Nasal epithelial gene expression identifies relevant asthma endotypes in the ATLANTIS study.

INTRODUCTION: Asthma is an inflammatory airways disease encompassing multiple phenotypes and endotypes. Several studies suggested gene expression in nasal epithelium to serve as a proxy for bronchial epithelium, being a non-invasive approach to investigate lung diseases. We hypothesised that molecular differences in upper airway epithelium reflect asthma-associated differences in the lower airways and are associated with clinical expression of asthma. METHODS: We analysed nasal epithelial gene expression data from 369 patients with asthma and 58 non-asthmatic controls from the Assessment of Small Airways Involvement in Asthma study. Unsupervised hierarchical clustering was performed on asthma-associated genes. Asthma-associated gene signatures were replicated in independent cohorts with nasal and bronchial brushes data by comparing Gene Set Variation Analysis scores between asthma patients and non-asthmatic controls. RESULTS: We identified 67 higher expressed and 59 lower expressed genes in nasal epithelium from asthma patients compared with controls (false discovery rate<0.05), including CLCA1, CST1 and POSTN, genes well known to reflect asthma in bronchial airway epithelium. Hierarchical clustering revealed several molecular asthma endotypes with distinct clinical characteristics, including an endotype with higher blood and sputum eosinophils, high fractional exhaled nitric oxide, and more severe small airway dysfunction, as reflected by lower forced expiratory flow at 50%. In an independent cohort, we demonstrated that genes higher expressed in the nasal epithelium reflect asthma-associated changes in the lower airways. CONCLUSION: Our results show that the nasal epithelial gene expression profile reflects asthma-related processes in the lower airways. We suggest that nasal epithelium may be a useful non-invasive tool to identify asthma endotypes and may advance personalised management of the disease.

Altered Extracellular Vesicle-Derived Protein and microRNA Signatures in Bronchoalveolar Lavage Fluid from Patients with Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease for which there is no cure. Accumulating research results suggest a role for extracellular vesicles (EVs) in the pathogenesis of COPD. This study aimed to uncover the involvement of EVs and their molecular cargo in the progression of COPD by identification of EV-associated protein and microRNA (miRNA) profiles. We isolated EVs from the bronchial alveolar lavage fluid (BALF) of 18 patients with COPD and 11 healthy controls using size-exclusion chromatography. EV isolates were characterized using nanoparticle tracking analysis and protein content. Proteomic analysis revealed a higher abundance of 284 proteins (log2FC > 1) and a lower abundance of 3 proteins (log2FC < -1) in EVs derived from patients with COPD. Ingenuity pathway analysis showed that proteins enriched in COPD-associated EVs trigger inflammatory responses, including neutrophil degranulation. Variances in surface receptors and ligands associated with COPD EVs suggest a preferential interaction with alveolar cells. Small RNAseq analysis identified a higher abundance of ten miRNAs and a lower abundance of one miRNA in EVs from COPD versus controls (Basemean > 100, FDR < 0.05). Our data indicate that the molecular composition of EVs in the BALF of patients with COPD is altered compared to healthy control EVs. Several components in COPD EVs were identified that may perpetuate inflammation and alveolar tissue destruction.

Low-Dose CT-derived Bronchial Parameters in Individuals with Healthy Lungs.

Background CT-derived bronchial parameters have been linked to chronic obstructive pulmonary disease and asthma severity, but little is known about these parameters in healthy individuals. Purpose To investigate the distribution of bronchial parameters at low-dose CT in individuals with healthy lungs from a Dutch general population. Materials and Methods In this prospective study, low-dose chest CT performed between May 2017 and October 2022 were obtained from participants who had completed the second-round assessment of the prospective, longitudinal Imaging in Lifelines study. Participants were aged at least 45 years, and those with abnormal spirometry, self-reported respiratory disease, or signs of lung disease at CT were excluded. Airway lumens and walls were segmented automatically. The square root of the bronchial wall area of a hypothetical airway with an internal perimeter of 10 mm (Pi10), luminal area (LA), wall thickness (WT), and wall area percentage were calculated. Associations between sex, age, height, weight, smoking status, and bronchial parameters were assessed using univariable and multivariable analyses. Results The study sample was composed of 8869 participants with healthy lungs (mean age, 60.9 years ± 10.4 [SD]; 4841 [54.6%] female participants), including 3672 (41.4%) never-smokers and 1197 (13.5%) individuals who currently smoke. Bronchial parameters for male participants were higher than those for female participants (Pi10, slope [ß] range = 3.49-3.66 mm; LA, ß range = 25.40-29.76 mm2; WT, ß range = 0.98-1.03 mm; all P < .001). Increasing age correlated with higher Pi10, LA, and WT (r2 range = 0.06-0.09, 0.02-0.01, and 0.02-0.07, respectively; all P < .001). Never-smoking individuals had the lowest Pi10 followed by formerly smoking and currently smoking individuals (3.62 mm ± 0.13, 3.68 mm ± 0.14, and 3.70 mm ± 0.14, respectively; all P < .001). In multivariable regression models, age, sex, height, weight, and smoking history explained up to 46% of the variation in bronchial parameters. Conclusion In healthy individuals, bronchial parameters differed by sex, height, weight, and smoking history; male sex and increasing age were associated with wider lumens and thicker walls. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Emrich and Varga-Szemes in this issue.

Sex differences in asthma control, lung function and exacerbations: the ATLANTIS study.

BACKGROUND: Asthma is a heterogeneous disease with a prevalence and severity that differs between male and female patients. QUESTION: What are differences between male and female patients with asthma with regard to asthma control, lung function, inflammation and exacerbations? METHODS: We performed a post hoc analysis in the ATLANTIS (Assessment of Small Airways Involvement in Asthma) study, an observational cohort study including patients with asthma from nine countries with a follow-up of 1 year during which patients were characterised with measures of large and small airway function, questionnaires, inflammation and imaging. We compared differences in baseline characteristics and longitudinal outcomes between male and female patients with asthma. RESULTS: 773 patients were enrolled; 450 (58%) of these were female. At baseline, female patients with asthma were in higher Global Initiative for Asthma (GINA) steps (p=0.042), had higher Asthma Control Questionnaire 6 (F: 0.83; M: 0.66, p<0.001) and higher airway resistance as reflected by uncorrected impulse oscillometry outcomes (ie, R5-R20: F: 0.06; M: 0.04 kPa/L/s, p=0.002). Male patients with asthma had more severe airway obstruction (forced expiratory volume in 1 s/forced vital capacity % predicted: F: 91.95; M: 88.33%, p<0.01) and more frequently had persistent airflow limitation (F: 27%; M: 39%, p<0.001). Blood neutrophils were significantly higher in female patients (p=0.014). With Cox regression analysis, female sex was an independent predictor for exacerbations. INTERPRETATION: We demonstrate that female patients are in higher GINA steps, exhibit worse disease control, experience more exacerbations and demonstrate higher airway resistance compared with male patients. The higher exacerbation risk was independent of GINA step and blood eosinophil level. Male patients, in turn, have a higher prevalence of persistent airflow limitation and more severe airflow obstruction. These findings show sex can affect clinical phenotyping and outcomes in asthma. TRIAL REGISTRATION NUMBER: NCT02123667.

Usability and feasibility of the Test of Adherence to Inhalers (TAI) Toolkit in daily clinical practice: The BANANA study.

The Test of Adherence to Inhalers (TAI) Toolkit links an adherence measurement instrument (the TAI) to proven effective interventions for different causes of non-adherence to inhaled medication. This study aimed to assess the usability and feasibility of the TAI Toolkit in clinical practice. The TAI Toolkit was piloted in eight primary and secondary care settings. Each study site included 10 patients with asthma and/or COPD and suspected non-adherence. Healthcare professionals (HCPs) recorded clinical data and TAI Toolkit outcomes. Data on usability and feasibility were collected in semi-structured interviews and with the System Usability Score (SUS). Of the included patients, 81% were non-adherent, and sporadic non-adherence was the most common (69%). The TAI Toolkit was valued with a mean SUS-score of 85.9 by the HCPs. They found the toolkit to 'be visually attractive', 'easy-to-use' and 'give insight into patients' adherence', thereby offering good potential for its use in clinical practice.

FixNCut: single-cell genomics through reversible tissue fixation and dissociation.

The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.

RSV-related Community COPD Exacerbations and Novel Diagnostics: A Binational Prospective Cohort Study.

RATIONALE: Respiratory syncytial virus (RSV) is a common global respiratory virus increasingly recognized as a major pathogen in frail older adults and as a cause of chronic obstructive pulmonary disease (COPD) exacerbations. There is no single test for RSV in adults with acceptable diagnostic accuracy. Trials of RSV vaccines have recently shown excellent safety and efficacy against RSV in older adults; defining the frequency of RSV-related community infections and COPD exacerbations is important for vaccine deployment decisions. OBJECTIVES: This prospective study aimed to establish the frequency of outpatient-managed RSV-related exacerbations of COPD in two well-characterized patient cohorts using a combination of diagnostic methods. METHODS: Participants were recruited at specialist clinics in London, UK and Groningen, NL from 2017 and observed for three consecutive RSV seasons, during exacerbations and at least twice yearly. RSV infections were detected by reverse transcription-polymerase chain reaction (RT-PCR) and serologic testing. MEASUREMENTS AND MAIN RESULTS: 377 patients with COPD attended 1,999 clinic visits and reported 310 exacerbations. There were 27 RSV-related exacerbations (8·7% of total); of these, seven were detected only on PCR, 16 only on serology and 4 by both methods. Increases in RSV specific N-protein antibody were as sensitive as antibody to pre-F or post-F for serodiagnosis of RSV related exacerbations. CONCLUSIONS: RSV is associated with 8.7% of outpatient managed COPD exacerbations in this study. Antibodies to RSV-N protein may have diagnostic value, potentially important in a vaccinated population. The introduction of vaccines that prevent RSV is expected to benefit patients with COPD. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

Alternative Splicing Is a Major Factor Shaping Transcriptome Diversity in Mild and Severe Chronic Obstructive Pulmonary Disease.

The role of alternative splicing in chronic obstructive pulmonary disease (COPD) is still largely unknown. We aimed to investigate the differences in alternatively splicing events between patients with mild-to-moderate and severe COPD compared with non-COPD control subjects and to identify splicing factors associated with aberrant alternative splicing in COPD. For this purpose, we performed genome-wide RNA-sequencing analysis of bronchial brushings from 23 patients with mild-to-moderate COPD, 121 with severe COPD, and 23 non-COPD control subjects. We found a significant difference in the frequency of alternative splicing events in patients with mild-to-moderate and severe COPD compared with non-COPD control subjects. There were from two to eight times (depending on event type) more differential alternative splicing events in the severe than in the mild-to-moderate stage. The severe COPD samples showed less intron retention and more exon skipping. It is interesting that the transcript levels of the top 10 differentially expressed splicing factors were significantly correlated with the percentage of many alternatively spliced transcripts in severe COPD. The aberrant alternative splicing in severe COPD was predicted to increase the overall protein-coding capacity of gene products. In conclusion, we observed large and significant differences in alternative splicing between bronchial samples of patients with COPD and control subjects, with more events observed in severe than in mild-to-moderate COPD. The changes in the expression of several splicing factors correlated with prevalence of alternative splicing in severe COPD. Alternative splicing can indirectly impact gene expression by changing the relative abundance of protein-coding isoforms potentially influencing pathophysiological changes. The results provide a better understanding of COPD-related alternative splicing changes.

Airway epithelial cell response to RSV is mostly impaired in goblet and multiciliated cells in asthma.

BACKGROUND: In patients with asthma, respiratory syncytial virus (RSV) infections can cause disease exacerbation by infecting the epithelial layer of the airways, inducing subsequent 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 causing the differences in the asthmatic bronchial epithelium in response to viral infection are poorly understood. METHODS: Here, we investigated the transcriptional response to RSV infection of primary bronchial epithelial cells (pBECs) from patients with asthma (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 3 days, cells were processed for single-cell RNA sequencing. RESULTS: A strong antiviral response to RSV was observed for all cell types, for all samples (p<1e-48). Most (1045) differentially regulated genes following RSV infection were found in cells transitioning to secretory cells. Goblet cells from patients with asthma showed lower expression of genes involved in the interferon response (false discovery rate <0.05), including OASL, ICAM1 and TNFAIP3. In multiciliated cells, an impairment of the signalling pathways involved in the response to RSV in asthma was observed. CONCLUSION: 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 multiciliated cells is impaired, highlighting the need for studying airway epithelial cells at high resolution in the context of asthma exacerbation.

Comparison of particles in exhaled air and multiple breath washout for assessment of small airway function in children with cystic fibrosis.

BACKGROUND: The introduction of modulator therapy for cystic fibrosis (CF) has led to an increased interest in the detection of small airway disease (SAD) as sensitive marker of treatment response. The particles in exhaled air (PExA) method, which records exhaled particle mass (PEx ng/L) and number (PExNR), detects SAD in adult patients. Our primary aim was to investigate if PExA outcomes in children with CF are different when compared to controls and associated with more severe disease. Secondary aims were to assess feasibility and repeatability of PExA in children with CF and to correlate PExA to multiple breath nitrogen washout (MBNW) as an established marker of SAD. METHODS: Thirteen healthy children (HC), 17 children with CF with normal lung function (CF-N) (FEV1 z-score ≥ -1.64) and six with airway obstruction (CF-AO) (FEV1 z-score < -1.64) between 8 and 18 years performed MBNW followed by PExA and spirometry. Children with CF repeated the measurements after 3 months. RESULTS: PEx ng/L and PExNR/L per liter of exhaled breath were similar between the three groups. The lung clearance index (LCI) was significantly higher in both CF-N and CF-AO compared to HC. All participants, except one, were able to perform PExA. Coefficient of variation for PEx ng/l was (median) 0.38, range 0-1.25 and PExNR/l 0.38, 0-1.09. Correlation between LCI and PEx ng/l was low, rs 0.32 (p = .07). CONCLUSION: PExA is feasible in children. In contrast to LCI, PExA did not differentiate healthy children from children with CF suggesting it to be a less sensitive tool to detect SAD.

Lack of a transcriptional response of primary bronchial epithelial cells from patients with asthma and controls to IL-33.

IL-33 and IL-1RL1 are well-replicated asthma genes that act in a single pathway toward type-2 immune responses. IL-33 is expressed by basal epithelial cells, and the release of IL-33 upon epithelial damage can activate innate lymphoid cells, T helper-2 cells, basophilic granulocytes, and mast cells through a receptor complex containing IL-1RL1. However, it is unknown how bronchial epithelial cells respond to IL-33, and whether this response is increased in the disease. We aimed to characterize the IL-33-driven transcriptomic changes in cultured primary bronchial epithelial cells from patients with asthma and healthy controls. Primary bronchial epithelial cells (PBECs) were obtained by bronchial brushing from six healthy control for air-liquid interface (ALI) cultures, whereas we selected eight healthy controls and seven patients with asthma for epithelial organoid cultures. We then stimulated the cultures for 24 h with recombinant IL-33 (rhIL33) at various concentrations with 1, 10, and 50 ng/mL for the ALI cultures and 20 ng/mL and 100 ng/mL for the organoid cultures, followed by RNA-sequencing and differential gene expression analysis. We did not detect any genome-wide significant differentially expressed genes after stimulation of PBECs with IL-33, irrespective of growth in three-dimensional (3-D) epithelial organoids or after differentiation in ALI cultures. These results were identical between PBECs obtained from patients with asthma or from healthy control subjects. We detected very low levels of IL-1RL1 gene expression in these airway epithelial cell cultures. We conclude that bronchial epithelial cells do not have a transcriptional response to IL-33, independent of their differentiation state. Hence, the airway epithelium acts as a source of IL-33 but does not seem to contribute to the response upon release of the alarmin after epithelial damage.NEW & NOTEWORTHY The IL-33/IL-1RL1 pathway stands as a formidable genetic predisposition for asthma, with ongoing clinical developments of various drugs designed to mitigate its influence in patients with asthma. The absence of a transcriptomic reaction to IL-33 within the bronchial epithelium holds significance in the pursuit of identifying biomarkers that can aid in pinpointing those individuals who would derive the greatest benefit from therapies targeting the IL-33 pathway.

3-D culture of human lung fibroblasts decreases proliferative and increases extracellular matrix remodeling genes.

Fibroblasts are the main producers of extracellular matrix (ECM) responsible for ECM maintenance and repair, a process often disrupted in chronic lung diseases. The accompanying mechanical changes adversely affect resident cells and overall lung function. Numerous models have been used to elucidate fibroblast behavior that are now evolving toward complex three-dimensional (3-D) models incorporating ECM, aiming to replicate the cells' native environment. Little is known about the cellular changes that occur when moving from two-dimensional (2-D) to 3-D cell culture. This study compared the gene expression profiles of primary human lung fibroblasts from seven subjects with normal lung function, that were cultured for 24 h on 2-D collagen I-coated tissue culture plastic and in 3-D collagen I hydrogels, which are commonly used to mimic ECM in various models, from contraction assays to intricate organ-on-a-chip models. Comparing 3-D with 2-D cell culture, 6,771 differentially expressed genes (2,896 up, 3,875 down) were found; enriched gene sets within the downregulated genes, identified through Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, were involved in the initiation of DNA replication which implied downregulation of fibroblast proliferation in 3-D. Observation of cells for 72 h in 2-D and 3-D environments confirmed the reduced progression through the cell cycle in 3-D. A focused analysis, examining the Hippo pathway and ECM-associated genes, showed differential patterns of gene expression in the 3-D versus 2-D culture. Altogether, the transcriptional response of fibroblasts cultured in 3-D indicated inhibition of proliferation, and alterations in Hippo and ECM pathways indicating a complete switch from proliferation to ECM remodeling.NEW & NOTEWORTHY With the introduction of complex three-dimensional (3-D) lung models, comes a need for understanding cellular behavior in these models. We compared gene expression profiles of human lung fibroblasts grown on two-dimensional (2-D) collagen I-coated surfaces with those in 3-D collagen I hydrogels. RNA sequencing and subsequent pathway analyses showed decreased proliferation, increased extracellular matrix (ECM) remodeling, and altered Hippo signaling and ECM deposition-related gene signatures. These findings highlight unique responses of fibroblasts in 3-D models.

An epithelial gene signature of trans-IL-6 signaling defines a subgroup of type 2-low asthma.

BACKGROUND: Asthma is stratified into type 2-high and type 2-low inflammatory phenotypes. Limited success has been achieved in developing drugs that target type 2-low inflammation. Previous studies have linked IL-6 signaling to severe asthma. IL-6 cooperates with soluble-IL-6Rα to activate cell signaling in airway epithelium. OBJECTIVE: We sought to study the role of sIL-6Rα amplified IL-6 signaling in airway epithelium and to develop an IL-6+ sIL-6Rα gene signature that may be used to select asthma patients who potentially respond to anti-IL-6 therapy. METHODS: Human airway epithelial cells were stimulated with combinations of IL-6, sIL-6Rα, and inhibitors, sgp130 (Olamkicept), and anti-IL-6R (Tocilizumab), to assess effects on pathway activation, epithelial barrier integrity, and gene expression. A gene signature was generated to identify IL-6 high patients using bronchial biopsies and nasal brushes. RESULTS: Soluble-IL-6Rα amplified the activation of the IL-6 pathway, shown by the increase of STAT3 phosphorylation and stronger gene induction in airway epithelial cells compared to IL-6 alone. Olamkicept and Tocilizumab inhibited the effect of IL-6 + sIL-6Rα on gene expression. We developed an IL-6 + sIL-6Rα gene signature and observed enrichment of this signature in bronchial biopsies but not nasal brushes from asthma patients compared to healthy controls. An IL-6 + sIL-6Rα gene signature score was associated with lower levels of sputum eosinophils in asthma. CONCLUSION: sIL-6Rα amplifies IL-6 signaling in bronchial epithelial cells. Higher local airway IL-6 + sIL-6Rα signaling is observed in asthma patients with low sputum eosinophils.

A bronchial gene signature specific for severe COPD that is retained in the nose.

Introduction: A subset of COPD patients develops advanced disease with severe airflow obstruction, hyperinflation and extensive emphysema. We propose that the pathogenesis in these patients differs from mild-moderate COPD and is reflected by bronchial gene expression. The aim of the present study was to identify a unique bronchial epithelial gene signature for severe COPD patients. Methods: We obtained RNA sequencing data from bronchial brushes from 123 ex-smokers with severe COPD, 23 with mild-moderate COPD and 23 non-COPD controls. We identified genes specific to severe COPD by comparing severe COPD to non-COPD controls, followed by removing genes that were also differentially expressed between mild-moderate COPD and non-COPD controls. Next, we performed a pathway analysis on these genes and evaluated whether this signature is retained in matched nasal brushings. Results: We identified 219 genes uniquely differentially expressed in severe COPD. Interaction network analysis identified VEGFA and FN1 as the key genes with the most interactions. Genes were involved in extracellular matrix regulation, collagen binding and the immune response. Of interest were 10 genes (VEGFA, DCN, SPARC, COL6A2, MGP, CYR61, ANXA6, LGALS1, C1QA and C1QB) directly connected to fibronectin 1 (FN1). Most of these genes were lower expressed in severe COPD and showed the same effect in nasal brushings. Conclusions: We found a unique severe COPD bronchial gene signature with key roles for VEGFA and FN1, which was retained in the upper airways. This supports the hypothesis that severe COPD, at least partly, comprises a different pathology and supports the potential for biomarker development based on nasal brushes in COPD.