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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.

ObjectiveInnate lymphoid cells-2 (ILC2) were shown to be involved in the development of lung or hepatic fibrosis. We sought to explore the functional and phenotypic heterogeneity of ILC2 in skin fibrosis within systemic sclerosis (SSc).MethodsBlood samples and skin biopsies from healthy donor or patients with SSc were analysed by immunostaining techniques. The fibrotic role of sorted ILC2 was studied in vitro on dermal fibroblast and further explored by transcriptomic approach. Finally, the efficacy of a new treatment against fibrosis was assessed with a mouse model of SSc.ResultsWe found that ILC2 numbers were increased in the skin of patients with SSc and correlated with the extent of skin fibrosis. In SSc skin, KLRG1− ILC2 (natural ILC2) were dominating over KLRG1+ ILC2 (inflammatory ILC2). The cytokine transforming growth factor-β (TGFβ), whose activity is increased in SSc, favoured the expansion of KLRG1- ILC2 simultaneously decreasing their production of interleukin 10 (IL10), which regulates negatively collagen production by dermal fibroblasts. TGFβ-stimulated ILC2 also increased myofibroblast differentiation. Thus, human KLRG1- ILC2 had an enhanced profibrotic activity. In a mouse model of SSc, therapeutic intervention-combining pirfenidone with the administration of IL10 was required to reduce the numbers of skin infiltrating ILC2, enhancing their expression of KLRG1 and strongly alleviating skin fibrosis.ConclusionOur results demonstrate a novel role for natural ILC2 and highlight their inter-relationships with TGFβ and IL10 in the development of skin fibrosis, thereby opening up new therapeutic approaches in SSc.

Pressure-volume (PV) curves constructed over the entire lung volume range can reliably detect functional changes in mouse models of lung diseases. In the present study, we constructed full-range PV curves in healthy and elastase-treated mice using either a classic manually operated technique or an automated approach using a computer-controlled piston ventilator [flexiVent FX; Scientific Respiratory Equipment (SCIREQ), Montreal, Quebec, Canada]. On the day of the experiment, subjects were anesthetized, tracheotomized, and mechanically ventilated. Following an initial respiratory mechanics scan and degassing of the lungs with 100% O2, full-range PV curves were constructed using either the classic or the automated technique. In control mice, superimposable curves were obtained, and statistical equivalence was attained between the two methodologies. In the elastase-treated ones, where significant changes in respiratory mechanics and lung volumes were expected, very small differences were observed between the two techniques, and the criteria for statistical equivalence were met in two out of four parameters assessed. The automated technique was adapted to rats and used to estimate the functional residual capacity (FRC) by volume subtraction. This novel approach generated FRC estimates consistent with the literature, with added accuracy relative to the existing method in diseased subjects. In conclusion, the automated technique generated full-range PV curves that were equivalent or very close to those obtained with the classic method under physiological or severe pathological conditions. The automation facilitated some technical aspects of the procedure, eased its use across species, and helped derive a more accurate estimate of FRC in preclinical models of respiratory disease. NEW & NOTEWORTHY Partial and full-range pressure-volume (PV) curves are frequently used to characterize lung disease models. Whereas automated techniques exist to construct partial PV curves, a manually operated approach is classically employed to build the full-range ones. In this study, the full-range PV curve technique was automated using a computer-controlled piston ventilator. The automation simplified the technique, facilitated its extension to other species, and inspired a novel way of estimating the functional residual capacity in laboratory rodents.

Background and Purpose Cysteinyl leukotrienes (CysLTs) are pro-inflammatory lipid mediators that exacerbate disease state in several asthma phenotypes including asthma induced by allergen, virus and exercise. However, the role of CysLTs in irritant-induced airway disease is not well characterized. The purpose of the current study was to investigate the effect of montelukast, a CysLT1 receptor antagonist, on parameters of irritant-induced asthma induced by inhalation of chlorine in the mouse. Experimental Approach BALB/c mice were exposed to chlorine in air (100 ppm, for 5 min). Montelukast (3 mg·kg−1) or the vehicle (1% methylcellulose) was administered 24 and 1 h prior to chlorine exposure and 1 h prior to outcome measurements. Twenty-four hours after exposure, responses to inhaled aerosolized methacholine, cell composition and an array of cytokines/chemokines in bronchoalveolar lavage (BAL) fluid were measured. Neutralizing antibodies against IL-6 and VEGF were administered prior to exposures. Key Results Montelukast reduced chlorine -induced airway hyperresponsiveness (AHR) to methacholine in the peripheral lung compartment as estimated from dynamic elastance, but not in large conducting airways. Montelukast treatment attenuated chlorine-induced macrophage influx, neutrophilia and eosinophilia in BAL fluid. Chlorine exposure increased VEGF, IL-6, the chemokines KC and CCL3 in BAL fluid. Montelukast treatment prevented chlorine-induced increases in VEGF and IL-6. Anti-IL-6 antibody inhibited chlorine-induced neutrophilia and reduced AHR. Conclusions and Implications Pre-treatment with montelukast attenuated chlorine-induced neutrophilia and AHR in mice. These effects are mediated, in part, via IL-6.

Inflammation is a response to injury and infection. Although protective under physiological conditions, excessive and persistent inflammation is linked to numerous diseases. As the lungs are continuously exposed to the external environment, the respiratory system is particularly liable to damage from inflammation. RelB is a member of the non-canonical NF-κB pathway that may control lung inflammation caused by cigarette smoke (CS), a leading cause of morbidity and mortality worldwide. Our lab has previously shown that RelB protects against CS-induced inflammation in vitro, leading us to hypothesize that RelB would protect against acute CS-induced pulmonary inflammation in vivo. We exposed wild-type (Relb+/+) and RelB-deficient mice (Relb-/-) mice to room air or to CS and found that CS exposure caused a sustained decrease in pulmonary granulocytes in Relb-/- mice that was predominated by a decrease in neutrophils. Pulmonary inflammation caused by other irritants, including chlorine, ovalbumin (OVA; to mimic features of asthma) and lipopolysaccharide (LPS) was not controlled by RelB. Differential cytokine analysis suggests that alterations in chemotactic cytokines do not fully account for the CS-specific decrease in neutrophils in Relb-/- mice. Flow cytometric analysis of the bronchoalveolar lavage and bone marrow cells also reveal that it is unlikely that the sustained decrease is caused by excessive cell death or decreased hematopoiesis from the bone marrow. Overall, our results indicate that RelB regulates acute CS-induced pulmonary inflammation. Understanding how RelB regulates CS-induced inflammation may potentiate the discovery of new therapeutic strategies for many of the inflammatory diseases caused by CS.

Background Inhaled oxidative toxicants present in ambient air cause airway epithelial injury, inflammation, and airway hyperresponsiveness. Effective adaptation to such environmental insults is essential for the preservation of pulmonary function, whereas failure or incomplete adaptation to oxidative injury can render the host susceptible to the development of airway disease. Objective We sought to explore the mechanisms of airway adaptation to oxidative injury. Methods For a model to study pulmonary adaptation to oxidative stress–induced lung injury, we exposed mice to repeated nose-only chlorine gas exposures. Outcome measures were evaluated 24 hours after the last chlorine exposure. Lung mechanics and airway responsiveness to methacholine were assessed by using the flexiVent. Inflammation and antioxidant responses were assessed in both bronchoalveolar lavage fluid and lung tissue. Using both loss or gain of function and genomic approaches, we further dissected the cellular and molecular mechanisms involved in pulmonary adaptation. Results Repeated exposures to oxidative stress resulted in pulmonary adaptation evidenced by abrogation of neutrophilic inflammation and airway hyperresponsiveness. This adaptation was independent of antioxidant mechanisms and regulatory T cells but dependent on residential alveolar macrophages (AMs). Interestingly, 5% of AMs expressed forkhead box P3, and depletion of these cells abolished adaptation. Results from transcriptomic profiling and loss and gain of function suggest that adaptation might be dependent on TGF-β and prostaglandin E2. Conclusion Pulmonary adaptation during oxidative stress–induced lung injury is mediated by a novel subset of forkhead box P3–positive AMs that limits inflammation, favoring airway adaptation and host fitness through TGF-β and prostaglandin E2.

Chlorine gas (Cl2) inhalation causes oxidative stress, airway epithelial damage, airway hyperresponsiveness (AHR), and neutrophilia. We evaluated the effect of neutrophil depletion on Cl2-induced AHR and its effect on the endogenous antioxidant response, and if eosinophils or macrophages influence Cl2-induced AHR. We exposed male Balb/C mice to 100 ppm Cl2 for 5 minutes. We quantified inflammatory cell populations in bronchoalveolar lavage (BAL), the antioxidant response in lung tissue by quantitative PCR, and nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear translocation by immunofluorescence. In vitro, NRF2 nuclear translocation in response to exogenous hypochlorite was assessed using a luciferase assay. Anti-granulocyte receptor-1 antibody or anti-Ly6G was used to deplete neutrophils. The effects of neutrophil depletion on IL-13 and IL-17 were measured by ELISA. Eosinophils and macrophages were depleted using TRFK5 or clodronate-loaded liposomes, respectively. AHR was evaluated with the constant-phase model in response to inhaled aerosolized methacholine. Our results show that Cl2 exposure induced neutrophilia and increased expression of NRF2 mRNA, superoxide dismutase-1, and heme-oxygenase 1. Neutrophil depletion abolished Cl2-induced AHR in large conducting airways and prevented increases in antioxidant gene expression and NRF2 nuclear translocation. Exogenous hypochlorite administration resulted in increased NRF2 nuclear translocation in vitro. After Cl2 exposure, neutrophils occupied 22 ± 7% of the luminal space in large airways. IL-17 in BAL was increased after Cl2, although this effect was not prevented by neutrophil depletion. Neither depletion of eosinophils nor macrophages prevented Cl2-induced AHR. Our data suggest the ability of neutrophils to promote Cl2-induced AHR is dependent on increases in oxidative stress and occupation of luminal space in large airways.

Introduction: Lung adaptation from pulmonary insults is an essential requirement for host fitness and survival. Lung epithelial injury and inflammation trigger oxidative stress which further damages the tracheobronchial tree. Failure or incomplete adaptation to oxidative injury favours the development of severe airway disease. Objectives: Establish a new model to explore airway adaptation to oxidative injury in order to elucidate key mechanisms for preserving lung function. Methods: To establish a model for the study of adaptation to oxidative stress induced-lung injury, we exposed mice to repeated chlorine gas exposures. Outcome measures were assessed 24h after the last chlorine exposure. Lung function mechanics were explored using FlexiVent. Inflammation and anti-oxidant responses were assessed in both bronchoalveolar lavage and lung tissue. Knock-out mice, depletion, adoptive transfer and gene array approaches were used to further explore the mechanistic. Results: Here we report a striking respiratory airway adaptation in mice after repeated chlorine exposures. Mice rapidly adapt by controlling the neutrophilic inflammation and airway hyperresponsiveness. After excluding antioxidant mechanisms and regulatory T cells, we found that adaptation was mediated by resident alveolar macrophages, programmed with a potent pro-phagocytic capacity. Notably, 5% of alveolar macrophages expressed Foxp3 and depletion of these cells abolished adaptation. Conclusions: This new model of adaptation to oxidative stress-induced lung injury lead us to discover a subset of alveolar macrophages Foxp3 + , which may provide new cellular mechanism in resolving inflammation leading to airway adaptation and host fitness. CIHR MOP-126131.

Rationale Chlorine gas (Cl2) is a potent oxidant and trigger of irritant induced asthma. We explored NF-E2–related factor 2 (Nrf2)-dependent mechanisms in the asthmatic response to Cl2, using Nrf2-deficient mice, buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and sulforaphane (SFN), a phytochemical regulator of Nrf2. Methods Airway inflammation and airway hyperresponsiveness (AHR) were assessed 24 and 48 h after a 5-min nose-only exposure to 100 ppm Cl2 of Nrf2-deficient and wild type Balb/C mice treated with BSO or SFN. Animals were anesthetized, paralyzed and mechanically ventilated (FlexiVent™) and challenged with aerosolized methacholine. Bronchoalveolar lavage (BAL) was performed and lung tissues were harvested for assessment of gene expression. Results Cl2 exposure induced a robust AHR and an intense neutrophilic inflammation that, although similar in Nrf2-deficient mice and wild-type mice at 24 h after Cl2 exposure, were significantly greater at 48 h post exposure in Nrf2-deficient mice. Lung GSH and mRNA for Nrf2-dependent phase II enzymes (NQO-1 and GPX2) were significantly lower in Nrf2-deficient than wild-type mice after Cl2 exposure. BSO reduced GSH levels and promoted Cl2-induced airway inflammation in wild-type mice, but not in Nrf2-deficient mice, whereas SFN suppressed Cl2-induced airway inflammation in wild-type but not in Nrf2-deficient mice. AHR was not affected by either BSO or SFN at 48 h post Cl2 exposure. Conclusions Nrf2-dependent phase II enzymes play a role in the resolution of airway inflammation and AHR after Cl2 exposure. Moderate deficiency of GSH affects the magnitude of acute inflammation but not AHR.

Asthma is characterized by the association of airway hyperresponsiveness (AHR), inflammation, and remodelling. The aim of the present article is to review the pivotal role of airway smooth muscle (ASM) in the pathophysiology of asthma. ASM is the main effector of AHR. The mechanisms of AHR in asthma may involve a larger release of contractile mediators and/or a lower release of relaxant mediators, an improved ASM cell excitation/contraction coupling, and/or an alteration in the contraction/load coupling. Beyond its contractile function, ASM is also involved in bronchial inflammation and remodelling. Whereas ASM is a target of the inflammatory process, it can also display proinflammatory and immunomodulatory functions, through its synthetic properties and the expression of a wide range of cell surface molecules. ASM remodelling represents a key feature of asthmatic bronchial remodelling. ASM also plays a role in promoting complementary airway structural alterations, in particular by its synthetic function.

Increased bronchial smooth muscle (BSM) mass is a key feature of airway remodeling that classically distinguishes severe from nonsevere asthma. Proliferation of BSM cells involves a specific mitochondria-dependent pathway in individuals with severe asthma. However, BSM remodeling and mitochondrial biogenesis have not been examined in nonsevere asthma.We aimed to assess whether an increase in BSM mass was also implicated in nonsevere asthma and its relationship with mitochondria and clinical outcomes.We enrolled 34 never-smoker subjects with nonsevere asthma. In addition, we recruited 56 subjects with nonsevere asthma and 19 subjects with severe asthma as comparative groups (COBRA cohort [Cohorte Obstruction Bronchique et Asthme; Bronchial Obstruction and Asthma Cohort; sponsored by the French National Institute of Health and Medical Research, INSERM]). A phenotypic characterization was performed using questionnaires, atopy and pulmonary function testing, exhaled nitric oxide measurement, and blood collection. Bronchial biopsy specimens were processed for immunohistochemistry and electron microscopy analysis. After BSM remodeling assessment, subjects were monitored over a 12-month period.We identified characteristic features of remodeling (BSM area26.6%) and increased mitochondrial number within BSM in a subgroup of subjects with nonsevere asthma. The number of BSM mitochondria was positively correlated with BSM area (r = 0.78; P0.001). Follow-up analysis showed that subjects with asthma with high BSM had worse asthma control and a higher rate of exacerbations per year compared with subjects with low BSM.This study reveals that BSM remodeling and mitochondrial biogenesis may play a critical role in the natural history of nonsevere asthma (Mitasthme study). Clinical trial registered with www.clinicaltrials.gov (NCT00808730).

Introduction: Chronic obstructive pulmonary disease (COPD) is characterized by peribronchial fibrosis. The chronic course of COPD is worsened by recurrent acute exacerbations. The aims of the study were to evaluate and understand the recruitment of blood fibrocytes in COPD patients during exacerbation. Methods: Using flow cytometry, we quantified circulating fibrocytes and characterized their chemokine receptors expression in 48 COPD patients during acute exacerbation (V1 COPD), two months after (V2 COPD), and 38 control subjects. The role of the chemokines CXCL12 and CCL11 in fibrocyte migration was investigated by a modified chemotaxis assay. Patients were followed for up to 3 years after V1. Results: We demonstrated a significant increased number of circulating fibrocytes in V1 COPD as compared to control subjects. The number of circulating fibrocytes decreased in V2 COPD. A high percentage of circulating fibrocytes during exacerbation was associated with increased risk of death. The percentage of fibrocytes at V2 was negatively correlated to FEV1, FVC, FEV1/FVC, TLCO and PaO 2 . Fibrocytes expressed in particular CXCR4 and CCR3, the chemokines receptors for CXCL12 and CCL11, respectively. Fibrocytes from V1 COPD had increased chemotactic migration in response to CXCL12 but not to CCL11 as compared to that from control subjects. Plerixafor, a CXCR4 antagonist, decreased fibrocyte migration to plasma of exacerbating COPD patients. Conclusion: Blood fibrocytes are recruited during COPD exacerbations and related to mortality and low lung function. The CXCL12/CXCR4 axis is involved in fibrocyte recruitment (“Firebrob” study; ClinicalTrials NCT01196832).

Rationale: Proliferation of bronchial smooth muscle (BSM) cells involves a specific mitochondria-dependent pathway in severe asthmatic patients. However, BSM remodeling and mitochondrial biogenesis have not been assessed in non-severe asthma. We aimed to assess whether a high BSM mass is implicated in non-severe asthma. Methods: We enrolled 30 never smokers subjects, with non-severe asthma. A phenotypic characterization was performed using questionnaires, atopy and pulmonary function testing, exhaled nitric oxide and blood collection. Bronchial-biopsy specimens obtained by fiberoptic bronchoscopy were processed for optic microscopy, immunohistochemistry and electron microscopy analysis. Following BSM remodeling assessment, participants were monitored over a 12 months period. Results: We identified characteristic features of BSM remodeling and mitochondrial biogenesis in non-severe asthmatics. Baseline characteristics were similar in patients with BSM remodeling (“high BSM group”) and subjects without remodeling (“low BSM group”). However, 12 months follow-up showed that Juniper asthma control score and the number of exacerbations were increased in asthmatics with high BSM as compared to low BSM patients. Furthermore, the number of BSM mitochondria was positively correlated to BSM area in non-severe asthma (r = 0.78; P Conclusions: This study reveals that BSM remodeling and mitochondrial biogenesis may play a key role in natural history of non-severe asthma (“Mitasthme” study; ClinicalTrials.gov number, NCT00808730).

Introduction: Asthma is a frequent respiratory disease characterized by an airway remodeling. Increase of bronchial smooth muscle (BSM) mass is a crucial feature of such a remodeling since it correlates with the decrease in lung function. The mechanisms of such an increased BSM mass are complex but involve an increased proliferation of asthmatic BSM cells, which involved an enhanced mitochondrial biogenesis. Major tumor suppressor protein p53 is a key cell regulator involved in cell proliferation and has also been implicated in mitochondrial biogenesis. However, the role of p53 in BSM cell proliferation and mitochondrial biogenesis has not been investigated so far. The objective was thus to evaluate the role of p53 in asthmatic BSM cell proliferation and mitochondrial biogenesis. Methods: We assessed the expression of p53 in vitro using flow cytometry and western blot but also ex vivo using laser microdissection and RT-PCR. Using shRNA lentivirus targeting p53, we assessed the role of p53 in both asthmatic and control BSM cells proliferation using both BrdU and cell counting. We analyzed the effect of the down regulation of p53 protein on the regulation of p21, Bax, TFAM and PGC-1a mRNA. Results: P53 expression was increased in asthmatic BSM both ex vivo and in vitro , with a decreased interaction with Mdm2. P53 is unable to inhibit the transcription of TFAM and PGC-1a in asthmatic BSM. As a consequence, p53 cannot slow down the increased mitochondrial biogenesis and the subsequent increased proliferation of asthmatic BSM cells. Conclusions: This work highlights p53 as a new potential therapeutic target against BSM remodeling in asthma.

Airway exposure to organic dust (OD) from swine confinement facilities induces airway inflammation dominated by neutrophils and airway hyperresponsiveness (AHR). One important neutrophilic innate defense mechanism is the induction of oxidative stress. Therefore, we hypothesized that neutrophils exacerbate airway dysfunction following OD exposure by increasing oxidant burden. BALB/C mice were given intranasal challenges with OD or PBS (1/day for 3 days). Mice were untreated or treated with a neutrophil-depleting antibody, anti-Ly6G, or the antioxidant dimethylthiourea (DMTU) prior to OD exposure. Twenty-four hours after the final exposure, we measured airway responsiveness in response to methacholine (MCh) and collected bronchoalveolar lavage fluid to assess pulmonary inflammation and total antioxidant capacity. Lung tissue was harvested to examine the effect of OD-induced antioxidant gene expression and the effect of anti-Ly6G or DMTU. OD exposure induced a dose-dependent increase of airway responsiveness, a neutrophilic pulmonary inflammation, and secretion of keratinocyte cytokine. Depletion of neutrophils reduced OD-induced AHR. DMTU prevented pulmonary inflammation involving macrophages and neutrophils. Neutrophil depletion and DMTU were highly effective in preventing OD-induced AHR affecting large, conducting airways and tissue elastance. OD induced an increase in total antioxidant capacity and mRNA levels of NRF-2-dependent antioxidant genes, effects that are prevented by administration of DMTU and neutrophil depletion. We conclude that an increase in oxidative stress and neutrophilia is critical in the induction of OD-induced AHR. Prevention of oxidative stress diminishes neutrophil influx and AHR, suggesting that mechanisms driving OD-induced AHR may be dependent on neutrophil-mediated oxidant pathways.

The bronchoconstrictive and proinflammatory properties of cysteinyl leukotrienes (cysLTs) in allergic asthma mediate their effects predominantly through the cysLT1 receptor (cysLT1R). However, the role of cysLTs and cysLT1R in innate immune-triggered asthma is largely unexplored. We explored the synthesis of cysLTs and cysLT1R as determinants of airway responses in an oxidative stress–induced model of irritant asthma. Wild-type (WT) mice exposed to 100 ppm Cl2 for 5 min had airway neutrophilia, increased cysLT production, and pulmonary expression of cysLT-related biosynthetic genes. CysLT1R-deficient (CysLTr1−/−) mice that were exposed to Cl2 demonstrated airway hyperresponsiveness to inhaled methacholine significantly greater than in WT BALB/c mice. Compared to WT mice, airway neutrophilia and keratinocyte chemoattractant production levels were higher in CysLTr1−/− mice and airway hyperresponsiveness was ameliorated using a granulocyte depletion Ab. CysLTr1−/− mice also demonstrated prolonged bronchial epithelial cell apoptosis following Cl2. WT mice showed increased antioxidant and NF erythroid 2–related factor 2 (Nrf2) gene expression, Nrf2 nuclear translocation in bronchial epithelial cells, and increased reduced glutathione/oxidized glutathione following Cl2 exposure whereas CysLTr1−/− mice did not. Furthermore, CysLTr1−/− mice demonstrated increased pulmonary E-cadherin expression and soluble E-cadherin shedding compared with WT mice. Loss of a functional cysLT1R results in aberrant antioxidant response and increased susceptibility to oxidative injury, apparently via a cysLT1R-dependent impairment of Nrf2 function.

Background Increase of bronchial smooth muscle (BSM) mass is a crucial feature of asthma remodeling. The mechanisms of such an increased BSM mass are complex but involve enhanced mitochondrial biogenesis, leading to increased proliferation of BSM cells in asthmatic patients. The major tumor suppressor protein p53 is a key cell regulator involved in cell proliferation and has also been implicated in mitochondrial biogenesis. However, the role of p53 in BSM cell proliferation and mitochondrial biogenesis has not been investigated thus far. Objective We sought to evaluate the role of p53 in proliferation of BSM cells in asthmatic patients and mitochondrial biogenesis. Methods The expression of p53 was assessed both in vitro by using flow cytometry and Western blotting and ex vivo by using RT-PCR after laser microdissection. The role of p53 was assessed with small hairpin RNA lentivirus in both asthmatic patients and control subjects with BSM cell proliferation by using 5-bromo-2′-deoxyuridine and cell counting and in the expression of p21, BCL2-associated X protein, mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Results Twenty-nine patients with moderate-to-severe asthma and 26 control subjects were enrolled in the study. p53 expression was increased in BSM from asthmatic patients both ex vivo and in vitro , with a decreased interaction with mouse double minute 2 homolog (Mdm2) and an increased phosphorylation of serine 20. p53 did not inhibit the transcription of both TFAM and PGC-1α in BSM cells from asthmatic patients. As a consequence, p53 is unable to slow the increased mitochondrial biogenesis and hence the subsequent increased proliferation of BSM cells in asthmatic patients. Conclusion This study suggests that p53 might act as a new potential therapeutic target against BSM remodeling in asthmatic patients.

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.

Despite the worldwide application of vaccination and other antiviral interventions, influenza A virus (IAV) infection still remains a serious threat to humans. The success of IAV infection is linked to its ability to cause lower airways infections, which activates alveolar macrophages and often leads to a cytokine storm, pneumonia and respiratory failure. At this stage, uncontrolled host inflammatory response is the major cause of death. Therefore, understanding the mechanisms of immune regulation during IAV infection is critical in preventing IAV-induced immunopathology and mortality. Although cytokines have been extensively studied in immunity to IAV infection, little is known about the role of eicosanoids. We have recently demonstrated that cyclooxygenase-derived PGE2 plays a deleterious role in protection against IAV infection by inhibiting antiviral type I interferon (IFN). Herein, we aimed to investigate the potential contribution of lipoxygenase (LOX)-derived lipid mediators in IAV-immunity using the 5-LOX (Alox5−/−) deficient mice. Our data indicate that despite reduced pulmonary IAV titers, Alox5−/− mice are more susceptible to infection and exogenous administration of stable lipoxin A4 restore protection to IAV in Alox5−/− mice. This increased susceptibility was coupled with enhanced immunopathology and decreased pulmonary function. Remarkably, the production of type I IFN and IL-10 were significantly reduced in the lungs of IAV-infected Alox5−/− mice as well as IAV-infected Alox5−/− macrophages. Collectively these findings identified a protective role of the LOX-derived lipids against IAV-induced immunopathology and may pave the way for novel anti-influenza treatments using stable bioactive lipids.

Background Chronic obstructive pulmonary disease (COPD) is characterized by peribronchial fibrosis. The chronic course of COPD is worsened by recurrent acute exacerbations. Objective The aim of the study was to evaluate the recruitment of blood fibrocytes in patients with COPD during exacerbations and, subsequently, to identify potential mechanisms implicated in such recruitment. Methods Using flow cytometry, we quantified circulating fibrocytes and characterized their chemokine receptor expression in 54 patients with COPD examined during an acute exacerbation (V1) and 2 months afterward (V2) and in 40 control subjects. The role of the chemokines CXCL12 and CCL11 in fibrocyte migration was investigated by using a chemotaxis assay. Patients were followed for up to 3 years after V1. Results We demonstrated a significantly increased number of circulating fibrocytes at V1 compared with control subjects. The number of circulating fibrocytes decreased at V2. A high percentage of circulating fibrocytes during exacerbation was associated with increased risk of death. The percentage of fibrocytes at V2 was negatively correlated with FEV 1 , forced vital capacity, FEV 1 /forced vital capacity ratio, transfer lung capacity of carbon monoxide, and Pao 2 . Fibrocytes highly expressed CXCR4 and CCR3, the chemokine receptors for CXCL12 and CCL11, respectively. Fibrocytes collected from patients with COPD at V1 had increased chemotactic migration in response to CXCL12 but not to CCL11 compared with those from control subjects. Plerixafor, a CXCR4 antagonist, decreased fibrocyte migration to plasma from patients with exacerbating COPD. Conclusion Blood fibrocytes are recruited during COPD exacerbations and related to mortality and low lung function. The CXCL12/CXCR4 axis is involved in such fibrocyte recruitment (Firebrob study; ClinicalTrials NCT01196832).

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.

Introduction Asthma is a frequent respiratory disease characterized by bronchial hyperresponsiveness, inflammation and remodeling. Bronchial remodeling corresponds to structural modification of the bronchial wall. Increase of smooth muscle mass is a crucial feature of such a remodeling in asthma. Indeed, it correlates with the decrease in lung function and bronchial thermoplasty, which is supposed to decrease BSM mass, was associated with a decrease in exacerbation rate. The mechanisms of such an increased BSM mass are complex and remain controversial. However, increased proliferation of asthmatic BSM cells has been proposed and largely documented both in vitro and in vivo. We previously demonstrated that such an increased BSM cell proliferation involved an increased mitochondrial biogenesis and mitochondrial mass through the increase of PGC-1α and Tfam expression. Major tumor suppressor protein p53 is a key cell regulator involved in cell proliferation and has also been implicated in mitochondrial biogenesis. However, the role of p53 in BSM cell proliferation and mitochondrial biogenesis has not been investigated so far. We thus hypothesized that, p53 can modulate BSM cell mitochondrial biogenesis and proliferation and that this modulation is disrupted in asthmatic cells. Methods Using shRNA lentivirus targeting p53, we assessed the role of p53 in both asthmatic and control BSM cells proliferation using both BrdU and cell counting. We analyzed the effect of the down-regulation of p53 protein on the regulation of p21, Bax, TFAM and PGC-1α mRNA. Finally, we assessed the expression of p53 in vitro using flow cytometry and western blot but also ex vivo using laser microdissection and RT-PCR. Results We found that, p53 is unable to inhibit the transcription of Tfam and PGC1α in asthmatic BSM. As a consequence, p53 cannot slow down the increased mitochondrial biogenesis and the subsequent increased proliferation of asthmatic BSM cells. We also found that p53 expression was increased in asthmatic BSM, which was related with a loss of Mdm2 activation by p53. Conclusions In conclusion, this work highlights p53 as a new potential therapeutic target against BSM remodeling in asthma.