Fibrocyte localisation to the ASM bundle in asthma: bidirectional effects on cell phenotype and behaviour.

OBJECTIVES: Airway hyper-responsiveness and persistent airflow obstruction contribute to asthma pathogenesis and symptoms, due in part to airway smooth muscle (ASM) hypercontractility and increased ASM mass. Fibrocytes have been shown to localise to the ASM in asthma however it is not known whether fibrocytes localise to the ASM in nonasthmatic eosinophilic bronchitis (NAEB) and chronic obstructive pulmonary disease (COPD). In addition, the potential consequences of fibrocyte localisation to ASM as regards asthma pathophysiology has not been widely studied. METHODS: Fibrocytes and proliferating cells were enumerated in ASM in bronchial tissue using immunohistochemistry. The effects of primary ASM and fibrocytes upon each other in terms of phenotype and behaviour following co-culture were investigated by assessing cell number, size, apoptotic status, phenotype and contractility in in vitro cell-based assays. RESULTS: Increased fibrocyte number in the ASM was observed in asthma versus NAEB, but not NAEB and COPD versus controls, and confirmed in asthma versus controls. ASM proliferation was not detectably different in asthmatics versus healthy controls in vivo. No difference in proliferation, apoptotic status or size of ASM was seen following culture with/without fibrocytes. Following co-culture with ASM from asthmatics versus nonasthmatics, fibrocyte smooth muscle marker expression and collagen gel contraction were greater. Following co-culture, fibrocyte CD14 expression was restored with the potential to contribute to asthma pathogenesis via monocyte-mediated processes dependent on the inflammatory milieu. CONCLUSION: Further understanding of mechanisms of fibrocyte recruitment to and/or differentiation within the ASM may identify novel therapeutic targets to modulate ASM dysfunction in asthma.

DP2 antagonism reduces airway smooth muscle mass in asthma by decreasing eosinophilia and myofibroblast recruitment.

Increased airway smooth muscle mass, a feature of airway remodeling in asthma, is the strongest predictor of airflow limitation and contributes to asthma-associated morbidity and mortality. No current drug therapy for asthma is known to affect airway smooth muscle mass. Although there is increasing evidence that prostaglandin D2 type 2 receptor (DP2) is expressed in airway structural and inflammatory cells, few studies have addressed the expression and function of DP2 in airway smooth muscle cells. We report that the DP2 antagonist fevipiprant reduced airway smooth muscle mass in bronchial biopsies from patients with asthma who had participated in a previous randomized placebo-controlled trial. We developed a computational model to capture airway remodeling. Our model predicted that a reduction in airway eosinophilia alone was insufficient to explain the clinically observed decrease in airway smooth muscle mass without a concomitant reduction in the recruitment of airway smooth muscle cells or their precursors to airway smooth muscle bundles that comprise the airway smooth muscle layer. We experimentally confirmed that airway smooth muscle migration could be inhibited in vitro using DP2-specific antagonists in an airway smooth muscle cell culture model. Our analyses suggest that fevipiprant, through antagonism of DP2, reduced airway smooth muscle mass in patients with asthma by decreasing airway eosinophilia in concert with reduced recruitment of myofibroblasts and fibrocytes to the airway smooth muscle bundle. Fevipiprant may thus represent a potential therapy to ameliorate airway remodeling in asthma.

In vitro, in silico and in vivo study challenges the impact of bronchial thermoplasty on acute airway smooth muscle mass loss.

Bronchial thermoplasty is a treatment for asthma. It is currently unclear whether its histopathological impact is sufficiently explained by the proportion of airway wall that is exposed to temperatures necessary to affect cell survival.Airway smooth muscle and bronchial epithelial cells were exposed to media (37-70°C) for 10 s to mimic thermoplasty. In silico we developed a mathematical model of airway heat distribution post-thermoplasty. In vivo we determined airway smooth muscle mass and epithelial integrity pre- and post-thermoplasty in 14 patients with severe asthma.In vitro airway smooth muscle and epithelial cell number decreased significantly following the addition of media heated to ≥65°C. In silico simulations showed a heterogeneous heat distribution that was amplified in larger airways, with <10% of the airway wall heated to >60°C in airways with an inner radius of ∼4 mm. In vivo at 6 weeks post-thermoplasty, there was an improvement in asthma control (measured via Asthma Control Questionnaire-6; mean difference 0.7, 95% CI 0.1-1.3; p=0.03), airway smooth muscle mass decreased (absolute median reduction 5%, interquartile range (IQR) 0-10; p=0.03) and epithelial integrity increased (14%, IQR 6-29; p=0.007). Neither of the latter two outcomes was related to improved asthma control.Integrated in vitro and in silico modelling suggest that the reduction in airway smooth muscle post-thermoplasty cannot be fully explained by acute heating, and nor did this reduction confer a greater improvement in asthma control.

Associations in asthma between quantitative computed tomography and bronchial biopsy-derived airway remodelling.

Airway remodelling in asthma remains poorly understood. This study aimed to determine the association of airway remodelling measured on bronchial biopsies with 1) lung function impairment and 2) thoracic quantitative computed tomography (QCT)-derived morphometry and densitometry measures of proximal airway remodelling and air trapping.Subjects were recruited from a single centre. Bronchial biopsy remodelling features that were the strongest predictors of lung function impairment and QCT-derived proximal airway morphometry and air trapping markers were determined by stepwise multiple regression. The best predictor of air trapping was validated in an independent replication group.Airway smooth muscle % was the only predictor of post-bronchodilator forced expiratory volume in 1 s (FEV1) % pred, while both airway smooth muscle % and vascularity were predictors of FEV1/forced vital capacity. Epithelial thickness and airway smooth muscle % were predictors of mean segmental bronchial luminal area (R2=0.12; p=0.02 and R2=0.12; p=0.015), whereas epithelial thickness was the only predictor of wall area % (R2=0.13; p=0.018). Vascularity was the only significant predictor of air trapping (R2=0.24; p=0.001), which was validated in the replication group (R2=0.19; p=0.031).In asthma, airway smooth muscle content and vascularity were both associated with airflow obstruction. QCT-derived proximal airway morphometry was most strongly associated with epithelial thickness and airway smooth muscle content, whereas air trapping was related to vascularity.

Fevipiprant, a prostaglandin D2 receptor 2 antagonist, in patients with persistent eosinophilic asthma: a single-centre, randomised, double-blind, parallel-group, placebo-controlled trial.

BACKGROUND: Eosinophilic airway inflammation is often present in asthma, and reduction of such inflammation results in improved clinical outcomes. We hypothesised that fevipiprant (QAW039), an antagonist of prostaglandin D2 receptor 2, might reduce eosinophilic airway inflammation in patients with moderate-to-severe eosinophilic asthma. METHODS: We performed a single-centre, randomised, double-blind, parallel-group, placebo-controlled trial at Glenfield Hospital (Leicester, UK). We recruited patients with persistent, moderate-to-severe asthma and an elevated sputum eosinophil count (≥2%). After a 2-week single-blind placebo run-in period, patients were randomly assigned (1:1) by the trial pharmacist, using previously generated treatment allocation cards, to receive fevipiprant (225 mg twice per day orally) or placebo, stratified by the use of oral corticosteroid treatment and bronchoscopy. The 12-week treatment period was followed by a 6-week single-blind placebo washout period. The primary outcome was the change in sputum eosinophil percentage from baseline to 12 weeks after treatment, analysed in the intention-to-treat population. All patients who received at least one dose of study drug were included in the safety analyses. This trial is registered with ClinicalTrials.gov, number NCT01545726, and with EudraCT, number 2011-004966-13. FINDINGS: Between Feb 10, 2012, and Jan 30, 2013, 61 patients were randomly assigned to receive fevipiprant (n=30) or placebo (n=31). Three patients in the fevipiprant group and four patients in the placebo group withdrew because of asthma exacerbations. Two patients in the fevipiprant group were incorrectly given placebo (one at the mid-treatment visit and one throughout the course of the study). They were both included in the fevipiprant group for the primary analysis, but the patient who was incorrectly given placebo throughout was included in the placebo group for the safety analyses. Between baseline and 12 weeks after treatment, sputum eosinophil percentage decreased from a geometric mean of 5·4% (95% CI 3·1-9·6) to 1·1% (0·7-1·9) in the fevipiprant group and from 4·6% (2·5-8·7) to 3·9% (CI 2·3-6·7) in the placebo group. Compared with baseline, mean sputum eosinophil percentage was reduced by 4·5 times in the fevipiprant group and by 1·3 times in the placebo group (difference between groups 3·5 times, 95% CI 1·7-7·0; p=0·0014). Fevipiprant had a favourable safety profile, with no deaths or serious adverse events reported. No patient withdrawals were judged by the investigator to be related to the study drug. INTERPRETATION: Fevipiprant reduces eosinophilic airway inflammation and is well tolerated in patients with persistent moderate-to-severe asthma and raised sputum eosinophil counts despite inhaled corticosteroid treatment. FUNDING: Novartis Pharmaceuticals, AirPROM project, and the UK National Institute for Health Research.

Airway smooth muscle NOX4 is upregulated and modulates ROS generation in COPD.

The burden of oxidative stress is increased in chronic obstructive pulmonary disease (COPD). However, whether the intra-cellular mechanisms controlling the oxidant/anti-oxidant balance in structural airway cells such as airway smooth muscle in COPD is altered is unclear. We sought to determine whether the expression of the NADPH oxidase (NOX)-4 is increased in airway smooth muscle in COPD both in vivo and primary cells in vitro and its role in hydrogen peroxide-induced reactive oxygen species generation. We found that in vivo NOX4 expression was up-regulated in the airway smooth muscle bundle in COPD (n = 9) and healthy controls with >20 pack year history (n = 4) compared to control subjects without a significant smoking history (n = 6). In vitro NOX4 expression was increased in airway smooth muscle cells from subjects with COPD (n = 5) compared to asthma (n = 7) and upregulated following TNF-α stimulation. Hydrogen peroxide-induced reactive oxygen species generation by airway smooth muscle cells in COPD (n = 5) was comparable to healthy controls (n = 9) but lower than asthma (n = 5); and was markedly attenuated by NOX4 inhibition. Our findings demonstrate that NOX4 expression is increased in vivo and in vitro in COPD and although we did not observe an intrinsic increase in oxidant-induced reactive oxygen species generation in COPD, it was reduced markedly by NOX4 inhibition supporting a potential therapeutic role for NOX4 in COPD.

Nociceptin/orphanin FQ (N/OFQ) modulates immunopathology and airway hyperresponsiveness representing a novel target for the treatment of asthma.

BACKGROUND AND PURPOSE: There is evidence supporting a role for the nociceptin/orphanin FQ (N/OFQ; NOP) receptor and its endogenous ligand N/OFQ in the modulation of neurogenic inflammation, airway tone and calibre. We hypothesized that NOP receptor activation has beneficial effects upon asthma immunopathology and airway hyperresponsiveness. Therefore, the expression and function of N/OFQ and the NOP receptor were examined in healthy and asthmatic human airway tissues. The concept was further addressed in an animal model of allergic asthma. EXPERIMENTAL APPROACH: NOP receptor expression was investigated by quantitative real-time PCR. Sputum N/OFQ was determined by RIA. N/OFQ function was tested using several assays including proliferation, migration, collagen gel contraction and wound healing. The effects of N/OFQ administration in vivo were studied in ovalbumin (OVA)-sensitized and challenged mice. KEY RESULTS: NOP receptors were expressed on a wide range of human and mouse immune and airway cells. Eosinophils expressed N/OFQ-precursor mRNA and their number correlated with N/OFQ concentration. N/OFQ was found in human sputum and increased in asthma. Additionally, in asthmatic human lungs N/OFQ immunoreactivity was elevated. NOP receptor activation inhibited migration of immunocytes and increased wound healing in airway structural cells. Furthermore, N/OFQ relaxed spasmogen-stimulated gel contraction. Remarkably, these findings were mirrored in OVA-mice where N/OFQ treatment before or during sensitization substantially reduced airway constriction and immunocyte trafficking to the lung, in particular eosinophils. N/OFQ also reduced inflammatory mediators and mucin production. CONCLUSIONS AND IMPLICATIONS: We demonstrated a novel dual airway immunomodulator/bronchodilator role for N/OFQ and suggest targeting this system as an innovative treatment for asthma.

Asthma therapy and its effect on airway remodelling.

Asthma remains a major health problem with significant morbidity, mortality and economic costs. In asthma, airway remodelling, which refers to all the microscopic structural changes seen in the airway tissue, has been recognised for many decades and remains one of the defining characteristics of the disease; however, it is still poorly understood. The detrimental pathophysiological consequences of some features of remodelling, like increased airway smooth muscle mass and subepithelial fibrosis, are well documented. However, whether targeting these by therapy would be beneficial is unknown. Although the prevailing thinking is that remodelling is an abnormal response to persistent airway inflammation, recent evidence, especially from studies of remodelling in asthmatic children, suggests that the two processes occur in parallel. The effects of asthma therapy on airway remodelling have not been studied extensively due to the challenges of obtaining airway tissue in the context of clinical trials. Corticosteroids remain the cornerstone of asthma therapy, and their effects on remodelling have been better studied than other drugs. Bronchial thermoplasty is the only asthma therapy to primarily target remodelling, although how it results in the apparent clinical benefits seen is not exactly clear. In this article we discuss the mechanisms of airway remodelling in asthma and review the effects of conventional and novel asthma therapies on the process.

Severe asthma: novel advances in the pathogenesis and therapy.

Asthma affects an estimated 300 million people worldwide and is severe in approximately 10% of sufferers. Asthma, especially severe asthma, is a heterogeneous disease that results from complex host-environment interactions. This review article outlines recent advances in both the understanding of pathogenesis and novel therapies. The pathogenesis of severe asthma can be broadly thought of in four domains: T(H)2 inflammation, non -T(H)2 inflammation, airway remodeling, and airway smooth muscle dysfunction. They can develop independently or partly as a consequence of each other. Interactions between these domains, their causation, and consequent impact upon disordered airway physiology and clinical expression are poorly understood. Recent advances in specific T(H)2- and non -T(H2)-targeted therapy, bronchial thermoplasty targeting airway remodeling and advances in therapies for airway smooth muscle dysfunction present new opportunities for treatment and inform our understanding of asthma pathogenesis. As our understanding of the pathogenesis increases, the need for individualized investigation, treatment, and management of asthma becomes more apparent.

Rationale and clinical results of inhibiting interleukin-5 for the treatment of severe asthma.

Severe asthma is responsible for considerable morbidity and a high proportion of the healthcare costs attributable to asthma. Management is not straightforward as the clinical, pathological and physiological features are heterogeneous and the relationships between these features are poorly understood. In recent years significant progress has been made in understanding this heterogeneity and eosinophilic asthma has emerged as a potentially clinically important phenotype because treatment with monoclonal antibodies against IL-5 is effective. This has required a change in our understanding of the role of eosinophilic airway inflammation in airways disease and the developments of reliable biomarkers of eosinophilic airway inflammation. We will review these developments and describe the clinical experience so far with treatment with monoclonal antibiotics against IL-5.

Origins of increased airway smooth muscle mass in asthma.

Asthma is characterized by both chronic inflammation and airway remodeling. Remodeling--the structural changes seen in asthmatic airways--is pivotal in the pathogenesis of the disease. Although significant advances have been made recently in understanding the different aspects of airway remodeling, the exact biology governing these changes remains poorly understood. There is broad agreement that, in asthma, increased airway smooth muscle mass, in part due to smooth muscle hyperplasia, is a very significant component of airway remodeling. However, significant debate persists on the origins of these airway smooth muscle cells. In this review article we will explore the natural history of airway remodeling in asthma and we will discuss the possible contribution of progenitors, stem cells and epithelial cells in mesenchymal cell changes, namely airway smooth muscle hyperplasia seen in the asthmatic airways.

Airway smooth muscle hypercontractility in asthma.

In recent years, asthma has been defined primarily as an inflammatory disorder with emphasis on inflammation being the principle underlying pathophysiological characteristic driving airway obstruction and remodelling. Morphological abnormalities of asthmatic airway smooth muscle (ASM), the primary structure responsible for airway obstruction seen in asthma, have long been described, but surprisingly, until recently, relatively small number of studies investigated whether asthmatic ASM was also fundamentally different in its functional properties. Evidence from recent studies done on single ASM cells and on ASM-impregnated gel cultures have shown that asthmatic ASM is intrinsically hypercontractile. Several elements of the ASM contraction apparatus in asthmatics and in animal models of asthma have been found to be different from nonasthmatics. These differences include some regulatory contractile proteins and also some components of both the calcium-dependent and calcium-independent contraction signalling pathways. Furthermore, oxidative stress was also found to be heightened in asthmatic ASM and contributes to hypercontractility. Understanding the abnormalities and mechanisms driving asthmatic ASM hypercontractility provides a great potential for the development of new targeted drugs, other than the conventional current anti-inflammatory and bronchodilator therapies, to address the desperate unmet need especially in patients with severe and persistent asthma.