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Objective: Bronchial thermoplasty is an effective intervention to improve respiratory symptoms and to reduce the rate of exacerbations in uncontrolled severe asthma. A reduction in airway smooth muscle is arguably the most widely discussed mechanisms accounting for these clinical benefits. Yet, this smooth muscle reduction should also translate into an impaired response to bronchodilator drugs. A pilot study was designed to address this question. Hypothesis: Thermoplasty decreases the response to a bronchodilator. Methods: Lung functions measured by spirometry and respiratory mechanics measured by oscillometry (tremoFlo, Thorasys) were examined pre- and post-bronchodilator (salbutamol, 400 μg) before and after at least 1 year of thermoplasty in 8 patients (7 women) with uncontrolled severe asthma. For each readout, a two-way ANOVA was used to assess the effect of thermoplasty, salbutamol, and their interaction. All procedures were approved by the IUCPQ Ethics Committee. Data: Consistent with previous studies, thermoplasty yielded no benefits in terms of baseline lung function and respiratory mechanics, despite improving symptoms based on two questionnaires assessing the level of asthma control (Asthma Control Questionnaire – ACQ-5, and Asthma Control Test – ACT-5). The response to salbutamol was not affected by thermoplasty based on spirometric readouts, including forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) and FEV1/FVC ratio. However, a significant interaction was observed between thermoplasty and salbutamol for two oscillometric readouts. The reactance of the respiratory system at 5 Hz (Xrs5) increased from -3.2 ±2.6 to -1.9 ± 1.9 cmH2O ·s/L in response to salbutamol pre-thermoplasty, versus from -2.8 ± 2.3 to -2.2 ± 1.9 cmH2O·s/L post-thermoplasty (interaction, p=0.04). Similarly, the reactance area (Ax) decreased from 32.6 ± 29.0 to 12.8 ±18.2 cmH2O/L in response to salbutamol pre-thermoplasty, versus 27.2 ± 29.4 to 15.7 ± 20.1 cmH2O/L post-thermoplasty (interaction, p=0.02). The salbutamol-induced changes in resistance at 5 Hz (Rrs5), resistance at 19 Hz (Rrs19), Rrs5 minus Rrs19, and resonant frequency were not significantly affected by thermoplasty. Summary of results: While spirometry was unable to detect changes in the response to salbutamol caused by thermoplasty, two oscillometric readouts, namely Xrs5 and Ax, confirmed that the bronchodilator response is attenuated post-thermoplasty. Conclusion: Despite its small size, this study demonstrates that thermoplasty attenuates the response to a bronchodilator. We argue that this result is a physiological proof of therapeutic efficacy, consistent with the well-described effect of thermoplasty in reducing the amount of airway smooth muscle. FQSR (Fondation Quebecoise en Sante Respiratoire); Canadian Institutes of Health Research (PJT-387910); FRQS (Fonds de recherche du Quebec - Sante); and Boston Scientific Inc This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Aim: This study aimed to characterize DNA methylation (DNA-me) in promoter region of IL33, IL1RL1 and CCL26 in asthma and their impacts on transcriptional activity in bronchial epithelial cells (BECs). Patients & methods: We performed bis-pyrosequencing, quantitative real-time PCR and sequencing in BECs from ten asthmatic and ten control individuals. Results: We detected lower DNA-me levels of IL33 and CCL26 in asthmatic than control BECs. No correlation was found between methylation and expression levels. Interestingly, carriers of a mutative allele in a haplotype within the promoter of IL33 had a lower IL33 DNA-me level and CCL26 gene expression correlated with eosinophil count. Conclusion: These findings highlight the importance of investigating both epigenetic and genetic mechanisms in understanding the epithelial immune response in asthma.

In an effort to reduce smoking-related diseases, alternative products such as e-cigarettes have been proposed. However, despite their growing popularity, the potential toxicity of e-cigarettes remains largely unknown. In this study, human gingival fibroblasts were repeatedly exposed to cigarette smoke condensate (CSC) and to nicotine-rich (NR) or nicotine-free (NF) e-vapor condensates for 60 min once a day for various time periods. They were then used to perform different analyses. Results indicate that cells exposed to CSC or NR condensates showed an altered morphology and a reduced proliferation rate, as ascertained by MTT and BrdU assays. Fibroblast cultures exposed to either CSC or e-vapor condensates also showed increased levels of TUNEL-positive apoptotic cells, compared to that recorded in the control. Furthermore, the cell scratch test revealed that repeated exposures to CSC or to e-vapor condensates delayed both fibroblast migration and wound healing. It should be noted that CSC was much more damageable to gingival fibroblasts than were the NR and NF e-vapor condensates. The representative chain of damage thus translates to CSC > NR e-vapor condensate > NF e-vapor condensate.

// Hesam Movassagh 1 , Lianyu Shan 1 , Jonathan S. Duke-Cohan 2 , Jamila Chakir 3 , Andrew J. Halayko 4, 5 , Latifa Koussih 1 and Abdelilah S. Gounni 1 1 Department of Immunology, Faculty of Health Sciences, College of Medicine, University of Manitoba, Winnipeg, MB, Canada 2 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Institutes of Medicine, Boston, MA, USA 3 Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie du Quebec, Universite´ Laval, Quebec City, QC, Canada 4 Department of Physiology & Pathophysiology, Faculty of Health Sciences, College of Medicine, University of Manitoba, Winnipeg, MB, Canada 5 Biology of Breathing Group, Children’s Hospital Research Institute of Manitoba, Winnipeg, MB, Canada Correspondence to: Abdelilah S. Gounni, email: abdel.gounni@umanitoba.ca Keywords: airway hyperresponsiveness; chronic allergic asthma; inflammation; remodeling; semaphorin 3E Received: May 30, 2017 Accepted: August 26, 2017 Published: October 27, 2017 ABSTRACT Guidance cues such as semaphorins are attractive novel therapeutic targets for allergic disorders. We have previously described an inhibitory effect of semaphorin 3E (Sema3E) on human airway smooth muscle cell function. We have further addressed a canonical role for Sema3E in acute model of allergic asthma in vivo . Considering the chronic nature of the disease, the potential implication of Sema3E to alleviate long-lasting deficits should be investigated. Expression of Sema3E in a chronic model of allergic asthma was assessed after exposure to house dust mite (HDM) as a clinically relevant allergen. Chronic features of allergic asthma including airway hyper-responsiveness (AHR), inflammation, and remodeling were studied in Sema3E-deficient mice. Additionally, the effect of exogenous Sema3E treatment was evaluated in prophylactic and therapeutic experimental models. We have demonstrated that expression of Sema3E is robustly suppressed in the airways upon chronic HDM exposure. Chronic allergic airway disease was significantly augmented in Sema3E-deficient mouse model which was associated with an increased AHR, remodeling, and Th2/Th17 inflammation. Intranasal Sema3E administration restored chronic deficits of allergic asthma in mice. Data from this study unveil a key regulatory role of Sema3E in chronic course of asthma via orchestration of impaired inflammatory and remodeling responses.

Background Bronchial fibroblasts play a key role in airway remodelling in asthma. They regulate epithelial cell functions such as proliferation through growth factors, cytokines, chemokines and exosomes. The role of exosomes in the communication between epithelial cells and fibroblasts by vehiculing these mediators in asthma remains to be determined. Objective To evaluate the role of exosomes released by bronchial fibroblasts on epithelial cell proliferation in severe asthma. Methods Exosomes were obtained from culture media of primary bronchial fibroblasts and characterized using Western blot, electron microscopy and flow cytometry. Uptake profile of fluorescent-labelled exosomes in epithelial cells was assessed by flow cytometry. Exosome cytokine content was analysed by Cytokine Arrays. Bronchial epithelial cell proliferation was evaluated by BrdU incorporation test. Exosome biogenesis/release was blocked using sphingomyelinase inhibitor. Plasmid transfection was used to modulate transforming growth factor beta 2 (TGF-β2) gene expression. Results We showed that bronchial fibroblasts secreted exosomes, which were internalized by bronchial epithelial cells. Exosomes of severe asthmatic subjects' fibroblasts showed a lower level of TGF-β2 and significantly increased the epithelial cell proliferation of both healthy and severe asthmatic subjects compared to healthy controls' exosomes. Overexpression of TGF-β2 in severe asthmatics' fibroblasts induced enhanced TGF-β2 in exosomes leading to a reduced proliferation of epithelial cells, whereas knockdown of TGF-β2 enhanced epithelial cell proliferation. Conclusion Our study shows that exosomes are involved in fine-tuning intercellular communication in asthma. Exosomes of severe eosinophilic asthmatics' fibroblasts can contribute to airway remodelling, at least in part, by modulating epithelial cell proliferation observed in severe asthma.

Atopy is characterized by the production of a high level of IgE in response to common allergens. We examined the RsaI-in2 polymorphism of the FceRI-β gene and its association with atopic asthma and comorbidity. The study population included 77 atopic asthmatics and 77 non-atopic, non-allergic and non-asthmatic controls. Atopic asthma was confirmed by the skin prick test and by IgE and interleukin IL-4 levels. RsaI-in2 polymorphism was determined by RFLP-PCR using the RsaI enzyme. A significant correlation was found between genotype BB and sensitization to house dust mites in the asthmatic subjects (p = 0.000). Results also reveal significant allele sharing in the subjects affected by eczema (p = 0.000) and allergic rhinitis (p = 0.01) during childhood. Furthermore, the subjects with heterozygous AB and homozygous BB genotypes displayed significant IgE levels > 1000 UI mL-1. IL-4 levels were also > 1000 pmol/ml for RsaI-in2 polymorphism genotype BB. Our findings show a strong association between genotype BB and the incidence of eczema and allergic rhinitis in childhood as well as increased IgE and IL-4 levels.

Background: Bronchial Thermoplasty (BT) reduces exacerbations and improves asthma quality of life questionnaire (AQLQ) scores in asthma [1]. Studies have shown reductions in airway smooth muscle (ASM) mass and reticular basement membrane (RBM) thickness after BT, but are limited by small sample size. Aims: To investigate airway remodeling responses to BT, and compare these to clinical outcomes. Methods: Pre- and post-BT data was pooled for 99 patients at 7 centres. ASM mass and RBM thickness were measured on bronchial biopsies, and compared to clinical outcomes. Results: Mean (SD) age was 48.4 years (11.7) and BMI was 29.0 kg/m2 (6.4). Baseline FEV1 was 68.8% predicted (23.1) and blood eosinophils were 0.25x10-9/L (0.26). 59 of 99 patients were taking maintenance oral Prednisolone (mean dose 25.7mg). In response to BT, mean (SD) annual exacerbation rate decreased from 6.4 (5.5) to 1.1 (1.7) (p= Conclusion: ASM mass and RBM thickness reduce rapidly following BT, and effects persist beyond 1 year. The observed improvements in airway remodeling parameters are not related to improvements in clinical outcomes. Other bronchial mucosal changes or mechanisms may play a role in the clinical response to BT. Reference: 1) Castro et al, AJRCCM. 2010;181(2):116-24

The molecular basis for the propensity of a small number of environmental proteins to provoke allergic responses is largely unknown. Herein, we report that mite group 13 allergens of the fatty acid-binding protein (FABP) family are sensed by an evolutionarily conserved acute-phase protein, serum amyloid A1 (SAA1), that promotes pulmonary type 2 immunity. Mechanistically, SAA1 interacted directly with allergenic mite FABPs (Der p 13 and Blo t 13). The interaction between mite FABPs and SAA1 activated the SAA1-binding receptor, formyl peptide receptor 2 (FPR2), which drove the epithelial release of the type-2-promoting cytokine interleukin (IL)-33 in a SAA1-dependent manner. Importantly, the SAA1–FPR2–IL-33 axis was upregulated in nasal epithelial cells from patients with chronic rhinosinusitis. These findings identify an unrecognized role for SAA1 as a soluble pattern recognition receptor for conserved FABPs found in common mite allergens that initiate type 2 immunity at mucosal surfaces. Smole and colleagues show that the soluble pattern recognition receptor serum amyloid A (SAA) recognizes several mite allergenic proteins, including Der p 13 and Blo t 13, which are conserved fatty acid-binding proteins. Such FABP–SAA1 binding triggers epithelial cell release of the type-2-promoting cytokine IL-33, which in turn drives IL-13 production and allergic syptoms.

Electronic cigarettes represent an increasingly significant proportion of today's consumable tobacco products. E-cigarettes contain several chemicals which may promote oral diseases. The aim of this study was to investigate the effect of e-cigarette vapor on human gingival epithelial cells. Results show that e-cigarette vapor altered the morphology of cells from small cuboidal form to large undefined shapes. Both single and multiple exposures to e-cigarette vapor led to a bulky morphology with large faint nuclei and an enlarged cytoplasm. E-cigarette vapor also increased L-lactate dehydrogenase (LDH) activity in the targeted cells. This activity was greater with repeated exposures. Furthermore, e-cigarette vapor increased apoptotic/necrotic epithelial cell percentages compared to that observed in the control. Epithelial cell apoptosis was confirmed by TUNEL assay showing that exposure to e-cigarette vapor increased apoptotic cell numbers, particularly after two and three exposures. This negative effect involved the caspase-3 pathway, the activity of which was greater with repeated exposure and which decreased following the use of caspase-3 inhibitor. The adverse effects of e-cigarette vapor on gingival epithelial cells may lead to dysregulated gingival cell function and result in oral disease. J. Cell. Physiol. 232: 1539-1547, 2017. © 2016 Wiley Periodicals, Inc.

Airway smooth muscle (ASM) hyperplasia is a key feature of airway remodeling in development of lung diseases such as asthma. Anomalous proliferation of ASM cells directly contributes to ASM hyperplasia. However, the molecular mechanisms controlling ASM cell proliferation are not completely understood. Semaphorins are versatile regulators of various cellular processes including cell growth and proliferation. The role of semaphorins in ASM cell proliferation has remained to be addressed. Here, we report that semaphorin 3A (Sema3A) receptor, neuropilin 1 (Nrp1), is expressed on human ASM cells (HASMC) isolated from healthy and asthmatic donors and treatment of these cells with exogenous Sema3A inhibits growth factor-induced proliferation. Sema3A inhibitory effect on HASMC proliferation is associated with decreased tyrosine phosphorylation of PDGFR, downregulation of Rac1 activation, STAT3 and GSK-3β phosphorylation. Bronchial sections from severe asthmatics displayed immunoreactivity of Nrp1, suggestive of functional contribution of Sema3A-Nrp1 axis in airway remodeling. Together, our data suggest Sema3A-Nrp1 signaling as a novel regulatory pathway of ASM hyperplasia.

Objective Upon use, e-cigarette aerosol comes in contact with various mucosal tissues, including the nasal epithelium, which may lead to nasal pathologies. We therefore assessed the effect of e-cigarettes on nasal epithelial cell and tissue behaviours. Methods Human primary nasal epithelial cells and engineered 3D nasal mucosa tissues were exposed or not to either e-cigarette aerosol or standard cigarette smoke. We then evaluated cell viability and lactate dehydrogenase (LDH) activity. With the tissues analysed tissue structure, the expression of Ki67 proliferating marker, and the secretion of pro-inflammatory cytokines by the engineered nasal mucosa. Results The nasal epithelial cells exposed to e-cigarettes displayed a larger cell size and a faint nucleus following exposure to e-cigarettes. This is supported by the increased levels of LDH activity following exposure to e-cigarettes, compared to that observed in the control. Tissues exposed to e-cigarette aerosol displayed a structural deregulation, with more large-sized cells, fewer Ki67-positive cells, and a reduced proliferation rate, compared to that observed in the non-exposed tissues. Cytokine measurements showed high levels of IL-6, IL-8, TNFα, and MCP-1, demonstrating that e-cigarettes activated pro-inflammatory cytokine responses. Conclusion E-cigarette aerosol showed adverse effects on nasal epithelial cells and nasal engineered mucosa tissue. These findings indicate that e-cigarettes could be a threat to nasal tissues and may impair the innate immune function of nasal epithelial cells.

The key factors underlying the development of allergic diseases-the propensity for a minority of individuals to develop dysfunctional responses to harmless environmental molecules-remain undefined. We report a pathway of immune counter-regulation that suppresses the development of aeroallergy and shrimp-induced anaphylaxis. In mice, signaling through epithelially expressed dectin-1 suppresses the development of type 2 immune responses through inhibition of interleukin-33 (IL-33) secretion and the subsequent recruitment of IL-13-producing innate lymphoid cells. Although this homeostatic pathway is functional in respiratory epithelial cells from healthy humans, it is dramatically impaired in epithelial cells from asthmatic and chronic rhinosinusitis patients, resulting in elevated IL-33 production. Moreover, we identify an association between a single-nucleotide polymorphism (SNP) in the dectin-1 gene loci and reduced pulmonary function in two cohorts of asthmatics. This intronic SNP is a predicted eQTL (expression quantitative trait locus) that is associated with reduced dectin-1 expression in human tissue. We identify invertebrate tropomyosin, a ubiquitous arthropod-derived molecule, as an immunobiologically relevant dectin-1 ligand that normally serves to restrain IL-33 release and dampen type 2 immunity in healthy individuals. However, invertebrate tropomyosin presented in the context of impaired dectin-1 function, as observed in allergic individuals, leads to unrestrained IL-33 secretion and skewing of immune responses toward type 2 immunity. Collectively, we uncover a previously unrecognized mechanism of protection against allergy to a conserved recognition element omnipresent in our environment.

Background: Corticosteroids (CS) and long acting b2-agonists (LABA) are mainstay of therapy for persistent asthma. Inhaled asthma medications are associated with oropharyngeal disorders and low salivary flow rate which may affect oral epithelial cell innate immunity. Objectives: To evaluate the effect of formoterol and mometasone on Candida albicans growth and transition, and on the expression of Toll-like receptors (TLR) and b-defensin expression by gingival epithelial cells. Methods: C. albicans (103 cells) were incubated with formoterol (10 or 50 ng/ml) or mometasone (10 or 50 ng/ml) separately or in combination, then the yeast growth and transition from blastospore to hyphe forms were evaluated. The effects of the mometasne or formoterol on the expression of TLR-2 and -4 as well as human b-defensin-2 expression by gingival epithelial cells were investigated. Results: Formoterol at 10 ng/ml promoted the growth of C. albicans as compared to the control or to the mometasone. Used separately or in combination, formoterol and mometasone increased the transition of C. albicans to hyphe form which may increase C. albicans pathogenesis. C. albicans growth and form changing may be due to the deregulation of the epithelial cell defense. Our results showed that the expression of TLR2 and 4 were down-regulated by formoterol as compared to mometasone. We also showed that single or combined molecules induced b-defensin-2 increase, but higher effect was obtained with mometasone. In conclusion: Our data show that CS and LABA promote in vitro C. albicans out growth and affect oral epithelial cell innate immunity (Study funded by the Fonds Emile-Beaulieu).

Activated bronchial epithelial cells (BEC) release various alarmins, including thymic stromal lymphopoietin (TSLP), that drive type 2 inflammation. We hypothesize that BEC-derived factors promote in situ eosinophil differentiation and maturation, a process that is driven by an IL-5-rich microenvironment in asthmatic airways. To assess the eosinophilopoietic potential of epithelial-derived factors, eosinophil/basophil colony forming units (Eo/B-CFU) were enumerated in 14-day methylcellulose cultures of blood-derived nonadherent mononuclear cells incubated with BEC supernatants (BECSN) from healthy nonatopic controls (n = 8), mild atopic asthmatics (n = 9), and severe asthmatics (n = 5). Receptor-blocking antibodies were used to evaluate the contribution of alarmins. Modulation of the mRNA expression of transcription factors that are crucial for eosinophil differentiation was evaluated. BECSN stimulated the clonogenic expansion of eosinophil progenitors in vitro. In the presence of IL-5, Eo/B-CFU numbers were significantly greater in cocultures of BESCN from severe asthmatics compared with other groups. This was attenuated in the presence of a TSLP (but not an IL-33) receptor-blocking antibody. Recombinant human TSLP (optimal at 100 pg/ml) stimulated Eo/B-CFU growth, which was significantly enhanced in the presence of IL-5 (1 ng/ml). Overnight culture of CD34+ cells with IL-5 and TSLP synergistically increased GATA-binding factor 2 and CCAAT/enhancer-binding protein α mRNA expression. The eosinophilopoietic potential of factors derived from BEC is increased in severe asthma. Our data suggest that TSLP is a key alarmin that is produced by BECs and promotes in situ eosinophilopoiesis in a type 2-rich microenvironment.

Background Bronchial fibroblasts are the main structural cells responsible for extracellular matrix production and turnover in lung tissue. They play a key role in airway remodelling in asthma through different cytokines including interleukin (IL-6). Objective To decipher IL-6 signalling in bronchial fibroblasts obtained from severe eosinophilic asthmatics compared to mild asthmatics and healthy controls. Methods Human bronchial fibroblasts were isolated from bronchial biopsies of mild and severe eosinophilic asthmatics and non-atopic healthy controls. IL-6 was assessed by qRT-PCR and ELISA. Phosphorylated STAT3, SHP2 and p38/MAPK were evaluated by Western blot. Chemical inhibitors for SHP2 and p38 were used. Fibroblast proliferation was evaluated by BrdU incorporation test. Results IL-6 release was significantly increased in fibroblasts from mild and severe asthmatics compared to healthy controls. Fibroblasts from severe asthmatics showed a reduced STAT3 activation compared to mild asthmatics and healthy controls. Constitutive activation of phosphatase SHP2 was found to negatively regulate IL-6 induced STAT3 phosphorylation in fibroblasts from severe asthmatics. This effect was accompanied by a decrease in fibroblast proliferation rate due to the activated p38/mitogen-activated protein kinase. SHP2 and p38/MAPK specific inhibitors (PHPS1 and SB212190) significantly induce a restoration of STAT3 phosphorylation, IL-6 target gene expression and cell proliferation. Conclusion These data show dysregulated IL-6 signalling in bronchial fibroblasts derived from severe eosinophilic asthmatic subjects involving the protein tyrosine phosphatase SHP2 and p38MAPK. Collectively, our data provides new insights into the mechanisms by which bronchial fibroblasts regulate airway remodelling in severe asthma.

Th17 cells play a critical role in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms by which these cells regulate the development of RA are not fully understood. We have recently shown that α2β1 integrin, the receptor of type I collagen, is the major collagen-binding integrin expressed by human Th17 cells. In this study, we examined the role of α2β1 integrin in Th17-mediated destructive arthritis in the murine model of collagen-induced arthritis (CIA). We found that α2β1 integrin is expressed on synovial Th17 cells from CIA mice and its neutralization with a specific mAb significantly reduced inflammation and cartilage degradation, and protected the mice from bone erosion. Blockade of α2β1 integrin led to a decrease in the number of Th17 cells in the joints and to a reduction of IL-17 levels in CIA mice. This was associated with an inhibition of receptor activator of NF-κB ligand levels and osteoclast numbers, and reduction of bone loss. We further show that α2β1 integrin is expressed on synovial Th17 cells from RA patients, and that its ligation with collagen costimulated the production of IL-17 by polarized human Th17 cells by enhancing the expression of retinoic acid receptor–related orphan receptor C through ERK and PI3K/AKT. Our findings provide the first evidence, to our knowledge, that α2β1 integrin is an important pathway in Th17 cell activation in the pathogenesis of CIA, suggesting that its blockade can be beneficial for the treatment of RA and other Th17-associated autoimmune diseases.

Background: Bronchial thermoplasty (BT) improves asthma symptoms by reducing the airway smooth muscle (ASM) mass. A durable efficacy and safety of BT on asthma control has been documented out to 5 years in patients with severe persistent asthma. The ASM reduction has been demonstrated in bronchial biopsies taken 3 weeks or 3 months post-BT. BT also decreased type 1 collagen deposition and sub-epithelial basement membrane thickening. The persistence of these structural changes over time with durable clinical benefits observed in clinical trials remains uncertain. Objectives: To assess long-term efficacy of BT on ASM mass and sub-epithelial collagen deposition. Methods: Nine subjects (age: 30-70 years; FEV1: 60-105% of predicted; ICS: 500-1500µg fluticasone equivalent) with persistent asthma underwent BT procedures. Bronchial biopsies were taken before (baseline), and at 3-14 weeks and 27-48 months after BT. Histology and immunohistochemistry studies were performed. Clinical evaluation was performed at 12 and ≥27 months post-BT. Results: ASM area decreased from 11.8% ±1.24% at baseline to 4.7± 0.95% at week 3 post BT and this decrease persisted at ≥27 months post-BT: 4.6±1.05%, P=0.0002 compared to baseline. The type I collagen deposition decreased from 6.7±0.4 µm at baseline to 4.5±0.5 µm 3 weeks post-BT and remained unchanged at long-term followup: 4.6±0.5 µm (P=0.003). At 12 and ≥27 months, BT improved significantly the Asthma Control Scoring Score compared to baseline and decreased the number of severe exacerbations. Conclusion: These data showed long-term (≥27 months) benefits from BT in terms of ASM mass reduction, sub-epithelial collagen deposition and asthma control.

Summary Hox genes encode transcription factors controlling complex developmental processes in various organs. Little is known, however, about how HOX proteins control cell fate. Herein, we demonstrate that the goblet cell metaplasia observed in lung airways from Hoxa5−/− mice originates from the transdifferentiation of Clara cells. Reduced CC10 expression in Hoxa5−/− embryos indicates that altered cell specification occurs prior to birth. The loss of Hoxa5 function does not preclude airway repair after naphthalene exposure, but the regenerated epithelium presents goblet cell metaplasia and less CC10-positive cells, demonstrating the essential role of Hoxa5 for correct differentiation. Goblet cell metaplasia in Hoxa5−/− mice is a FOXA2-independent process. However, it is associated with increased Notch signaling activity. Consistent with these findings, expression levels of activated NOTCH1 and the effector gene HEY2 are enhanced in patients with chronic obstructive pulmonary disease. In vivo administration of a γ-secretase inhibitor attenuates goblet cell metaplasia in Hoxa5−/− mice, highlighting the contribution of Notch signaling to the phenotype and suggesting a potential therapeutic strategy to inhibit goblet cell differentiation and mucus overproduction in airway diseases. In summary, the loss of Hoxa5 function in lung mesenchyme impacts on epithelial cell fate by modulating Notch signaling.

Neutrophils and eosinophils are important sources of bioactive lipids from the 5- and the 15-lipoxygenase (LO) pathways. Herein, we compared the effectiveness of humans eosinophils and eosinophil-depleted neutrophils to synthesize 15-LO metabolites using a cocktail of different 15-LO substrates as well as their sensitivities to eight documented 15-lipoxygenase inhibitors. The treatment of neutrophils and eosinophils with linoleic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and arachidonyl-ethanolamide, led to the synthesis of 13-HODE, 15-HETrE, 15-HETE, 15-HEPE, 14-HDHA/17-HDHA, and 15-hydroxy-AEA. Neutrophils and eosinophils also metabolized the endocannabinoid 2-arachidonoyl-glycerol into 15-HETE-glycerol, although this required 2-arachidonoyl-glycerol hydrolysis inhibition. Neutrophils and eosinophils differed in regard to dihomo-γ-linolenic acid and linoleic acid utilization with 15-HETrE/13-HODE ratios of 0.014 ± 0.0008 and 0.474 ± 0.114 for neutrophils and eosinophils respectively. 15-LO metabolite synthesis by neutrophils and eosinophils also differed in regard to their relative production of 17-HDHA and 14-HDHA.The synthesis of 15-LO metabolites by neutrophils was concentration-dependent and rapid, reaching a plateau after one minute. While investigating the biosynthetic routes involved, we found that eosinophil-depleted neutrophils express the 15-lipoxygenase-2 but not the 15-LO-1, in contrast to eosinophils which express the 15-LO-1 but not the 15-LO-2. Moreover, 15-LO metabolite synthesis by neutrophils was not inhibited by the 15-LO-1 inhibitors BLX769, BLX3887, and ML351. However, 15-LO product synthesis was partially inhibited by 100 μM NDGA. Altogether, our data indicate that the best 15-LO-1 inhibitors in eosinophils are BLX3887, BLX769, NDGA and ML351 and that the synthesis of 15-LO metabolites by neutrophils does not involve the 15-LO-1 nor the phosphorylation of 5-LO on Ser-663 but is rather the consequence of 15-LO-2 or another unidentified 15-LO.

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-α1I promoter was evaluated by transient transfections. Transforming growth factor-β (TGF-β) 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 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 interaction. Methods 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 α5β1 were evaluated by flow cytometry. Bronchial fibroblasts were stimulated with rsCD40L. Neutralisation was performed using neutralizing antibodies anti-CD40L and anti-α5. 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 α5β1 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 α5β1 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/α5β1. These results suggest that T cells and structural cells crosstalk in asthma may maintain local mucosal inflammation.

Cysteinyl leukotrienes (CysLTs) contribute to allergic and inflammatory diseases through CysLT 1 -R. We aimed to assess CysLT 1 -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 CysLT 1 -R, 5-lipoxygenase (5-LO), and eotaxin mRNA assessments. At baseline, there was no difference in mediator levels between groups. An increase in CysLT 1 -R mRNA ( p =0.04) and a trend towards an increase in 5-LO and eotaxin ( p =0.06 for both) at 24h post-nasal challenge were observed. Following "low dose" lung allergen challenge, there was a trend towards an increase in CysLT 1 -R ( p =0.07). In conclusion, CysLT 1 -R gene expression changes can be detected in sputum following allergen challenges. No difference was observed between groups, suggesting that changes in CysLT 1 -R expression occur whether or not the subject has concurrent asthma.

Summary Background Corticosteroid insensitivity in asthmatics is associated with an increased expression of glucocorticoid receptor-β (GR-β) in many cell types. T-helper type 17 (Th17) cytokine (IL-17A and F) expressions increase in mild and in difficult-to-treat asthma. We hypothesize that IL-17A and F cytokines alone or in combination, induce the expression of GR-β in bronchial epithelial cells. Objectives To confirm the expression of the GR-β and IL-17 cytokines in the airways of normal subjects and mild asthmatics and to examine the effect of cytokines IL-17A and F on the expression of GR-β in bronchial epithelial cells obtained from normal subjects and asthmatic patients. Methods The expression of IL-17A and F, GR-α and GR-β was analysed in bronchial biopsies from mild asthmatics and normal subjects by Q-RT-PCR. Immunohistochemistry for IL-17 and GR-β was performed in bronchial biopsies from normal and asthmatic subjects. The expression of IL-6 in response to IL-17A and F and dexamethasone was determined by Q-RT-PCR using primary airway epithelial cells from normal and asthmatic subjects. Results We detected significantly higher levels of IL-17A mRNA expression in the bronchial biopsies from mild asthmatics, compared with normal. GR-α expression was significantly lower in the biopsies from asthmatics compared with controls. The expression of IL-17F and GR-β in biopsies from asthmatics was not significantly different from that of controls. Using primary epithelial cells isolated from normal subjects and asthmatics, we found an increased expression of GR-β in response to IL-17A and F in the cells from asthmatics (P0.05). This effect was only partially significant in the normal cells. Dexamethasone significantly decreased the IL-17-induced IL-6 expression in cells from normal individuals but not in those from asthmatics (P0.05). Conclusion Evidence of an increased GR-β expression in epithelial cells following IL-17 stimulation suggests a possible role for Th17-associated cytokines in the mechanism of steroid hypo-responsiveness in asthmatic subjects. Cite this as: A. Vazquez-Tello, A. Semlali, J. Chakir, J. G. Martin, D. Y. Leung, D. H. Eidelman and Q. Hamid, Clinical & Experimental Allergy, 2010 (40) 1312–1322.

Bacteria, moulds, endotoxin and quartz from respirable dust of agricultural and industrial buildings are typically incriminated for the respiratory health decline of exposed workers despite that dust being an undefined mixture and quantification methods of aerosolized bacteria, moulds or endotoxin not being standardized yet. We developed an in vitro alveolar epithelial cell system in which biological activities of peat moss factories' dust might be correlated to bacteria, mould, endotoxin and quartz concentrations of the analyzed samples. Following exposure, interleukin-8 protein secretion, necrosis and apoptosis of the exposed A549 cells were monitored respectively with ELISA on cell supernatants, trypan blue exclusion and DNA fragmentation detection by flow cytometry. Respirable dust was collected with liquid impingers and respirable quartz with 10mm Dorr-Oliver cyclones. We quantified mesophilic bacteria, mesophilic moulds and endotoxins from liquid impinger samples. No correlation was observed between biological activities of dust and bacteria, mould, endotoxin or quartz concentrations under our experimental conditions. Our speculation is that simple measurements, such as dust concentrations, may not be adequate indicators of the human respiratory health hazard for a given environment.

To cite this article: Semlali A, Jacques E, Rouabhia M, Milot J, Laviolette M, Chakir J. Regulation of epithelial cell proliferation by bronchial fibroblasts obtained from mild asthmatic subjects. Allergy 2010; 65: 1438–1445. Abstract Background: Bronchial epithelium is considered a key player in coordinating airway wall remodelling. The function of epithelial cells can be modulated by the underlying fibroblasts through autocrine and paracrine mechanisms. Objective: To investigate the effect of phenotypic changes in bronchial fibroblasts from asthmatic subjects on epithelial cell proliferation. Methods: Epithelial cells and fibroblasts derived from bronchial biopsies of asthmatic and healthy controls were cultured in an engineered model. Proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromid (MTT). Epidermal growth factor receptor (EGFR), cyclin-dependent kinase inhibitors p21 and p27 were measured by western blots. Total and active forms of transforming growth factor (TGF)-β1 were measured using ELISA and bioassay. TGF-β was inhibited using a recombinant TGF-β soluble receptor II protein. Results: Proliferation of epithelial cells from asthmatics (AE) is increased when cells were cultured with fibroblasts from normal controls (NF). Fibroblasts from asthmatics (AF) significantly decreased the proliferation of epithelial cells from healthy subjects (NE). Activation of p21, p27, EGFR and TGF-β1 reflects the proliferation data by decreasing in AE cultured with NF and increasing in NE cultured with AF. Neutralization of TGF-β increased proliferation of epithelial cells cultured in the asthmatic model. Conclusion: Fibroblasts from asthmatic subjects regulate epithelial cell prolifearation, and TGF-β signalling may represent one of the pathway involved in these interactions.

Background House dust mite (HDM) induces allergic asthma in sensitized individuals, although the mechanisms by which HDM is sensed and recognized by the airway mucosa, leading to dendritic cell (DC) recruitment, activation, and subsequent T H 2-mediated responses, are unknown. Objective We sought to define the pathways by which HDM activates respiratory epithelium to induce allergic airway responses. Methods Using a human airway epithelial cell line (16HBE14o-), we studied secretion of the DC chemokine CCL20 after exposure to HDM or other allergens, investigated components of the HDM responsible for the induction of chemokine release, and examined activation of signaling pathways. Central findings were also confirmed in primary human bronchial cells. Results We demonstrate that exposure of airway epithelium to HDM results in specific and rapid secretion of CCL20, a chemokine attractant for immature DCs. The induction of CCL20 secretion is dose and time dependent and quite specific to HDM because other allergens, such as ragweed pollen and cockroach antigen, fail to significantly induce CCL20 secretion. Induction of CCL20 secretion is not protease or Toll-like receptor 2/4 dependent but, interestingly, relies on β-glucan moieties within the HDM extract, as evidenced by the ability of other β-glucans to competitively inhibit its secretion and by the fact that disruption of these structures by treatment of HDM with β-glucanase significantly reduces subsequent chemokine secretion. Conclusion Taken together, our results describe a novel mechanism for specific pattern recognition of HDM-derived β-glucan moieties, which initiates allergic airway inflammation and, through recruitment of DCs, might link innate pattern recognition at the airway surface with adaptive immune responses.