Epithelial cell proliferation contributes to airway remodeling in severe asthma.
ABSTRACT: Despite long-term therapy with corticosteroids, patients with severe asthma develop irreversible airway obstruction.To evaluate if there are structural and functional differences in the airway epithelium in severe asthma associated with airway remodeling.In bronchial biopsies from 21 normal subjects, 11 subjects with chronic bronchitis, 9 subjects with mild asthma, and 31 subjects with severe asthma, we evaluated epithelial cell morphology: epithelial thickness, lamina reticularis (LR) thickness, and epithelial desquamation. Levels of retinoblastoma protein (Rb), Ki67, and Bcl-2 were measured, reflecting cellular proliferation and death. Terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) was used to study cellular apoptosis.Airway epithelial and LR thickness was greater in subjects with severe asthma compared with those with mild asthma, normal subjects, and diseased control subjects (p=0.009 and 0.033, respectively). There was no significant difference in epithelial desquamation between groups. Active, hypophosphorylated Rb expression was decreased (p=0.002) and Ki67 was increased (p<0.01) in the epithelium of subjects with severe asthma as compared with normal subjects, indicating increased cellular proliferation. Bcl-2 expression was decreased (p<0.001), indicating decreased cell death suppression. There was a greater level of apoptotic activity in the airway biopsy in subjects with severe asthma as compared with the normal subjects using the TUNEL assay (p=0.002), suggesting increased cell death.In subjects with severe asthma, as compared with subjects with mild asthma, normal subjects, and diseased control subjects, we found novel evidence of increased cellular proliferation in the airway contributing to a thickened epithelium and LR. These changes may contribute to the progressive decline in lung function and airway remodeling in patients with severe asthma.
Project description:RATIONALE AND OBJECTIVES:Previous cross-sectional studies have demonstrated that airway wall thickness and air trapping are greater in subjects with severe asthma than in those with mild-to-moderate asthma. However, a better understanding of how airway remodeling and lung density change over time is needed. This study aimed to evaluate predictors of airway wall remodeling and change in lung function and lung density over time in severe asthma. MATERIALS AND METHODS:Phenotypic characterization and quantitative multidetector-row computed tomography (MDCT) of the chest were performed at baseline and ?2.6 years later in 38 participants with asthma (severe n = 24 and mild-to-moderate n = 14) and nine normal controls from the Severe Asthma Research Program. RESULTS:Subjects with severe asthma had a significant decline in postbronchodilator forced expiratory volume in 1 second percent (FEV1%) predicted over time (P < .001). Airway wall thickness measured by MDCT was increased at multiple airway generations in severe asthma compared to mild-to-moderate asthma (wall area percent [WA%]: P < .05) and normals (P < .05) at baseline and year 2. Over time, there was an increase in WA% and wall thickness percent (WT%) in all subjects (P = .030 and .009, respectively) with no change in emphysema-like lung or air trapping. Baseline prebronchodilator FEV1% inversely correlated with WA% and WT% (both P < .05). In a multivariable regression model, baseline WA%, race, and health care utilization were predictors of subsequent airway remodeling. CONCLUSIONS:Severe asthma subjects have a greater decline in lung function over time than normal subjects or those with mild-to-moderate asthma. MDCT provides a noninvasive measure of airway wall thickness that may predict subsequent airway remodeling.
Project description:Airway bacterial infections are a major problem in lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Increased Th2 cytokines, such as IL-13, are observed in lung diseases and may contribute to bacterial infections. How Th2 cytokines affect bacterial infection remains unknown. MUC18, an adhesion molecule shown to be involved in the pathogenesis of malignant melanoma, has been recently identified in airway epithelial cells of patients with COPD. We investigated MUC18 regulation by IL-13 and the role of MUC18 in bacterial adherence to epithelial cells. Human airway tissues, brushed bronchial epithelial cells from normal subjects and subjects with asthma, and epithelial cell lines (e.g., HEK293 cells) were used to study the regulation of MUC18 by IL-13 and the involvement of MUC18 in bacterial (e.g., Mycoplasma pneumoniae [Mp] and nontypeable Haemophilus influenzae [NTHi]) adherence to epithelial cells. Asthmatic bronchial epithelium expressed higher levels of MUC18 than normal bronchial epithelium. IL-13 increased MUC18 in cultured bronchial epithelial cells from normal subjects and particularly from subjects with asthma. IL-13-induced MUC18 expression may be modulated in part through transcription factor specificity protein 1. Overexpression of human MUC18 in HEK293 cells increased cell-associated Mp and NTHi levels. Moreover, MUC18 was shown to directly interact with Mp and NTHi. These results for the first time show that an allergic airway milieu (e.g., IL-13) increases MUC18 expression, which may contribute to increased bacterial infection/colonization in asthma and other lung diseases.
Project description:<h4>Background</h4>Previously, we found that mast cell tryptases and carboxypeptidase A3 (CPA3) are differentially expressed in the airway epithelium in asthmatic subjects. We also found that asthmatic subjects can be divided into 2 subgroups ("T(H)2 high" and "T(H)2 low" asthma) based on epithelial cell gene signatures for the activity of T(H)2 cytokines.<h4>Objectives</h4>We sought to characterize intraepithelial mast cells (IEMCs) in asthma.<h4>Methods</h4>We performed gene expression profiling in epithelial brushings and stereology-based quantification of mast cell numbers in endobronchial biopsy specimens from healthy control and asthmatic subjects before and after treatment with inhaled corticosteroids (ICSs). We also performed gene expression and protein quantification studies in cultured airway epithelial cells and mast cells.<h4>Results</h4>By means of unsupervised clustering, mast cell gene expression in the airway epithelium related closely to the expression of IL-13 signature genes. The levels of expression of mast cell genes correlate positively with lung function improvements with ICSs. IEMC density was 2-fold higher than normal in subjects with T(H)2-high asthma compared with that seen in subjects with T(H)2-low asthma or healthy control subjects (P = .015 for both comparisons), and these cells were characterized by expression of tryptases and CPA3 but not chymase. IL-13 induced expression of stem cell factor in cultured airway epithelial cells, and mast cells exposed to conditioned media from IL-13-activated epithelial cells showed downregulation of chymase but no change in tryptase or CPA3 expression.<h4>Conclusion</h4>IEMC numbers are increased in subjects with T(H)2-high asthma, have an unusual protease phenotype (tryptase and CPA3 high and chymase low), and predict responsiveness to ICSs. IL-13-stimulated production of stem cell factor by epithelial cells potentially explains mast cell accumulation in T(H)2-high asthmatic epithelium.
Project description:<h4>Rationale</h4>The hallmarks of allergic asthma are airway inflammation, obstruction, and remodeling. Airway remodeling may lead to irreversible airflow obstruction with increased morbidity and mortality. Despite advances in the treatment of asthma, the mechanisms underlying airway remodeling are still poorly understood. We reported that insulin-like growth factor (IGF) binding proteins (IGFBPs) contribute to extracellular matrix deposition in idiopathic pulmonary fibrosis; however, their contribution to airway remodeling in asthma has not been established.<h4>Objectives</h4>We hypothesized that IGFBP-3 is overexpressed in asthma and contributes to airway remodeling.<h4>Methods</h4>We evaluated levels of IGFBP-3 in tissues and bronchoalveolar lavage fluid from patients with asthma at baseline and 48 hours after allergen challenge, in reparative epithelium in an in vitro wounding assay, and in conditioned media from cytokine- and growth factor-stimulated primary epithelial cells.<h4>Measurements and main results</h4>IGFBP-3 levels and distribution were evaluated by Western blot, ELISA, and immunofluorescence. IGFBP-3 is increased in vivo in the airway epithelium of patients with asthma compared with normal control subjects. The concentration of IGFBP-3 is increased in the bronchoalveolar lavage fluid of patients with asthma after allergen challenge, its levels are increased in reparative epithelium in an in vitro wounding assay and in the conditioned medium of primary airway epithelial cell cultures stimulated with IGF-I.<h4>Conclusions</h4>Our results suggest that one mechanism of allergic airway remodeling is through the secretion of the profibrotic IGFBP-3 from IGF-I-stimulated airway epithelial cells during allergic inflammation.
Project description:Airway remodeling in asthma is characterized by reticular basement membrane (RBM) thickening, likely related to epithelial structural and functional changes. Gene expression profiling of the airway epithelium might identify genes involved in bronchial structural alterations. We analyzed bronchial wall geometry (computed tomography (CT)), RBM thickness (histology), and the bronchial epithelium transcriptome profile (gene expression array) in moderate to severe persistent (<i>n</i> = 21) vs. no persistent (<i>n</i> = 19) airflow limitation asthmatics. RBM thickness was similar in the two studied subgroups. Among the genes associated with increased RBM thickness, the most essential were those engaged in cell activation, proliferation, and growth (e.g., <i>CDK20</i>, <i>TACC2</i>, <i>ORC5</i>, and <i>NEK5</i>) and inhibiting apoptosis (e.g., higher mRNA expression of <i>RFN34</i>, <i>BIRC3</i>, <i>NAA16</i>, and lower of <i>RNF13</i>, <i>MRPL37</i>, <i>CACNA1G</i>). Additionally, RBM thickness correlated with the expression of genes encoding extracellular matrix (ECM) components (<i>LAMA3</i>, <i>USH2A</i>), involved in ECM remodeling (<i>LTBP1</i>), neovascularization (<i>FGD5</i>, <i>HPRT1</i>), nerve functioning (<i>TPH1</i>, <i>PCDHGC4</i>), oxidative stress adaptation (<i>RIT1</i>, <i>HSP90AB1</i>), epigenetic modifications (<i>OLMALINC</i>, <i>DNMT3A</i>), and the innate immune response (<i>STAP1</i>, <i>OAS2</i>). Cluster analysis revealed that genes linked with RBM thickness were also related to thicker bronchial walls in CT. Our study suggests that the pro-fibrotic profile in the airway epithelial cell transcriptome is associated with a thicker RBM, and thus, may contribute to asthma airway remodeling.
Project description:BACKGROUND:Eosinophilic inflammation is implicated in asthma. Eotaxin 1-3 regulate eosinophil trafficking into the airways along with other chemotactic factors. However, the epithelial and bronchoalveolar lavage (BAL) cell expression of these chemokines in relation to asthma severity and eosinophilic phenotypes has not been addressed. OBJECTIVE:To measure the expression of the three eotaxin isoforms in bronchoscopically obtained samples and compare them with clinically relevant parameters between normal subjects and patients with asthma. METHODS:Normal subjects and patients with asthma of varying severity recruited through the Severe Asthma Research Program underwent clinical assessment and bronchoscopy with airway brushing and BAL. Eotaxin 1-3 mRNA/protein were measured in epithelial and BAL cells and compared with asthma severity, control and eosinophilic inflammation. RESULTS:Eotaxin-2 and eotaxin-3 mRNA and eotaxin-2 protein were increased in airway epithelial brushings from patients with asthma and were highest in cases of severe asthma (p values 0.0155, 0.0033 and 0.0006, respectively), with eotaxin-2 protein increased with age at onset. BAL cells normally expressed high levels of eotaxin-2 mRNA/protein but BAL fluid levels of eotaxin-2 were lowest in severe asthma. Epithelial eotaxin-2 and eotaxin-3 mRNA/protein was associated with sputum eosinophilia, lower forced expiratory volume in 1 s and more asthma exacerbations. Airway epithelial cell eotaxin-2 protein differed by asthma severity only in those with late onset disease, and tended to be highest in those with late onset eosinophilic asthma. CONCLUSIONS:Epithelial eotaxin-2 and 3 are increased in asthma and severe asthma. Their expression may contribute to luminal migration of eosinophils, especially in later onset disease, asthma control and severity.
Project description: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.
Project description:Severe asthma (SA) remains poorly understood. Mast cells (MC) are implicated in asthma pathogenesis, but it remains unknown how their phenotype, location, and activation relate to asthma severity.To compare MC-related markers measured in bronchoscopically obtained samples with clinically relevant parameters between normal subjects and subjects with asthma to clarify their pathobiologic importance.Endobronchial biopsies, epithelial brushings, and bronchoalveolar lavage were obtained from subjects with asthma and normal subjects from the Severe Asthma Research Program (N = 199). Tryptase, chymase, and carboxypeptidase A (CPA)3 were used to identify total MC (MC(Tot)) and the MC(TC) subset (MCs positive for both tryptase and chymase) using immunostaining and quantitative real-time polymerase chain reaction. Lavage was analyzed for tryptase and prostaglandin D2 (PGD2) by ELISA.Submucosal MC(Tot) (tryptase-positive by immunostaining) numbers were highest in "mild asthma/no inhaled corticosteroid (ICS) therapy" subjects and decreased with greater asthma severity (P = 0.002). In contrast, MC(TC) (chymase-positive by immunostaining) were the predominant (MC(TC)/MC(Tot) > 50%) MC phenotype in SA (overall P = 0.005). Epithelial MC(Tot) were also highest in mild asthma/no ICS, but were not lower in SA. Instead, they persisted and were predominantly MC(TC). Epithelial CPA3 and tryptase mRNA supported the immunostaining data (overall P = 0.008 and P = 0.02, respectively). Lavage PGD2 was higher in SA than in other steroid-treated groups (overall P = 0.02), whereas tryptase did not differentiate the groups. In statistical models, PGD2 and MC(TC)/MC(Tot) predicted SA.Severe asthma is associated with a predominance of MC(TC) in the airway submucosa and epithelium. Activation of those MC(TC) may contribute to the increases in PGD2 levels. The data suggest an altered and active MC population contributes to SA pathology.
Project description:BACKGROUND: The link between upper and lower airways in patients with both asthma and allergic rhinitis is still poorly understood. As the biological complexity of these disorders can be captured by gene expression profiling we hypothesized that the clinical expression of rhinitis and/or asthma is related to differential gene expression between upper and lower airways epithelium. OBJECTIVE: Defining gene expression profiles of primary nasal and bronchial epithelial cells from the same individuals and examining the impact of allergic rhinitis with and without concomitant allergic asthma on expression profiles. METHODS: This cross-sectional study included 18 subjects (6 allergic asthma and allergic rhinitis; 6 allergic rhinitis; 6 healthy controls). The estimated false discovery rate comparing 6 subjects per group was approximately 5%. RNA was extracted from isolated and cultured epithelial cells from bronchial brushings and nasal biopsies, and analyzed by microarray (Affymetrix U133+ PM Genechip Array). Data were analysed using R and Bioconductor Limma package. For gene ontology GeneSpring GX12 was used. RESULTS: The study was successfully completed by 17 subjects (6 allergic asthma and allergic rhinitis; 5 allergic rhinitis; 6 healthy controls). Using correction for multiple testing, 1988 genes were differentially expressed between healthy lower and upper airway epithelium, whereas in allergic rhinitis with or without asthma this was only 40 and 301 genes, respectively. Genes influenced by allergic rhinitis with or without asthma were linked to lung development, remodeling, regulation of peptidases and normal epithelial barrier functions. CONCLUSIONS: Differences in epithelial gene expression between the upper and lower airway epithelium, as observed in healthy subjects, largely disappear in patients with allergic rhinitis with or without asthma, whilst new differences emerge. The present data identify several pathways and genes that might be potential targets for future drug development.
Project description:The fungal allergen, Alternaria, is specifically associated with severe asthma, including life-threatening exacerbations. To better understand the acute innate airway response to Alternaria, naive wild-type (WT) mice were challenged once intranasally with Alternaria. Naive WT mice developed significant bronchoalveolar lavage eosinophilia following Alternaria challenge when analyzed 24 h later. In contrast to Alternaria, neither Aspergillus nor Candida induced bronchoalveolar lavage eosinophilia. Gene microarray analysis of airway epithelial cell brushings demonstrated that Alternaria-challenged naive WT mice had a >20-fold increase in the level of expression of found in inflammatory zone 1 (FIZZ1/Retnla), a resistin-like molecule. Lung immunostaining confirmed strong airway epithelial FIZZ1 expression as early as 3 h after a single Alternaria challenge that persisted for ?5 d and was significantly reduced in STAT6-deficient, but not protease-activated receptor 2-deficient mice. Bone marrow chimera studies revealed that STAT6 expressed in lung cells was required for epithelial FIZZ1 expression, whereas STAT6 present in bone marrow-derived cells contributed to airway eosinophilia. Studies investigating which cells in the nonchallenged lung bind FIZZ1 demonstrated that CD45(+)CD11c(+) cells (macrophages and dendritic cells), as well as collagen-1-producing CD45(-) cells (fibroblasts), can bind to FIZZ1. Importantly, direct administration of recombinant FIZZ1 to naive WT mice led to airway eosinophilia, peribronchial fibrosis, and increased thickness of the airway epithelium. Thus, Alternaria induces STAT6-dependent acute airway eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness. This may provide some insight into the uniquely pathogenic aspects of Alternaria-associated asthma.