Inhibition of PI3Kδ Differentially Regulates Poly I:C- and Human Metapneumovirus-Induced PD-L1 and PD-L2 Expression in Human Bronchial Epithelial Cells.

Bronchial epithelial cells are front sentinels eliciting innate and adaptive immunity to respiratory viral pathogens. Recognition of viral double-stranded RNA induces antiviral interferon (IFN) responses in bronchial epithelial cells. Co-inhibitory molecules programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2) were also induced on bronchial epithelial cells, which bind programmed cell death 1 on T cell and inhibit the function of virus-specific cytotoxic T lymphocyte. A previous study showed that antiviral type I IFN increased PD-L1 and PD-L2 expression in cultured melanoma cells. However, it remains unknown whether antiviral IFNs affect PD-L1 and PD-L2 expression in bronchial epithelial cells. In addition, we previously reported that inhibition of PI3Kδ signaling enhanced antiviral IFN responses in human primary bronchial epithelial cells (PBECs). Here we assessed the effect of exogenous IFNs or a selective PI3Kδ inhibitor IC87114 on PD-L1 and PD-L2 in PBECs stimulated with a synthetic double-stranded RNA poly I:C or human metapneumovirus. Treatment with IFNß or IFNλ increased PD-L1 and PD-L2, and IFNß or IFNλ treatment plus poly I:C further increased both expressions. Treatment with IC87114 or transfection with siRNA targeting PI3K p110δ enhanced poly I:C-induced gene and protein expression of PD-L2, whereas IC87114 suppressed poly I:C-induced PD-L1. IC87114 enhanced poly I:C-induced gene expression of IFNß, IFNλ, and IFN-regulated genes via increased TBK1 and IRF3 phosphorylation. Transfection with siIRF3 counteracted the enhancement of poly I:C-induced PD-L2 by IC87114, whereas IC87114 suppressed poly I:C-induced PD-L1 regardless of transfection with siNC or siIRF3. Similar effects of IC87114 on PD-L1 and PD-L2 expression were observed in human metapneumovirus-infected PBECs. We showed for the first time that type I and type III IFNs induced the expression of PD-L1 and PD-L2 in PBECs. Our findings suggest that during viral infections, inhibition of PI3Kδ differentially regulates PD-L1 and PD-L2 expression in bronchial epithelial cells.

Airflow limitation and tongue microbiota in community-dwelling elderly individuals.

Numerous oral indigenous microorganisms are constantly introduced into the stomach via the laryngopharynx, and a portion of these microorganisms irregularly reaches the lower airways and lungs. This study investigated the association between airflow limitation and the status of tongue microbiota, which is a primary source of ingested oral bacterial populations. The study population consisted of 484 community-dwelling adults aged 70-80 years inhabiting Hisayama town, Japan, who underwent a regular health examination including dental examination and spirometry test in 2016. The bacterial density and composition of their tongue microbiota were determined using a previously used 16S rRNA gene to understand their relationship with oral health conditions. The present cross-sectional study compared the tongue microbiota status between elderly individuals with airflow limitation and those with normal airflow. The total bacterial density of the tongue microbiota of individuals with airflow limitation was significantly higher than that of individuals with normal airflow. Logistic regression analysis demonstrated that a high-biomass tongue microbiota was significantly associated with airflow limitation after adjustment for smoking intensity and other covariates (adjusted OR 1.61, 95% CI 1.01-2.60). Of the predominant commensals, higher amounts of Prevotella melaninogenica and Actinomyces odontolyticus were associated with a higher prevalence of airflow limitation. These results indicate that increased bacterial burden in the tongue microbiota is associated with a higher prevalence of airflow limitation.

Incense smoke-induced oxidative stress disrupts tight junctions and bronchial epithelial barrier integrity and induces airway hyperresponsiveness in mouse lungs.

Recent clinical studies have suggested that inhalation of incense smoke (IS) may result in impaired lung function and asthma. However, there is little experimental evidence to link IS with airway hyperresponsiveness (AHR) and bronchial epithelial barrier function. Using mouse and cell culture models, we evaluated the effects of IS exposure on AHR, expression of multiple epithelial tight junction (TJ)- and adherens junction-associated mRNAs and proteins in the lungs, and the barrier function of bronchial epithelial cells assessed by transepithelial electronic resistance (TEER). Exposure of BALB/c mice to IS increased AHR and inflammatory macrophage recruitment to BALF; reduced claudin-1, -2, -3, -7, -10b, -12, -15, and -18, occludin, zonula occludens-1 [ZO-1], and E-cadherin mRNA expression; and caused discontinuity of claudin-2 and ZO-1 protein immunostaining in lung tissue. IS extract dose-dependently decreased TEER and increased reactive oxygen species production in bronchial epithelial cell cultures. Treatment with N-acetyl-L-cysteine, but not glucocorticosteroids or long-acting ß2-agonists, prevented the detrimental effects of IS. IS exposure can be problematic for respiratory health, as evidenced by AHR, increased recruitment of inflammatory macrophages and disruption of TJ proteins in the lung, and damage to epithelial barrier function. However, antioxidants may be useful for the treatment of IS-induced airway dysfunction.

Mycobacterium avium pleuritis with multiple nodules in the pleura.

As opposed to tuberculosis, pleurisy hardly develops in patients with nontuberculous mycobacteria (NTM) infection. In spite of increasing prevalence of NTM infection, little is known about thoracoscopic or pathological findings of the NTM-infected pleura. We now report the first case of NTM pleuritis with multiple granulomatous nodules in the pleura. A 74-year-old woman was admitted to our hospital due to massive effusion of the left thoracic cavity. The analysis of pleural fluid showed lymphocytic exudative effusions with increased levels of adenosine deaminase, although culture of the pleural fluid was negative. The patient accordingly underwent thoracoscopy, which revealed multiple pleural nodules. Biopsy of the nodules demonstrated epithelioid cell granulomas without caseous necrosis. In addition, culture of the biopsy specimens confirmed infection by Mycobacterium avium. As culture of pleural fluid often fails to detect NTM pathogens, demonstration of pleural nodules during thoracoscopy can contribute to prompt diagnosis and treatment of NTM pleuritis.

Pleuropulmonary Paragonimiasis with Multiple Nodules in the Pleura.

An asymptomatic 47-year-old woman was admitted with pleural effusion and pulmonary infiltrates 1 month after ingesting raw wild boar and deer meat. Both her blood and pleural fluid were eosinophilic. Thoracoscopy revealed multiple nodules of the pleura, and biopsy samples of the nodules showed necrosis with epithelioid cell granulomas. An enzyme-linked immunosorbent assay was positive for antibodies against Paragonimus westermani, and the patient was successfully treated with praziquantel. This is the first reported case of pulmonary or pleuropulmonary paragonimiasis where several pleural nodules were observed. The detection of pleural nodules on thoracoscopy can contribute to the prompt and accurate diagnosis of paragonimiasis.

A measuring method for occupancy of immune checkpoint inhibitors in the cell surface.

Monoclonal antibodies, including immune-checkpoint inhibitors, are becoming popular in treatments of many cancers and connective tissue diseases. However, little is known about how long the antibodies combine with antigens on targeted cells or how this duration of binding associates with therapeutic efficacy or potential adverse events. Here, we show the principle and the results of a feasible method for measuring the antibodies' occupancy on the targeted cells using two different detecting antibodies in conjunction with different fluorochromes. Nivolumab occupancy was measured using two detecting antibodies, MIH4 and EH12.2, which are commercially available in vitro (programmed cell death-1 [PD-1] expressing the cell line MIT9 and human T cells) and in T cells from patients treated with nivolumab. Our method has potential for use as a simple and feasible monitoring system in the clinical setting.

Immune-checkpoint profiles for T cells in bronchoalveolar lavage fluid of patients with immune-checkpoint inhibitor-related interstitial lung disease.

Immune-checkpoint inhibitors (ICIs) have improved clinical outcomes and are becoming a standard treatment for many cancer types. However, these drugs also induce immune-related adverse events, among which interstitial lung disease (ILD) is potentially fatal. The underlying mechanism of ILD induction by ICIs is largely unknown. With the use of flow cytometry, we determined the expression levels of the immune-checkpoint proteins PD-1, TIM-3, TIGIT, LAG-3 and PD-L1 in T cells of bronchoalveolar lavage fluid (BALF) from patients with ICI-related ILD and compared them with those for patients with sarcoidosis or with ILD related to connective tissue disease or cytotoxic drug use. The proportions of CD8+ T cells positive for both PD-1 and TIM-3 or for TIGIT in BALF were significantly higher for ICI-related ILD patients than for those with other types of ILD. A prominent increase in the proportion of PD-1+PD-L1+ cells among CD8+ T cells was also apparent in BALF of a patient with a fatal case of ICI-related ILD, and the proportion of such cells was positively correlated with the grade of ICI-related ILD. Our data reveal the immune-checkpoint profiles of T cells in ICI-related ILD and may provide mechanistic insight into the development of this adverse event.

Inhibition of PI3Kδ Enhances Poly I:C-Induced Antiviral Responses and Inhibits Replication of Human Metapneumovirus in Murine Lungs and Human Bronchial Epithelial Cells.

Viral infections of the airway can exacerbate respiratory diseases, such as asthma or chronic obstructive pulmonary disease (COPD), and accelerate disease progression. Phosphoinositide 3-kinase (PI3K)δ, a class 1A PI3K, has been studied as a potential target for achieving anti-oncogenic and anti-inflammatory effects. However, the role of PI3Kδ in antiviral responses is poorly understood. Using a synthetic double-stranded RNA poly I:C and a selective PI3Kδ inhibitor IC87114, we investigated the role of PI3Kδ signaling in poly I:C-induced expression of the T lymphocyte-inhibitory molecule programmed death 1 ligand 1 (PD-L1), inflammatory responses and antiviral interferon (IFN) responses. C57BL/6N mice were treated with IC87114 or vehicle by intratracheal (i.t.) instillation followed by i.t. administration of poly I:C. Poly I:C increased PD-L1 expression on epithelial cells, lymphocytes, macrophages, and neutrophils in the lungs and IC87114 suppressed poly I:C-induced PD-L1 expression on epithelial cells and neutrophils possibly via inhibition of the Akt/mTOR signaling pathway. IC87114 also attenuated poly I:C-induced increases in numbers of total cells, macrophages, neutrophils and lymphocytes, as well as levels of KC, IL-6 and MIP-1ß in bronchoalveolar lavage fluid. Gene expression of IFNß, IFNλ2 and IFN-stimulated genes (ISGs) were upregulated in response to poly I:C and a further increase in gene expression was observed following IC87114 treatment. In addition, IC87114 enhanced poly I:C-induced phosphorylation of IRF3. We assessed the effects of IC87114 on human primary bronchial epithelial cells (PBECs). IC87114 decreased poly I:C-induced PD-L1 expression on PBECs and secretion of IL-6 and IL-8 into culture supernatants. IC87114 further enhanced poly I:C- induced increases in the concentrations of IFNß and IFNλ1/3 in culture supernatants as well as upregulated gene expression of ISGs in PBECs. Similar results were obtained in PBECs transfected with siRNA targeting the PIK3CD gene encoding PI3K p110δ, and stimulated with poly I:C. In human metapneumovirus (hMPV) infection of PBECs, IC87114 suppressed hMPV-induced PD-L1 expression and reduced viral replication without changing the production levels of IFNß and IFNλ1/3 in culture supernatants. These data suggest that IC87114 may promote virus elimination and clearance through PD-L1 downregulation and enhanced antiviral IFN responses, preventing prolonged lung inflammation, which exacerbates asthma and COPD.

Effects of cigarette smoke on barrier function and tight junction proteins in the bronchial epithelium: protective role of cathelicidin LL-37.

BACKGROUND: Airway epithelial barrier function is maintained by the formation of tight junctions (TJs) and adherens junctions (AJs). Inhalation of cigarette smoke causes airway epithelial barrier dysfunction and may contribute to the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). We assessed the effects of cigarette smoke on barrier function and expression of multiple TJ and AJ proteins in the bronchial epithelium. We also examined whether treatment with glucocorticosteroids (GCSs), long-acting ß2-agonists (LABAs), and human cathelicidin LL-37 can protect against cigarette smoke extract (CSE)-induced barrier dysfunction. METHODS: Calu-3 cells cultured at the air-liquid interface were pretreated with or without GCSs, LABAs, GCSs plus LABAs, or LL-37, and subsequently exposed to CSE. Barrier function was assessed by transepithelial electronic resistance (TEER) measurements. Gene and protein expression levels of TJ and AJ proteins were analyzed by quantitative PCR and western blotting, respectively. Immunofluorescence staining of TJ and AJ proteins was performed. RESULTS: CSE decreased TEER and increased permeability in a concentration-dependent manner. CSE suppressed gene expression of claudin-1, claudin-3, claudin-4, claudin-7, claudin-15, occludin, E-cadherin, junctional adhesion molecule-A (JAM-A) and zonula occludens-1 (ZO-1) within 12 h post-CSE exposure, while suppressed protein expression levels of occludin at 12 h. CSE-treated cells exhibited discontinuous or attenuated immunostaining for claudin-1, claudin-3, claudin-4, occludin, ZO-1, and E-cadherin compared with untreated cells. GCS treatment partially restored CSE-induced TEER reduction, while LABA treatment had no effect. GCS and LABA combination treatment had no additive effect on CSE-induced TEER reduction and gene suppression of TJ and AJ proteins. Human cathelicidin LL-37 counteracted CSE-induced TEER reduction and prevented disruption of occludin and ZO-1. LL-37 also attenuated CSE-induced decreases in gene and protein expression levels of occludin. CONCLUSIONS: CSE caused airway epithelial barrier dysfunction and simultaneously downregulated multiple TJ and AJ proteins. GCS and LABA combination treatment had no additive effect on CSE-induced TEER reduction. LL-37 counteracted CSE-induced TEER reduction and prevented disruption of occludin and ZO-1. Use of LL-37 to counteract airway epithelial barrier dysfunction may have significant benefits for respiratory diseases such as asthma and COPD.

Trends in the prevalence of airflow limitation in a general Japanese population: two serial cross-sectional surveys from the Hisayama Study.

OBJECTIVES: Chronic obstructive airway disease, which is characterised by airflow limitation, is a major burden on public health. Reductions in environmental pollution in the atmosphere and workplace and a decline in the prevalence of smoking over recent decades may have affected the prevalence of airflow limitation in Japan. The present epidemiological study aimed to evaluate trends in the prevalence of airflow limitation and in the influence of risk factors on airflow limitation in a Japanese community. DESIGN: Two serial cross-sectional surveys. SETTING: Data from the Hisayama Study, a population-based prospective study that has been longitudinally conducted since 1961. PARTICIPANTS: A total of 1842 and 3033 residents aged ≥40 years with proper spirometric measurements participated in the 1967 and 2012 surveys, respectively. MAIN OUTCOME MEASURES: Airflow limitation was defined as forced expiratory volume in 1 s/forced vital capacity <70% by spirometry. For each survey, the age-adjusted prevalence of airflow limitation was evaluated by sex. ORs and population attributable fractions of risk factors on the presence of airflow limitation were compared between surveys. RESULTS: The age-standardised prevalence of airflow limitation decreased from 1967 to 2012 in both sexes (from 26.3% to 16.1% in men and from 19.8% to 10.5% in women). Smoking was significantly associated with higher likelihood of airflow limitation in both surveys, although the magnitude of its influence was greater in 2012 than in 1967 (the multivariable-adjusted OR was 1.63 (95% CI 1.19 to 2.24) in 1967 and 2.26 (95% CI 1.72 to 2.99) in 2012; p=0.007 for heterogeneity). Accordingly, the population attributable fraction of smoking on airflow limitation was 33.5% in 2012, which was 1.5-fold higher than that in 1967 (21.1%). CONCLUSIONS: The prevalence of airflow limitation was decreased over 45 years in Japan, but the influence of smoking on airflow limitation increased with time.

Periodontal status and lung function decline in the community: the Hisayama study.

This study aimed to determine whether periodontal status is related to a decline in lung function in a general Japanese population. We followed a total of 1,650 community-dwelling individuals (≥40 years) without chronic obstructive pulmonary disease, with at least one teeth, for 3 years. Periodontal status was assessed at baseline by clinical attachment loss (CAL) and probing pocket depth (PPD) at two sites for each tooth, and the mean values were calculated for each subject. Lung function was measured at baseline and follow-up using spirometry, and longitudinal decline in forced expiratory volume in one second (FEV1) was calculated. Multivariate Poisson regression with robust error variance was used to estimate risk ratio (RR). After adjusting for potential confounders including smoking status, there was a tendency for the adjusted RR of developing rapid lung function decline (≥160 mL/3years, the highest quartile of the distribution of FEV1 declines) to increase as mean CAL levels increased (P trend = 0.039). Likewise, a positive association was observed between mean PPD levels and RR of developing rapid lung function decline (P trend = 0.047). Our findings suggest deterioration of periodontal status could be a risk factor for rapid lung function decline in the general Japanese population.

Interleukin-22 attenuates double-stranded RNA-induced upregulation of PD-L1 in airway epithelial cells via a STAT3-dependent mechanism.

Double-stranded RNA derived from viruses induces host immune responses. PD-L1, also known as B7-H1, is an immune-checkpoint molecule associated with the escape of viruses from host immune systems, which plays a role in the persistence of viral infection, resulting in exacerbations of underlying diseases such as asthma and chronic obstructive pulmonary disease. Interleukin (IL)-22 is produced from various immune cells and has protective properties on mucosal tissue. The binding of IL-22 to IL-22 receptor induces STAT3 activation. We investigated the effect of IL-22 on the expression in airway epithelial cells in vitro and in mouse lungs in vivo after the stimulation with an analog of viral double-stranded RNA, polyinosinic-polycytidylic acid (poly I:C). Stimulation with poly I:C upregulated PD-L1 expression on BEAS-2B cells. This upregulation of PD-L1 was attenuated by IL-22 administration. STAT3 phosphorylation was induced by IL-22 and poly I:C. Treatment of cells with STAT3 siRNA abolished the effect of IL-22 on the poly I:C-induced upregulation of PD-L1. This upregulation of PD-L1 was also attenuated by IL-11, a cytokine inducing STAT3 phosphorylation, in BEAS-2B cells. In mouse lung cells in vivo, IL-22 suppressed poly I:C-induced upregulation of PD-L1. These results suggest that IL-22 attenuates virus-induced upregulation of PD-L1 in airway epithelial cells via a STAT3-dependent mechanism.

Association of Airflow Limitation With Carotid Atherosclerosis in a Japanese Community　- The Hisayama Study.

BACKGROUND: There has been no large-scale observational study examining the association between chronic obstructive pulmonary disease (COPD) or airflow limitation and carotid atherosclerosis in the general population across a wide range of generations in Asia. In the present study we assessed the association between airflow limitation and carotid intima-media thickness (IMT) in a general Japanese population, with consideration of a comprehensive array of cardiovascular risk factors.Methods and Results:In all, 2,099 community-dwelling Japanese subjects were included in the study. Airflow limitation was defined by spirometry. Maximum and mean IMT values were measured using carotid ultrasonography. Among the subjects, 352 (16.8%) had airflow limitation. The geometric mean values of maximum IMT and mean IMT were significantly higher in subjects with than without airflow limitation (1.27 vs. 1.18 mm, respectively, for maximum IMT; 0.73 mm vs. 0.72 mm, respectively, for mean IMT) and increased with the severity of airflow limitation after adjustment for conventional risk factors, including smoking habits and serum high-sensitivity C-reactive protein. It should be noted that the magnitude of these associations was greater in the middle-aged (40-64 years) than elderly (≥65 years) subgroup. CONCLUSIONS: The findings of the present study suggest that airflow limitation is a significant risk factor for carotid atherosclerosis, especially in midlife, in the general Japanese population.

Induction of PD-L1 Expression by the EML4-ALK Oncoprotein and Downstream Signaling Pathways in Non-Small Cell Lung Cancer.

PURPOSE: Therapies targeted to the immune checkpoint mediated by PD-1 and PD-L1 show antitumor activity in a subset of patients with non-small cell lung cancer (NSCLC). We have now examined PD-L1 expression and its regulation in NSCLC positive for the EML4-ALK fusion gene. EXPERIMENTAL DESIGN: The expression of PD-L1 at the protein and mRNA levels in NSCLC cell lines was examined by flow cytometry and by reverse transcription and real-time PCR analysis, respectively. The expression of PD-L1 in 134 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis. RESULTS: The PD-L1 expression level was higher in NSCLC cell lines positive for EML4-ALK than in those negative for the fusion gene. Forced expression of EML4-ALK in Ba/F3 cells markedly increased PD-L1 expression, whereas endogenous PD-L1 expression in EML4-ALK-positive NSCLC cells was attenuated by treatment with the specific ALK inhibitor alectinib or by RNAi with ALK siRNAs. Furthermore, expression of PD-L1 was downregulated by inhibitors of the MEK-ERK and PI3K-AKT signaling pathways in NSCLC cells positive for either EML4-ALK or activating mutations of the EGFR. Finally, the expression level of PD-L1 was positively associated with the presence of EML4-ALK in NSCLC specimens. CONCLUSIONS: Our findings that both EML4-ALK and mutant EGFR upregulate PD-L1 by activating PI3K-AKT and MEK-ERK signaling pathways in NSCLC reveal a direct link between oncogenic drivers and PD-L1 expression.

Validation of a COPD screening questionnaire and establishment of diagnostic cut-points in a Japanese general population: the Hisayama study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is highly prevalent worldwide. COPD is a treatable disease and it is important to identify COPD subjects, highlighting the need for an efficient screening measure. Although the COPD screening questionnaire (COPD Population Screener, COPD-PS) was developed as a screening tool, its validity is not clear in population-based studies. This study determines the validity of the COPD-PS in the general Japanese population. METHODS: All registered residents living in the town of Hisayama aged above 40 were solicited to participate in a health check-up in 2012. All subjects aged 40-79 without physician-diagnosed asthma or lung resection were recruited, and 2357 subjects with the COPD-PS recorded and valid spirometry measurements were analyzed. Persistent airflow obstruction (AO) was defined by post-bronchodilator FEV1/FVC < 0.7. The sensitivity and specificity of the COPD-PS score for identifying AO was assessed by logistic regression analysis. RESULTS: The prevalence of AO in this population was 6.5%. The overall area under the receiver operating characteristic (ROC) curve for the continuous COPD-PS score was 0.748. A cut-point of 4-points is recommended, resulting in a sensitivity of 67.1% and specificity of 72.9% with an area under the ROC curve of 0.70. The positive predictive value was 14.6% and negative predictive value was 97.0%. CONCLUSIONS: The COPD-PS appears to be an adequate measure for large scale screening of possible airflow obstruction requiring further testing with spirometry.

Prevalence of asthma with airflow limitation, COPD, and COPD with variable airflow limitation in older subjects in a general Japanese population: the Hisayama Study.

BACKGROUND: Elucidating the prevalence of asthma and chronic obstructive pulmonary disease (COPD) is important for designing a public health strategy. Recent studies have discriminated a phenotype of COPD with variable airflow limitation (COPD-VAL) associated with asthma-COPD overlap syndrome. Its prevalence remains uncertain. The age and occupational distributions in the town of Hisayama and in Japan are nearly identical. Each disease's prevalence was estimated for the town's residents. METHODS: In 2008, town residents (≥ 40 years) were solicited to participate in a health checkup. Individuals with abnormal spirometry (forced expiratory volume in 1s/forced vital capacity [FEV1/FVC]<70% and/or %FVC<80%) were recommended for further evaluations. Two pulmonologists in a blinded fashion reviewed their medical records, including bronchodilator reversibility. Individuals with airflow limitation were classified as having asthma, COPD, COPD-VAL, or other diseases. The prevalence of each disease was then estimated. RESULTS: A total of 2100 residents (43.4% of residents in the age group) completed spirometry. In 455 residents with abnormal spirometry, 190 residents had further evaluations, and the medical records of 174 residents were reviewed. The prevalence of asthma with airflow limitation, COPD, and COPD-VAL, were 2.0%, 8.4%, and 0.9%, respectively. The prevalence of COPD and COPD-VAL were higher in men and smokers than in women and never-smokers. The prevalence of COPD, but not COPD-VAL or asthma, increased with age. CONCLUSION: The prevalence of asthma with airflow limitation, COPD, and COPD-VAL were estimated in a population of residents (≥ 40 years) in Hisayama.

Viral infections in asthma and COPD.

Airway viral infections are associated with the pathogenesis of asthma and COPD. It has been argued that respiratory syncytial virus (RSV) infection in infancy is a probable causal factor in the development of pediatric asthma. RSV infections tend to induce Th2-biased immune responses in the host airways. RSV infection, atopy, and low pulmonary function in neonates may work synergistically toward the development of pediatric asthma. Human rhinovirus (HRV) is a representative virus associated with the exacerbation of asthma in both children and adults. Viral infections trigger innate immune responses including granulocytic inflammation and worsen the underlying inflammation due to asthma and COPD. The innate immune responses involve type-I and -III interferon (IFN) production, which plays an important role in anti-viral responses, and the airway epithelia of asthmatics reportedly exhibit defects in the virus-induced IFN responses, which renders these individuals more susceptible to viral infection. A similarly impaired IFN response is seen in COPD, and several investigators propose that latent adenoviral infection may be involved in COPD development. Persistent RSV infections were detected in a sub-population of patients with COPD and were associated with the accelerated decline of lung function. The virus-induced upregulation of co-inhibitory molecules in the airway epithelium partly accounts for the persistent infections. Experimental animal models for virus-asthma/COPD interactions have shed light on the underlying immune mechanisms and are expected to help develop novel approaches to treat respiratory diseases.

Cigarette smoke impairs phagocytosis of apoptotic neutrophils by alveolar macrophages via inhibition of the histone deacetylase/Rac/CD9 pathways.

Efferocytosis, which is the homeostatic phagocytosis of apoptotic cells, prevents the release of toxic intracellular contents and subsequent tissue damage. Impairment of efferocytosis was reported in alveolar macrophages (AMs) of patients with chronic obstructive pulmonary disease (COPD), a common disease caused by smoking. In COPD, histone deacetylase (HDAC) activity is reduced in AMs. We investigated whether the reduction of HDAC activity is associated with the impairment of efferocytosis. Murine AMs were collected by bronchoalveolar lavage and their ability to efferocytose apoptotic human polymorphonuclear leukocytes was assessed. Pre-treatment of AMs with cigarette smoke extract (CSE) or trichostatin A (TSA), an HDAC inhibitor, suppressed efferocytosis and CSE reduced HDAC activity. TSA inhibited the activity of Rac, a key mediator of efferocytosis. These TSA-induced impairments were restored by treatment of AMs with aminophylline, a potent activator of HDAC. To further elucidate the underlying mechanism, we explored a role of CD9 in TSA-induced impairment of efferocytosis. CD9 is a transmembrane protein of the tetraspanin family that facilitates the uptake of several pathogens and other material. TSA profoundly down-regulated the expression of CD9 on AMs. The expression of CD9 was partly down-regulated by the Rac inhibitor. Pretreatment with an anti-CD9 mAb or CD9 small interfering RNA inhibited efferocytosis, which was attributable to the reduced binding of AMs to apoptotic cells. These results suggest that smoking impairs efferocytosis via inhibition of HDAC/Rac/CD9 pathways. Aminophylline/theophylline is effective in restoring the impairment of efferocytosis and might have benefit for the treatment of patients with COPD.

PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells.

Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB.