Keiichiro Akamatsu

Activation of Toll-Like Receptor 3 Augments Myofibroblast Differentiation

Airway remodeling is observed in the airways of patients with asthma, and differentiation of fibroblasts to myofibroblasts plays a critical role in the progress of airway remodeling. Viral infection induces not only the disease development and exacerbations but also airway remodeling. The aim of this study was to evaluate whether the activation of Toll-like receptor 3 (TLR3) can affect the differentiation of fibroblasts to myofibroblasts and the extracellular matrix (ECM) protein production. Human fetal lung fibroblasts (HFL-1) and adult lung fibroblasts were treated with a synthetic double-stranded RNA, polyinosine-polycytidylic acid (poly[I:C]) and the expression of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblast differentiation, was evaluated. The release of transforming growth factor-beta(1) (TGF-beta(1)) and ECM protein production were assessed. The effect of anti-TGF-beta antibody on the alpha-SMA and ECM production was also assessed. Poly(I:C) significantly augmented the alpha-SMA expression (P < 0.01) and release of TGF-beta(1) (P < 0.01) compared with control. Bafilomycin, an inhibitor of TLR3 signaling, diminished poly(I:C)-augmented TGF-beta(1) release. Anti-TGF-beta(1) antibody inhibited the poly(I:C)-augmented alpha-SMA expression. Poly(I:C) enhanced translocation of nuclear factor-kB (NF-kappaB) and interferon regulatory factor-3 (IRF-3) into the nucleus. Poly(I:C)-augmented TGF-beta(1) release was almost completely blocked by NF-kappaB inhibitors, but not by silencing IRF-3. The production of fibronectin and collagen I expression were significantly increased by poly(I:C) (P < 0.01) and they were inhibited by anti-TGF-beta antibody. These results suggest that activation of TLR3 can affect the differentiation to myofibroblasts and enhance ECM production via the NF-kappaB-TGF-beta(1)-dependent pathway.

Oxidative stress enhances toll-like receptor 3 response to double-stranded RNA in airway epithelial cells.

Virus infections are a major cause of chronic obstructive pulmonary disease (COPD) exacerbations. Recently, Toll-like receptor 3 (TLR3) has been demonstrated to react to double-stranded RNA (dsRNA) and to be involved in the immune responses after viral infections. In the present study, we examined whether oxidative stress, which is involved in the pathogenesis of COPD, enhances the responses of TLR3 in airway epithelial cells. The effect of hydrogen peroxide (H(2)O(2)) on the release of IL-8 from BEAS-2B cells and primary human bronchial epithelial cells after stimulation with polyinosine-polycytidylic acid [poly(I:C)], a synthetic analog of viral dsRNA and a ligand for TLR3, and the signal transduction were examined. One hundred to 150 muM H(2)O(2) significantly potentiated the release of IL-8 from the epithelial cells after stimulation with 10 microg/ml poly(I:C). The H(2)O(2)-augmented IL-8 release was inhibited by treatment with N-acetylcysteine. One hundred micromoles of H(2)O(2) enhanced the translocation of nuclear factor (NF)-kappaB p65, but not that of interferon regulatory factor-3 (IRF-3), into the nucleus and the NF-kappaB DNA binding activity after poly(I:C) stimulation, which effect was inhibited not by the silencing of IRF-3 but by MG132, a proteasome inhibitor, or dexamethasone. One hundred micromoles of H(2)O(2) potentiated the TLR3 expression on the airway epithelial cells treated with poly(I:C). These data suggest that oxidative stress augments the response of TLR3 in airway epithelial cells via NF-kappaB and that this effect might be partly mediated by the enhancement of TLR3 expression. Modulation of this pathway may be a therapeutic target for viral-induced exacerbations of COPD.

Oxidative stress augments toll-like receptor 8 mediated neutrophilic responses in healthy subjects.

BackgroundExcessive oxidative stress has been reported to be generated in inflamed tissues and contribute to the pathogenesis of inflammatory lung diseases, exacerbations of which induced by viral infections are associated with toll-like receptor (TLR) activation. Among these receptors, TLR8 has been reported as a key receptor that recognizes single-strand RNA virus. However, it remains unknown whether TLR8 signaling is potentiated by oxidative stress. The aim of this study is to examine whether oxidative stress modulates TLR8 signaling in vitro.MethodsHuman peripheral blood neutrophils were obtained from healthy non-smokers and stimulated with TLR 7/8 agonist imidazoquinoline resiquimod (R848) in the presence or absence of hydrogen peroxide (H2O2). Neutrophilic responses including cytokine release, superoxide production and chemotaxis were examined, and the signal transduction was also analyzed.ResultsActivation of TLR8, but not TLR7, augmented IL-8 release. The R848-augmented IL-8 release was significantly potentiated by pretreatment with H2O2 (p < 0.01), and N-acetyl-L-cysteine reversed this potentiation. The combination of H2O2 and R848 significantly potentiated NF-kB phosphorylation and IkBα degradation. The H2O2-potentiated IL-8 release was suppressed by MG-132, a proteosome inhibitor, and by dexamethasone. The expressions of TLR8, myeloid differentiation primary response gene 88 (MyD88), and tumor necrosis factor receptor-associated factor 6 (TRAF6) were not affected by H2O2.ConclusionTLR8-mediated neutrophilic responses were markedly potentiated by oxidative stress, and the potentiation was mediated by enhanced NF-kB activation. These results suggest that oxidative stress might potentiate the neutrophilic inflammation during viral infection.

Peroxynitrite augments fibroblast-mediated tissue remodeling via myofibroblast differentiation

Irreversible airflow limitation in asthma is associated with airway remodeling in which the differentiation of fibroblasts to myofibroblasts plays a pivotal role. In asthmatic airways, excessive production of reactive nitrogen species (RNS) has been observed. The aim of this study is to evaluate whether peroxynitrite, one of the RNS, can affect the differentiation of fibroblasts to myofibroblasts. Human fetal lung fibroblasts were treated with various concentrations of authentic peroxynitrite or a peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1), and the expressions of alpha-smooth muscle actin (alpha-SMA) and desmin, markers of myofibroblast differentiation, were evaluated. The releases of transforming growth factor-beta(1) (TGF-beta(1)) and ECM proteins including fibronectin and collagen I were assessed. To clarify the mechanism in this differentiation, the effect of anti-TGF-beta antibody or NF-kappaB inhibitors on the alpha-SMA expression and ECM production was assessed. Peroxynitrite and SIN-1 significantly augmented the alpha-SMA expression compared with control in a concentration-dependent manner (P < 0.01 and P < 0.05, respectively). Peroxynitrite significantly increased desmin and TGF-beta(1) production (P < 0.01). Peroxynitrite enhanced the translocation of NF-kappaB into the nucleus confirmed by immunocytostaining and immunoblotting. Peroxynitrite-augmented alpha-SMA expression was blocked by NF-kappaB inhibitors, MG132 and caffeic acid phenethyl ester (CAPE), and anti-TGF-beta antibody. CAPE completely inhibited the peroxynitrite-augmented TGF-beta(1) release. The production of fibronectin and collagen I was significantly increased by peroxynitrite (P < 0.01) and inhibited by anti-TGF-beta antibody. These results suggest that RNS can affect the differentiation to myofibroblasts and excessive ECM production via a NF-kappaB-TGF-beta(1)-dependent pathway.

Exhaled Nitric Oxide Cutoff Values for Asthma Diagnosis According to Rhinitis and Smoking Status in Japanese Subjects

BACKGROUND Measurement of the exhaled nitric oxide fraction (FE(NO)) has been proposed as a useful diagnostic test for asthma. However, most of the data concerning the FE(NO) cutoff values for the diagnosis of asthma have not yet examined using standard procedures. Furthermore, there is no detailed study that investigated the cutoff values that takes into account patient factors that influence the FE(NO) levels. METHODS FE(NO) was measured in 142 steroid-naive asthmatics and 224 control subjects using an online electrochemical nitric oxide analyzer in accordance with the current guidelines. Subjects without respiratory symptoms and normal spirometric parameters were included in the control group. Asthma was diagnosed on the basis of the presence of significant airway reversibility and/or airway hyperresponsiveness during clinical follow up 6 months after FE(NO) measurements. RESULTS FE(NO) was significantly higher in asthmatic patients compared with control subjects (p < 0.01). Based on all study subjects, the receiver operating characteristic curves indicated that the cutoff value of FE(NO) 22 parts per billion (ppb) was associated with the highest combination of sensitivity (90.8%) and specificity (83.9%). Multivariate analysis showed allergic rhinitis, current smoking, and asthma were significant factors influencing the FE(NO) levels. The cutoff values of FE(NO) to discriminate asthma from non-asthma ranged from 18 to 28 ppb depending on rhinitis and smoking status. CONCLUSIONS The cutoff values presented may be useful for the interpretation of FE(NO) values in the clinical practice.

Associated demographics of persistent exhaled nitric oxide elevation in treated asthmatics

The fraction of exhaled nitric oxide (FENO) is reduced by anti‐inflammatory treatment in asthma. However, the FENO level is also regulated by individual demographics and there is considerable variation among clinically stable patients.

Cigarette smoke augments the expression and responses of toll‐like receptor 3 in human macrophages

Background and objective:  Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD). Recently, toll‐like receptor 3 (TLR3) was shown to recognize pathogen‐associated molecular patterns, especially viral‐derived double‐stranded RNA, and to be involved in immune responses. However, the effects of cigarette smoke on TLR3 remain unclear. In this study, it was examined whether cigarette smoke affects the expression and responses of TLR3 in human macrophages.

25-hydroxycholesterol enhances cytokine release and toll-like receptor 3 response in airway epithelial cells

Background25-hydroxycholesterol (25-HC) is one of the oxysterols, which are oxidized derivatives of cholesterol, and has been reported to be involved in the pathogenesis of atherosclerosis and Alzheimer’s disease. In lung, the possible involvement of 25-HC in airway diseases has been revealed. In the present study, we examined whether 25-HC affects the release of cytokines and also modulates the responses of toll-like receptor 3 (TLR3) in airway epithelial cells.MethodsThe effect of 25-HC on the release of cytokines from primary human bronchial epithelial cells after stimulation with or without polyinosine-polycytidylic acid [poly(I:C)], a ligand for TLR3, and the signal transduction were examined.Results25-HC significantly potentiated the release of interleukin-8 (IL-8) and IL-6 from the cells. This effect was more potent compared with that of other oxysterols, 22-HC and 27-HC. GW3965 and TO901317, synthetic agonists of liver X receptors that are receptors for oxysterols, did not augment the IL-8 release. 25-HC enhanced the nuclear factor-kappa B (NF-κB) DNA binding activity and translocation of phosphorylated c-Jun into the nucleus. The release of IL-8 was inhibited by the NF-κB inhibitor, caffeic acid phenethyl ester (CAPE), an inhibitor of nuclear factor kappa-B alpha (IκBα) inhibitor, BAY 11–7085, and an inhibitor of nuclear factor kappa-B kinase-2 (IKK-2) inhibitor, SC-514, but not by a c-Jun N-terminal kinase (JNK) inhibitory peptide, L-JNKi1. 25-HC significantly potentiated IL-8 release in poly(I:C)-treated cells and the augmentation was inhibited by CAPE, BAY 11–7085, and SC-514. Furthermore, 25-HC potentiated the translocation of interferon regulatory factor 3 into the nucleus and the release of interferon-beta (IFN-β) in poly(I:C)-treated cells.ConclusionsThese data demonstrated that 25-HC augments the release of IL-8 and IL-6 via NF-κB signalling pathway and enhances the release of IL-8 and IFN-β after stimulation of TLR3 in airway epithelial cells. 25-HC may be involved in the neutrophilic airway inflammation through the stimulant effect of IL-8 and IL-6 release and also potentiate the TLR3-mediated innate immunity in airway diseases.

Poor Sensitivity of Symptoms in Early Detection of COPD

The prevalence of chronic obstructive pulmonary disease (COPD) has been increasing. However, COPD is often underdiagnosed. The objective of this study was to determine how many outpatients had persistent airflow limitation and could be diagnosed as COPD by post-bronchodilator spirometry. We also evaluated whether the newly diagnosed patients had any symptoms. All outpatients with liver or general diseases over 40 years old who regularly visited to our hospital were tested for pulmonary function by spirometry. Patients with airflow limitation by the first screening spirometry had further examinations including post-bronchodilator spirometry and chest radiograph by pulmonary specialists. A total of 288 patients accepted a first spirometry. The most common chronic diseases of these patients were chronic hepatitis (33.7%), fatty liver (26.4%), liver cirrhosis (8.3%), diabetes (3.5%) and hypertension (3.1%). Approximately half of the patients had a smoking history. 44 of 288 patients (15.3%) showed airflow limitation by pre-bronchodilator spirometry. Of these, 8 patients did not show airflow limitation by a repeat pre-bronchodilator spirometry nor did 5 patients by post-bronchodilator spirometry. The rest were diagnosed as COPD (80.6%), asthma (16.1%) and bronchiectasis (3.2%). The prevalence of COPD was 8.7%. Approximately half of the patients (13/25, 52.0%) diagnosed as COPD had never complained of any respiratory symptoms. Because symptoms such as dyspnea on exertion, cough and sputum are less sensitive for the diagnosis of COPD, the propagation of spirometry in a general practice/setting should be recommended for establishing the diagnosis rate of COPD rather than relying on the presence of respiratory symptoms.

Validation of a compact motion sensor for the measurement of physical activity in patients with chronic obstructive pulmonary disease.

Background: The DynaPort Activity Monitor (DAM) has been reported to be useful to evaluate the activity in healthy subjects and patients with chronic obstructive pulmonary disease (COPD). However, it is difficult to estimate the activity of COPD patients using DAM, because its battery works only for several hours and sensors should be worn at two parts of the body. A newly developed compact, single-position triaxial accelerometer (Actimarker) can measure the activity for >1 month, but has not been validated for COPD patients. Objectives: The validity of the Actimarker was evaluated in COPD patients. Methods: In study 1, the validity of the device was tested in 14 stable COPD patients by comparing it with DAM. In study 2, the influence of the weather on activity was examined. In study 3, the number of measurement days required to ensure repeatability was determined. Results: The durations of activity measured by the Actimarker and DAM were significantly correlated at intensity values ≧2.0, ≧2.5 and ≧3.0 METs. The duration of activity on rainy days was significantly shorter than that on non-rainy days. The values of intraclass correlation coefficients were >0.8 in 3-, 4- or 5-day measurements, and there was no systematic bias at any number of days or intensities with Bland-Altman plots. Conclusions: The validity of the Actimarker was confirmed, and repeatability was obtained when the data from at least 3 non-rainy weekdays were analyzed. Actimarker appears to be useful as a simplified method to evaluate the physical activity of COPD patients.