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Background Bronchial thermoplasty (BT) is a novel technique used in the treatment of subjects with severe refractory asthma. Radiofrequency is provided to airway walls during bronchoscopy in order to reduce airway remodeling. Several clinical studies have reported an improvement in subjects’ symptoms following BT. However, how BT affects the airway architectures and inflammatory mediators in the airways has not been yet fully elucidated. Methods Fourteen subjects with severe asthma were recruited in this study according to the criteria of ATS severe asthma definition. The study subjects undertook bronchial biopsy during the bronchoscopy procedure at baseline and 6 weeks after the initial BT treatment. The obtained samples were stained with antibodies for α-smooth muscle actin (α-SMA); protein gene product (PGP) 9.5, a specific nerve marker; von Willebrand factor (vWF), a marker for blood vessels; interleukin-17A (IL-17A) and transforming growth factor-β1 (TGF-β1). Results The expression of α-SMA and PGP9.5 were significantly reduced post-BT. There was no significant difference in the number of blood vessels between baseline and post-BT. In addition, BT did not affect the production of IL-17A and TGF-β1 in the airways. The changes in the expression of α-SMA and PGP9.5 had no significant correlation with the improvement of pulmonary function. Conclusion and Clinical Relevance: This study suggests that BT reduces airway smooth muscle mass and the airway innervation without affecting vasculature and the production of inflammatory mediators in the airways of subjects with severe asthma.

Immunoglobulin E (IgE) plays an important role in the pathogenesis of asthma and anti-IgE therapy was approved for treating patients with severe persistent allergic asthma. The exact target sites of anti-IgE therapy are not well characterized; however, it has been proposed that the therapeutic effects of anti-IgE come from its intervention in the airway remodeling process. To gain insights on how anti-IgE therapy improves asthma symptoms, we aimed to validate the expression of FcεRI on airway epithelial cells and demonstrate its role in airway remodeling. The expression of FcεRI was measured (1) in situ in bronchial biopsy tissues of asthmatic and control subjects using immunohistochemistry and (2) in vitro in primary bronchial epithelial cells obtained from asthmatic subjects, at baseline and after treatment with human IgE, using qPCR and flow cytometry. FcεRI expression in situ was detected only in a very small number of cells in the epithelium of bronchial biopsies of asthmatic and control subjects. In vitro measurement revealed no expression of the receptor both at baseline and after stimulation with IgE. The release of transforming growth factor—beta (TGF)-β and thymic stromal lymphopoietin (TSLP) were examined by ELISA in bronchial epithelial cells after crosslinking of IgE. No significant differences in TSLP and TGF-β protein levels were detected between stimulated and unstimulated cells. Hence, our data conclusively indicate that bronchial epithelial cells have negligible expression of functional high affinity receptor for IgE. Taken together, anti-IgE therapy is very likely to exert its therapeutic effects via other structural cell types.

Background Asthma is a heterogenous disease characterized by chronic inflammation and airway remodeling. An increase in the severity of airway remodeling is associated with a more severe form of asthma. There is increasing interest in the epithelial to mesenchymal transition process and mechanisms involved in the differentiation and repair of the airway epithelium, especially as they apply to severe asthma. Growing evidence suggests that Epithelial-Mesenchymal transition (EMT) could contribute to airway remodeling and fibrosis in asthma. Severe asthmatic patients with remodeled airways have a neutrophil driven inflammation. Neutrophils are an important source of TGF-β1, which plays a role in recruitment and activation of inflammatory cells, extracellular matrix (ECM) production and fibrosis development, and is a potent inducer of EMT. Objective As there is little data examining the contribution of neutrophils and/or their mediators to the induction of EMT in airway epithelial cells, the objective of this study was to better understand the potential role of neutrophils in severe asthma in regards to EMT. Methods We used an in vitro system to investigate the neutrophil-epithelial cell interaction. We obtained peripheral blood neutrophils from severe asthmatic patients and control subjects and examined for their ability to induce EMT in primary airway epithelial cells. Results Our data indicate that neutrophils from severe asthmatic patients induce changes in morphology and EMT marker expression in bronchial epithelial cells consistent with the EMT process when co-cultured. TGF-β1 levels in the culture medium of severe asthmatic patients were increased compared to that from co-cultures of non-asthmatic neutrophils and epithelial cells. Conclusions and clinical relevance As an inducer of EMT and an important source of TGF-β1, neutrophils may play a significant role in the development of airway remodeling and fibrosis in severe asthmatic airways.

Background Fibrosis, particularly excessive collagen deposition, presents a challenge for treating asthmatic individuals. At present, no drugs can remove or reduce excessive collagen in asthmatic airways. Hence, the identification of pathways involved in collagen deposition would help to generate therapeutic targets to interfere with the airway remodeling process. Autophagy, a cellular degradation process, has been shown to be dysregulated in various fibrotic diseases, and genetic association studies in independent human populations have identified autophagy-related 5 (ATG5) to be associated with asthma pathogenesis. Hence, the dysregulation of autophagy may contribute to fibrosis in asthmatic airways. Objective This study aimed to determine if (1) collagen deposition in asthmatic airways is associated with ATG5 expression and (2) ATG5 protein expression is associated with asthma per se and severity. Methods Gene expression of transforming growth factor beta 1, various asthma-related collagen types [collagen, type I, alpha 1; collagen, type II, alpha 1; collagen, type III, alpha 1; collagen, type V, alpha 1 (COL5A1) and collagen, type V, alpha 2], and ATG5 were measured using mRNA isolated from bronchial biopsies of refractory asthmatic subjects and assessed for pairwise associations. Protein expression of ATG5 in the airways was measured and associations were assessed for asthma per se, severity, and lung function. Main results In refractory asthmatic individuals, gene expression of ATG5 was positively associated with COL5A1 in the airways. No association was detected between ATG5 protein expression and asthma per se, severity, and lung function. Conclusion and clinical relevance Positive correlation between the gene expression patterns of ATG5 and COL5A1 suggests that dysregulated autophagy may contribute to subepithelial fibrosis in the airways of refractory asthmatic individuals. This finding highlights the therapeutic potential of ATG5 in ameliorating airway remodeling in the difficult-to-treat refractory asthmatic individuals.

Summary Background Intravenous immunoglobulin (IVIG) has potent anti-inflammatory and immune-modulating properties. IVIG has been utilized as a steroid-sparing agent in severe asthma, but the results of clinical trials have been conflicting. Objective To determine whether IVIG is able to attenuate bronchial reactivity, pulmonary inflammation and T cell function using a murine model of allergic airways disease. Methods BALB/c or C57BL/6 mice were sensitized to ovalbumin (OVA) or a phosphate-buffered saline control using local nasal sensitization, and then received five intranasal challenges on days 28–32 before sacrifice. Mice were treated intraperitoneally with either IVIG (1–2 g/kg) or equivalent human serum albumin 24 h before the first OVA challenge. Bronchial reactivity to methacholine was examined using the FlexiVent small animal ventilator. We evaluated pulmonary histology, mRNA from lung digests for T-helper type 2 (Th2)-related genes and bronchoalveolar lavage for cell counts and cytokines. Splenocytes were utilized to study OVA-induced cell proliferation, cytokine production and dendritic cell maturation. Results IVIG markedly attenuated the perivascular and peribronchial pulmonary inflammation, and decreased bronchial hyperresponsiveness to methacholine. IVIG treatment of splenocytes from sensitized animals diminished cellular proliferation to OVA, whereas IVIG treatment in vivo markedly attenuated OVA-driven splenocyte proliferation. This is accompanied by diminished IL-13 and TNF-α levels in splenocyte culture, decreased expression of Jagged-1, increased Delta-4 and decreased GATA-3 mRNA levels, signs that IVIG has suppressed the expected Th2 response that accompanies repeated allergen exposure. Increased regulatory T cells were found in draining pulmonary lymph nodes in IVIG-treated mice but not in controls. Conclusions and Clinical Relevance IVIG was effective in ameliorating allergic airway disease in our model. IVIG may be a promising adjunct therapy requiring further study for patients with severe asthma. Cite this as: G. N. Kaufman, A. H. Massoud, S. Audusseau, A.-A. Banville-Langelier, Y. Wang, J. Guay, J. A. Garellek, W. Mourad, C. A. Piccirillo, C. McCusker and B. D. Mazer, Clinical & Experimental Allergy, 2011 (41) 718–728.

Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder marked by relative resistance to steroids. Inflammation and apoptosis have been suggested to be important mechanisms for COPD. Interleukin (IL)-17 superfamily has been associated with chronic inflammation and diminished responses to steroids. It is reasonable to consider that IL-17 may play a role in the pathogenesis of COPD. In this study, we examined IL-17 expression in mice exposed to cigarette smoke (CS) and investigated the contribution of IL-17 to CS-induced inflammation and alveolar cell apoptosis in IL-17−/− mice. After exposing wild-type and IL-17−/− mice to mainstream CS for 4 wk, IL-17A, but not IL-17F, expression was increased in mice upon CS exposure. Neutrophil infiltration in the lungs of IL-17−/− mice was significantly decreased. In IL-17−/− mice, there is reduced expression of IL-6, macrophage inflammatory protein-2, and matrix metalloproteinase-12 compared with wild-type mice after CS exposure. The number of apoptotic type II alveolar cells was significantly increased in CS-exposed wild-type mice but not in IL-17−/− mice. The effect of IL-17A on type II alveolar cell apoptosis was confirmed in vitro through either addition of IL-17A or transient knockdown of IL-17A by small-interfering RNA transfection in type II alveolar cells. These findings suggest that IL-17A plays an important role in the inflammatory response to CS exposure through increased multiple inflammatory mediators. Moreover, IL-17 may also contribute to type II alveolar cell apoptosis. This study opens a new option in targeting IL-17A to modulate inflammatory response to CS and may be the bases for new therapy for COPD.

Cigarette smoke is the principal cause of chronic obstructive pulmonary disease (COPD), a disorder characterized by airway inflammation. As epithelial cells are the first line of defense against foreign material, the response of normal epithelial cells to smoke has been extensively studied. However, little is known about how epithelial cells derived from COPD patients respond to ongoing smoke exposure. This study was aimed at comparing the intracellular response of normal human bronchial/tracheal epithelial cells (NHBE) and COPD-diseased human bronchial/tracheal epithelial cells (DHBE) to cigarette smoke. NHBE and DHBE cells were treated with cigarette smoke condensate (CSC) for 24 h. IL-8 production was measured by ELISA and western blot was used to measure TLR4 expression. Cells were pretreated with CLI-095, a TLR4 inhibitor, or the signaling pathway inhibitors PD184352, Helenalin, or PI-103, which inhibit the ERK1/2, NF-κB and PI3K pathways, respectively. NHBE cells increased IL-8 production in a dose-dependent manner in response to CSC while DHBE cells did not show any significant difference and had a much lower production of IL-8 in response to CSC compared to NHBE cells. There was no change in TLR4 expression with CSC exposure. CLI-095 and PD184352 attenuated IL-8 secretion, indicating that CSC-induced inflammation is both TLR4- and ERK1/2-dependent. These results demonstrate that NHBE and DHBE cells differentially respond to cigarette smoke. DHBE cells exhibit a dampened IL-8 release, indicating that COPD is associated with a reduced capacity of airway epithelial cells to respond to foreign material.

Background: Eosinophilic airway inflammation is heterogeneous in asthma. We recently described a distinct subtype of asthma defined by the expression of genes inducible by Th2 cytokines in bronchial epithelium. This gene signature, which includes periostin, is present in approximately half of asthmatics, and correlates with eosinophilic airway inflammation. However, identification of this subtype depends on invasive airway sampling, hence non-invasive biomarkers of this phenotype are desirable. Objective: Identify systemic biomarkers of eosinophilic airway inflammation. Methods: We measured fractional exhaled nitric oxide (FeNO) and peripheral blood eosinophil, periostin, YKL-40, and IgE levels and compared these biomarkers to airway eosinophilia in 5 cohorts of asthmatics across a range of severity (N=150). Results: We replicated our previous finding of a three-gene bronchial epithelial Th2 signature in a subset of asthmatics and found that peripheral blood periostin levels were highly correlated to the gene signature. Blood periostin is significantly elevated in asthmatics with evidence of eosinophilic airway inflammation relative to those with minimal eosinophilic airway inflammation despite inhaled corticosteroid (ICS) treatment across a range of disease severity. A logistic regression model including sex, age, body mass index (BMI), IgE, blood eosinophils, FeNO, and serum periostin in 59 severe asthmatics showed that, of these indices, serum periostin was the single best predictor of airway eosinophilia (p=0.007). Conclusions: Periostin is a systemic biomarker of airway eosinophilia in asthma and has potential utility in patient selection for emerging asthma therapeutics targeting Th2 inflammation.

Beryllium (Be) is used in several forms: pure metal, beryllium oxide, and as an alloy with copper, aluminum, or nickel. Beryllium oxide, beryllium metal, and beryllium alloys are the main forms present in the workplace, with inhalation being the primary route of exposure. Cases of workers with sensitization or chronic beryllium disease challenge the scientific community for a better understanding of Be toxicity. Therefore, a toxicological inhalation study using a murine model was performed in our laboratory in order to identify the toxic effects related to different particle sizes and chemical forms of Be. This article attempts to provide information regarding the relative effectiveness of the environmental monitoring and exposure protection program that was enacted to protect staff (students and researchers) in this controlled animal beryllium inhalation exposure experiment. This includes specific attention to particle migration control through intensive housekeeping and systematic airborne and surface monitoring. Results show that the protective measures applied during this research have been effective. The highest airborne Be concentration in the laboratory was less than one-tenth of the Quebec OEL (occupational exposure limit) of 0.15 μg/m3. Considering the protection factor of 103 of the powered air-purifying respirator used in this research, the average exposure level would be 0.03 × 10— 4 μg/m3, which is extremely low. Moreover, with the exception of one value, all average Be concentrations on surfaces were below the Quebec Standard guideline level of 3 μg/100 cm2 for Be contamination. Finally, all beryllium lymphocyte proliferation tests for the staff were not higher than controls.

This study aimed to determine the toxicity and toxicokinetic of three Be chemical species A total of 120 mice (four groups of 30) were nose-only exposed. The first group was used as a control while the three others were exposed to 250 μg m−3 of fine particles of three different Be species (Be metal, Be–F; Be oxide, BeO–F; Be aluminium, BeAl–F). Exposure lasted over three consecutive weeks, five days per week and 6 h per day. Blood and several tissues were collected one week after exposure. Urines were collected before the beginning of exposure, at the end of every week of exposure and one week after exposure. Results showed that urine concentrations were different from one Be species to another and that excretion continued after the end of exposure. Except for BeO–F, where Be urine concentrations were stable during the three weeks of exposure, concentrations of Be–F and BeAl–F reached a peak after the first week. According to particle size, BeO–F obtained the highest theoretical pulmonary deposition rate, which partially led to the highest Be lung concentration. This group also presented the lowest urine concentration but that did not lead to more severe lung inflammation. Moreover, even if BeAl–F obtained the lowest percentage theoretical pulmonary deposition, it showed the highest Be urinary concentration, the lowest Be lung concentration and the lowest lung toxicity. In this specific case, a high Be concentration in urine did not reflect a high exposure or a severe toxic effect. Copyright © 2010 John Wiley & Sons, Ltd.

Beryllium is used in a wide variety of industries. Chronic beryllium disease is the most common occupational disease among workers following exposure to Be. The objective of this study was to determine the immunologic effects of two different particle sizes of Be metal

Background IL-17E is a new T H 2 cytokine that promotes airway eosinophilia in mice. IL-17E proinflammatory activity has been proposed to involve induction of cytokine and chemokine production. Recruitment of inflammatory cells may be mediated by tissue-resident cells. Objective This study aimed to evaluate whether fibroblasts represent a target of IL-17E for the production of eosinophil active mediators in the lung. Methods Expression of IL-17B receptor (IL-17BR), a receptor for IL-17E, was evaluated by immunofluorescent staining, Western blot, and real-time PCR in human primary lung fibroblasts. Mediator production was analyzed by using real-time PCR and ELISA after stimulation of fibroblasts with IL-17E alone or in combination with TNF-α and TGF-β 1 . Expression of IL-17E and of eosinophil major basic protein was evaluated by immunohistochemistry in bronchial biopsies from subjects with asthma. Results Human primary lung fibroblasts constitutively expressed IL-17BR. IL-17BR mRNA levels were increased in cells stimulated with TNF-α and decreased with TGF-β 1 . IL-17E slightly upregulated CC chemokine ligand (CCL)–5, CCL-11, GM-CSF, and CXC chemokine ligand (CXCL)–8 mRNA in fibroblasts. Moreover, IL-17E and TNF-α synergistically induced GM-CSF and CXCL-8 mRNA. IL-17E also potentiated the upregulation of CXCL-8 transcripts observed with TGF-β 1 . In contrast, TGF-β 1 decreased IL-17E–induced CCL-11 mRNA. The capacity of IL-17E to enhance GM-CSF and CXCL-8 responses to TNF-α was accompanied by production and secretion of both proteins by lung fibroblasts. Finally, IL-17E was detected in asthma in eosinophil-infiltrated bronchial submucosa. Conclusion IL-17E may contribute to the induction and maintenance of eosinophilic inflammation in the airways by acting on lung fibroblasts. This study supports a role for IL-17E in asthma pathophysiology.

BackgroundEosinophilic airway inflammation is heterogeneous in asthmatic patients. We recently described a distinct subtype of asthma defined by the expression of genes inducible by T(H)2 cytokines in bronchial epithelium. This gene signature, which includes periostin, is present in approximately half of asthmatic patients and correlates with eosinophilic airway inflammation. However, identification of this subtype depends on invasive airway sampling, and hence noninvasive biomarkers of this phenotype are desirable.ObjectiveWe sought to identify systemic biomarkers of eosinophilic airway inflammation in asthmatic patients.MethodsWe measured fraction of exhaled nitric oxide (Feno), peripheral blood eosinophil, periostin, YKL-40, and IgE levels and compared these biomarkers with airway eosinophilia in asthmatic patients.ResultsWe collected sputum, performed bronchoscopy, and matched peripheral blood samples from 67 asthmatic patients who remained symptomatic despite maximal inhaled corticosteroid treatment (mean FEV(1), 60% of predicted value; mean Asthma Control Questionnaire [ACQ] score, 2.7). Serum periostin levels are significantly increased in asthmatic patients with evidence of eosinophilic airway inflammation relative to those with minimal eosinophilic airway inflammation. Alogistic regression model, including sex, age, body mass index, IgE levels, blood eosinophil numbers, Feno levels, and serum periostin levels, in 59 patients with severe asthma showed that, of these indices, the serum periostin level was the single best predictor of airway eosinophilia (P= .007).ConclusionPeriostin is a systemic biomarker of airway eosinophilia in asthmatic patients and has potential utility inpatient selection for emerging asthma therapeutics targetingT(H)2 inflammation.

Background Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder marked by relative resistance to steroids. The IL-17 superfamily, which mediates cross-talk between the adaptive and innate immune systems, has been associated with diminished responses to steroids. Increasing evidence supports elevated IL-17 expression in the lung of COPD subjects. However, whether cells of the immune system (systemic) and/or local lung cells are contributing to the elevated IL-17 remains unclear. To address this issue, we utilized a human parenchymal lung tissue explant culture system with cigarette smoke exposure to investigate the expression of IL-17 and the mechanisms involved. Methods Parenchymal lung tissue removed from 10 non-COPD and 8 COPD patients was sectioned and cultured with different concentrations of cigarette smoke extract (CSE) for 3 or 6 hours. Tissue viability was evaluated by LDH (lactate dehydrogenase) in culture supernatants. Western blot and real-time PCR were performed to evaluate IL-17A/F expression. To investigate the mechanisms, pharmacological inhibitors for MAPK p38, ERK1/2, NF-κB and PI3K pathways were added into the culture media. Results No tissue damage was observed after the cigarette smoke exposure for 3 h or 6 h compared with the control media. At the protein level, the expression of both IL-17A (2.4 ± 0.6 fold) and IL-17 F (3.7 ± 0.7 fold) in the tissue from non-COPD subjects was significantly increased by 5% of CSE at 3 h. For COPD subjects, IL-17A/F expression were significantly increased only at 6 h with 10% of CSE (IL-17A: 4.2 ± 0.8 fold; IL-17 F: 3.3 ± 0.8 fold). The increased expression of IL-17A/F is also regulated at the mRNA level. The inhibitors for NF-κB and PI3K pathways significantly inhibited CSE-induced IL-17A/F expression from lung tissue of non-COPD subjects. Conclusions We found the evidence that the expression of both IL-17A and IL-17 F is increased by the cigarette smoke exposure in explants from both non-COPD and COPD subjects, supporting that local lung cells contribute IL-17 production. The elevated IL-17A/F expression is dependent on NF-κB and PI3K pathways. These observations add to the growing evidence which suggests that Th17 cytokines play a significant role in COPD.

Background Interleukin-33 (IL-33) is a novel cytokine that triggers inflammatory immune responses, but evidence of its role in human asthma, a common allergic airway disease, is lacking. There is also a paucity of information regarding which cells express IL-33, and what conditions promote its expression. We sought to investigate whether IL-33 is expressed in the lung tissue from patients with asthma.