Background Exposure to tobacco smoke (ETS) induces epigenetic modifications including DNA methylation [1]. In asthma, it has been shown that those modifications affect immune cell differentiation by downregulating expression of specific pro-inflammatory cytokines [2-4]. Interleukin 1 (IL-1) is recognized to be increased in asthma [5] and by cigarette smoke [5,6]. Based on previous genetic association [7,8] and DNA methylation signature of receptors in asthma and/or atopy the aim of this study is to evaluate the changes in expression and methylation pattern induced by ETS for IL-1 subunit alpha (IL-1A) and beta (IL-1B), receptors type I (IL-1R1), type II (IL-1R2) and antagonist (IL-1RA) and for interleukin 33 (IL-33) in lung tissue.

BACKGROUND:Airway inflammation is an important characteristic of asthma and has been associated with airway remodelling and bronchial hyperreactivity. The mucosal microenvironment composed of structural cells and highly specialised extracellular matrix is able to amplify and promote inflammation. This microenvironment leads to the development and maintenance of a specific adaptive response characterized by Th2 and Th17. Bronchial fibroblasts produce multiple mediators that may play a role in maintaining and amplifying this response in asthma.OBJECTIVE:To investigate the role of bronchial fibroblasts obtained from asthmatic subjects and healthy controls in regulating Th17 response by creating a local micro-environment that promotes this response in the airways.METHODS:Human bronchial fibroblasts and CD4(+)T cells were isolated from atopic asthmatics and non-atopic healthy controls. CD4(+)T were co-cultured with bronchial fibroblasts of asthmatic subjects and healthy controls. RORc gene expression was detected by qPCR. Phosphorylated STAT-3 and RORγt were evaluated by western blots. Th17 phenotype was measured by flow cytometry. IL-22, IL17, IL-6 TGF-β and IL1-β were assessed by qPCR and ELISA.RESULTS:Co-culture of CD4(+)T cells with bronchial fibroblasts significantly stimulated RORc expression and induced a significant increase in Th17 cells as characterized by the percentage of IL-17(+)/CCR6(+) staining in asthmatic conditions. IL-17 and IL-22 were increased in both normal and asthmatic conditions with a significantly higher amount in asthmatics compared to controls. IL-6, IL-1β, TGF-β and IL-23 were significantly elevated in fibroblasts from asthmatic subjects upon co-culture with CD4(+)T cells. IL-23 stimulates IL-6 and IL-1β expression by bronchial fibroblasts.CONCLUSION:Interaction between bronchial fibroblasts and T cells seems to promote specifically Th17 cells profile in asthma. These results suggest that cellular interaction particularly between T cells and fibroblasts may play a pivotal role in the regulation of the inflammatory response in asthma.

BACKGROUND:Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-β1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFβ1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects.METHODS:We used simvastatin (1-15 μM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 μM) and farnesyl transferase (FT; FTI-277, 10 μM) were used to determine whether GGT1 and FT contribute to TGFβ1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 μM) or farnesylpyrophosphate (30 μM).RESULTS:Immunoblotting revealed concentration-dependent simvastatin inhibition of TGFβ1 (2.5 ng/ml, 48 h)-induced fibronectin. This was prevented by exogenous mevalonate, or isoprenoids (geranylgeranylpyrophosphate or farnesylpyrophosphate). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting fundamental involvement of GGT1 in TGFβ1-induced signaling. Asthmatic fibroblasts exhibited greater TGFβ1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin.CONCLUSIONS:We conclude that TGFβ1-induced fibronectin expression in airway fibroblasts relies on activity of GGT1 and availability of isoprenoids. Our results suggest that targeting regulators of isoprenoid-dependent signaling holds promise for treating airway wall fibrosis.

Cysteinyl leukotrienes (CysLTs) contribute to allergic and inflammatory diseases through CysLT1-R. We aimed to assess CysLT1-R mRNA expression in induced sputum of rhinitics with or without asthma before and following allergen challenges. Both groups underwent nasal and “low dose” lung allergen challenges. Asthmatics also underwent “standard” lung challenge. Sputum was obtained before and at different time-points following the challenges for CysLT1-R, 5-lipoxygenase (5-LO), and eotaxin mRNA assessments. At baseline, there was no difference in mediator levels between groups. An increase in CysLT1-R mRNA (p=0.04) and a trend towards an increase in 5-LO and eotaxin (p=0.06 for both) at 24 h post-nasal challenge were observed. Following “low dose” lung allergen challenge, there was a trend towards an increase in CysLT1-R (p=0.07). In conclusion, CysLT1-R gene expression changes can be detected in sputum following allergen challenges. No difference was observed between groups, suggesting that changes in CysLT1-R expression occur whether or not the subject has concurrent asthma.

Asthma is characterized by airway remodeling associated with an increase in the deposition of ECM proteins such as type I collagen. These components are mainly produced by fibroblasts. Inhaled corticosteroids are considered the cornerstone of asthma therapy. Despite substantial evidence as to the anti-inflammatory action of corticosteroids, their effect on controlling ECM protein deposition in the airways is not completely understood. This study determined the effect of dexamethasone (Dex) on collagen production by bronchial fibroblasts derived from asthmatic and healthy subjects. Expression of procollagen mRNA in fibroblasts from asthmatics and normal controls was determined by quantitative PCR. Regulation of the procollagen-alpha(1)I promoter was evaluated by transient transfections. Transforming growth factor-beta (TGF-beta) protein expression was determined by ELISA. Protein expression of glucocorticoid receptor (GR) and interaction with activator protein-1 (AP-1), a collagen regulatory transcription factor, was assessed by Western blots, coimmunoprecipitations, and EMSA. AP-1 overexpression was performed by transient transfection using c-Fos/c-Jun expression plasmids. Dex significantly downregulated procollagen production and promoter activity in normal fibroblasts but had no effect on asthmatic fibroblasts. AP-1 and GR interaction increased after Dex stimulation in asthmatic fibroblasts. AP-1 overexpression in control fibroblasts abrogated collagen gene response to Dex. These results show that Dex failed to reduce collagen production in fibroblasts from asthmatic subjects. This impaired response may be related to AP-1 overexpression in these cells.

BACKGROUND:Thymic stromal lymphopoietin (TSLP) plays a pivotal role in the initiation of allergic airway inflammation. This cytokine is produced by several cell types, including human epithelial cells. OBJECTIVE:We sought to determine the effect of TSLP on proliferation and repair of epithelial cells isolated from asthmatic patients and healthy subjects. METHODS:Expression of TSLP receptor (TSLPR) and its response to inhaled corticosteroids was evaluated on bronchial biopsy specimens of healthy control subjects and asthmatic patients by means of immunohistochemistry. TSLPR, TSLP, and IL-13 mRNA expression was determined by means of quantitative PCR, and protein expression was measured by means of ELISA and Western blotting in epithelial cells isolated from asthmatic subjects compared with those isolated from healthy control subjects. The effect of TSLP on cell proliferation and wound healing was performed. RESULTS:TSLPR is expressed by bronchial epithelial cells in bronchial biopsy specimens and in cultured cells, with no difference between asthmatic patients and healthy control subjects. Inhaled corticosteroids did not affect this expression. TSLP mRNA and protein levels were significantly higher in epithelial cells isolated from asthmatic patients compared with those from healthy control subjects. TSLP stimulated IL-13 production by bronchial epithelial cells. TSLP induced airway epithelial cell proliferation and enhanced epithelial injury repair. This effect was abrogated with IL-13 neutralization. CONCLUSIONS:Our data indicate that epithelial cells express TSLPR and that TSLP induces bronchial epithelial cell proliferation and increases injury repair through IL-13 production. This suggests that TSLP and IL-13 loops play a homeostatic role on epithelial cell proliferation and repair.

Objective/purposeIn asthma, CD4+T cells are selectively recruited into the bronchial mucosa.CD4+ T cells consist of different subsets that express lineage specific transcription factors and play different roles either in initiating and supporting the development of immune response, but also in orchestrating and regulating them.The aim of our study was to evaluate the effect of T cells-bronchial fibroblasts interaction on CD4+T cell phenotype.

Background. Allergic asthma is characterized by infiltration of inflammatory cells Into the airways T cell-derived cytokines regulate both airway inflammation and remodelling. In the human airways, T cell-fibroblast interactions may have a role in regulating inflammation and remodelling.Objectives To evaluate the effect of bronchial fibroblast-T cell interaction on profibrogenic cytokine release and determine the nature of the molecules involved in this interactionMethods: Human bronchial fibroblasts obtained from healthy and asthmatic donors were co-cultured with purified T cells derived from peripheral blood of the same subjects. IL-6 mRNA and protein levels were measured by real time PCR and ELISA. CD40, CD40L and alpha 5 beta 1 were evaluated by flow cytometry Bronchial fibroblasts were stimulated with rsCD40L Neutralisation was performed using neutralizing antibodies anti-CD40L and anti-alpha 5.Results: Contact of T cells with bronchial fibroblasts up-regulated IL-6 at both gene and protein levels This effect was significantly higher in fibroblasts from asthmatics than those from controls Blocking CD40L and alpha 5 beta 1 integrin showed a significant inhibition of IL-6 expression in asthmatics but not in healthy controls. Stimulation of fibroblasts with recombinant soluble CD40L up-regulated IL-6 production in asthmatics but not in controls Adhesion to fibronectin. a alpha 5 beta 1 integrin ligand, is increased in fibroblasts from asthmatics compared to fibroblasts from controls.Conclusion: These results showed that interaction of bronchial fibroblasts with T cells increases the production of profibrogenic cytokine IL-6. In asthmatic condition this interaction involves CD40L/alpha 5 beta 1 These results suggest that T cells and structural cells crosstalk in asthma may maintain local mucosal inflammation. (C) 2010 Elsevier Ltd All rights reserved

Background: Inflammatory changes such as subepithelial edema and excessive inflammatory cell infiltration have been observed in uvular tissues of obstructive sleep apnea (OSA) subjects. The levels of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin-6 are elevated in the serum of apneic patients and have been proposed as mediators of muscle weakness. TNF-alpha has been shown to affect diaphragm contractility in mice and rabbit in vivo.Objectives: To assess total and compartmental TNF-alpha expression in uvular tissues of apneic and nonapneic patients.Methods: Uvular tissues were collected from 14 snorers without sleep disorders breathing, 14 subjects with OSA (OSA I group) whose body mass index (BMI) was similar to that of snorers, and 12 additional obese OSA subjects (OSA 2 group) who underwent an uvulopalatopharyngoplasty. Sections were examined using immunohistochemistry and Western blot analysis. TNF-alpha expression was evaluated in the musculus uvulae (MU), epithelial layer, and perimuscular tissues from proximal uvular sections.Results: TNF-alpha was more highly expressed in whole uvular protein extracts of apneic groups than in snorers ([mean +/- SEM] snorers, 100.5 +/- 3.0%; OSA I group, 127.1 +/- 6.9%; OSA 2 group, 140.7 +/- 11.0%; p = 0.01). In the muscular area, TNF-alpha levels were higher in the more obese OSA subjects than in the other two groups (snorers, 100.3 +/- 3%; OSA I group, 107.4 +/- 0.7%; OSA 2 group, 124.1 +/- 4.2%; p = 0.007). In the muscular area, TNF-alpha was correlated with BMI, but no relationship was found with the apnea-hypopnea index.Conclusions: We conclude that MU is the major TNF-alpha source in uvular tissue and that TNF-alpha is more highly expressed in the heaviest OSA patients compared to less obese OSA patients and nonapneic snorers. (CHEST 2008; 134:911-918)

In asthma, bronchial mucosal tissues are chronically infiltrated by activated inflammatory cells (mainly lymphocytes and eosinophils) that have the capacity to produce a wide range of cytokines (IL-4, IL-5) and mediators (leucotrienes, prostaglandins, etc.). These molecules may significantly change the functional and phenotypic behavior of resident cells (epithelial cells, fibroblasts, smooth muscle cells, etc.) In addition to their role in tissue repair, resident cells are an important source of cytokines. They play an active role in modulating the immune response, suggesting that they may be involved in the maintenance and chronicity of the inflammatory response. These cells are also involved in structural changes observed in asthma, such as subepithelial fibrosis [3]. The alteration in the structure of the bronchial tree appears to be one of the factors involved in the persistence of bronchial hyperreactivity [4]. This review will focus on the role of structural cells on airway remodeling and their response to asthma medication.

BACKGROUND:Interaction between lymphocytes and structural cells has been proposed as a key factor in regulating inflammation in asthma.OBJECTIVE:This study was designed to investigate the effect of epithelial cells and fibroblasts on T-lymphocyte survival by using a 3-dimensional tissue-engineered model.METHODS:Engineered human bronchial mucosal tissues were produced by using fibroblasts, epithelial cells, and autologous T cells from asthmatic and healthy donors. T-cell apoptosis and apoptotic marker expression by T cells were evaluated by using the terminal deoxynucleotidyl transferase biotinylated d-UTP nick end-labeling technique and immunofluorescence, respectively. Cytokines implicated in T-cell survival were measured by means of ELISA in culture supernatants.RESULTS:We demonstrated histologically that we were able to generate a well-structured engineered bronchial mucosa by using epithelial cells, fibroblasts, and T cells cultured from healthy and asthmatic subjects. Structural cells from asthmatic subjects cultured in this model induced a significant decrease in the ability of T cells to undergo apoptosis represented by a decrease in DNA fragmentation and proapoptotic molecule expression (Bcl-2-associated X protein and Fas ligand). Structural cells from healthy control subjects have no effect. Among cytokines measured in the supernatants, only TGF-beta(1) was significantly increased in the model derived from cells of asthmatic subjects.CONCLUSION:These results support the concept that bronchial structural cells might play a critical role in the regulation of inflammation in asthma by increasing the survival of T lymphocytes. The results also further validated the model as a tool for investigating the interaction between inflammatory and structural cells.

Epithelial damage is an important pathophysiologic feature of asthma. Bronchial epithelium damage results in release of growth factors such as transforming growth factor (TGF)-beta(1) that may affect epithelial cell proliferation. The objective of our study is to evaluate the importance of TGF-beta(1) in regulating epithelial cell repair in asthma. We evaluated the effect of TGF-beta(1) on epidermal growth factor (EGF)-induced proliferation and downstream signaling in epithelial cells obtained from subjects with asthma compared with cells from healthy subjects. Cell proliferation was evaluated by bromodeoxyuridine incorporation. EGF receptor (EGFR), mitogen-activated protein kinase, TGF-beta receptors, Smads, Smad anchor for receptor activation (SARA), and cyclin-dependant kinase inhibitors were evaluated by Western blot. TGF-beta(1) and receptor expression were measured by RT-PCR and by enzyme-linked immunosorbent assay. Proliferation of epithelial cells at baseline and after EGF stimulation was significantly reduced in cells derived from subjects with asthma compared with cells obtained from healthy control subjects. EGF-induced ERK1/2 phosphorylation was reduced in epithelial cells from subjects with asthma compared with cells from healthy control subjects. This was paralleled with a reduced EGFR phosphorylation. Addition of TGF-beta(1) significantly decreased EGF-induced cell proliferation. TGF-beta(1) production was higher in asthmatic epithelial cells compared with normal cells. This was supported by a high expression of pSmad 3 and SARA in cells derived from individuals with asthma compared with normal subjects. Cycline-dependent kinase inhibitors were highly expressed in asthmatic compared with normal cells. Inhibition of TGF-beta(1) signaling in asthmatic epithelial cells restored EGFR, ERK1/2 phosphorylation, and cell proliferation induced by EGF. Our results suggest that TGF-beta restrains EGFR phosphorylation and downstream signaling in bronchial epithelial cells.

Background The purpose of this study was to assess low molecular weight heparin auto-injection skills of self-supporting patients, taught by a nurse through a rapid demonstration in an emergency department. Methods The study was a prospective, multicentre study, carried out in emergency departments. It included all ambulatory attending patients over 18 years of age, who were given a lower limb cast in an emergency ward and provided with limited orthopaedic treatment. Eligible patients who accepted self-injection training were given a quick demonstration by the nurse in the emergency ward. A questionnaire about the perception of the patient was completed at the beginning of the treatment and at the end of the treatment. A surveillance chart for recording injections and side effects was also given to the patient, and platelet counts were performed twice weekly. Findings Two hundred and fourteen patients have been assessed. Forty-four patients (20.5%) were judged inappropriate for training (n=19) or refused it (n=25). Primary perception of the self-injection method showed absence of fear of injections among patients in 43.7% of cases. Training was successful in 88% of cases. Twenty-one patients out of 170 (12%) gave up after completing the training. Questionnaires given at the end of the treatment found a generally favourable opinion on self-injections among 73.26% of patients (n=86). Compliance was good – 95.5% of patients completed all their injections. Platelet counts were considered appropriate only in 52.5% of cases. Interpretation It seems possible to extend the practice of self-injection to other types of injections prescribed after discharge from the emergency department, such as preventive low molecular weight heparin for surgical or medical purposes and curative ambulatory low molecular weight heparin treatment for deep vein thrombosis.