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 remodeling; Airway smooth muscle; Asthma; Fibrocytes; Mesenchymal stem cells
CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma.
IFN-inducible protein 10; mRNA levels and stability; NK-κB; p38; ERK
Vascular Endothelial Growth Factor (VEGF), a key angiogenic molecule, is aberrantly expressed in several diseases including asthma where it contributes to bronchial vascular remodelling and chronic inflammation. Asthmatic human airway smooth muscle (HASM) cells hypersecrete VEGF but the mechanism is unclear. Here we defined the mechanism in HASM cells from non-asthmatic (NA) and asthmatic (A) patients. We found that asthmatic cells lacked a repression complex at the VEGF promoter which was present in non-asthmatic cells. Recruitment of G9A, trimethylation of histone H3 at lysine 9 (H3K9me3) and a resultant decrease in RNA polymerase II (RNA pol II) at the VEGF promoter was critical to repression of VEGF secretion in non-asthmatic cells. At the asthmatic promoter H3K9me3 was absent due to failed recruitment of G9a; RNA pol II binding, in association with TAF1, was increased, H3K4me3 was present and Sp1 binding was exaggerated and sustained. In contrast DNA methylation and histone acetylation were similar in A and NA cells. This is the first study to show that airway cells in asthma have altered epigenetic regulation of remodelling gene(s). Histone methylation at genes such as VEGF may be an important new therapeutic target.
Rationale: Exacerbations of chronic obstructive pulmonary disease (COPD) and responses to treatment are heterogeneous.
Objectives: Investigate the usefulness of blood eosinophils to direct corticosteroid therapy during exacerbations.
Methods: Subjects with COPD exacerbations were entered into a randomized biomarker-directed double-blind corticosteroid versus standard therapy study. Subjects in the standard arm received prednisolone for 2 weeks, whereas in the biomarker-directed arm, prednisolone or matching placebo was given according to the blood eosinophil count biomarker. Both study groups received antibiotics. Blood eosinophils were measured in the biomarker-directed and standard therapy arms to define biomarker-positive and -negative exacerbations (blood eosinophil count > and ≤ 2%, respectively). The primary outcome was to determine noninferiority in health status using the chronic respiratory questionnaire (CRQ) and in the proportion of exacerbations associated with a treatment failure between subjects allocated to the biomarker-directed and standard therapy arms.
Measurements and Main Results: There were 86 and 80 exacerbations in the biomarker-directed and standard treatment groups, respectively. In the biomarker-directed group, 49% of the exacerbations were not treated with prednisolone. CRQ improvement after treatment in the standard and biomarker-directed therapy groups was similar (0.8 vs. 1.1; mean difference, 0.3; 95% confidence interval, 0.0–0.6; P = 0.05). There was a greater improvement in CRQ in biomarker-negative exacerbations given placebo compared with those given prednisolone (mean difference, 0.45; 95% confidence interval, 0.01–0.90; P = 0.04). In biomarker-negative exacerbations, treatment failures occurred in 15% given prednisolone and 2% of those given placebo (P = 0.04).
Conclusions: The peripheral blood eosinophil count is a promising biomarker to direct corticosteroid therapy during COPD exacerbations, but larger studies are required.
Clinical trial registered with www.controlled-trials.com (ISRCTN92422949).
chronic obstructive pulmonary disease; exacerbations; prednisolone; infection; eosinophils
Severe asthma is associated with fixed airway obstruction attributable to inflammation, copious luminal mucus, and increased airway smooth muscle (ASM) mass. Paradoxically, studies demonstrated that the hypertrophic and hyperplastic ASM characteristic of severe asthma has reduced contractile capacity. We compared the G-protein–coupled receptor (GPCR)–induced Ca2+ mobilization and expression of GPCRs and signaling proteins related to procontractile signaling in ASM derived postmortem from subjects who died of nonrespiratory causes, with cells from subjects who died of asthma. Despite the increased or comparable expression of contraction-promoting GPCRs (bradykinin B2 or histamine H1 and protease-activated receptor 1, respectively) in asthmatic ASM cells relative to cells from healthy donors, asthmatic ASM cells exhibited reduced histamine-induced Ca2+ mobilization and comparable responses to bradykinin and thrombin, suggesting a postreceptor signaling defect. Accordingly, the expression of regulator of G-protein signaling–5 (RGS5), an inhibitor of ASM contraction, was increased in cultured, asthmatic ASM cells and in bronchial smooth muscle bundles of both human subjects with asthma and allergen-challenged mice, relative to those of healthy human subjects or naive mice. The overexpression of RGS5 impaired the release of Ca2+ to thrombin, histamine, and carbachol, and reduced the contraction of precision-cut lung slices to carbachol. These results suggest that increased RGS5 expression contributes to decreased myocyte shortening in severe and fatal asthma.
asthma; bronchial smooth muscle; signal transduction; G-protein–coupled receptors
Severe asthma is a heterogeneous condition. Airway remodelling is a feature of severe asthma and can be determined by the assessment of high-resolution computed tomography (HRCT) scans. The aim of this study was to assess whether airway remodelling is restricted to specific subphenotypes of severe asthma.
A retrospective analysis was performed of HRCT scans from subjects who had attended a single-centre severe asthma clinic between 2003 and 2008. The right upper lobe apical segmental bronchus (RB1) dimensions were measured and the clinical and sputum inflammatory characteristics associated with RB1 geometry were assessed by univariate and multivariate regression analyses. Longitudinal sputum data were available and were described as area under the time curve (AUC). Comparisons were made in RB1 geometry across subjects in four subphenotypes determined by cluster analysis, smokers and non-smokers, and subjects with and without persistent airflow obstruction.
Ninety-nine subjects with severe asthma and 16 healthy controls were recruited. In the subjects with severe asthma the RB1 percentage wall area (%WA) was increased (p=0.009) and lumen area (LA)/body surface area (BSA) was decreased (p=0.008) compared with controls but was not different across the four subphenotypes. Airway geometry was not different between smokers and non-smokers and RB1 %WA was increased in those with persistent airflow obstruction. RB1 %WA in severe asthma was best associated with airflow limitation and persistent neutrophilic airway inflammation (model R2=0.27, p=0.001).
Airway remodelling of proximal airways occurs in severe asthma and is associated with impaired lung function and neutrophilic airway inflammation.
Severe asthma; airway remodelling; computed tomography; airway inflammation; quantitative analysis; asthma phenotypes; cluster analysis; imaging/CT MRI etc
Colonization of the airways by filamentous fungi can occur in asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis. A recent study found IgE sensitization to Aspergillus fumigatus to be associated with reduced lung function. Significantly higher rates of A. fumigatus were detected in sputum from asthmatics sensitized to this fungus compared to non-sensitized asthmatics. The rate of positive cultures was far higher than equivalent historical samples analysed by the local clinical laboratory following protocols recommended by the UK Health Protection Agency (HPA). This study compares the HPA procedure with our sputum processing method, whereby sputum plugs are separated from saliva and aliquots of approximately 150 mg are inoculated directly onto potato dextrose agar. A total of 55 sputum samples from 41 patients with COPD were analyzed, comparing fungal recovery of five dilutions of sputa on two media. Isolation of A. fumigatus in culture was significantly higher using the research approach compared to the HPA standard method for mycological investigations (P < 0.001). There was also a significant difference in the recovery rate of A. fumigatus (P < 0.05) between media. This highlights the need for a standardized approach to fungal detection which is more sensitive than the method recommended by the HPA
Aspergillus fumigatus; yeast; culture methods; respiratory samples; fungal growth media
Purpose of Review
Asthma is a global burden, affecting 5% of the general adult population, of whom approximately 5-10% suffer from severe asthma. Severe asthma is a complex heterogeneous disease entity, with high morbidity and mortality. Increasingly novel techniques in computerised tomography are being used to understand the pathophysiology of severe asthma. The utility and clinical implications of these CT techniques are the focus of this review.
Novel qualitative and quantitative CT imaging techniques have enabled us to study the large airway architecture in detail, assess the small airway structure, and perform functional analysis of regional ventilation.
Despite advances in CT imaging techniques, there is an urgent need for both proof-of-concept studies, large cross-sectional and longitudinal clinical trials in severe asthma to validate and clinically correlate imaging derived measures. This will extend our current understanding of the pathophysiology of severe asthma, and unravel the structure-function relationship, with the potential to discover novel severe asthma phenotypes, predict mortality, morbidity, and response to existing and novel pharmacological and non-pharmacological therapies.
Severe Asthma; Airway disease; Computed Tomography
Chronic obstructive pulmonary disease is a common condition and a major cause of mortality. COPD is characterized by irreversible airflow obstruction. The physiological abnormalities observed in COPD are due to a combination of emphysema and obliteration of the small airways in association with airway inflammation. The predominant cells involved in this inflammatory response are CD8+ lymphocytes, neutrophils, and macrophages. Although eosinophilic airway inflammation is usually considered a feature of asthma, it has been demonstrated in large and small airway tissue samples and in 20%–40% of induced sputum samples from patients with stable COPD. This airway eosinophilia is increased in exacerbations. Thus, modifying eosinophilic inflammation may be a potential therapeutic target in COPD. Eosinophilic airway inflammation is resistant to inhaled corticosteroid therapy, but does respond to systemic corticosteroid therapy, and the degree of response is related to the intensity of the eosinophilic inflammation. In COPD, targeting treatment to normalize the sputum eosinophilia reduced the number of hospital admissions. Whether controlling eosinophilic inflammation in COPD patients with an airway eosinophilia will modify disease progression and possibly alter mortality is unknown, but warrants further investigation.
COPD; sputum eosinophilia; corticosteroids
The asthma-associated gene urokinase plasminogen activator receptor (uPAR) may be involved in epithelial repair and airway remodelling. These processes are not adequately targeted by existing asthma therapies. A fuller understanding of the pathways involved in remodelling may lead to development of new therapeutic opportunities. uPAR expression in the lung epithelium of normal subjects and patients with asthma was investigated and the contribution of uPAR to epithelial wound repair in vitro was studied using primary bronchial epithelial cells (NHBECs).
Bronchial biopsy sections from normal subjects and patients with asthma were immunostained for uPAR. NHBECs were used in a scratch wound model to investigate the contribution of the plasminogen pathway to repair. The pathway was targeted via blocking of the interaction between urokinase plasminogen activator (uPA) and uPAR and overexpression of uPAR. The rate of wound closure and activation of intracellular signalling pathways and matrix metalloproteinases (MMPs) were measured.
uPAR expression was significantly increased in the bronchial epithelium of patients with asthma compared with controls. uPAR expression was increased during wound repair in monolayer and air-liquid interface-differentiated NHBEC models. Blocking the uPA–uPAR interaction led to attenuated wound repair via changes in Erk1/2, Akt and p38MAPK signalling. Cells engineered to have raised levels of uPAR showed attenuated repair via sequestration of uPA by soluble uPAR.
The uPAR pathway is required for efficient epithelial wound repair. Increased uPAR expression, as seen in the bronchial epithelium of patients with asthma, leads to attenuated wound repair which may contribute to the development and progression of airway remodelling in asthma. This pathway may therefore represent a potential novel therapeutic target for the treatment of asthma.
Asthma; urokinase plasminogen activator receptor; bronchial epithelial cells; wound repair; airway epithelium; asthma genetics; COPD mechanisms; allergic lung disease; asthma pharmacology; COPD exacerbations; COPD pathology; COPD pharmacology; allergic lung disease; asthma genetics
Rationale: The importance of Aspergillus fumigatus sensitization and colonization of the airways in patients with asthma is unclear.
Objectives: To define the relationship between the clinical and laboratory features of A. fumigatus–associated asthma.
Methods: We studied 79 patients with asthma (89% classed as GINA 4 or 5) classified into 3 groups according to A. fumigatus sensitization: (1) IgE-sensitized (immediate cutaneous reactivity > 3 mm and/or IgE > 0.35 kU/L); (2) IgG-only–sensitized (IgG > 40 mg/L); and (3) nonsensitized. These were compared with 14 healthy control subjects. Sputum culture was focused toward detection of A. fumigatus and compared with clinical assessment data.
Measurements and Main Results: A. fumigatus was cultured from 63% of IgE-sensitized patients with asthma (n = 40), 39% of IgG-only–sensitized patients with asthma (n = 13), 31% of nonsensitized patients with asthma (n = 26) and 7% of healthy control subjects (n = 14). Patients sensitized to A. fumigatus compared with nonsensitized patients with asthma had lower lung function (postbronchodilator FEV1 % predicted, mean [SEM]: 68 [±5]% versus 88 [±5]%; P < 0.05), more bronchiectasis (68% versus 35%; P < 0.05), and more sputum neutrophils (median [interquartile range]: 80.9 [50.1–94.1]% versus 49.5 [21.2–71.4]%; P < 0.01). In a multilinear regression model, A. fumigatus–IgE sensitization and sputum neutrophil differential cell count were important predictors of lung function (P = 0.016), supported by culture of A. fumigatus (P = 0.046) and eosinophil differential cell count (P = 0.024).
Conclusions: A. fumigatus detection in sputum is associated with A. fumigatus–IgE sensitization, neutrophilic airway inflammation, and reduced lung function. This supports the concept that development of fixed airflow obstruction in asthma is consequent upon the damaging effects of airway colonization with A. fumigatus.
asthma; lung function; Aspergillus fumigatus; induced sputum; neutrophil
COPD is a heterogeneous disease characterized by airflow obstruction and diagnosed by lung function. CT imaging is emerging as an important, noninvasive tool in phenotyping COPD. However, the use of CT imaging in defining the disease heterogeneity above lung function is not fully known.
Seventy-five patients with COPD (58 men, 17 women) were studied with CT imaging and with measures of airway inflammation. Airway physiology and health status were also determined.
The presence of emphysema (EM), bronchiectasis (BE), and bronchial wall thickening (BWT) was found in 67%, 27%, and 27% of subjects, respectively. The presence of EM was associated with lower lung function (mean difference % FEV1, −20%; 95% CI, −28 to −11; P < .001). There was no difference in airway inflammation, exacerbation frequency, or bacterial load in patients with EM alone or with BE and/or BWT ± EM. The diffusing capacity of the lung for carbon monoxide/alveolar volume ratio was the most sensitive and specific parameter in identifying EM (area under the receiver operator characteristic curve, 0.87; 95% CI, 0.79-0.96). Physiologic cluster analysis identified three clusters, two of which were EM predominant and the third characterized by a heterogeneous combination of EM and BE.
The application of CT imaging can be useful as a tool in the multidimensional approach to phenotyping patients with COPD.
Background. Asthma and obesity are common; however the impact of obesity upon asthma remains uncertain. Objectives. To assess relationships between obesity and fat mass with airway inflammation, lung function, and disease control in patients with refractory asthma. Methods. 151 refractory asthma patients were characterised for measures of airway inflammation, lung function, Juniper asthma control questionnaire (JACQ), body mass index (BMI), and fat mass index (FMI) derived from dual energy X-ray absorptiometry. Patients were reassessed over 12 months. Results. 74% of patients had an elevated BMI. BMI and FMI correlated (r = 0.9, P < .001). FMI and JACQ correlated in men (r = 0.3, P = .01). After 12 months 23% lost weight. Weight change over 12 months correlated with FEV1 change (r = −0.3, P = .03), but not with change in JACQ or exacerbations. Conclusion. Increased fat mass is common in refractory asthma and is associated with asthma symptom control in men. Loss of weight is associated with improvement in lung function in refractory asthma.
Antibiotic overuse in respiratory illness is common and is associated with drug resistance and hospital-acquired infection. Biomarkers that can identify bacterial infections may reduce antibiotic prescription. We aimed to compare the usefulness of the biomarkers procalcitonin and C-reactive protein (CRP) in patients with pneumonia or exacerbations of asthma or COPD.
Patients with a diagnosis of community-acquired pneumonia or exacerbation of asthma or COPD were recruited during the winter months of 2006 to 2008. Demographics, clinical data, and blood samples were collected. Procalcitonin and CRP concentrations were measured from available sera.
Sixty-two patients with pneumonia, 96 with asthma, and 161 with COPD were studied. Serum procalcitonin and CRP concentrations were strongly correlated (Spearman rank correlation coefficient [rs] = 0.56, P < .001). Patients with pneumonia had increased procalcitonin and CRP levels (median [interquartile range] 1.27 ng/mL [2.36], 191 mg/L ) compared with those with asthma (0.03 ng/mL [0.04], 9 mg/L ) and COPD (0.05 ng/mL [0.06], 16 mg/L ). The area under the receiver operating characteristic curve (95% CI) for distinguishing between patients with pneumonia (antibiotics required) and exacerbations of asthma (antibiotics not required), for procalcitonin and CRP was 0.93 (0.88-0.98) and 0.96 (0.93-1.00). A CRP value > 48 mg/L had a sensitivity of 91% (95% CI, 80%-97%) and specificity of 93% (95% CI, 86%-98%) for identifying patients with pneumonia.
Procalcitonin and CRP levels can both independently distinguish pneumonia from exacerbations of asthma. CRP levels could be used to guide antibiotic therapy and reduce antibiotic overuse in hospitalized patients with acute respiratory illness.
Asthma and COPD are characterized by airway dysfunction and inflammation. Neutrophilic airway inflammation is a common feature of COPD and is recognized in asthma, particularly in severe disease. The T helper (Th) 17 cytokines IL-17A and IL-17F have been implicated in the development of neutrophilic airway inflammation, but their expression in asthma and COPD is uncertain.
We assessed IL-17A and IL-17F expression in the bronchial submucosa from 30 subjects with asthma, 10 ex-smokers with mild to moderate COPD, and 27 nonsmoking and 14 smoking control subjects. Sputum IL-17 concentration was measured in 165 subjects with asthma and 27 with COPD.
The median (interquartile range) IL-17A cells/mm2 submucosa was increased in mild to moderate asthma (2.1 [2.4]) compared with healthy control subjects (0.4 [2.8]) but not in severe asthma (P = .04). In COPD, IL-17A+ cells/mm2 submucosa were increased (0.5 [3.7]) compared with nonsmoking control subjects (0 ) but not compared with smoking control subjects (P = .046). IL-17F+ cells/mm2 submucosa were increased in severe asthma (2.7 [3.6]) and mild to moderate asthma (1.6 [1.0]) compared with healthy controls subjects (0.7 [1.4]) (P = .001) but was not increased in subjects with COPD. IL-17A and IL-17F were not associated with increased neutrophilic inflammation, but IL-17F was correlated with the submucosal eosinophil count (rs = 0.5, P = .005). The sputum IL-17 concentration in COPD was increased compared with asthma (2 [0-7] pg/mL vs 0 [0-2] pg/mL, P < .0001) and was correlated with post-bronchodilator FEV1% predicted (r = −0.5, P = .008) and FEV1/FVC (r = −0.4, P = .04).
Our findings support a potential role for the Th17 cytokines IL-17A and IL-17F in asthma and COPD, but do not demonstrate a relationship with neutrophilic inflammation.
Human lung mast cells (HLMC) express the Ca2+-activated K+ channel KCa3.1, which opens following IgE-dependent activation. This hyperpolarises the cell membrane and potentiates both Ca2+ influx and degranulation. In addition, blockade of KCa3.1 profoundly inhibits HLMC migration to a variety of diverse chemotactic stimuli. KCa3.1 activation is attenuated by the β2adrenoceptor through a Gαs-coupled mechanism independent of cyclic AMP. Adenosine is an important mediator that both attenuates and enhances HLMC mediator release through the Gαs-coupled A2A and A2B adenosine receptors, respectively. We show that at concentrations that inhibit HLMC degranulation (10–5–10–3 M), adenosine closes KCa3.1 both dose-dependently and reversibly. KCa3.1 suppression by adenosine was reversed partially by the selective adenosine A2A receptor antagonist ZM241385 but not by the A2B receptor antagonist MRS1754, and the effects of adenosine were mimicked by the selective A2A receptor agonist CGS21680. Adenosine also opened a depolarising current carried by non-selective cations. As predicted from the role of KCa3.1 in HLMC migration, adenosine abolished HLMC chemotaxis to asthmatic airway smooth muscle-conditioned medium. In summary, the Gαs-coupled adenosine A2A receptor closes KCa3.1, providing a clearly defined mechanism by which adenosine inhibits HLMC migration and degranulation. A2A receptor agonists with channel-modulating function may be useful for the treatment of mast cell-mediated disease.
Adenosine; Chemotaxis; Ion channel; KCa3.1; Mast cell
There is a need for more objective outcome measures for chronic cough. In this pilot study we sought to investigate the utility of the mannitol challenge as a cough-provocation test in non-asthmatic chronic cough. We studied 16 healthy controls and 13 subjects with chronic cough. We assessed cough severity using a visual analogue score, capsaicin cough sensitivity, health status using the Leicester Cough Questionnaire and the dose of mannitol to cause 2 (C2) or 5 (C5) coughs. In all of the subjects with chronic cough and 6 of the controls we assessed the 1-week repeatability of the mannitol challenge. We found that in those subjects with chronic cough the geometric mean (logSEM) mannitol C2 and C5 was heightened compared to controls (C2: 4 (0.2) versus 16 (0.1); p = 0.04 and C5: 63 (0.1) versus 251 (0.1); p = 0.04). Cough visual analogue score, capsacin-induced cough sensitivity and health status were also altered in chronic cough compared to healthy controls, but in those subjects with chronic cough none of these outcomes was correlated with the mannitol C2 or C5. The repeatability of the mannitol challenge assessed by intraclass correlation was C2 = 0.53 and C5 = 0.59. A cut-off in the dose of mannitol of 62 mg/ml for C2 and 550 mg/ml for C5 had a sensitivity of 69 and 62% and specificity of 69 and 81% respectively to distinguish chronic coughers from healthy controls. In conclusion, the mannitol challenge my have potential as a novel cough challenge test and further work is required to extend our findings and to assess whether it has utility in different causes of chronic cough.
Despite asthma and chronic obstructive pulmonary disease being widely regarded as heterogeneous diseases, a consensus for an accurate system of classification has not been agreed. Recent studies have suggested that the recognition of subphenotypes of airway disease based on the pattern of airway inflammation may be particularly useful in increasing our understanding of the disease. The use of non-invasive markers of airway inflammation has suggested the presence of four distinct phenotypes: eosinophilic, neutrophilic, mixed inflammatory and paucigranulocytic asthma. Recent studies suggest that these subgroups may differ in their etiology, immunopathology and response to treatment. Importantly, novel treatment approaches targeted at specific patterns of airway inflammation are emerging, making an appreciation of subphenotypes particularly relevant. New developments in phenotyping inflammation and other facets of airway disease mean that we are entering an era where careful phenotyping will lead to targeted therapy.
asthma; COPD; eosinophil; inflammation; neutrophil
Severe asthma is a heterogeneous disease and the relationship between airway inflammation and airway remodelling is poorly understood. We sought to define sputum mediator profiles in severe asthmatics categorised by CT-determined airway geometry and sputum differential cell counts.
In a single centre cross-sectional observational study we recruited 59 subjects with severe asthma that underwent sputum induction and thoracic CT. Quantitative CT analysis of the apical segment of the right upper lobe (RB1) was performed. Forty-one mediators in sputum samples were measured of which 21 mediators that were assessable in >50% of samples were included in the analyses.
Independent of airway geometry, sputum MMP9 and IL-1β were elevated in those groups with a high sputum neutrophil count while sputum ICAM was elevated in those subjects with a low sputum neutrophil count. In contrast, sputum CCL11, IL-1α and fibrinogen were different in groups stratified by both sputum neutrophil count and airway geometry. Sputum CCL11 concentration was elevated in subjects with a low sputum neutrophil count and high luminal and total RB1 area, whereas sputum IL1α was increased in subjects with a high sputum neutrophil count and low total RB1 area. Sputum fibrinogen was elevated in those subjects with RB1 luminal narrowing and in those subjects with neutrophilic inflammation without luminal narrowing.
We have demonstrated that sputum mediator profiling reveals a number of associations with airway geometry. Whether these findings reflect important biological phenotypes that might inform stratified medicine approaches requires further investigation.
Asthma; Remodelling; RB1 bronchus
Chronic mast cell activation is a characteristic feature of asthma. BEAS-2B human airway epithelial cells (AEC) profoundly inhibit both constitutive and IgE-dependent human lung mast cell (HLMC) histamine release. The aim of this study was to examine the regulation of HLMC degranulation by primary AEC from healthy and asthmatic subjects, and investigate further the inhibitory mechanism.
HLMC were co-cultured with both BEAS-2B and primary AEC grown as monolayers or air-liquid interface (ALI) cultures.
Both constitutive and IgE-dependent HLMC histamine release were attenuated by BEAS-2B, primary AEC monolayers and ALI cultures. This occurred in the absence of HLMC-AEC contact indicating the presence of a soluble factor. Unlike healthy ALI AEC, asthmatic ALI-AEC did not significantly reduce constitutive histamine release. AEC inhibitory activity was transferable in primary AEC monolayer supernatant, but less active than with Transwell co-culture, suggesting that the inhibitory factor was labile. The AEC inhibitory effects were attenuated by both AEC wounding and pertussis toxin, indicating the involvement of a G0/Gi receptor coupled mechanism. Solid phase extraction of lipids (<10 kDa) removed the AEC inhibitory activity. The lipid derivatives resolvin D1 and D2 and lipoxin A4 attenuated HLMC histamine release in a dose-dependent fashion but were not detectable in co-culture supernatants.
Primary AEC suppress HLMC constitutive and IgE-dependent histamine secretion through the release of a soluble, labile lipid mediator(s) that signals through the G0/Gi receptor coupled mechanism. Manipulation of this interaction may have a significant therapeutic role in asthma.
In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.
During pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs) function in the presence and absence of IL-13 and TNFα, cytokines known to be important in asthma.
Expression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS) were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL) samples from healthy subjects and those with asthma.
PCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL)-13 and tumor necrosis factor (TNFα) stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFα had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL) fluid derived from healthy subjects as well as from those with asthma.
Taken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a potential role for inflammation-induced changes in oxytocin receptor signaling in the regulation of airway hyper-responsiveness in asthma.