Maarten van Den Berge

Background: Itepekimab, a monoclonal antibody being investigated for the treatment of COPD, binds to pro-inflammatory cytokine IL-33 and inhibits signaling. Aim: An exploratory, open-label, 12-week, phase 2a mechanistic study was conducted to evaluate the effect of itepekimab on airway inflammation and structure in former smokers (FS) and current smokers (CS) with COPD. Methods: Endobronchial biopsies, bronchial brushings, and bronchoabsorption fluid (BF) were collected at baseline and Week 12 of itepekimab (300 mg q2w) from FS (n=23) and CS (n=24) with COPD who had ≥1 moderate or severe exacerbation within 5 years of screening. Analyses included transcriptional profiling, proteomics, and histology/immunohistology of immune cells and epithelial changes. Results: At baseline, biopsies and brushings from FS and CS were transcriptionally distinct. FS showed higher baseline protein levels of IL-33, MCP-4, and TARC (CCL17) in BF along with increased neutrophil infiltration and ciliated epithelial cells in biopsies compared to CS. Gene sets modified by itepekimab in FS biopsies were significantly enriched in CS biopsies after treatment; FS brushing-derived gene sets were not significantly enriched in CS brushings. Type 2 and non–type-2 inflammatory mediators were reduced by itepekimab in FS as measured by decreased CXCL8, IRAK4, TARC, eotaxin-1, and IL5RA in BF and decreased airway eosinophils and neutrophils in biopsies. Ciliated epithelial cells were significantly increased in FS biopsies at Week 12 vs baseline. Conclusion: This study underscores the molecular and cellular heterogeneity of COPD. Itepekimab induced transcriptomic changes in both FS and CS. Itepekimab reduced type 2 and non–type-2 inflammation and increased epithelial barrier repair in FS within 12 weeks of treatment.

1 1.1 Background: Many longitudinal omics studies contain only a small number of repeated measurements collected before, during, or after an intervention. Existing approaches, including mixed-effects models and generalized additive models, estimate temporal effects but do not generally provide a discrete representation of trajectory topology that can be queried directly across experimental groups. 1.2 Methods: We developed LongOmicsTraj, an open-source R package for topology-based representation and querying of short longitudinal omics trajectories. The framework encodes the direction of change between adjacent visits as up , down , or flat , with the ordered sequence defining an Ordinal Trajectory State (OTS). LongOmicsTraj operates downstream of trajectory estimation and can therefore be applied to empirical summaries or model-derived visit-level estimates, including those from linear mixed-effects models, generalized additive models, and polynomial regression, following a maSigPro-style time-course formulation [1]. OTS labels provide a common representation for topology-based querying, cross-group comparison, and evaluation of higher-level representations such as trajectory clusters. We evaluated the framework using controlled simulations and bronchial biopsy transcriptomic data from the GLUCOLD corticosteroid intervention study (GEO accession GSE36221 ), measured at baseline, 6 months, and 30 months. The biological analysis compared continued inhaled corticosteroid (ICS) treatment, ICS withdrawal after 6 months, and placebo. 1.3 Results: In simulations, LongOmicsTraj recovered predefined stable, monotonic, transient, rebound, and oscillatory trajectories with high accuracy when longitudinal signal was sufficiently clear, with performance declining under high-noise conditions and depending partly on the upstream estimator. In GLUCOLD, comparator-aware topology queries reduced 20,358 measured transcripts to 168 genes showing a corticosteroid response that was maintained during continued treatment, reversed following withdrawal, and was not reproduced under placebo. The selected genes included established corticosteroid-response genes and were enriched for immune-cell migration, chemotaxis, cell adhesion, and extracellular-matrix organisation. Topology-aware evaluation of FlexMix trajectory clusters additionally revealed substantial within-cluster temporal heterogeneity, with topology purities of approximately 46–60%. 1.4 Conclusions: LongOmicsTraj provides a compact, directly queryable representation of temporal direction and order in short longitudinal omics studies. It complements existing longitudinal estimation and clustering methods by making trajectory structure explicit, enabling structured cross-group queries and quantification of temporal heterogeneity within trajectory clusters.

Cellular senescence is increasingly recognized as a hallmark of Chronic Obstructive Pulmonary Disease (COPD), with higher levels in lung fibroblasts from COPD patients. Upon senescence, both hypomethylation and hypermethylation have been described, but not in COPD-derived fibroblasts yet. This study investigated whether altered DNA methylation can be a driver of fibroblasts senescence in COPD. Genome wide gene expression and DNA methylation data was generated from primary lung fibroblasts of 11 COPD stage IV patients and 10 matched controls. Gene expression of six well-known senescence genes was compared between COPD and control. COPD-associated senescence genes were correlated with their related CpG sites in an expression quantitative trait methylation (eQTM) analysis. Methylation levels of significant eQTMs were compared between COPD and control fibroblasts. A causal relationship between altered DNA methylation and senescence was validated in 5-Aza-2'-deoxycytidine (5-Aza-2'-dC)-treated primary lung fibroblasts. Gene expression of CDKN1A, CDKN2A, and CDKN2B was higher, while LMNB1 expression was lower in COPD-derived fibroblasts compared to control. A total of 19 eQTMs were found for the COPD-associated senescence genes CDKN1A (9), CDKN2A (1), and LMNB1 (9). Among these, seven CpG sites (four for CDKN1A and three for LMNB1) exhibited differential methylation between COPD and control. Treatment with 5-Aza-2'-dC led to global demethylation and increased senescence and importantly, confirmed the association between senescence and hypomethylation of the COPD-associated CpG site cg04924375. Altered DNA methylation is linked to fibroblast senescence in COPD and seven CpG sites are identified as potential epigenetic regulators of the senescence genes CDKN1A and LMNB1.

Analysis of RNA-seq data of respiratory samples has contributed much to our understanding of lung disease. However, bulk RNA-seq data are dependent on both cell type composition and the transcriptional activity of these samples’ constituent cells, which complicates interpretation. Cell type deconvolution is frequently used to estimate cell type proportions of bulk transcriptomic gene expression data and improve interpretation of bulk transcriptomics data. However, accuracy of the estimated cell type proportions reported after deconvolution is unknown, which may have a negative impact on the validity of the conclusions drawn. Here, we present UnBlender, a pipeline that enables respiratory scientists to perform cell type deconvolution and routinely evaluate deconvolution accuracy of their approach. UnBlender allows for custom cell type deconvolution tailored to the research question at hand, using consensus cell type labels and validating the approach to promote accurate, reproducible results.

Communication with airway fibroblasts promotes mucus production by COPD-derived airway epithelial cells. This mechanism, partly mediated by fibroblast-derived proinflammatory IL-6, may contribute to chronic mucus hypersecretion in COPD.

In asthma, augmented airway wall smooth muscle (ASM) bulk is a major remodeling feature, promoted by increased transforming growth factor (TGF)‐β1 and connective tissue growth factor (CTGF). Runt‐related transcription factor‐2 (RUNX2) represses TGF‐β1‐induced CTGF through interactions with SMAD3. This study aimed to investigate the expression and role of RUNX2 in asthmatic and nonasthmatic ASM cells. mRNA and protein were detected by microarray, PCR, and western blot in nonasthmatic and asthmatic ASM cells. Immunohistochemistry identified RUNX2 in lung tissues from asthmatic patients and nonasthmatic subjects. Different RUNX2 isoforms were transfected into immortalized‐asthmatic ASM cells, and markers of inflammation and airway remodeling were measured. RUNX2 alternatively spliced forms were examined in bronchial biopsies from asthmatic and healthy subjects. The abundance of RUNX2 was decreased in isolated ASM cells from asthmatic compared with nonasthmatic subjects. The ASM layer around airways in lung tissue sections from asthmatic and nonasthmatic patients had a heterogeneous pattern of RUNX2 protein detection. TGF‐β1 stimulation increased RUNX2/RUNX2 variant 1 mRNA in nonasthmatic but not asthmatic ASM cells, facilitating SMAD3 activation and nuclear translocation in asthmatic ASM cells. RUNX2 isoform overexpression in immortalized asthmatic ASM cells failed to alter markers of inflammation (IL‐6) but significantly reduced markers of remodeling (CTGF), ASM cell hypertrophy (GSK‐3β and desmin), and proliferation (pSer ⁷⁹⁵ Rb and α‐tubulin). In bronchial biopsies, RUNX2 mRNA splicing was higher in asthmatic patients compared with healthy subjects. These data suggest RUNX2 plays a role in the homeostasis of healthy airways. Restoring RUNX2 may provide a new therapeutic approach for asthma.

Rationale: Variations in age-related lung function decline are associated with genetic and environmental factors. The genetic variants contributing to this decline remain largely unknown, limiting the understanding of individual susceptibility and potential interventions. Objectives: This study aims to uncover the genetic factors associated with lung function decline, using the Lifelines cohort study. Methods: Longitudinal data covering 3 visits and approximately 15 years follow up from 165,138 individuals were available. Genotyping and longitudinal spirometry data were present for 24,749 subjects aged 25 or older at baseline. A three-step approach was used. First, lung function change over time was estimated using a linear-mixed effect model. Second, a genome-wide association study for the estimated change was conducted, adjusting for age, sex, smoking load and the first 10 principal components. Third, single nucleotide polymorphisms (SNPs) with p-value−5 were analysed using a linear-mixed effect model. Selected SNPs were tested in two independent cohorts (N=1,376 and N=27,249), followed by meta-analysis. Measurements and Main Results: Among the included individuals, 19,722 had spirometry at two timepoints and 5,027 at three, with median [IQR] follow-up of 8.1 [4.4, 10.4] and 12.1 [11, 13.6] years, respectively. We identified 67 variants suggestively associated with lung function decline (p?5 ). Rs150094594, was significant (P=0.007) in a replication cohort with consistent effect direction. This intergenic variant, not previously reported, reached genome-wide significance in the meta-analysis (rs150094594:T, β(SE): 15.9(2.7) ml/year, p-value=3.9×10 ⁻⁹ ). Conclusions: This study shows a role of genetics in lung function decline, emphasizing the importance of exploring the interplay between genetic and environmental factors.

Rationale: The clinical characteristics of persistent airflow limitation (PAL) were explored in patients aged ≥12 years with physician-assigned diagnoses of asthma, asthma plus chronic obstructive pulmonary disease (COPD), or COPD in the NOVEL Observational longiTudinal studY (NOVELTY) cohort. The NOVELTY study is a prospective study conducted in primary and secondary care in 18 countries. Objectives: To determine the proportion of patients with PAL at baseline, their baseline characteristics, and the stability and prognostic utility of PAL during follow-up. Methods: PAL was defined as post–bronchodilator forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC) ratio less than the lower limit of the normal range (European Respiratory Society [ERS]/American Thoracic Society [ATS]) or as <0.7 (Global Initiative for Chronic Obstructive Lung Disease [GOLD] criteria). Results: We studied 9081 patients over 3 years (asthma: 4754; asthma + COPD; 1147; COPD: 3180). Baseline prevalence of PAL was 24.2% and 29.2% (asthma), 63.3% and 74.1% (asthma + COPD), and 65.4% and 75.8% (COPD) using ERS/ATS and GOLD criteria, respectively. Patients with PAL had markedly worse symptom burden and a history of more frequent moderate and severe exacerbations. In patients with asthma, PAL was associated with higher blood eosinophils and fractional exhaled nitric oxide (FeNO) values; 60% had never smoked. Of patients with PAL at baseline 84% continued to meet PAL criteria at Year 3. Irrespective of physician diagnosis, PAL was a marker of increased risk of moderate and severe exacerbations and poor symptom control during the 3-year follow-up. Conclusions: PAL is a stable trait associated with more severe disease and poor outcomes in adults with a physician-assigned diagnosis of asthma and/or COPD. Clinical Trial Registration (if any): NOVELTY: NCT02760329.

Airway wall thickness, assessed by HRCT, is increased in patients with asthma compared to healthy controls, but reverted towards normal in those with complete remission of their asthma https://bit.ly/4jaM2HV