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 (H2O2) 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 μM H2O2 significantly potentiated the release of IL-8 from the epithelial cells after stimulation with 10 μg/ml poly(I:C). The H2O2-augmented IL-8 release was inhibited by treatment with N-acetylcysteine. One hundred micromoles of H2O2 enhanced the translocation of nuclear factor (NF)-κB p65, but not that of interferon regulatory factor-3 (IRF-3), into the nucleus and the NF-κB 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 H2O2 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-κB 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.

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 α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, was evaluated. The release of transforming growth factor-β1 (TGF-β1) and ECM protein production were assessed. The effect of anti-TGF-β antibody on the α-SMA and ECM production was also assessed. Poly(I:C) significantly augmented the α-SMA expression (P < 0.01) and release of TGF-β1 (P < 0.01) compared with control. Bafilomycin, an inhibitor of TLR3 signaling, diminished poly(I:C)-augmented TGF-β1 release. Anti–TGF-β1 antibody inhibited the poly(I:C)-augmented α-SMA expression. Poly(I:C) enhanced translocation of nuclear factor-kB (NF-κB) and interferon regulatory factor-3 (IRF-3) into the nucleus. Poly(I:C)-augmented TGF-β1 release was almost completely blocked by NF-κB 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-β antibody. These results suggest that activation of TLR3 can affect the differentiation to myofibroblasts and enhance ECM production via the NF-κB–TGF-β1–dependent pathway.

25-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. The 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. 25-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. These 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.

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. 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. 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. The cutoff values presented may be useful for the interpretation of FE(NO) values in the clinical practice.