δ, 42 mL; 95% CI, -35 to 120 mL), and 52 (Δ, 41 mL; 95% CI, -37 to 120 mL). IND/MF high-dose exhibited numerically greater improvement in ACQ-7 score versus SAL/FLU high-dose at Weeks 26 (Δ, -0.215; 95% CI, -0.385 to -0.044) and 52 (Δ, -0.176; 95% CI, -0.348 to -0.005). Improvements in trough FEV1 and ACQ-7 score were comparable between IND/MF medium-dose and SAL/FLU high-dose. IND/MF high- and medium-dose showed reductions in moderate or severe (45%; 30%), severe (39%; 41%), and all (9%; 25%) exacerbations, respectively, versus SAL/FLU high-dose over 52 weeks. All treatments were well tolerated. CONCLUSIONS: Once-daily, single-inhaler IND/MF high-dose improved lung function with better asthma control, reduced asthma exacerbations with comparable safety versus twice-daily SAL/FLU high-dose. IND/MF medium-dose showed comparable outcomes to SAL/FLU high-dose at a reduced steroid dose.

Incidence and risk factors for hyperkalaemia in patients treated for COVID-19 with nafamostat mesylate.

WHAT IS KNOWN AND OBJECTIVE: Nafamostat mesylate (NM) is used clinically in combination with antiviral drugs to treat coronavirus disease (COVID-19). One of the adverse events of NM is hyperkalaemia due to inhibition of the amiloride-sensitive sodium channels (ENaC). The incidence and risk factors for hyperkalaemia due to NM have been studied in patients with pancreatitis but not in COVID-19. COVID-19 can be associated with hypokalaemia or hyperkalaemia, and SARS-CoV-2 is thought to inhibit ENaC. Therefore, frequency and risk factors for hyperkalaemia due to NM may differ between COVID-19 and pancreatitis. Hyperkalaemia may worsen the respiratory condition of patients. The objective of this study was to determine the incidence and risk factors for hyperkalaemia in COVID-19 patients treated with favipiravir, dexamethasone and NM. METHODS: This retrospective study reviewed the records of hospitalized COVID-19 patients treated with favipiravir and dexamethasone, with or without NM, between March 2020 and January 2021. Multivariable logistic regression analysis was performed to identify the risk factors for hyperkalaemia. RESULTS AND DISCUSSION: Of 45 patients who received favipiravir and dexamethasone with NM for the treatment of COVID-19, 21 (47%) experienced hyperkalaemia. The duration of NM administration was a significant predictor of hyperkalaemia (odds ratio: 1.55, 95% confidence interval: 1.04-2.31, p = 0.031). The receiver-operating characteristic curve analysis determined that the cut-off value for predicting the number of days until the onset of hyperkalaemia was 6 days and the area under the curve was 0.707. WHAT IS NEW AND CONCLUSION: This study revealed that the incidence of hyperkalaemia is high in patients treated for COVID-19 with NM, and that the duration of NM administration is a key risk factor. When NM is administered for the treatment of COVID-19, it should be discontinued within 6 days to minimize the risk of hyperkalaemia.

miR103a-3p in extracellular vesicles from FcεRI-aggregated human mast cells enhances IL-5 production by group 2 innate lymphoid cells.

BACKGROUND: Mast cells (MCs) are key regulators of IgE-mediated allergic inflammation. Cell-derived extracellular vesicles (EVs) contain bioactive compounds such as microRNAs. EVs can transfer signals to recipient cells, thus using a novel mechanism of cell-to-cell communication. However, whether MC-derived EVs are involved in FcεRI-mediated allergic inflammation is unclear. OBJECTIVE: We sought to investigate the effect of EVs derived from FcεRI-aggregated human MCs on the function of human group 2 innate lymphoid cells (ILC2s). METHODS: Human cultured MCs were sensitized with and without IgE for 1 hour and then incubated with anti-IgE antibody, IL-33, or medium alone for 24 hours. EVs in the MC supernatant were isolated by using ExoQuick-TC. RESULTS: Coculture of ILC2s with EVs derived from the FcεRI-aggregated MCs significantly enhanced IL-5 production and sustained upregulation of IL-5 mRNA expression in IL-33-stimulated ILC2s, but IL-13 production and IL-13 mRNA expression were unchanged. miR103a-3p expression was upregulated in IL-33-stimulated ILC2s that had been cocultured with EVs derived from anti-IgE antibody-stimulated MCs. Transduction of an miR103a-3p mimic to ILC2s significantly enhanced IL-5 production by IL-33-stimulated ILC2s. miR103a-3p promoted demethylation of an arginine residue of GATA3 by downregulating protein arginine methyltransferase 5 (PRMT5) mRNA. Reduction of protein arginine methyltransferase 5 expression in ILC2s by using a small interfering RNA technique resulted in upregulation of IL-5 production by IL-33-stimulated ILC2s. Furthermore, the level of miR103a-3p expression was significantly higher in EVs from sera of patients with atopic dermatitis than in EVs from nonatopic healthy control subjects. CONCLUSION: Eosinophilic allergic inflammation may be exacerbated owing to ILC2 activation by MC-derived miR103a-3p.

Circulating microRNA-15b-5p as a biomarker for asthma-COPD overlap.

BACKGROUND: It remains unclear how to characterize different subtypes of asthma and chronic obstructive pulmonary disease (COPD). We previously described serum periostin and chitinase-3-like protein 1 (YKL-40) as useful markers for asthma-COPD overlap (ACO). MicroRNAs (miRNAs) are now recognized as markers for identifying the pathophysiological features in several diseases. This study aimed to identify circulating miRNAs that could discriminate patients with ACO from patients with asthma or COPD. METHODS: This study included two independent cohorts. First, we screened 84 miRNAs for expression levels in patients with ACO (n = 6) or asthma (n = 6) using a quantitative real-time PCR array. The miRNAs showing at least a 2-fold difference in the discovery phase were analyzed in 30 patients each with asthma, COPD, or ACO in the replication phase. The diagnostic accuracy was evaluated using the area under the receiver operating characteristic curve (AUROC). RESULTS: Nine miRNAs were identified in the discovery phase. Five of these miRNAs (miR-148a-3p, miR-15b-5p, miR-223-3p, miR-23a-3p, and miR-26b-5p) had lower levels in ACO patients and could discriminate between ACO patients and patients with either asthma or COPD. miR-15b-5p was the most accurate miRNA for the discrimination of patients with ACO (AUROC, 0.71). Moreover, the combined assessment of miR-15b-5p, serum periostin, and YKL-40 (AUROC, 0.80) improved diagnostic accuracy for ACO compared with the combined model of periostin and YKL-40 (AUROC, 0.69). CONCLUSIONS: Circulating miR-15b-5p is a potential marker for identifying patients with ACO. By elucidating the molecular pathways controlled by miRNAs, we may better understand the pathophysiology of ACO.

High serum free IL-18 is associated with decreased omalizumab efficacy: findings from a 2-year omalizumab treatment study.

OBJECTIVE: Omalizumab is more effective in severe allergic patients with eosinophilic asthma than those with non-eosinophilic asthma. IL-18, a unique cytokine involved in allergic but non-eosinophilic inflammation, might be associated with the latter condition. We aimed to clarify the roles of IL-18 related pathways in insufficient response to omalizumab treatment. METHODS: Patients with severe allergic asthma who completed 2-year omalizumab treatments at Kyoto University Hospital were included in this study (UMIN000002389). Associations between pretreatment levels of serum free IL-18 in addition to other mediators and asthma phenotypes including responses to omalizumab treatment were analyzed. Changes in serum free IL-18, periostin and total IgE levels during the treatment were also examined. RESULTS: Twenty-seven patients (19 females, average age of 55.7 years) were examined. Fifteen incomplete responders who experienced exacerbations in the second year, were significantly and more frequently obese and showed significantly earlier asthma onset, lower blood eosinophils and more exacerbations before omalizumab treatment than complete responders. Significantly more patients showed high baseline serum free IL-18 levels (≥141 pg/mL, a threshold for the highest tertile) among the incomplete responders than complete responders. Patients with high serum free IL-18 levels shared similar characteristics with incomplete responders, showing significant reductions in serum total IgE levels during omalizumab treatment. Finally, serum free IL-18 levels negatively correlated with serum periostin levels at baseline and in change ratios. CONCLUSIONS: High baseline serum free IL-18 levels may predict reduced omalizumab efficacy in severe allergic patients with type-2 low asthma, regarding reduction of exacerbations.

Long-term outcomes in extensive disease small cell lung cancer patients treated without immune checkpoint inhibitors.

OBJECTIVE: Immune checkpoint inhibitors (ICIs) combined with chemotherapy have been approved as first-line treatment for patients with untreated extensive disease-small cell lung cancer (ED-SCLC). However, there are few reports about the long-term outcomes in patients with ED-SCLC treated without ICIs. Thus, we analyzed the long-term outcomes in patients with ED-SCLC. METHODS: We retrospectively examined the medical records of patients with SCLC who were treated at our hospital between September 2002 and September 2019. The main inclusion criteria were as follows: (i) histological or cytological confirmation of SCLC, (ii) diagnosed with ED-SCLC and (iii) received chemotherapy, not including ICIs, as the first-line treatment. To assess the trends of treatment outcomes, we compared the survival outcomes between 2002-2010 (early) and 2011-2019 (late) groups. RESULTS: A total of 314 patients were included in this study. Patient characteristics at the time of first-line treatment were as follows: median age was 69 years; 82% of the patients were male and 70% had a performance status of 0 or 1. The median follow-up time of overall survival (OS) was 7.4 years, and 89% of the patients died. The median progression-free survival and survival time were 4.9 and 12.1 months, respectively. Five-year survival rate was 2%. There was no significant difference in survival between the early and late groups. CONCLUSIONS: We found that the long-term outcomes in ED-SCLC patients treated without ICIs were poor. Prior to the approval of ICI treatment for ED-SCLC, there was no improvement in the OS for ~20 years.

Detection of programmed cell death-ligand 1 using 22C3 antibody in patients with unresectable stage III non-small cell lung cancer receiving chemoradiotherapy.

BACKGROUND: The expression of programmed cell death-ligand 1 (PD-L1) is a biomarker for administering immune check point inhibitors in patients with advanced stage non-small cell lung cancer. Although the consolidation therapy of durvalumab after definitive chemoradiotherapy has become the new standard of care for patients with unresectable stage III non-small cell lung cancer, the prevalence and prognostic role of PD-L1 expression in this population remain unclear. METHODS: We retrospectively reviewed data from patients with unresectable stage III non-small cell lung cancer who received definitive chemoradiotherapy at our institution between 2012 and 2017. Levels of PD-L1 were assessed using 22C3 antibody, and associations of progression-free and overall survival rates with PD-L1 statuses at a tumor proportion score cutoff of 1% were analyzed. RESULTS: Among the 104 patients enrolled, PD-L1 statuses were as follows: tumor proportion score < 1%, 73 (70.2%); 1-49%, 21 (20.2%); and ≥ 50%, 10 (9.6%). The number of patients with stage III non-small cell lung cancer with pretreatment PD-L1 tumor proportion score ≥ 1% was less than the number with advanced stage disease. There was no association between patient characteristics and PD-L1 status, and no significant differences were observed in progression-free and overall survival rates relative to PD-L1 status. CONCLUSION: Expression of PD-L1 in patients with stage III non-small cell cancer before chemoradiotherapy should be assessed because of the low prevalence of tumors with tumor proportion scores ≥ 1%. Further studies are needed to clarify whether durvalumab improves survival after definitive chemoradiotherapy, irrespective of tumor PD-L1 expression.

Cluster analysis identifies a pathophysiologically distinct subpopulation with increased serum leptin levels and severe obstructive sleep apnea.

PURPOSE: To investigate the different pathophysiologies of obstructive sleep apnea (OSA) phenotypes using cluster analysis. Differences between leptin/adiponectin levels in the resulting OSA phenotypes were also examined. METHODS: In total, 1057 OSA patients were selected, and a retrospective survey of clinical records, polysomnography results, and blood gas data was conducted. Patients were grouped into four clusters by their OSA severity, PaCO2, body mass index (BMI), and sleepiness. A k-means cluster analysis was performed, resulting in a division into four subpopulations. The Tukey or Games-Howell tests were used for intergroup comparisons. RESULTS: Among the 20 clinical OSA items, four common factors (Epworth Sleepiness Scale [ESS], BMI, Apnea-Hypopnea Index [AHI], and PaCO2) were extracted by principal component analysis, and a cluster analysis was performed using the k-means method, resulting in four distinct phenotypes. The Clusters 1 (middle age, symptomatic severe OSA) and 4 (young, obese, symptomatic very severe OSA) exhibited high leptin levels. C-reactive protein levels were also elevated in Cluster 4, indicating a different pathophysiological background. No apparent differences between clusters were observed regarding adiponectin/leptin ratios and adiponectin levels. Classification into groups based on phenotype showed that Epworth Sleepiness Scale [ESS] score and disease severity were not correlated, suggesting that sleepiness is affected by multiple elements. CONCLUSIONS: The existence of multiple clinical phenotypes suggests that different pathophysiological backgrounds exist such as systemic inflammation and metabolic disorder. This classification may be used to determine the efficacy of continuous positive airway pressure treatment that cannot be determined by the AHI.

Association of upper and lower airway eosinophilic inflammation with response to omalizumab in patients with severe asthma.

Background: The anti-immunoglobulin E monoclonal antibody, omalizumab, is used to treat severe asthma and has the potential to ameliorate airway inflammation. However, the effect of omalizumab in ameliorating upper airway inflammation has not been fully elucidated. Objective: We investigated the association of upper and lower airway inflammation with the response to omalizumab treatment. Methods: We used the Global Evaluation of Treatment Effectiveness to assess the efficacy of omalizumab in treating 16 patients with severe asthma. We also investigated the symptom score, short-acting ß-agonist inhaler use, pulmonary function, biomarkers, computed tomography scans, and nasal mucosa pathology at omalizumab initiation and after four months of treatment. Results: When the fraction of exhaled nitric oxide (FeNO) and the percentage of sputum eosinophil were used as indicators of lower airway inflammation, positive correlations were found between CD20 B-cell, mast cell, and eosinophil counts in the nasal mucosa. Improved asthma symptoms were observed in 12 of the 16 severe asthma cases. The FeNO and eosinophil levels in the nasal tissue, prior to the administration of omalizumab were predictors of the response to asthma treatment. Conclusions: These findings suggest heterogeneity among people with severe asthma. In addition, the phenotype associated with response to omalizumab, leading to improvement in asthma symptoms, comprises upper airway eosinophilia and high FeNO levels.

Molecular techniques for respiratory diseases: MicroRNA and extracellular vesicles.

miRNA are a class of evolutionarily conserved non-coding 19- to 22-nt regulatory RNA. They affect various cellular functions through modulating the transcriptional and post-transcriptional levels of their target mRNA by changing the stability of protein-coding transcripts or attenuating protein translation. miRNA were discovered in the early 1990s, and they have been the focus of new research in both basic and clinical medical sciences. Today, it has become clear that specific miRNA are linked to the pathogenesis of respiratory diseases, as well as cancer and cardiovascular disease. In addition, EV, including exosomes, which are small membrane-bound vesicles secreted by cells, were found to contain various functional miRNA that can be used for diagnostic and therapeutic purposes. As body fluids, such as blood and respiratory secretions, are major miRNA sources in the body, EV carrying extracellular miRNA are considered potentially useful for the diagnosis and assessment of pathological conditions, as well as the treatment of respiratory or other diseases. Although research in the field of lung cancer is actively progressing, studies in other respiratory fields have emerged recently as well. In this review, we provide an update in the topics of miRNA and EV focused on airway inflammatory diseases, such as asthma and COPD, and explore their potential for clinical applications on respiratory diseases.

Increased extracellular vesicle miRNA-466 family in the bronchoalveolar lavage fluid as a precipitating factor of ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a life-threatening disease; however, its treatment has not yet been fully established. The progression of ARDS is considered to be mediated by altered intercellular communication between immune and structural cells in the lung. One of several factors involved in intercellular communication is the extracellular vesicle (EV). They act as carriers of functional content such as RNA molecules, proteins, and lipids and deliver cargo from donor to recipient cells. EVs have been reported to regulate the nucleotide-binding oligomerization like receptor 3 (NLRP3) inflammasome. This has been identified as the cellular machinery responsible for activating inflammatory processes, a key component responsible for the pathogenesis of ARDS. METHODS: Here, we provide comprehensive genetic analysis of microRNAs (miRNAs) in EVs, demonstrating increased expression of the miRNA-466 family in the bronchoalveolar lavage fluid of a mouse ARDS model. RESULTS: Transfection of bone marrow-derived macrophages (BMDMs) with miRNA-466 g and 466 m-5p resulted in increased interleukin-1 beta (IL-1ß) release after LPS and ATP treatment, which is an established in vitro model of NLRP3 inflammasome activation. Moreover, LPS-induced pro-IL-1ß expression was accelerated by miRNA-466 g and 466 m-5p in BMDMs. CONCLUSIONS: These findings imply that miRNA-466 family molecules are secreted via EVs into the airways in an ARDS model, and this exacerbates inflammation through the NLRP3 inflammasome. Our results suggest that the NLRP3 inflammasome pathway, regulated by extracellular vesicle miRNA, could act as a therapeutic target for ARDS.

Recent Advances in Allergy Research Using Humanized Mice.

The prevalence rates of allergic diseases are increasing worldwide, particularly in industrial countries. To date, many mouse models have been generated for allergy research; studies conducted using these models have suggested the importance of cross-talk between immune cells and tissue-resident non-immune cells in the onset of allergic diseases. However, there are several differences between the immune systems of rodents and humans, and human studies are limited. Thus, mice reconstituted with human immune cells are a novel tool for the preclinical evaluation of the efficacy and safety of developing drugs. Genetic technologies for generating humanized mice have improved markedly in recent years. In this review, we will discuss recent progress in allergy research using humanized mice and introduce our recent humanized mouse model of airway inflammation in human immune cells.

Role of airway epithelial barrier dysfunction in pathogenesis of asthma.

Bronchial asthma is characterized by persistent cough, increased sputum, and repeated wheezing. The pathophysiology underlying these symptoms is the hyper-responsiveness of the airway along with chronic airway inflammation. Repeated injury, repair, and regeneration of the airway epithelium following exposure to environmental factors and inflammation results in histological changes and functional abnormalities in the airway mucosal epithelium; such changes are believed to have a significant association with the pathophysiology of asthma. Damage to the barrier functions of the airway epithelium enhances mucosal permeability of foreign substances in the airway epithelium of patients with asthma. Thus, epithelial barrier fragility is closely involved in releasing epithelial cytokines (e.g., TSLP, IL-25, and IL-33) because of the activation of airway epithelial cells, dendritic cells, and innate group 2 innate lymphoid cells (ILC2). Functional abnormalities of the airway epithelial cells along with the activation of dendritic cells, Th2 cells, and ILC2 form a single immunopathological unit that is considered to cause allergic airway inflammation. Here we use the latest published literature to discuss the potential pathological mechanisms regarding the onset and progressive severity of asthma with regard to the disruption of the airway epithelial function.

Asthma and COPD overlap pathophysiology of ACO.

Asthma and COPD appear as a result of different mechanisms triggered by different pathogeneses and although they present different features and symptoms of airway inflammation and airway obstruction, there are also cases that present the features of both asthma and COPD. This type of pathology is known as asthma-COPD overlap syndrome (ACOS). Asthma-COPD overlap is identified in clinical practice by the features that it shares with both asthma and COPD. This is not a definition, but a description for clinical use, as asthma-COPD overlap includes several different clinical phenotypes and there are likely to be several different underlying mechanisms". In this paper, the disease that shares several features of both asthma and COPD will be referred to as asthma-COPD overlap (ACO). In this article, we describe the pathogenesis of ACO for understanding the mechanism in asthma and COPD overlap.

NDRG1 is important to maintain the integrity of airway epithelial barrier through claudin-9 expression.

Impairment of epithelial barrier integrity caused by environmental triggers is associated with the pathogenesis of airway inflammation. Using human airway epithelial cells, we attempted to identify molecule(s) that promote airway epithelial barrier integrity. Microarray analyses were conducted using the Affimetrix human whole genome gene chip, and we identified the N-myc downstream-regulated gene 1 (NDRG1) gene, which was induced during the development of the epithelial cell barrier. Immunohistochemical analysis revealed strong NDRG1 expression in ciliated epithelial cells in nasal tissues sampled from patients with chronic rhinosinusitis (CRS), and the low expression of NDRG1 was observed in goblet cells or damaged epithelial cells. NDRG1 gene knockdown with its specific siRNA decreased the transepithelial electrical resistance and increased the dextran permeability. Immunocytochemistry revealed that NDRG1 knockdown disrupted tight junctions of airway epithelial cells. Next, we analyzed the effects of NDRG1 knockdown on the expression of tight and adhesion junction molecules. NDRG1 knockdown significantly decreased only claudin-9 expression, but did not decrease other claudin family molecules, such as E-cadherin, and ZO-1, -2, or -3. Knockdown of claudin-9 markedly impaired the barrier function in airway epithelial cells. These results suggest that NDRG1 is important for the barrier integrity in airway epithelial cells.