The effects of inhaled corticosteroids on healthy airways.

BACKGROUND: The effects of inhaled corticosteroids (ICS) on healthy airways are poorly defined. OBJECTIVES: To delineate the effects of ICS on gene expression in healthy airways, without confounding caused by changes in disease-related genes and disease-related alterations in ICS responsiveness. METHODS: Randomized open-label bronchoscopy study of high-dose ICS therapy in 30 healthy adult volunteers randomized 2:1 to (i) fluticasone propionate 500 mcg bd daily or (ii) no treatment, for 4 weeks. Laboratory staff were blinded to allocation. Biopsies and brushings were analysed by immunohistochemistry, bulk RNA sequencing, DNA methylation array and metagenomics. RESULTS: ICS induced small between-group differences in blood and lamina propria eosinophil numbers, but not in other immunopathological features, blood neutrophils, FeNO, FEV1, microbiome or DNA methylation. ICS treatment upregulated 72 genes in brushings and 53 genes in biopsies, and downregulated 82 genes in brushings and 416 genes in biopsies. The most downregulated genes in both tissues were canonical markers of type-2 inflammation (FCER1A, CPA3, IL33, CLEC10A, SERPINB10 and CCR5), T cell-mediated adaptive immunity (TARP, TRBC1, TRBC2, PTPN22, TRAC, CD2, CD8A, HLA-DQB2, CD96, PTPN7), B-cell immunity (CD20, immunoglobulin heavy and light chains) and innate immunity, including CD48, Hobit, RANTES, Langerin and GFI1. An IL-17-dependent gene signature was not upregulated by ICS. CONCLUSIONS: In healthy airways, 4-week ICS exposure reduces gene expression related to both innate and adaptive immunity, and reduces markers of type-2 inflammation. This implies that homeostasis in health involves tonic type-2 signalling in the airway mucosa, which is exquisitely sensitive to ICS.

Nicotinic receptors in airway disease.

With continued smoking of tobacco products and expanded use of nicotine delivery devices worldwide, understanding the impact of smoking and vaping on respiratory health remains a major global unmet need. Although multiple studies have shown a strong association between smoking and asthma, there is a relative paucity of mechanistic understanding of how elements in cigarette smoke impact the airway. Recognizing that nicotine is a major component in both smoking and vaping products, it is critical to understand the mechanisms by which nicotine impacts airways and promotes lung diseases such as asthma. There is now increasing evidence that α7 nicotinic acetylcholine receptors (α7nAChRs) are critical players in nicotine effects on airways, but the mechanisms by which α7nAChR influences different airway cell types have not been widely explored. In this review, we highlight and integrate the current state of knowledge regarding nicotine and α7nAChR in the context of asthma and identify potential approaches to alleviate the impact of smoking and vaping on the lungs.

Functional role of glial-derived neurotrophic factor in a mixed allergen murine model of asthma.

Our previous study showed that glial-derived neurotrophic factor (GDNF) expression is upregulated in asthmatic human lungs, and GDNF regulates calcium responses through its receptor GDNF family receptor α1 (GFRα1) and RET receptor in human airway smooth muscle (ASM) cells. In this study, we tested the hypothesis that airway GDNF contributes to airway hyperreactivity (AHR) and remodeling using a mixed allergen mouse model. Adult C57BL/6J mice were intranasally exposed to mixed allergens (ovalbumin, Aspergillus, Alternaria, house dust mite) over 4 wk with concurrent exposure to recombinant GDNF, or extracellular GDNF chelator GFRα1-Fc. Airway resistance and compliance to methacholine were assessed using FlexiVent. Lung expression of GDNF, GFRα1, RET, collagen, and fibronectin was examined by RT-PCR and histology staining. Allergen exposure increased GDNF expression in bronchial airways including ASM and epithelium. Laser capture microdissection of the ASM layer showed increased mRNA for GDNF, GFRα1, and RET in allergen-treated mice. Allergen exposure increased protein expression of GDNF and RET, but not GFRα1, in ASM. Intranasal administration of GDNF enhanced baseline responses to methacholine but did not consistently potentiate allergen effects. GDNF also induced airway thickening, and collagen deposition in bronchial airways. Chelation of GDNF by GFRα1-Fc attenuated allergen-induced AHR and particularly remodeling. These data suggest that locally produced GDNF, potentially derived from epithelium and/or ASM, contributes to AHR and remodeling relevant to asthma.NEW & NOTEWORTHY Local production of growth factors within the airway with autocrine/paracrine effects can promote features of asthma. Here, we show that glial-derived neurotrophic factor (GDNF) is a procontractile and proremodeling factor that contributes to allergen-induced airway hyperreactivity and tissue remodeling in a mouse model of asthma. Blocking GDNF signaling attenuates allergen-induced airway hyperreactivity and remodeling, suggesting a novel approach to alleviating structural and functional changes in the asthmatic airway.

Piezo channels in stretch effects on developing human airway smooth muscle.

The use of respiratory support strategies such as continuous positive airway pressure in premature infants can substantially stretch highly compliant perinatal airways, leading to airway hyperreactivity and remodeling in the long term. The mechanisms by which stretch detrimentally affects the airway are unknown. Airway smooth muscle cells play a critical role in contractility and remodeling. Using 18-22-wk gestation human fetal airway smooth muscle (fASM) as an in vitro model, we tested the hypothesis that mechanosensitive Piezo (PZ) channels contribute to stretch effects. We found that PZ1 and PZ2 channels are expressed in the smooth muscle of developing airways and that their expression is influenced by stretch. PZ activation via agonist Yoda1 or stretch results in significant [Ca2+]i responses as well as increased extracellular matrix production. These data suggest that functional PZ channels may play a role in detrimental stretch-induced airway changes in the context of prematurity.NEW & NOTEWORTHY Piezo channels were first described just over a decade ago and their function in the lung is largely unknown. We found that piezo channels are present and functional in the developing airway and contribute to intracellular calcium responses and extracellular matrix remodeling in the setting of stretch. This may improve our understanding of the mechanisms behind development of chronic airway diseases, such as asthma, in former preterm infants exposed to respiratory support, such as continuous positive airway pressure (CPAP).

Functional α7 nicotinic receptors in human airway smooth muscle increase intracellular calcium concentration and contractility in asthmatics.

Although nicotinic acetylcholine receptors (nAChRs) are commonly associated with neurons in the brain and periphery, recent data indicate that they are also expressed in non-neuronal tissues. We recently found the alpha7 (α7nAChR) subunit is highly expressed in human airway smooth muscle (hASM) with substantial increase in asthmatics, but their functionality remains unknown. We investigated the location and functional role of α7nAChRs in hASM cells from normal versus mild-moderate asthmatic patients. Immunostaining and protein analyses showed α7nAChR in the plasma membrane including in asthmatics. In asthmatic hASM, patch-clamp recordings revealed significantly higher functional homomeric α7nAChR channels. Real-time fluorescence imaging showed nicotine, via α7nAChR, increases intracellular Ca2+ ([Ca2+]i) independent of ACh effects, particularly in asthmatic hASM, while cellular traction force microscopy showed nicotine-induced contractility including in asthmatics. These results indicate functional homomeric and heteromeric nAChRs that are increased in asthmatic hASM, with pharmacology that likely differ owing to different subunit interfaces that form the orthosteric sites. nAChRs may represent a novel target in alleviating airway hyperresponsiveness in asthma.NEW & NOTEWORTHY Cigarette smoking and vaping exacerbate asthma. Understanding the mechanisms of nicotine effects in asthmatic airways is important. This study demonstrates that functional alpha7 nicotinic acetylcholine receptors (α7nAChRs) are expressed in human airway smooth muscle, including from asthmatics, and enhance intracellular calcium and contractility. Although a7nAChRs are associated with neuronal pathways, α7nAChR in smooth muscle suggests inhaled nicotine (e.g., vaping) can directly influence airway contractility. Targeting α7nAChR may represent a novel approach to alleviating airway hyperresponsiveness in asthma.

Airway smooth muscle in contractility and remodeling of asthma: potential drug target mechanisms.

INTRODUCTION: Asthma is characterized by enhanced airway contractility and remodeling where airway smooth muscle (ASM) plays a key role, modulated by inflammation. Understanding the mechanisms by which ASM contributes to these features of asthma is essential for the development of novel asthma therapies. AREAS COVERED: Inflammation in asthma contributes to a multitude of changes within ASM including enhanced airway contractility, proliferation, and fibrosis. Altered intracellular calcium ([Ca2+]i) regulation or Ca2+ sensitization contributes to airway hyperreactivity. Increased airway wall thickness from ASM proliferation and fibrosis contributes to structural changes seen with asthma. EXPERT OPINION: ASM plays a significant role in multiple features of asthma. Increased ASM contractility contributes to hyperresponsiveness, while altered ASM proliferation and extracellular matrix production promote airway remodeling both influenced by inflammation of asthma and conversely even influencing the local inflammatory milieu. While standard therapies such as corticosteroids or biologics target inflammation, cytokines, or their receptors to alleviate asthma symptoms, these approaches do not address the underlying contribution of ASM to hyperresponsiveness and particularly remodeling. Therefore, novel therapies for asthma need to target abnormal contractility mechanisms in ASM and/or the contribution of ASM to remodeling, particularly in asthmatics resistant to current therapies.

Cellular senescence is increased in airway smooth muscle cells of elderly persons with asthma.

Senescent cells can drive age-related tissue dysfunction partially via a senescence-associated secretory phenotype (SASP) involving proinflammatory and profibrotic factors. Cellular senescence has been associated with a structural and functional decline during normal lung aging and age-related diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Asthma in the elderly (AIE) represents a major healthcare burden. AIE is associated with bronchial airway hyperresponsiveness and remodeling, which involves increased cell proliferation and higher rates of fibrosis, and resistant to standard therapy. Airway smooth muscle (ASM) cells play a major role in asthma such as remodeling via modulation of inflammation and the extracellular matrix (ECM) environment. Whether senescent ASM cells accumulate in AIE and contribute to airway structural or functional changes is unknown. Lung tissues from elderly persons with asthma showed greater airway fibrosis compared with age-matched elderly persons with nonasthma and young age controls. Lung tissue or isolated ASM cells from elderly persons with asthma showed increased expression of multiple senescent markers including phospho-p53, p21, telomere-associated foci (TAF), as well as multiple SASP components. Senescence and SASP components were also increased with aging per se. These data highlight the presence of cellular senescence in AIE that may contribute to airway remodeling.

Feno differentiates epithelial gene expression clusters: Exploratory analysis from the MESOS randomized controlled trial.

BACKGROUND: Understanding how asthma biomarkers relate to gene expression signatures could help identify drivers of pathogenesis. OBJECTIVE: This post hoc exploratory analysis of the phase II tralokinumab trial MESOS (ClinicalTrials.gov identifier NCT02449473) aimed to profile baseline airway inflammation in patients with moderate-to-severe asthma. METHODS: The T2 and T17 gene expression signatures, 3-gene mean and 5-gene mean, were calculated through transcriptomic analysis of baseline bronchial brushing samples. Clustering analysis using these signatures identified 3 distinct inflammatory subgroups: T2LOW/T17HIGH (n = 33), T2HIGH/T17LOW (n = 10), and T2LOW/T17LOW (n = 27). RESULTS: Fractional exhaled nitric oxide (Feno) levels were highest for T2HIGH/T17LOW and lowest for T2LOW/T17HIGH (median = 52.0 [range 42.5-116.3] and median = 18.8 [range 6.6-128.6] ppb, respectively; P = .003). High Feno levels were strongly correlated with high T2 gene expression (Spearman ρ = 0.5537; P < .001). Individual genes differentially expressed in patients with elevated levels of Feno, blood and bronchial submucosal eosinophil counts, and IgE level were explored, with cystatin SN (CST1) being the most upregulated gene in all subgroups (4.49- to 34.42-fold upregulation across clinically defined subgroups with high biomarker expression). CONCLUSION: Feno level may be useful to differentiate patients with T2 or T17 gene expression. Elevated Feno levels are associated with high CST1 expression.

Airway remodelling rather than cellular infiltration characterizes both type2 cytokine biomarker-high and -low severe asthma.

BACKGROUND: The most recognizable phenotype of severe asthma comprises people who are blood eosinophil and FeNO-high, driven by type 2 (T2) cytokine biology, which responds to targeted biological therapies. However, in many people with severe asthma, these T2 biomarkers are suppressed but poorly controlled asthma persists. The mechanisms driving asthma in the absence of T2 biology are poorly understood. OBJECTIVES: To explore airway pathology in T2 biomarker-high and -low severe asthma. METHODS: T2 biomarker-high severe asthma (T2-high, n = 17) was compared with biomarker-intermediate (T2-intermediate, n = 21) and biomarker-low (T2-low, n = 20) severe asthma and healthy controls (n = 28). Bronchoscopy samples were processed for immunohistochemistry, and sputum for cytokines, PGD2 and LTE4 measurements. RESULTS: Tissue eosinophil, neutrophil and mast cell counts were similar across severe asthma phenotypes and not increased when compared to healthy controls. In contrast, the remodelling features of airway smooth muscle mass and MUC5AC expression were increased in all asthma groups compared with health, but similar across asthma subgroups. Submucosal glands were increased in T2-intermediate and T2-low asthma. In spite of similar tissue cellular inflammation, sputum IL-4, IL-5 and CCL26 were increased in T2-high versus T2-low asthma, and several further T2-associated cytokines, PGD2 and LTE4 , were increased in T2-high and T2-intermediate asthma compared with healthy controls. CONCLUSIONS: Eosinophilic tissue inflammation within proximal airways is suppressed in T2 biomarker-high and T2-low severe asthma, but inflammatory and structural cell activation is present, with sputum T2-associated cytokines highest in T2 biomarker-high patients. Airway remodelling persists and may be important for residual disease expression beyond eosinophilic exacerbations. Registered at ClincialTrials.gov: NCT02883530.

Effect of tezepelumab on airway inflammatory cells, remodelling, and hyperresponsiveness in patients with moderate-to-severe uncontrolled asthma (CASCADE): a double-blind, randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Tezepelumab is a human monoclonal antibody that blocks the activity of thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine. In phase 2b and 3 studies, tezepelumab significantly reduced exacerbations versus placebo in patients with severe uncontrolled asthma, irrespective of baseline levels of type 2 inflammatory biomarkers. We investigated the mechanism of action of tezepelumab by assessing its effects on airway inflammatory cells, airway remodelling, and airway hyperresponsiveness. METHODS: CASCADE was an exploratory, double-blind, randomised, placebo-controlled, parallel-group, phase 2 study done in 27 medical centres in Canada, Denmark, Germany, the UK, and the USA. Adults aged 18-75 years with uncontrolled, moderate-to-severe asthma were randomly assigned (1:1) to receive tezepelumab 210 mg or placebo administered subcutaneously every 4 weeks for a planned 28 weeks, extended to up to 52 weeks if COVID-19-related disruption delayed participants' end-of-treatment assessments. Randomisation was balanced and stratified by blood eosinophil count. The primary endpoint was the change from baseline to the end of treatment in the number of airway submucosal inflammatory cells in bronchoscopic biopsy samples. Eosinophils, neutrophils, CD3+ T cells, CD4+ T cells, tryptase+ mast cells, and chymase+ mast cells were evaluated separately. This endpoint was also assessed in subgroups according to baseline type 2 inflammatory biomarker levels, including blood eosinophil count. Airway remodelling was assessed via the secondary endpoints of change from baseline in reticular basement membrane thickness and epithelial integrity (proportions of denuded, damaged, and intact epithelium). Exploratory outcomes included airway hyperresponsiveness to mannitol. All participants who completed at least 20 weeks of study treatment, had an end-of-treatment visit up to 8 weeks after the last dose of study drug, and had evaluable baseline and end-of-treatment bronchoscopies were included in the primary efficacy analysis. All participants who received at least one dose of study drug were included in the safety analyses. This study is registered with ClinicalTrials.gov, NCT03688074. FINDINGS: Between Nov 2, 2018, and Nov 16, 2020, 250 patients were enrolled, 116 of whom were randomly assigned (59 to tezepelumab, 57 to placebo). 48 in the tezepelumab group and 51 in the placebo group completed the study and were assessed for the primary endpoint. Treatment with tezepelumab resulted in a nominally significantly greater reduction from baseline to the end of treatment in airway submucosal eosinophils versus placebo (ratio of geometric least-squares means 0·15 [95% CI 0·05-0·41]; nominal p<0·0010), with the difference seen across all baseline biomarker subgroups. There were no significant differences between treatment groups in the other cell types evaluated (ratio of geometric least-squares means: neutrophils 1·36 [95% CI 0·94-1·97]; CD3+ T cells 1·12 [0·86-1·46]; CD4+ T cells 1·18 [0·90-1·55]; tryptase+ mast cells 0·83 [0·61-1·15]; chymase+ mast cells 1·19 [0·67-2·10]; all p>0·10). In assessment of secondary endpoints, there were no significant differences between treatment groups in reticular basement membrane thickness and epithelial integrity. In an exploratory analysis, the reduction in airway hyperresponsiveness to mannitol was significantly greater with tezepelumab versus placebo (least-squares mean change from baseline in interpolated or extrapolated provoking dose of mannitol required to induce ≥15% reduction in FEV1 from baseline: tezepelumab 197·4 mg [95% CI 107·9 to 286·9]; placebo 58·6 mg [-30·1 to 147·33]; difference 138·8 [14·2 to 263·3], nominal p=0·030). Adverse events were reported in 53 (90%) patients in the tezepelumab group and 51 (90%) patients in the placebo group, and there were no safety findings of concern. INTERPRETATION: The improvements in asthma clinical outcomes observed in previous studies with tezepelumab are probably driven, at least in part, by reductions in eosinophilic airway inflammation, as shown here by reduced airway eosinophil counts regardless of baseline blood eosinophil count. Tezepelumab also reduced airway hyperresponsiveness to mannitol, indicating that TSLP blockade might have additional benefits in asthma beyond reducing type 2 airway inflammation. FUNDING: AstraZeneca and Amgen.

Human Lung Mast Cells Impair Corticosteroid Responsiveness in Human Airway Smooth Muscle Cells.

The mechanisms underlying corticosteroid insensitivity in severe asthma have not been elucidated although some indirect clinical evidence points toward a role of mast cells. Here, we tested the hypothesis that mast cells can drive corticosteroid insensitivity in airway smooth muscle cells, a key player in asthma pathogenesis. Conditioned media from resting or FcεR1-activated human lung mast cells were incubated with serum-deprived ASM cells (1:4 dilution, 24 h) to determine their impact on the anti-inflammatory action of fluticasone on ASM cell chemokine expression induced by TNFα (10 ng/ml). Conditioned media from FcεR1-activated mast cells (but not that from non-activated mast cells or control media) significantly reduced the ability of 100 nM fluticasone to suppress ASM TNFα-dependent CCL5 and CXCL10 production at both mRNA and protein levels. In contrast, fluticasone inhibition of CXCL-8 production by TNFα was still preserved in the presence of activated mast cell conditioned media. Transcriptomic analysis validated by individual qPCR assays revealed that activated mast cell conditioned media dramatically reduced the number of anti-inflammatory genes induced by fluticasone in ASM cells. Our study demonstrates for the first time that conditioned media from FcεR1-activated mast cells blunt the anti-inflammatory action of corticosteroids in ASM cells by altering their transactivation properties. Because infiltration of mast cells within the ASM bundles is a defining feature of asthma, mast cell-derived mediators may contribute to the glucocorticoid insensitivity present in severe asthma.