Vulnerability to acid reflux of the airway epithelium in severe asthma

BackgroundSevere asthma is associated with multiple co-morbidities, including gastro-oesophageal reflux disease (GORD) which can contribute to exacerbation frequency and poor quality of life. Since epithelial dysfunction is an important feature in asthma, we hypothesised that in severe asthma the bronchial epithelium is more susceptible to the effects of acid reflux.MethodsWe developed an in vitro model of GORD using differentiated bronchial epithelial cells (BECs) from normal or severe asthmatic donors exposed to a combination of pepsin, acid pH, and bile acids using a multiple challenge protocol (MCP-PAB). We also analysed bronchial biopsies and undertook RNA-sequencing of bronchial brushings from controls and severe asthmatics without or with GORD.ResultsExposure of BECs to the MCP-PAB caused structural disruption, increased permeability, IL-33 expression, inflammatory mediator release and changes in gene expression for multiple biological processes. Cultures from severe asthmatics were significantly more affected than those from healthy donors. Analysis of bronchial biopsies confirmed increased IL-33 expression in severe asthmatics with GORD. RNA-sequencing of bronchial brushings from this group identified 15 of the top 37 dysregulated genes found in MCP-PAB treated BECs, including genes involved in oxidative stress responses.ConclusionsBy affecting epithelial permeability, GORD may increase exposure of the airway submucosa to allergens and pathogens, resulting in increased risk of inflammation and exacerbations. Clinical implication: These results suggest the need for research into alternative therapeutic management of GORD in severe asthma.

Decreased Systemic and Airway Sirtuin 1 Expression in Adults With Bronchiectasis

Aim: Whether accelerated aging, reflected by sirtuin 1 (SIRT1) expression, is implicated in bronchiectasis remains largely unknown. We sought to determine the patterns of SIRT1 and other aging markers in systemic circulation and airways and their expression levels associated with bronchiectasis severity and exacerbation.Methods: We enrolled 132 patients with bronchiectasis and 50 healthy subjects in a prospective cohort study to profile aging markers in systemic circulation and recruited 36 patients with bronchiectasis and 32 disease controls (idiopathic pulmonary fibrosis or tumors) in a cross-sectional study to profile aging markers in bronchial epithelium of both large-to-medium and small airways. We profiled aging marker expression from peripheral blood mononuclear cells and enumerated the positively stained cells for detection of aging marker expression in bronchial epithelium.Results: Compared with healthy controls, the relative telomere length (median: 0.88 vs. 0.99, p = 0.009), SIRT1 (median: 0.89 vs. 0.99, p = 0.002), and Ku80 (median: 0.87 vs. 0.96, p < 0.001) expression levels were consistently lower in the peripheral blood mononuclear cells among patients with bronchiectasis and modestly discriminated patients with bronchiectasis from healthy controls. No remarkable changes in SIRT1, telomere length, or Ku70 were identified at onset of exacerbation. Within the bronchial epithelium, the percentage of positively stained cells was lower for SIRT1 (median: 25.1 vs. 57.2%, p < 0.05) and numerically lower for p16 (median: 40.0 vs. 45.1%) and p21 (median: 28.9 vs. 35.9%) in patients with bronchiectasis than in disease controls (p > 0.05).Conclusion: SIRT1 was downregulated in systemic circulation and bronchiectatic airways, which was independent of disease severity and lung function impairment.

Role of Toll-like receptors in COVID-19 pathogenesis

Introduction. This review summarizes the available information on the role of Toll-like receptors (TLRs) in the pathogenesis of the novel coronavirus infection COVID-19 induced by SARS-CoV-2. The exact pathogenesis of COVID-19 and the role of each component of innate and adaptive immunity are still unknown. Aim. Discussion of the possible role of TLRs in the immune response in COVID-19 infection. Results. Analysis of the literature in the PubMed database showed that the mechanism of penetration of SARS-CoV-2 and lysis of type II alveolocytes is the binding of the spike S-glycoprotein or capsid protein M of the virus to the receptor-binding domain of ACE2 on the surface of epithelial cells. Migration and infiltration of inflammatory cells leads to overactivation of TLR4 on the surface of alveolocytes and bronchial epithelium, shifting the process to MyD88-dependent acute inflammatory signaling and hypersecretion of proinflammatory cytokines that cause a “cytokine storm” and the development of severe complications of COVID-19 infection, in particular, acute respiratory infections, respiratory distress syndrome, pathology of internal organs, and, in some cases, the death of the patient. Conclusion. It is possible to suggest that TLRs have an impact on the immune response in COVID19 infection. Both antagonists and agonists of TLRs, depending on their type, can be examined to determine the therapeutic and negative effects of COVID-19 infection. Further research is needed to investigate TLRs and pathways for activating cytokine expression, as they indicate a direct relationship with mortality and virus susceptibility. Bioinformatic research can also help to better understand the interaction of TLRs with proteins and RNA of the SARS-CoV-2.

Imiquimod Boosts Interferon Response, and Decreases ACE2 and Pro-Inflammatory Response of Human Bronchial Epithelium in Asthma

BackgroundBoth anti-viral and anti-inflammatory bronchial effects are warranted to treat viral infections in asthma. We sought to investigate if imiquimod, a TLR7 agonist, exhibits such dual actions in ex vivo cultured human bronchial epithelial cells (HBECs), targets for SARS-CoV-2 infectivity.ObjectiveTo investigate bronchial epithelial effects of imiquimod of potential importance for anti-viral treatment in asthmatic patients.MethodsEffects of imiquimod alone were examined in HBECs from healthy (N=4) and asthmatic (N=18) donors. Mimicking SARS-CoV-2 infection, HBECs were stimulated with poly(I:C), a dsRNA analogue, or SARS-CoV-2 spike-protein 1 (SP1; receptor binding) with and without imiquimod treatment. Expression of SARS-CoV-2 receptor (ACE2), pro-inflammatory and anti-viral cytokines were analyzed by RT-qPCR, multiplex ELISA, western blot, and Nanostring and proteomic analyses.ResultsImiquimod reduced ACE2 expression at baseline and after poly(I:C) stimulation. Imiquimod also reduced poly(I:C)-induced pro-inflammatory cytokines including IL-1β, IL-6, IL-8, and IL-33. Furthermore, imiquimod increased IFN-β expression, an effect potentiated in presence of poly(I:C) or SP1. Multiplex mRNA analysis verified enrichment in type-I IFN signaling concomitant with suppression of cytokine signaling pathways induced by imiquimod in presence of poly(I:C). Exploratory proteomic analyses revealed potentially protective effects of imiquimod on infections.ConclusionImiquimod triggers viral resistance mechanisms in HBECs by decreasing ACE2 and increasing IFN-β expression. Additionally, imiquimod improves viral infection tolerance by reducing viral stimulus-induced epithelial cytokines involved in severe COVID-19 infection. Our imiquimod data highlight feasibility of producing pluripotent drugs potentially suited for anti-viral treatment in asthmatic subjects.

Cystic Fibrosis: Systems Biology Analysis from Homozygous p.Phe508del Variant Patients’ Samples Reveals Perturbations in Tissue-Specific Pathways

Cystic fibrosis (CF) is an autosomal recessive disorder, caused by diverse genetic variants for the CF transmembrane conductance regulator (CFTR) protein. Among these, p.Phe508del is the most prevalent variant. The effects of this variant on the physiology of each tissue remains unknown. This study is aimed at predicting cell signaling pathways present in different tissues of fibrocystic patients, homozygous for p.Phe508del. The study involved analysis of two microarray datasets, E-GEOD-15568 and E-MTAB-360 corresponding to the rectal and bronchial epithelium, respectively, obtained from the ArrayExpress repository. Particularly, differentially expressed genes (DEGs) were predicted, protein-protein interaction (PPI) networks were designed, and centrality and functional interaction networks were analyzed. The study reported that p.Phe508del-mutated CFTR-allele in homozygous state influenced the whole gene expression in each tissue differently. Interestingly, gene ontology (GO) term enrichment analysis revealed that only “neutrophil activation” was shared between both tissues; however, nonshared DEGs were grouped into the same GO term. For further verification, functional interaction networks were generated, wherein no shared nodes were reported between these tissues. These results suggested that the p.Phe508del-mutated CFTR-allele in homozygous state promoted tissue-specific pathways in fibrocystic patients. The generated data might further assist in prediction diagnosis to define biomarkers or devising therapeutic strategies.

Nonspecific binding of common anti-CFTR antibodies in ciliated cells of human airway epithelium

There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR.

Airway Wall Remodeling in Childhood Asthma—A Personalized Perspective from Cell Type-Specific Biology

Airway wall remodeling is a pathology occurring in chronic inflammatory lung diseases including asthma, chronic obstructive pulmonary disease, and fibrosis. In 2017, the American Thoracic Society released a research statement highlighting the gaps in knowledge and understanding of airway wall remodeling. The four major challenges addressed in this statement were: (i) the lack of consensus to define “airway wall remodeling” in different diseases, (ii) methodologic limitations and inappropriate models, (iii) the lack of anti-remodeling therapies, and (iv) the difficulty to define endpoints and outcomes in relevant studies. This review focuses on the importance of cell-cell interaction, especially the bronchial epithelium, in asthma-associated airway wall remodeling. The pathology of “airway wall remodeling” summarizes all structural changes of the airway wall without differentiating between different pheno- or endo-types of asthma. Indicators of airway wall remodeling have been reported in childhood asthma in the absence of any sign of inflammation; thus, the initiation event remains unknown. Recent studies have implied that the interaction between the epithelium with immune cells and sub-epithelial mesenchymal cells is modified in asthma by a yet unknown epigenetic mechanism during early childhood.

Epithelial RIG-I inflammasome activation suppresses antiviral immunity and promotes inflammatory responses in virus-induced asthma exacerbations and COVID-19

Rhinoviruses (RV) and inhaled allergens, such as house dust mite (HDM) are the major agents responsible for asthma onset, its life-threatening exacerbations and progression to severe disease. The role of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in exacerbations of asthma or the influence of preexisting viral or allergic airway inflammation on the development of coronavirus disease 2019 (COVID-19) is largely unknown. To address this, we compared molecular mechanisms of HDM, RV and SARS-CoV-2 interactions in experimental RV infection in patients with asthma and healthy individuals. RV infection was sensed via retinoic acid-inducible gene I (RIG-I) helicase, but not via NLR family pyrin domain containing 3 (NLRP3), which led to subsequent apoptosis-associated speck like protein containing a caspase recruitment domain (ASC) recruitment, oligomerization and RIG-I inflammasome activation. This phenomenon was augmented in bronchial epithelium in patients with asthma, especially upon pre-exposure to HDM, which itself induced a priming step, pro-IL-1β release and early inhibition of RIG-I/TANK binding kinase 1/IκB kinase ϵ/type I/III interferons (RIG-I/TBK1/IKKϵ/IFN-I/III) responses. Excessive activation of RIG-I inflammasomes was partially responsible for the alteration and persistence of type I/III IFN responses, prolonged viral clearance and unresolved inflammation in asthma. RV/HDM-induced sustained IFN I/III responses initially restricted SARS-CoV-2 replication in epithelium of patients with asthma, but even this limited infection with SARS-CoV-2 augmented RIG-I inflammasome activation. Timely inhibition of the epithelial RIG-I inflammasome and reduction of IL-1β signaling may lead to more efficient viral clearance and lower the burden of RV and SARS-CoV-2 infection.

CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

The conducting airways are lined by distinct cell types, comprising basal, secretory, ciliated, and rare cells, including ionocytes, solitary cholinergic chemosensory cells, and solitary and clustered (neuroepithelial bodies) neuroendocrine cells. Airway neuroendocrine cells are in clinical focus since they can give rise to small cell lung cancer. They have been implicated in diverse functions including mechanosensation, chemosensation, and regeneration, and were recently identified as regulators of type 2 immune responses via the release of the neuropeptide calcitonin gene-related peptide (CGRP). We here assessed the expression of the chemokine CXCL13 (B cell attracting chemokine) by these cells by RT-PCR, in silico analysis of publicly available sequencing data sets, immunohistochemistry, and immuno-electron microscopy. We identify a phenotype of neuroendocrine cells in the naïve mouse, producing the chemokine CXCL13 predominantly in solitary neuroendocrine cells of the tracheal epithelium (approx. 70% CXCL13+) and, to a lesser extent, in the solitary neuroendocrine cells and neuroepithelial bodies of the intrapulmonary bronchial epithelium (< 10% CXCL13+). In silico analysis of published sequencing data of murine tracheal epithelial cells was consistent with the results obtained by immunohistochemistry as it revealed that neuroendocrine cells are the major source of Cxcl13-mRNA, which was expressed by 68–79% of neuroendocrine cells. An unbiased scRNA-seq data analysis of overall gene expression did not yield subclusters of neuroendocrine cells. Our observation demonstrates phenotypic heterogeneity of airway neuroendocrine cells and points towards a putative immunoregulatory role of these cells in bronchial-associated lymphoid tissue formation and B cell homeostasis.