Background Tight junctions (TJs) form a barrier on the apical side of neighboring epithelial cells in the bronchial mucosa. Changes in their integrity might play a role in asthma pathogenesis by ...enabling the paracellular influx of allergens, toxins, and microbes to the submucosal tissue. Objective The regulation of bronchial epithelial TJs by TH 2 cells and their cytokines and their involvement in epigenetic regulation of barrier function were investigated. Methods The expression, regulation, and function of TJs were determined in air-liquid interface (ALI) cultures of control and asthmatic primary human bronchial epithelial cells (HBECs) by means of analysis of transepithelial electrical resistance, paracellular flux, mRNA expression, Western blotting, and immunofluorescence staining. Results HBECs from asthmatic patients showed a significantly low TJ integrity in ALI cultures compared with HBECs from healthy subjects. TH 2 cell numbers and levels of their cytokines, IL-4 and IL-13, decreased barrier integrity in ALI cultures of HBECs from control subjects but not in HBECs from asthmatic patients. They induced a physical separation of the TJs of adjacent cells in immunofluorescence staining of the TJ molecules occludin and zonula occludens-1. We observed that expression of histone deacetylases (HDACs) 1 and 9, and Silent information regulator genes (sirtuins SIRTs) 6 and 7 were significantly high in HBECs from asthmatic patients. IL-4 and IL-13 significantly increased the expression of HDACs and SIRTs. The role of HDAC activation on epithelial barrier leakiness was confirmed by HDAC inhibition, which improved barrier integrity through increased synthesis of TJ molecules in epithelium from asthmatic patients to the level seen in HBECs from control subjects. Conclusion Our data demonstrate that barrier leakiness in asthmatic patients is induced by TH 2 cells, IL-4, and IL-13 and HDAC activity. The inhibition of endogenous HDAC activity reconstitutes defective barrier by increasing TJ expression.
Human rhinoviruses (HRV) are frequent cause of asthma exacerbations, however the influence of airway inflammation on the severity of viral infection is poorly understood. Here, we investigated how ...cytokine-induced remodeling of airway epithelium modulates antiviral response. We analyzed gene expression response in in vitro differentiated bronchial epithelium exposed to cytokines and next infected with HRV16. IL-13-induced mucous cell metaplasia (MCM) was associated with impaired ciliogenesis and induction of antiviral genes, resulting in lower susceptibility to HRV. Epithelial-mesenchymal transition caused by TGF-β was associated with increased virus replication and boosted innate response. Moreover, HRV infection per se caused transient upregulation of MCM markers and growth factors, followed by low-level virus replication and shedding. Our data suggest that the outcome of HRV infection depends on the type of lower airway inflammation and the extent of epithelial damage. Type-2 inflammation (eosinophilic asthma) may induce antiviral state of epithelium and decrease virus sensitivity, while growth factor exposure during epithelial repair may facilitate virus replication and inflammatory response. Additionally, responses to HRV were similar in cells obtained from asthma patients and control subjects, which implicates that antiviral mechanisms are not intrinsically impaired in asthma, but may develop in the presence of uncontrolled airway inflammation.
Churg-Strauss syndrome (CSS) is a rare systemic vasculitis associated with eosinophilia and asthma. We assessed the local immune response in airways of CSS patients with different activity of the ...disease.
Concentration of IL-5, CCL17, CCL22 and CCL26 (ELISA) together with cell expression of T-helper-related genes (real-time PCR array) were measured in bronchoalveolar lavage fluid (BALF) sampled from 11 patients with active CSS, 11 patients with CSS in remission and 9 control subjects with bronchial asthma.
In active CSS, both BALF and blood eosinophil counts were increased (P<0.01). BALF cells in active disease were characterized by an increased expression of Th2 and regulatory-type transcripts: STAT6, STAT3, GATA3, IL4, IL5 and IL10 as compared with asthmatics, and STAT5A, CCR4, FOXP3, IL4, IL5 and IL10 when compared with inactive CSS. There was significant increase in BALF concentration of IL-5 and CCL26 in exacerbation of CSS. CCR4-active chemokines were detected more frequently in active disease. We found a strong positive correlation between clinical parameters of disease activity (BVAS, eosinophilia) and expression of IL4, IL5, IL10 and STAT5A.
These results indicate that as compared with asthma, active-CSS patients have much stronger local Th2 response in the airways. Airway cells may contribute to lung eosinophilia in CSS by producing IL-5 and eosinophil active chemokines.
...bronchial epithelial cells are at the forefront of tissue defense and the innate immune response, preventing invasion of tissues as a physical barrier. CpG-2006 stimulation remarkably restored the ...impaired TJ immunostaining of bronchial epithelial ALI cultures from asthmatic patients (Fig 2, B, and see Fig E5, B), which is consistent with the TER and FITC-dextran flux measurements shown above. ...improvement of the barrier in bronchial epithelial cells by CpG-2006 seems to be mediated by enhanced expression of TJ-related molecules and their appropriate distribution on cell-cell borders.
Rhinoviruses and allergens, such as house dust mite are major agents responsible for asthma exacerbations. The influence of pre-existing airway inflammation on the infection with severe acute ...respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. We analyse mechanisms of response to viral infection in experimental in vivo rhinovirus infection in healthy controls and patients with asthma, and in in vitro experiments with house dust mite, rhinovirus and SARS-CoV-2 in human primary airway epithelium. Here, we show that rhinovirus infection in patients with asthma leads to an excessive RIG-I inflammasome activation, which diminishes its accessibility for type I/III interferon responses, leading to their early functional impairment, delayed resolution, prolonged viral clearance and unresolved inflammation in vitro and in vivo. Pre-exposure to house dust mite augments this phenomenon by inflammasome priming and auxiliary inhibition of early type I/III interferon responses. Prior infection with rhinovirus followed by SARS-CoV-2 infection augments RIG-I inflammasome activation and epithelial inflammation. Timely inhibition of the epithelial RIG-I inflammasome may lead to more efficient viral clearance and lower the burden of rhinovirus and SARS-CoV-2 infections.
The objective of our study was to evaluate the T‐helper (Th) and regulatory T (Treg) cell profile in ANCA‐positive granulomatosis with polyangiitis (GPA) and its relation to disease activity. In a ...prospective study, we studied two groups of GPA patients: (i) disease flare (active‐GPA, BVAS>6, n = 19), (ii) sustained remission (≥ 1‐year prior enrollment, inactive‐GPA, BVAS = 0, n = 18). 24 age‐sex matched healthy subjects served as controls. Active‐GPA patients were followed for 6 months and reevaluated during remission (early remission; n = 13). We analyzed subsets of Th‐cells (flow cytometry), production of signature cytokines by in vitro stimulated lymphocytes, and broad spectrum of serum cytokines (Luminex). In all GPA patients we observed expansion of effector Th17 cells, and increased production of IL‐17A by in vitro stimulated T cells, as compared to controls. Disease flare was characterized by marked reduction in Treg cells, whereas in sustained remission we showed expansion of both Treg and Th2 subset. Finally, analyzing the cytokine profile, we identified CCL23 and LIGHT, as potential biomarkers of active disease. We conclude that in GPA, expansion of Treg and Th2 lymphocytes in parallel to increased Th17 response is a characteristic feature of sustained remission. In contrast, Treg cells are markedly decreased in disease flare.
We evaluated functional subsets of T‐helper cells in granulomatosis with polyangiitis (GPA) patients. Disease flare was characterized by expansion of Th17 and marked reduction in Treg cells. We showed suppression of Th17 responses early after induction therapy, while sustained remission was linked with additional expansion of Treg and Th2 subsets.
Hypoxic conditions induce the expression of hypoxia-inducible factors (HIFs) that allow cells to adapt to the changing conditions and alter the expression of a number of genes including the cystic ...fibrosis transmembrane conductance regulator (CFTR).
is a low abundance mRNA in airway epithelial cells even during normoxic conditions, but during hypoxia its mRNA expression decreases even further.
In the current studies, we examined the kinetics of hypoxia-induced changes in
mRNA and protein levels in two human airway epithelial cell lines, Calu-3 and 16HBE14o-, and in normal primary bronchial epithelial cells. Our goal was to examine the posttranscriptional modifications that affected CFTR expression during hypoxia. We utilized in silico predictive protocols to establish potential miRNAs that could potentially regulate
message stability and identified miR-200b as a candidate molecule.
Analysis of each of the epithelial cell types during prolonged hypoxia revealed that
expression decreased after 12 h during a time when miR-200b was continuously upregulated. Furthermore, manipulation of the miRNA levels during normoxia and hypoxia using miR-200b mimics and antagomirs decreased and increased
mRNA levels, respectively, and thus established that miR-200b downregulates
message levels during hypoxic conditions.
The data suggest that miR-200b may be a suitable target for modulating CFTR levels in vivo.
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 (
= 21) vs. no persistent (
= 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.,
,
,
, and
) and inhibiting apoptosis (e.g., higher mRNA expression of
,
,
, and lower of
,
,
). Additionally, RBM thickness correlated with the expression of genes encoding extracellular matrix (ECM) components (
,
), involved in ECM remodeling (
), neovascularization (
,
), nerve functioning (
,
), oxidative stress adaptation (
,
), epigenetic modifications (
,
), and the innate immune response (
,
). 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.
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•CTGF contributes to fibroblasts-to-myofibroblasts transition in bronchial asthma.•TGF-β1-induced FMT in bronchial fibroblasts of asthmatics is enhanced by CTGF.•CTGF silencing ...potently decreases TGF-β1-induced FMT in bronchial fibroblasts.
Fibroblast to myofibroblast transition (FMT) contributes to bronchial wall remodelling in persistent asthma. Among other numerous factors involved, transforming growth factor type β (TGF-β) plays a pivotal role. Recently it has been demonstrated that connective tissue growth factor (CTGF), a matricellular protein, combines with TGF-β in the pathomechanism of many fibrotic disorders. However, it is not clear whether this interaction takes place in asthma as well.
Primary cultures of human bronchial fibroblasts from asthmatic and non-asthmatic subjects were used to investigate the impact of CTGF and TGF-β1 on the fibroblast to myofibroblast transition. The combined activity of TGF-β1 and CTGF resulted in an average of 90% of FMT accomplished in cell lines derived from asthmatics. In this group FMT was highly dependent on the presence of CTGF produced by the cells, as shown by gene silencing experiments with the specific siRNA.
Results support the important role of CTGF biosynthesis in the asthmatic bronchi amplifying FMT. This is evidenced by inhibition of TGF-β1-induced FMT following CTGF silencing in asthmatic bronchial fibroblasts. CTGF is produced by fibroblasts and contributes to the FMT phenomenon in positive loop-back, inducing and boosting TGF-β1 triggered FMT. Thus, CTGF is a promising target for pharmacological intervention in secondary prevention of bronchial remodelling in asthma.
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