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In patients with asthma, respiratory syncytial virus (RSV) infections can cause disease exacerbations by infecting the epithelial layer of the airways, inducing an innate and adaptive immune response. The type-I interferon antiviral response of epithelial cells upon RSV infection is found to be reduced in asthma in most -but not all-studies. Moreover, the molecular mechanisms that cause the differences in the asthmatic bronchial epithelium in response to viral infection are poorly understood.Here, we investigated the transcriptional response to RSV infection of primary bronchial epithelial cells (pBECs) from asthma patients(n=8) and healthy donors(n=8). The pBECs obtained from bronchial brushes were differentiated in air-liquid interface conditions and infected with RSV. After three days, cells were processed for single-cell RNA sequencing.A strong antiviral response to RSV was observed for all cell types present, from both asthma patients and healthy donors. Most differentially regulated genes following RSV infection were found in cells transitioning from basal to secretory. Goblet cells from asthma patients showed lower expression of genes involved in the interferon response. In multiciliated cells, an impairment of the signaling pathways involved in the response to RSV in asthma was observed, including no enrichment of the type-III interferon response.Our results highlight that the response to RSV infection of the bronchial epithelium in asthma and healthy airways was largely similar. However, in asthma, the response of goblet and the multiciliated cells was impaired, highlighting the need for studying airway epithelial cells at high resolution in the context of asthma exacerbations.What is already know on this topicThe airway epithelium response to RSV is altered in asthma. However, literature remains conflicted about the exact changes in the antiviral response, and the mechanisms causing these changes are yet to be found.What this study addsThis study describes extensively the response of the bronchial epithelial cells (BECs) to RSV for both healthy subjects and asthma patients, at a single-cell resolution. It highlights the major overlap between healthy and asthma in the antiviral response to RSV. It allows the identification of specific genes and cell types that show a different behavior in response to RSV in asthma compared to healthy.How this study might affect research, practice or policyOur study indicates that goblet and multiciliated cells are the most relevant BECs to further investigate in the context of drug development for RSV-induced asthma exacerbation. It also suggests that focusing research on the cross-talk between the epithelial and the immune cells, or into investigating a potential delayed response in asthma would be the best way forward into understanding the mechanisms involved in the asthma response to RSV.

Background and objective: Severe COPD patients can significantly benefit from bronchoscopic lung volume reduction (BLVR) treatments with coils or endobronchial valves. However, the potential impact of BLVR on survival is less understood. Therefore, our aim was to investigate the survival rate in patients who are evaluated for BLVR treatment and whether there is a difference in survival rate between patients who undergo BLVR treatment and patients who do not. Methods: We included patients with COPD who visited our hospital for a consultation evaluating their eligibility for BLVR treatment and who performed pulmonary function tests during this visit. Furthermore, vital status was verified. Results: In total 1471 patients were included (63% female, mean age 61 years). A total of 531 patients (35%) died during follow-up and the median survival time of the total population was 2694 days (95% confidence interval(CI) 2462–2926) which is approximately 7.4 years. The median survival time of patients who were treated with BLVR was significantly longer compared to patients who were not treated with BLVR (3133 days versus 2503 days, p < 0.001), and BLVR was found to be an independent predictor of survival when adjusting for other survival-influencing factors such as age, gender or severity of disease. Conclusions: Our results suggest that bronchoscopically reducing lung volume in patients with severe hyperinflation may lead to a survival benefit for a population with a severely reduced life expectancy.

Respiratory disease, characterized by changes in the cells of the lung, can affect molecular phenotype of cells and the intercellular interactions, resulting in a disbalance in the relative proportions of individual cell types. Understanding these changes is essential to understand the pathophysiology of lung disease. Conventional 'bulk' RNA-sequencing (RNA-seq), analyzing the entire transcriptome of the tissue sample, provides information about average expression levels of each gene in the mixed cell population; whereas it does not consider the cellular heterogeneity in samples composed of more than one cell type 1 . Single-cell RNA-seq (scRNA-seq) assesses the transcriptome of a complex biological sample with single-cell resolution, allowing identification of the relative frequency of discrete cell-types and analysis of their transcriptomes 1 . Nevertheless, analyzing the transcriptomic signature in large numbers of patients by scRNA-Seq is currently limited by its high costs. Mast cells are key regulatory cells driving the inflammatory process in asthma2 . Since they can be quantified by immunohistochemical staining for validation purposes, we used mast cells as an example of a rare cell population to assess the validity of our deconvolution approach. Recently, a number of bulk RNA-seq deconvolution methods have become available 3 , for instance of two deconvolution methods, namely support vector regression (SVR) 4 , the machine-learning method implemented in CYBERSORT, and Non-Negative Least Square (NNLS) 5 , using a matrix of cell-type selective genes identified with AutoGeneSc 6 . Both approaches are designed to estimate relative proportion of the main, common cell types present in the sample. When we used these methods to estimate the number of mast cells, we found a poor correlation with the number of mast cells stained by immunohistochemistry in the biopsies, suggesting the CIBERSORT and NNLS are less reliable in the case of rare cell types. We explored the possibility to use scRNA-Seq data from small numbers of subjects to specifically interrogate the relative cell type frequency of a rare cell population in a bulk RNA-Seq dataset obtained from a large asthma cohort.

Antiviral therapies are urgently needed to treat and limit the development of severe COVID-19 disease. Ivermectin, a broad-spectrum anti-parasitic agent, has been shown to have anti-SARS-CoV-2 activity in Vero cells at a concentration of 5 µM. These in vitro results triggered the investigation of ivermectin as a treatment option to alleviate COVID-19 disease. In April 2021, the World Health Organization stated, however, the following: “the current evidence on the use of ivermectin to treat COVID-19 patients is inconclusive”. It is speculated that the in vivo concentration of ivermectin is too low to exert a strong antiviral effect. Here, we performed a head-to head comparison of the antiviral activity of ivermectin and a structurally related, but metabolically more stable, moxidectin in multiple in vitro models of SARS-CoV-2 infection, including physiologically relevant human respiratory epithelial cells. Both moxidectin and ivermectin exhibited antiviral activity in Vero E6 cells. Subsequent experiments revealed that the compounds predominantly act on a step after virus cell entry. Surprisingly, however, in human airway-derived cell models, moxidectin and ivermectin failed to inhibit SARS-CoV-2 infection, even at a concentration of 10 µM. These disappointing results calls for a word of caution in the interpretation of anti-SARS-CoV-2 activity of drugs solely based on Vero cells. Altogether, these findings suggest that, even by using a high-dose regimen of ivermectin or switching to another drug in the same class are unlikely to be useful for treatment against SARS-CoV-2 in humans.

Introduction: Most guidelines recommend long-acting bronchodilators over short-acting bronchodilators for patients with chronic obstructive pulmonary disease (COPD). The available evidence for the guidelines was based on dry powder or pressurized metered dose inhalers, but not nebulizations. Nevertheless, there is considerable, poorly evidenced based, use of short acting nebulized bronchodilators. Methods: This was an investigator initiated, randomized, active controlled, cross-over, double-blind and double-dummy single centre study in patients with stable COPD. The active comparators were indacaterol/glycopyrronium 110/50 μg as Ultibro® via Breezhaler® (IND/GLY) and salbutamol/ipratropium 2,5/0,5 mg via air driven nebulization (SAL/IPR), both given as a single dose on separate days. The primary end point was the area under the FEV1 curve from baseline till 6 h. Secondary end points included change in Borg dyspnoea score, adverse events and change in hyperinflation measured by the inspiratory capacity. Results: A total of 33 COPD patients completed the trial and were evaluable, most of them were ex-smokers. The difference between the tested regimens for the primary endpoint, FEV1 AUC 0–6 h, 2965 ± 1544 mL (mean ± SD) for IND/GLY versus 3513 ± 1762 mL for SAL/IPR, was not significant (P = 0.08). The peak in FEV1 was higher and was reached faster with SAL/IPR compared to IND/GLY. No other significant differences were detected for the secondary endpoints including the Borg score, or adverse events. Conclusion: Among patients with stable COPD, dry powder long-acting single inhalation of a LABA and a LAMA (IND/GLY) was not superior compared to nebulized short-acting salbutamol plus ipratropium (SAL/IPR) in its bronchodilating effects over 6 h.The effects of the nebulization kicked in faster and peaked higher. The observed differences may be caused by the difference in dosing between the two regimens. The improvement in Borg dyspnoea score did not favour the nebulization. Long-term outcomes were not assessed in this study.

The chapters presented in this thesis provide an overview of what is known about asthma remission, further characterize of complete asthma remission, elaborate on the asthma- obesity complexity, apply cutting edge techniques to endotype asthma and COPD as well as novel devices to analyze airway remodeling and small airways dysfunction.Various conclusions have been made:I. In order to elucidate the pathophysiological state of asthma remission, future studies should focus on complete asthma remission, since this phenomenon is likely to yield superior prognostic and scientific impact. This is of interest, since elucidation of the pathophysiology of asthma remission could potentially lead to new treatment options for asthma.II. To clearly predict asthma remission later in life, we need to integrate biomarkers with clinical features at asthma-onset.III. Measuring particles of exhaled air correlates with large, and indirectly, small airways parameters, in asthmatics, clinical-, complete asthma remission subjects, and healthy controls.IV. Transcriptomic bronchial cell typing (e.g. single-cell RNA-sequencing) characterizes the landscape of lung-resident structural and inflammatory cells and their interactions, enabling us to identify differences in proportions and transcriptional output of cells between asthmatics and healthy.V. Optical coherence tomography enables us to quantify extracellular matrix components in the airway wall, such as collagen. This now allows for future studies ‘in vivo’ to explore the clinical characteristics and the underlying pathobiology related to airway remodeling in asthma and asthma remission.VI. The asthma-obesity syndrome is a common combination of diseases with its own distinct pathophysiological processes.VII. There will presumably be no room for serum periostin in COPD clinical decision- making.VIII.Transcriptomic profiling can be implemented as a biomarker for COPD patient prognosis.Unavoidably, aforementioned conclusions lead to new questions and recommendations for future studies. These recommendations include:I. To expand our knowledge on asthma remission by implementing single-cell RNA-sequencing on blood samples, bronchial- and nasal brushes, and bronchial biopsies of subjects with clinical and complete asthma remission, while comparing to asthmatics and healthy controls.II. To test single-nucleus RNA-sequencing in the ARMSTRONG study. This method enables sequencing of frozen biopsy samples of former datasets, consequently extending the number of subjects.III. To analyze exhaled, aberrant proteins linked to asthma, instead of merely counting exhaled particles.IV. To compare metabolomic breathprints of various asthma severities.V. To introduce novel methods in small airways disease-phenotypes in asthma, whichenable visualization of airtrapping and gas exchange, such as functional MRI. VI. To study the effects of leptin and adiponectin in the asthma-obesity syndrome. Specifying the eligibility for bariatric surgery in patients with severe asthma andmorbid obesity, in order to treat this phenotype more safely.VII. To analyze the presence of airway remodeling – defined by optical coherence tomography and histological parameters - in complete and clinical asthmaremission, compared to asthmatics and healthy controls.VIII.To correlate optical coherence tomography-defined airway wall remodelingparameters with both fixed airway obstruction and single-cell RNA sequencing inflammatory cell types or proportions.

Asthma is a chronic disease, characterised by variable airflow obstruction and airway inflammation [1]. Small airways are thought to be a major site of pathology in asthma [2, 3]. There are different tools to assess small airways dysfunction (SAD), such as spirometry, body plethysmography, impulse oscillometry (IOS), multiple-breath nitrogen washout (MBNW), alveolar fraction of exhaled nitric oxide (FENO) and gas trapping assessed by high-resolution computed tomography (CT). However, there is no golden standard and some tests are difficult to perform [2, 3]., PExA mass can distinguish asthmatics from healthy individuals. Subjects with complete, but not clinical, asthma remission exhale more PExA mass compared to asthma. Higher PExA mass was associated with better function of both the small and large airways. http://bit.ly/2znHABg

Introduction: Airway diseases are characterized by structural airway remodeling, including extracellular matrix (ECM) changes. Although a key feature, no diagnostic test currently assesses airway remodeling other than invasive airway biopsies. Real-time optical coherence tomography (OCT) generates infrared-based cross-sectional images of airway segments. Previously we reported that OCT identifies and quantifies airway wall layers. We hypothesized that OCT is able to detect collagen deposition areas within the airway wall ECM. Methods: In 5 lobectomy specimens 13 airways were dissected, marked with needles for matching histology and imaged by OCT. Sections were stained for total collagen (TC; pico sirius red) and collagen type I A1 (CA1; antibody). Color deconvolution was used to quantify TC or CA1 area (ImageJ). OCT images were segmented by light scattering intensity after thresholding, attenuation and roll-off correction. Areas were measured in mm2 and defined as % of the airway wall. Results: 23 airway OCT images with matched histology sections stained for TC and CA1 were analyzed (Fig. 1A-F). OCT area showed a significant correlation with TC area (r=0.52, p=0.01) and a non-significant correlation with CA1 area (r=0.31; p=0.12). Conclusion: OCT scattering area appears to correspond to collagen within the airway wall. Therefore, OCT may represent an imaging technique of choice to assess airway remodeling.

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.

Asthma is a chronic respiratory condition, which is highly prevalent worldwide. Although no cure is currently available, it is well recognized that some asthma patients can spontaneously enter remission of the disease later in life. Asthma remission is characterized by absence of symptoms and lack of asthma-medication use. Subjects in asthma remission can be divided into two groups: those in clinical remission and those in complete remission. In clinical asthma remission, subjects still have a degree of lung functional impairment or bronchial hyperresponsiveness, while in complete asthma remission, these features are no longer present. Over longer periods, the latter group is less likely to relapse. This remission group is of great scientific interest due to the higher potential to find biomarkers or biological pathways that elicit or are associated with asthma remission. Despite the fact that the definition of asthma remission varies between studies, some factors are reproducibly observed to be associated with remitted asthma. Among these are lower levels of inflammatory markers, which are lowest in complete remission. Additionally, in both groups some degree of airway remodeling is present. Still, the pathological disease state of asthma remission has been poorly investigated. Future research should focus on at least two aspects: further characterisation of the small airways and airway walls in order to determine histologically true remission, and more thorough biological pathway analyses to explore triggers that elicit this phenomenon. Ultimately, this will result in pharmacological targets that provide the potential to steer the course of asthma towards remission. (C) 2019 Elsevier Inc. All rights reserved.

COPD is primarily diagnosed, staged and monitored by phenotypical parameters (e.g. pulmonary function test), even though it is well known that these hardly reflect differences in underlying pathology. Genome-wide gene-expression profiling presents a new depth, which could offer an addition to endotyping (distinct pathobiological mechanism). The aim of this study was to investigate whether whole genome-gene expression profiling in bronchial biopsies can be used to identify different endotypes of COPD. COPD patients were 45-75 years old, had an FEV1/FVC As starting point for cluster-analysis, we used a published 98 genes COPD signature from bronchial brushes. Of these 98 genes, 93 were detected in our RNA-seq dataset and therefore used for unbiased ConcensusClusterPlus clustering. Analysis demonstrated that based on least inter-group consensus, 2 clusters were optimal; COPD Airway Gene Expressed 1 (CAGE1)-cluster (n=39) and CAGE2-cluster (n=17) (Fig. 1A-B). The main differences between the clusters included higher levels of sputum lymphocytes and CD4+ and CD8+ T-cells in biopsies (Fig. 1C). Lung function decline (LFD) between 0.5 to 7.5 years was greater in CAGE2, i.e. 74.3ml vs. 53.2ml, P=0.005 (fig. 1D), which was unable to be predicted by baseline inflammatory cell counts. Smoking status did not affect these outcomes. In conclusion, clustering based on gene-expression profiling identified a COPD phenotype with higher bronchial CD8+ T-cell numbers and accelerated LFD.

Asthma is a chronic inflammatory disease with structural changes in the airway wall, including goblet cell metaplasia, loss of ciliated epithelial cells, basement membrane thickening and airway smooth muscle hypertrophy and hyperplasia. To characterize the cellular composition of airway wall in asthma, and identify the associated molecular changes in each cell type individually, we performed single-cell RNA sequencing of airway wall biopsies from asthma patients and matched healthy controls. Airway wall biopsies from subsegmental carinae (3rd to 6th generation) were obtained by bronchoscopy from patients with asthma (confirmed by provocation test) and matched healthy controls. Suspensions of live biopsy cells were partitioned using a 10XGenomics Chromium controller. We generated and sequenced single-cell libraries for over 15000 cells. Cluster analysis revealed presence of at least 20 discrete cell types, including known structural and inflammatory airway wall cells. We identify a novel airway epithelial cell type characterized by expression of Foxi1 and high levels of CFTR and V-ATPase subunits indicating a role in regulating Cl- and H+ transport across the epithelial barrier. Direct comparison of airway wall cells from asthma patients and healthy controls revealed selective loss of well-differentiated ciliated cells accompanied by an increase in basal cells (both proliferative and activated) in asthma. Moreover, specific subsets of inflammatory cells were observed selectively in asthma. Integration of cell-type specific gene expression patterns and GWAS data for allergy and asthma identified the cell types and interactions that contribute to disease pathogenesis.

Although Th2 driven inflammation is present in COPD, it is not clearly elucidated which COPD patients are affected. Since periostin is associated with Th2 driven inflammation and inhaled corticosteroid (ICS)-response in asthma, it could function as a biomarker in COPD. The aim of this study was to analyze if serum periostin is elevated in COPD compared to healthy controls, if it is affected by smoking status, if it is linked to inflammatory cell counts in blood, sputum and endobronchial biopsies, and if periostin can predict ICS-response in COPD patients. Serum periostin levels were measured using Elecsys Periostin immunoassay. Correlations between periostin and inflammatory cell count in blood, sputum and endobronchial biopsies were analyzed. Additionally, the correlation between serum periostin levels and treatment responsiveness after 6 and 30 months was assessed using i.e. ΔFEV1% predicted, ΔCCQ score and ΔRV/TLC ratio. Forty-five COPD smokers, 25 COPD past-smokers, 22 healthy smokers and 23 healthy never-smokers were included. Linear regression analysis of serum periostin showed positive correlations age (B = 0.02, 95%CI 0.01–0.03) and FEV1% predicted (B = 0.01, 95%CI 0.01–0.02) in healthy smokers, but not in COPD patients In conclusion, COPD -smokers and -past-smokers have significantly higher periostin levels compared to healthy smokers, yet periostin is not suitable as a biomarker for Th2-driven inflammation or ICS-responsiveness in COPD. Electronic supplementary material The online version of this article (10.1186/s12931-018-0818-8) contains supplementary material, which is available to authorized users.

PURPOSE OF REVIEW: Obesity is a worldwide epidemic with a prevalence that has tripled in the last two decades. Worldwide, more than 1.5 billion adults are overweight and more than 500 million obese. Obesity has been suggested to be a risk factor for the development of more difficult-to-control asthma. Although the mechanisms underlying the asthma-obesity relationship are not fully understood, several possible explanations have been put forward. These will be reviewed in this manuscript as well as the implications for the treatment of overweight and obese asthma patients.RECENT FINDINGS: Insulin resistance is a possible factor contributing to the asthma-obesity relationship and the effect is independent of other components of the metabolic syndrome such as hypertriglyceridemia, hypertension, hyperglycemia, and systemic inflammation. Obesity has important effects on airway geometry, by especially reducing expiratory reserve volume causing obese asthmatics to breathe at low lung volumes. Furthermore, obesity affects the type of inflammation in asthma and is associated with reduced inhaled corticosteroids treatment responsiveness.SUMMARY: Obesity induces the development of asthma with a difficult-to-control phenotype. Treatment targeting insulin resistance may be beneficial in obese asthma patients, especially when they have concomitant diabetes. Systemic corticosteroids should be avoided as much as possible as they are not very effective in obese asthma and associated with side-effects like diabetes, weight gain, and osteoporosis.

Asthma remission is rare but strictly defined complete asthma remission is persistent in most cases http://ow.ly/g0ZK30b1ACC

SummaryHuman lungs enable efficient gas exchange, and form an interface with the environment which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in health. We report location-dependent airway epithelial cell states, and a novel subset of tissue-resident memory T cells. In lower airways of asthma patients, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report presence of pathogenic effector Th2 cells in asthma, and find evidence for type-2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identify a shift from airway structural cell communication in health to a Th2-dominated interactome in asthma.