Asthmatic Bronchial Smooth Muscle Cells Research Articles

Anti-inflammatory and relaxation effects of Ulmus pumilla L. on EGF-inflamed bronchial epithelial and asthmatic bronchial smooth muscle cells

Anti-inflammatory and relaxation effects of Ulmus pumilla L. on EGF-inflamed bronchial epithelial and asthmatic bronchial smooth muscle cells

Asthmatic bronchial smooth muscle increases rhinovirus replication within the bronchial epithelium.

Rhinovirus (RV) infection of the bronchial epithelium is implicated in the vast majority of severe asthma exacerbations. Interestingly, the susceptibility of bronchial epithelium to RV infection is increased in persons with asthma. Bronchial smooth muscle (BSM) remodeling is an important feature of severe asthma pathophysiology, and its reduction using bronchial thermoplasty has been associated with a significant decrease in the exacerbation rate. We hypothesized that asthmatic BSM can play a role in RV infection of the bronchial epithelium. Using an original co-culture model between bronchial epithelium and BSM cells, we show that asthmatic BSM cells increase RV replication in bronchial epithelium following RV infection. These findings are related to the increased production of CCL20 by asthmatic BSM cells. Moreover, we demonstrate an original downregulation of the activity of the epithelial protein kinase RNA-activated (PKR) antiviral pathway. Finally, we identify a direct bottom-up effect of asthmatic BSM cells on bronchial epithelium susceptibility to RV infection.

Asthmatic bronchial smooth muscle decreases epithelial response to rhinovirus infection through a CCL-20/EIF2AK2 dependent pathway

Asthma is a very frequent airway disease affecting 6 to 20% of the population of western European countries. The disease is characterized by an increase of bronchial smooth muscle (BSM) mass correlated with the severity of asthma. Recent clinical investigations have shown a link between BSM mass and the exacerbations rate. Exacerbations are mainly the consequence from inhalation of aero-contaminant acting on bronchial epithelium cells with the most important trigger being the human rhinovirus (RV). However, the role of the BSM in infections of the epithelium by the RV is still unknown. Asthmatic and non-asthmatic BSM cells and bronchial epithelial cells (BEC) were obtained from patient biopsies (COBRA cohort). BEC were cultured and differentiated in Air-Liquide- Interface. RV infection was measured using qPCR. Cytokines concentrations were assessed using ELISA assays. Proteomic and transcriptomic data were analyzed using IPA (Qiagen). Using a co-culture model between BEC and BSM cells, we found an increased RV replication in BEC induced by asthmatic BSM cells. These findings were related to an increased production of CCL20 by asthmatic BSM cells. Moreover, we demonstrated a down regulation of the epithelial PKR/eIF2α antiviral pathway through this BSM-related CCL20 overproduction. Using both in vitro and ex vivo experiments, we demonstrated, for the first time, a direct bottom-up effect of BSM cells in severe asthmatic patients on BE susceptibility to RV infection. Asthmatic BSM cells are able to increase RV infection in BEC. Proteomic analysis highlight modified expression of CCL20 in BSM cells, showing that asthmatic BSM cells increase RV replication in BE through CCL-20 production.

Human Rhinovirus induces specific bronchial smooth muscle cell migration toward bronchial epithelium in severe asthma through the CXCL10/CXCR3-A axis

Severe asthmatic patients are characterized by an increase in both exacerbation frequency and bronchial smooth muscle (BSM) mass. Rhinovirus (RV) infection of the bronchial epithelium (BE) is the main trigger of asthma exacerbations. A decreased distance between BE and BSM mass is associated with the severity of asthma. We hypothesized that RV infection of the BE increased the migration of asthmatic BSM cells, specifically. Serums, biopsies or BSM cells were obtained from 86 severe asthmatic patients and 31 non-asthmatic subjects. BE cells from non-asthmatic subjects were cultured in air-liquid interface and exposed to RV-16. Migration of BSM cells was assessed in response to BE supernatant using chemotaxis assays and live microscopy. Chemokines concentrations were analyzed by transcriptomic and ELISA. Immunocytochemistry, western blot and flow cytometry were used to quantify CXCR3 isoforms distribution. CXCR3 downstream signaling pathways were assessed by calcium imaging and western blot. BSM cells from severe asthmatic patients harbor a specific migration toward RV-infected BE whereas those from non-asthmatic subjects do not. This specific migration is driven by BE CXCL10 production that is increased both in vitro in response to RV infection and in vivo in serum from exacerbating severe asthmatic patients. The mechanism is related to both decreased expression and activation of CXCR3-B specific isoform in BSM cells from severe asthmatic patients. We demonstrate a new mechanism of BSM remodeling in severe asthmatic patients following RV exacerbation. This study highlights the CXCL10/CXCR3-B axis as a new potential therapeutic target in severe asthma.

Asthmatic bronchial smooth muscle migration towards rhinovirus-infected epithelium through a CXCR3 isoform-dependant pathway

Asthma is the most common chronic respiratory disease. In severe patients, one of the main feature of the physiopathology is the increased bronchial smooth muscle (BSM) mass, which is related to frequency and severity of asthma exacerbations. Exacerbations are mainly triggered by rhinovirus infection of the epithelium. In addition, histology analysis has shown that distance between BSM cells and epithelium decrease proportionally to the severity of the disease. We thus hypothesis that rhinovirus infection of the epithelium increase the migration of asthmatic BSM cells. Primary BSM cells from control and asthmatic patients were obtained from bronchial biopsies. Epithelial cells were cultivated in an air-liquid Interface. After complete differentiation, epithelium were exposed to RVA-16 (MOI 0,1) and epithelium supernatant were collected 24 h post infection. BSM migration was assessed in response to epithelium supernatant in a chemotaxis assay for 24 h using live microscopy. Transcriptomics and ELISA were used to analysed chemokine content within the epithelial supernatant. Immunohistochemistry and flow cytometry were used to quantify CXCR3 isoforms distribution ( i.e CXCR3 A and B). Finally, western blot, cAMP assay and Ca2+ fluorometric probes were used to assess CXCR3 activation pathway. BSM cells from asthmatics migrates towards RV-infected epithelium whereas such a chemotactic effect was not observed in control BSM cells. Analysis of epithelium supernatant show that CXCL9 and CXCL10 were both induced by RVA-16. Interestingly, blocking of CXCR3 receptor, which is activated, by both CXCL9 and 10 abolished asthmatic BSM migration. They are two main isoforms of CXCR3 (A and B) which are described as activator and inhibitor of cell migration reciprocally. Interestingly, CXCR3-B expression and activation pathway through PKC and cAMP are increased in control BSM cells whereas CXCR3-A signalling pathway through calcium and Raf-1 activation are increase in asthmatic BSM cells. Asthmatic BSM cells specifically migrate toward RV-infected epithelium through the activation of CXCR3-A. This new mechanism could be a new therapeutic target against BSM remodelling in asthma.

Rhinoviral stimuli, epithelial factors and ATP signalling contribute to bronchial smooth muscle production of IL-33.

BackgroundBronchial smooth muscle cells (BSMCs) from severe asthmatics have been shown to overexpress the Th2-driving and asthma-associated cytokine IL-33. However, little is known regarding factors involved in BSMC production of IL-33. Rhinovirus (RV) infections cause asthma exacerbations, which exhibit features of Th2-type inflammation. Here, we investigated the effects of epithelial-derived media and viral stimuli on IL-33 expression in human BSMCs.MethodsPrimary human BSMCs from healthy (n = 3) and asthmatic (n = 3) subjects were stimulated with conditioned media from primary human bronchial epithelial cells (BECs), double-stranded (ds)RNA, dsRNA/LyoVec, or infected with RV. BSMCs were also pretreated with the purinergic receptor antagonist suramin. IL-33 expression was analysed by RT-qPCR and western blot and ATP levels were determined in cell supernatants.ResultsRV infection and activation of TLR3 by dsRNA increased IL-33 mRNA and protein in healthy and asthmatic BSMCs. These effects were inhibited by dexamethasone. BSMC expression of IL-33 was also increased by stimulation of RIG-I-like receptors using dsRNA/LyoVec. Conditioned media from BECs induced BSMC expression of IL-33, which was further enhanced by dsRNA. BEC-derived medium and viral-stimulated BSMC supernatants exhibited elevated ATP levels. Blocking of purinergic signalling with suramin inhibited BSMC expression of IL-33 induced by dsRNA and BEC-derived medium.ConclusionsRV infection of BSMCs and activation of TLR3 and RIG-I-like receptors cause expression and production of IL-33. Epithelial-released factor(s) increase BSMC expression of IL-33 and exhibit positive interaction with dsRNA. Increased BSMC IL-33 associates with ATP release and is antagonised by suramin. We suggest that epithelial-derived factors contribute to baseline BSMC IL-33 production, which is further augmented by RV infection of BSMCs and stimulation of their pathogen-recognising receptors.

House dust mites induce proliferation of severe asthmatic smooth muscle cells via an epithelium-dependent pathway.

Asthma is a frequent airway disease, and asthma control determinants have been associated with indoor allergen sensitization. The most frequent allergens are house dust mites (HDM), which act in vivo on the bronchial epithelial layer. Severe asthma has also been associated with bronchial remodeling and more specifically with increased mass of bronchial smooth muscle (BSM). However, the relationship between HDM stimulation of the bronchial epithelial layer and BSM remodeling is unknown. To evaluate whether epithelial stimulation with HDM induces BSM cell proliferation in subjects with severe asthma. A total of 22 subjects with severe asthma and 27 subjects with no asthma were recruited. We have developed an in vitro culture model combining an epithelium layer in air-liquid interface (ALI) interacting with BSM. We assessed BSM proliferation using BrdU incorporation. We explored the role of epithelium-derived mediators using reverse-transcriptase polymerase chain reaction (RT-PCR) and ELISA in vitro and in vivo. Finally, leukotrienes receptor expression was assessed in vitro by flow cytometry and RT-PCR and ex vivo by laser microdissection and RT-PCR. We found that epithelial stimulation by HDM selectively increased the proliferation of asthmatic BSM cells and not that of nonasthmatic cells. The mechanism involved epithelial protease-activated receptor-2-dependent production of leukotrienes C4 associated with an overexpression of leukotrienes receptor CysLTR1 by asthmatic BSM cells in vitro and ex vivo. This work demonstrates the selective role of HDM on BSM remodeling in patients with severe asthma and points out different therapeutic targets at epithelial and smooth muscle levels.

House dust mites induce asthmatic smooth muscle cell proliferation through epithelium- and leukotrienes-dependent pathways

Introduction Asthma is a very frequent airway disease that affects 5 to 20% of the whole population worldwide. Whereas the mortality of asthma has been dramatically decreased within the last 10 years, uncontrolled asthma still induces major consequences on morbidity, quality of life, and economic burden. Asthma control determinants have been associated to indoor allergens sensitization. The most frequent indoor allergen is the house dust mites (HDM), which act in vivo on bronchial epithelial layer. Uncontrolled asthma has also been associated with bronchial remodeling and more specifically to the increased mass of bronchial smooth muscle (BSM). It is now well admitted that, BSM remodeling is related with an increase of asthmatic BSM cell proliferation both in vitro and in vivo. However, the relationship between HDM stimulation of the bronchial epithelial layer and BSM remodeling is unknown. Methods We have developed an in vitro culture model of epithelium in air liquid interface (ALI) and BSM interaction. We assessed BSM proliferation using BrdU incorporation. We explored the role of leukotrienes in such a proliferation. We assessed the expression of enzymes involved in the leukotrienes synthesis pathway using RT-PCR. Leukotrienes release was assessed by Elisa in vitro on epithelial cell supernatant and in vivo in mouse broncho-alveolar lavage fluid. Finally, leukotrienes receptor expression was assessed both in vitro by flow cytometry and ex vivo by laser microdissection and RT-PCR. Results We found that epithelial stimulation by HDM increased the proliferation of asthmatic BSM cells and not that of non-asthmatics. The mechanisms involved an epithelial production of leukotrienes C4 and an over-expression of leukotrienes receptor CysLTR1 by asthmatic BSM cells both in vitro and in vivo. Conclusions This work demonstrates the role of HDM on BSM remodeling that occurs only in asthmatic patients and highlights different therapeutic targets at both epithelial and smooth muscle levels.

Protease Activated Receptor-2 Expression and Function in Asthmatic Bronchial Smooth Muscle

Asthmatic bronchial smooth muscle (BSM) is characterized by structural remodeling associated with mast cell infiltration displaying features of chronic degranulation. Mast cell-derived tryptase can activate protease activated receptor type-2 (PAR-2) of BSM cells. The aims of the present study were (i) to evaluate the expression of PAR-2 in both asthmatic and non asthmatic BSM cells and, (ii) to analyze the effect of prolonged stimulation of PAR-2 in asthmatic BSM cells on cell signaling and proliferation.BSM cells were obtained from both 33 control subjects and 22 asthmatic patients. PAR-2 expression was assessed by flow cytometry, western blot and quantitative RT-PCR. Calcium response, transduction pathways and proliferation were evaluated before and following PAR-2 stimulation by SLIGKV-NH2 or trypsin for 1 to 3 days.Asthmatic BSM cells expressed higher basal levels of functional PAR-2 compared to controls in terms of mRNA, protein expression and calcium response. When PAR-2 expression was increased by means of lentivirus in control BSM cells to a level similar to that of asthmatic cells, PAR-2-induced calcium response was then similar in both types of cell. However, repeated PAR-2 stimulations increased the proliferation of asthmatic BSM cells but not that of control BSM cells even following lentiviral over-expression of PAR-2. Such an increased proliferation was related to an increased phosphorylation of ERK in asthmatic BSM cells.In conclusion, we have demonstrated that asthmatic BSM cells express increased baseline levels of functional PAR-2. This higher basal level of PAR-2 accounts for the increased calcium response to PAR-2 stimulation, whereas the increased proliferation to repeated PAR-2 stimulation is related to increased ERK phosphorylation.

Counterpoint: Alterations in airway smooth muscle phenotype do not cause airway hyperresponsiveness in asthma

We have been assigned the task of showing that “alterations in airway smooth muscle phenotype DO NOT cause the airways hyperresponsiveness of asthma.” In the following paragraphs we will briefly review the overwhelming lack of evidence that any fundamental change in the intrinsic properties of

House dust mite extract downregulates C/EBP in asthmatic bronchial smooth muscle cells

Reduced translation of CEBPA mRNA has been associated with increased proliferation of bronchial smooth muscle (BSM) cells of asthma patients. Here, we assessed the effect of house dust mite (HDM) extracts on the cell proliferation ([(3)H]-thymidine incorporation), inflammation (interleukin (IL)-6 release) and upstream translation regulatory proteins of CCAAT/enhancer-binding protein (C/EBP)α in human BSM cells of healthy controls and asthmatic patients. HDM extract significantly increased IL-6 protein and proliferation of BSM cells of asthma patients only. HDM extract reduced the C/EBPα expression in BSM cells of asthma patients, which coincided with significantly increased levels of calreticulin (CRT) protein, an inhibitor of CEBPA mRNA translation. HDM extract elicited both protease-dependent and -independent responses, which were mediated via protease-activated receptor (PAR)2 and CRT, respectively. In conclusion, HDM extract reduced CEBPA mRNA translation, specifically in asthmatic BSM cells, and 1) upregulated CRT, 2) activated PAR2, and increased 3) IL-6 expression and 4) the proliferation of asthmatic BSM cells. Hence, HDM exposure contributes to inflammation and remodelling by a nonimmune cell-mediated mechanism via a direct interaction with BSM cells. These findings may potentially explain several pathological features of this disease, in particular BSM cell hyperplasia.

Mast cell adhesion to bronchial smooth muscle in asthma specifically depends on CD51 and CD44 variant 6

Mast cells infiltrate the bronchial smooth muscle (BSM) in asthmatic patients, but the mechanism of mast cell adhesion is still unknown. The adhesion molecules CD44 (i.e. hyaluronate receptor) and CD51 (i.e. vitronectin receptor) are widely expressed and bind to many extracellular matrix (ECM) proteins. The aims of the study are (i) to identify the role of ECM in mast cell adhesion to BSM and (ii) to examine the role of CD51 and CD44 in this adhesion. Human lung mast cells, human mast cell line (HMC-1), and BSM cells from control donors or asthmatic patients were cultured in the presence/absence of various cytokines. Mast cell-BSM interaction was assessed using (3)H-thymidine-pulsed mast cells, confocal immunofluorescence, or electron microscopy. Adhesion molecules expression and collagen production on both cell types were evaluated by quantitative RT-PCR, western blot, and flow cytometry. Mast cell adhesion to BSM cells mostly involved type I collagen of the ECM. Such an adhesion was increased in normal BSM cells under inflammatory condition, whereas it was maximal in asthmatic BSM cells. Blockade of either CD51 or CD44 significantly decreased mast cell adhesion to BSM. At the molecular level, protein and the transcriptional expression of type I collagen, CD51 or CD44 remained unchanged in asthmatic BSM cells or in mast cells/BSM cells under inflammatory conditions, whereas that of CD44 variant isoform 6 (v6) was increased. Mast cell-BSM cell adhesion involved collagen, CD44, and CD51, particularly under inflammatory conditions. CD44v6 expression is increased in asthmatic BSM cells.

Bronchial smooth muscle remodeling involves calcium-dependent enhanced mitochondrial biogenesis in asthma.

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by different patterns of airway remodeling, which all include an increased mass of bronchial smooth muscle (BSM). A remaining major question concerns the mechanisms underlying such a remodeling of BSM. Because mitochondria play a major role in both cell proliferation and apoptosis, we hypothesized that mitochondrial activation in BSM could play a role in this remodeling. We describe that both the mitochondrial mass and oxygen consumption were higher in the BSM from asthmatic subjects than in that from both COPD and controls. This feature, which is specific to asthma, was related to an enhanced mitochondrial biogenesis through up-regulation of peroxisome proliferator-activated receptor γ coactivator (PGC)–1α, nuclear respiratory factor-1, and mitochondrial transcription factor A. The priming event of such activation was an alteration in BSM calcium homeostasis. BSM cell apoptosis was not different in the three groups of subjects. Asthmatic BSM was, however, characterized by increased cell growth and proliferation. Both characteristics were completely abrogated in mitochondria-deficient asthmatic BSM cells. Conversely, in both COPD and control BSM cells, induction of mitochondrial biogenesis reproduced these characteristics. Thus, BSM in asthmatic patients is characterized by an altered calcium homeostasis that increases mitochondrial biogenesis, which, in turn, enhances cell proliferation, leading to airway remodeling.

Dysfunctional interaction of C/EBPalpha and the glucocorticoid receptor in asthmatic bronchial smooth-muscle cells.

Increased proliferation of bronchial smooth-muscle cells may lead to increased muscle mass in the airways of patients with asthma. The antiproliferative effect of glucocorticoids in bronchial smooth-muscle cells in subjects without asthma is mediated by a complex of the glucocorticoid receptor and the CCAAT/enhancer binding protein alpha (C/EBPalpha). We examined the signaling pathway controlling the inhibitory effect of glucocorticoids on cell proliferation and interleukin-6 synthesis in bronchial smooth-muscle cells of subjects with asthma and those without asthma. Lines of bronchial smooth-muscle cells were established from cells from 20 subjects with asthma, 8 subjects with emphysema, and 26 control subjects. Cell proliferation was determined by means of cell counts and [3H]thymidine incorporation. Signal transduction was studied by means of an electrophoretic DNA mobility-shift assay, a supershift electrophoretic-mobility assay, immunoblotting, use of C/EBPalpha antisense oligonucleotides, and use of a human C/EBPalpha expression vector. Interleukin-6 release was determined by means of an enzyme-linked immunosorbent assay. Glucocorticoids activated the glucocorticoid receptor and inhibited serum-induced secretion of interleukin-6 in bronchial smooth-muscle cells from both subjects with asthma and those without asthma; however, glucocorticoids inhibited proliferation only in bronchial smooth-muscle cells from subjects without asthma. C/EBPalpha protein was detected by immunoblotting in all bronchial smooth-muscle cells from subjects without asthma but not in those with asthma, whereas the protein was expressed in lymphocytes from both groups of subjects. C/EBPalpha antisense oligonucleotides or the glucocorticoid-receptor inhibitor mifepristone reversed the antiproliferative effect of glucocorticoids in bronchial smooth-muscle cells from subjects without asthma. When bronchial smooth-muscle cells from subjects with asthma were transiently transfected with an expression vector for human C/EBPalpha, two forms of the protein were expressed, and subsequent administration of glucocorticoids inhibited cell proliferation. We hypothesize that a cell-type-specific absence of C/EBPalpha is responsible for the enhanced proliferation of bronchial smooth-muscle cells derived from subjects with asthma and that it explains the failure of glucocorticoids to inhibit proliferation in vitro.

Changes in biophysical and biochemical properties of single bronchial smooth muscle cells from asthmatic subjects.

Whether contractility of bronchial smooth muscle cells (BSMC) from asthmatic subjects is significantly altered has never been validated. We tested the hypothesis that such BSMC show increased contractility. Cells were isolated from endobronchial biopsies. BSMC shortening was measured under an inverted microscope. Statistically significant increases in maximum shortening capacity (Delta L max) and velocity (Vo) were found in asthmatic BSMC compared with normal cells. Mean Delta L max in asthmatic BSMC was 39.05 +/- 1.99% (SE) of resting cell length compared with 28.6 +/- 1.1% in normal cells; mean Vo was 7.2 +/- 0.8% of resting cell length/s in asthmatic cells and 5.23 +/- 0.46% in normal cells. To investigate the mechanism of the increased contractility, we measured mRNA abundance of smooth muscle types of myosin light chain kinase (smMLCK) and myosin heavy chain. RT-PCR data revealed that smMLCK mRNA was higher in asthmatic BSMC (0.106 +/- 0.021 arbitrary densitometric units, n = 7) than in control cells (0.04 +/- 0.008, n = 11; P < 0.05). Messages for myosin heavy chain isoforms showed no difference. Increased kinase message content is an index of the mechanism for the increased velocity and capacity of shortening we report.