Biology subjects

Timens, W.

Publications and source records attributed to Timens, W..

At least 19 recordsLinked to original sources

bioRxiv2026

A repair-associated bronchial epithelial differentiation trajectory through KRT14+ basal and hillock-like cells drives airway inflammation and remodelling in childhood-onset asthma

The bronchial epithelium in asthma is vulnerable to damage and has impaired barrier function, but the mechanisms by which it contributes to airway inflammation and remodelling remain unclear. Here, we dissect these epithelial and immunological disease mechanisms by establishing a comprehensive single cell atlas of the bronchial wall from 21 patients with childhood-onset asthma and 25 matched healthy controls. We identify a novel asthma-associated non-canonical epithelial differentiation trajectory in which a repair-associated KLF4+ basal cell subset differentiates into KRT13+ hillock-like cells through a proliferative KRT14+ intermediate. In vitro cultured matched primary bronchial epithelial cells show that this trajectory is retained in absence of exogenous factors. We find that IL-13 induces hillock-like cell differentiation into CEACAM5hi goblet cells, driving goblet cell metaplasia. Repair-associated basal cells and transitioning CEACAM5hi hillock-like cells strongly contribute to airway inflammation and remodelling. In turn, dendritic cells and mast cells promote a state of highly active epithelial differentiation, which shows increased multiciliated cell fate decisions, in concordance with an increase in multiciliated cell death observed in asthma. Proportions of the epithelial cells of the non-canonical differentiation trajectory are associated with clinical outcomes such as disease severity, FeNO, and small airway function.

immunology↗

bioRxiv2026

Mechanosensitive Piezo Channels Contribute to Airway Changes in Chronic Obstructive Pulmonary Disease

As an intrinsically mechanosensitive organ, the lung experiences a range of mechanical forces. Chronic obstructive pulmonary disease (COPD) involves abnormal macroscopic cellular and extracellular matrix (ECM) changes that impact mechanical properties of the lung. Mechanosensitive Piezo1/2 channels are expressed in the lung including on airway smooth muscle cells (ASM) that mediate cellular responses to stretch and ECM biomechanics. The expression and roles of Piezos in COPD lung ASM are not known. We hypothesized that Piezo expression and activation are altered in COPD lung ASM influencing ECM regulation. Distribution of Piezo proteins in ASM and epithelium of small airways of COPD stage II and IV vs. non-COPD controls was assessed using immunohistochemistry and ImageJ (n=10-17/group). Isolated ASM cells from control (n=6) vs. COPD stage II and IV patients (n=3 each stage) were exposed to stretch or the Piezo1 agonist Yoda1 followed by measurement of ECM gene and protein expression. Less Piezo2 staining was observed in COPD IV patients compared to controls, with lesser area and intensity of staining in the epithelial layer, and lower intensity of staining in ASM and small airways as a whole. Fura-2-based imaging of ASM Ca2+ showed lower influx after Yoda1 exposure in COPD II compared to control and COPD IV. Gene expression of Piezo1 increased upon stretching in controls but not in COPD ASM, while Piezo2 protein expression decreased with stretching in all groups. Yoda1 treatment resulted in decreased collagen1, fibulin1 and periostin gene and collagen 1 and periostin protein expression in ASM. Overall, these results support a role for Piezo activation in abnormal ECM-ASM cell crosstalk in COPD.

physiology↗

bioRxiv2025

A novel molecular interaction in bronchiolitis obliterans syndrome in lung transplantation patients: the role of SERPINA3 and osteoprotegerin

Background and objectiveChronic lung allograft dysfunction, significantly limits survival after lung transplantation. The obstructive phenotype bronchiolitis obliterans syndrome (BOS) is characterized by the abnormal activation of epithelium in airways accompanied by fibrotic changes with excessive deposition of extracellular matrix, resulting in narrowing and ultimately obliteration of airways. Mast cells, characterized by mediators such as tryptase and chymase, play a role in lung fibrosis. The proteins, osteoprotegerin (OPG) and SERPIN family member A 3 (SERPINA3) have previously been associated with progression of lung fibrosis. Tryptase-mast cells can produce OPG, while chymase released from mast cells interacts with SERPINA3. This study aimed to investigate if and how SERPINA3, OPG and tryptase/chymase-positive mast cells are related to fibrotic airway obliteration, and potentially show an association with BOS severity. MethodsSERPINA3 levels in serum from patients with or without BOS were examined using ELISA. In lung tissue from patients with BOS, we identified non-cartilaginous airways and classified them into normal, partially and completely obstructed airways. Immunohistochemistry was used for detection of SERPINA3, OPG, chymase and tryptase. Colocalization of, and interactions between, SERPINA3, OPG and tryptase were assessed by immunofluorescence, proximity ligation assay, and AlphaFold modeling. ResultsSERPINA3 levels in serum from BOS patients were higher compared to non-BOS patients. A low percentage of SERPINA3 and OPG was detected in partially and completely obstructed airways. Cells positive for OPG and SERPINA3 colocalized with tryptase-mast cells in airways from BOS lung tissue. OPG colocalized with SERPINA3 and their staining positively correlated in partially obstructed airways. In completely obstructed airways, OPG, SERPINA3 and tryptase staining areas all positively correlated with each other. ConclusionSERPINA3 and OPG are expressed by mast cells and these proteins may form a complex in lung tissue with potential for contributing to airway remodeling in BOS.

pathology↗

bioRxiv2025

Receptor agonist of NFκB signaling ligand directs lung epithelial cell expansion through RANK signaling

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally, with progressive emphysema driven by repeated epithelial damage and impaired repair. Recently, we found that secretion of the osteokine "receptor agonist of nuclear factor {kappa}B signaling ligand" (RANKL) is higher in lung fibroblasts from patients with COPD compared to control and that RANKL represses lung epithelial cell death. However, the underlying mechanism, its cross-species conservation and the involved epithelial cell types remain unclear. To investigate how RANKL affects lung epithelial cells, we used primary lung organoids from human and mouse epithelial cells and showed that RANKL supplementation increased alveolar organoid forming capacity in these cultures compared to vehicle-treated controls. In elastase-treated mice, RANKL was able to rescue the elastase-induced loss of epithelial cells compared to vehicle-treated controls and it augmented the proportion of epithelial cells in transitional states expressing Krt8 and MHCII. Using A549 epithelial cells to investigate whether RANK or the alternative receptor for RANKL, "leucine-rich repeat-containing G-protein coupled receptor 4" (LGR4) were responding to RANKL treatment, we found that RANKL most likely acts through RANK These results suggest that RANKL may enhance stem cell survival of primarily alveolar epithelial cells in both mice and humans. We therefore conclude that RANKL is another osteokine, in addition to periostin, osteopontin, osteoglycin, and osteoprotegerin, that has a role in lung tissue repair and that its signaling pathway could be explored for therapeutic applications. New & NoteworthyRANKL enhances human and murine alveolar epithelial cell expansion. Using organoids and an elastase injury model, we show RANKL promotes alveolar epithelial type II cell expansion and rescues transitional cell type loss. RANKL/RANK signaling therefore emerges as a conserved lung regenerative pathway and may be a potential therapeutic target for diseases like COPD with impaired epithelial repair.

cell biology↗

bioRxiv2024

Compositional changes of the lung extracellular matrix in acute respiratory distress syndrome

BackgroundAcute respiratory distress syndrome (ARDS) is pathologically characterized by diffuse alveolar damage (DAD) and is associated with high morbidity and mortality rates. Remodeling of the extracellular matrix (ECM), which is pivotal for both tissue repair and organ recovery, may play a large role in persistent ARDS. This study investigated the compositional changes in the ECM in different DAD stages in ARDS. MethodsParaffin-embedded lung sections collected during autopsy or from post-transplant lungs were obtained from patients with ARDS (n=28) admitted to the University Medical Center Groningen between 2010-2020. Sections were stained histochemically, and immunohistochemically for collagen III 1 chain (Col IIIa1), IV 3 chain (Col IVa3), VI 1 chain (Col VIa1), periostin (PSTN), lumican (LUM), and fibronectin (FN). The sections were divided into 118 regions based on DAD stages (54 early vs 64 advanced). The differences in the expression of selected proteins were compared between DAD stages or across ARDS duration (<7days, 7-14days, >14days). The fiber pattern of Col VIa1 was analyzed using CellProfiler. ResultsHigher tissue density, lower proportional areas of Col IIIa1, Col IVa3, and LUM, and more concentrated Col VIa1 fibers were observed in the advanced DAD stage than in the early DAD stage. Areas with higher proportions of total collagen and FN, and lower proportional areas of Col IIIa1, Col IVa3, and LUM were detected in lung regions from patients with ARDS >14days duration. ConclusionsThese findings revealed proportional changes in ECM components, strongly suggesting that dynamic changes in ECM proteins play a role in pathophysiology in ARDS during progression.

molecular biology↗

bioRxiv2024

Fibroblast-derived osteoglycin promotes epithelial cell repair

There is an urgent need for innovative pharmacological treatments targeting defective epithelial repair in chronic diseases, such as chronic obstructive pulmonary disease. The mesenchymal niche is a critical regulator in epithelial stem cell activation during repair. We hypothesized that secreted factors in this interaction are potent drug targets. Utilizing a cutting-edge proteomics-guided drug discovery strategy, we explored the lung fibroblast secretome to uncover impactful drug targets. Our lung organoid assays identified several regenerative ligands, with the secreted matrix protein osteoglycin (OGN) surprisingly showing the most profound effects. Transcriptomic analyses revealed that OGN enhances alveolar progenitor cell differentiation, boosts reactive oxygen species detoxification, reduces cellular senescence, and strengthens fibroblast-epithelial crosstalk. Critically, OGN expression was diminished in COPD patients and smoke-exposed mice. An active fragment of OGN, encompassing leucine-rich repeat regions 4-7, demonstrated regenerative potential akin to full-length OGN. This fragment significantly ameliorated elastase-induced lung injury precision-cut lung slices and improved lung function in vivo. These findings highlight lung fibroblast-derived OGN as a pivotal secreted protein for alveolar epithelial growth, positioning its active fragment as a promising therapeutic for epithelial repair in individuals with accelerated lung tissue damage.

pharmacology and toxicology↗

bioRxiv2023

The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease.

Extracellular matrix (ECM) remodeling has been implicated in the irreversible obstruction of airways and destruction of alveolar tissue in chronic obstructive pulmonary disease (COPD). Studies investigating differences in the lung ECM in COPD have mainly focused on some collagens and elastin, leaving an array of ECM components unexplored. We investigated the differences in the ECM landscape comparing severe-early onset (SEO-) COPD and moderate COPD to control lung tissue for collagen type I chain 1 (COL1A1), COL6A1, COL6A2, COL14A1, fibulin 2 and 5 (FBLN2, FBLN5), latent transforming growth factor-beta binding protein 4 (LTBP4), lumican (LUM), versican (VCAN), decorin (DCN), and elastin (ELN) using image analysis and statistical modelling. Percentage area and/or mean intensity of expression of LUM in the parenchyma, and COL1A1, FBLN2, LTBP4, DCN, and VCAN in the airway walls, was proportionally lower in COPD compared to controls. Lowered levels of most ECM proteins were associated with decreasing FEV1 measurements, indicating a relationship with disease severity. Furthermore, we identified six unique ECM signatures where LUM and COL6A1 in parenchyma and COL1A1, FBLN5, DCN, and VCAN in airway walls appear essential in reflecting the presence and severity of COPD. These signatures emphasize the need to examine groups of proteins to represent an overall difference in the ECM landscape in COPD, that are more likely to be related to functional effects, than individual proteins. Our study revealed differences in the lung ECM landscape between control and COPD and between SEO and moderate COPD signifying distinct pathological processes in the different subgroups. NEW & NOTEWORTHYOur study identified COPD-associated differences in the lung ECM composition. We highlight the compartmental differences in the ECM landscape in different subtypes of COPD. The most prominent differences were observed for severe-early onset COPD. Moreover, we identified unique ECM signatures that describe airway walls and parenchyma providing insight into the intertwined nature and complexity of ECM changes in COPD that together drive ECM remodeling and may contribute to disease pathogenesis.

molecular biology↗

bioRxiv2023

Role of RANKL in Alveolar Epithelial Cell Regeneration: Potential Novel Contributor to Lung Tissue Repair

Receptor activator for NF-{kappa}{beta} (RANK) ligand (RANKL) is found in lung tissue and elevated in lung diseases like chronic obstructive pulmonary disease (COPD), cystic fibrosis and silica-induced pulmonary fibrosis. RANKL is a well-known stimulator of bone tissue degradation, which may explain the association between these lung diseases and osteoporosis. However, RANKL is also reported to be involved in epithelial cell regeneration in breast and thymus. We hypothesized that RANKL, which is produced directly in lung tissue, is involved in the regeneration of lung epithelial cells. Therefore, we aimed to clarify the specific role of RANKL in this process. Using an organoid model of lung epithelial development by co-culturing primary EpCAM+ lung epithelial cells with fibroblasts, we found higher numbers of alveolar organoids after soluble RANKL treatment compared to control. Importantly, this effect was similar in human RANKL-treated organoids derived from epithelial cells isolated from lung tissue of COPD patients. The effect of RANKL was abrogated upon addition of osteoprotegerin, the soluble inhibitor of RANKL. We also found that RANKL stimulated phosphorylation of Akt suggesting involvement of its receptor RANK in the signaling pathway. Moreover, in vivo RANKL administration resulted in more type II alveolar epithelial cells in lungs of mice with silica-induced pulmonary fibrosis. In conclusion, we found that RANKL promotes type II alveolar epithelial cell regeneration and may therefore be a novel contributor to lung tissue repair. New and NoteworthyOur study provides compelling evidence demonstrating an as of yet unknown function of receptor activator for NF-{kappa}{beta} ligand (RANKL) in lung tissue regeneration. We found that RANKL plays a role in the regeneration of lung epithelial cells, particularly type II alveolar epithelial cells. This may also have clinical implications as promotion of alveolar epithelial cell regeneration may enhance lung tissue repair, an important target in patients with lung diseases like COPD and fibrosis.

cell biology↗

bioRxiv2023

Strong paracrine effects of SASP from senescence-induced severe early-onset COPD-derived fibroblasts

BackgroundLung fibroblasts from Severe Early-Onset (SEO-)COPD patients exhibit increased cellular senescence with higher levels of senescence-associated secretory phenotype (SASP) protein secretion. Yet, the impact of senescent fibroblasts, and their SASP, on surrounding fibroblasts in SEO-COPD lungs remains unclear. AimTo identify the effect of the SASP secreted by senescent SEO-COPD-derived fibroblasts on surrounding lung fibroblasts. MethodsCellular senescence was induced in lung fibroblasts derived from seven SEO-COPD patients (age[≤]53 years, FEV1<40% predicted), and conditioned medium (CM) containing the SASP, was collected (senescent CM). CM from untreated fibroblasts was used as control. Fibroblasts were stimulated with senescent and control CM, and with tissue plasminogen activator (t-PA), a previously identified COPD-associated SASP protein. Effects on paracrine senescence, inflammation, and extracellular matrix (ECM) regulation were assessed. ResultsStimulation with senescent CM increased the percentage of Senescence-associated beta-galactosidase positive fibroblasts and decreased p16, p21 & LMNB1 expression (p<0.05). T-PA did not affect these markers. Senescent CM increased IL-8 gene expression, increased IL-6 secretion, and strongly increased IL-8 secretion compared to control CM. T-PA slightly decreased IL-6 and IL-8 secretion. Additionally, senescent CM and t-PA stimulation both reduced decorin secretion. Senescent CM reduced FN1 gene expression, while DCN and MMP2 expression remained unaffected. T-PA did not affect ECM gene expression. ConclusionThe SASP from senescence-induced SEO-COPD-derived fibroblasts has a strong paracrine effect on untreated fibroblasts, suggesting that senescent lung fibroblasts contribute to chronic inflammation and ECM dysregulation. These findings imply involvement of senescent fibroblasts in abnormal lung ageing and possibly disease pathology in COPD.

cell biology↗

bioRxiv2023

E-cigarette vapour induces cellular senescence in primary lung fibroblasts and may contribute to lung pathology

Cellular senescence has been recognized to play a role in COPD pathophysiology. Non-aerosolized E-liquid treatment of a lung fibroblast foetal cell line resulted in cellular senescence induction, but this has not been assessed using E-vapour exposure and using primary lung fibroblasts. Therefore, we investigated whether E-vapour exposure induces cellular senescence in primary human lung fibroblasts and whether this affects their tissue repair function. Primary human lung fibroblasts were stimulated with E-cigarette vapour extracts, and cigarette smoke extract (CSE) and Paraquat (PQ) as positive controls. IL-8 secretion was measured to confirm a stimulatory response. Multiple senescence markers (p16, p21, SA-{beta}-gal, and proliferation) were assessed and the tissue repair function was assessed using a scratch assay. Finally, we tried to validate our findings in an E-cigarette-exposed mouse model. Upon stimulation with CSE, PQ, and E-vapour extracts, cellular senescence was induced, which seemed dose-dependent and nicotine-independent. E-vapour-induced senescence resulted in an impaired tissue repair function. No significant difference was observed in the E-cigarette-exposed mouse model. In this study, we identify E-cigarette vapours potential to induce cellular senescence in primary human lung fibroblasts and that this affects their tissue repair function, which further adds to the identified risks of E-cigarette use.

cell biology↗

bioRxiv2023

Three dimensional fibrotic extracellular matrix directs microenvironment fiber remodeling by fibroblasts

Idiopathic pulmonary fibrosis (IPF), for which effective treatments are limited, results in excessive and disorganized deposition of an aberrant extracellular matrix (ECM). An altered ECM microenvironment is postulated to contribute to disease perpetuation in a feed-forward manner through inducing profibrotic behavior by lung fibroblasts, the main producers and regulators of ECM. Here, we examined this hypothesis in a 3D in vitro model system by growing primary human lung fibroblasts in ECM-derived hydrogels from non-fibrotic (control) or IPF lung tissue. Culture of fibroblasts in fibrotic hydrogels did not trigger a change in the overall amount of collagen or glycosaminoglycans but did cause a drastic change in fiber organization compared to culture in control hydrogels. Mechanical properties of fibrotic hydrogels were modified by fibroblasts while control hydrogels were not. These results illustrate how the 3D microenvironment plays a crucial role in directing cells to exhibit pro-fibrotic responses by providing biochemical and/or biomechanical cues.

pathology↗

bioRxiv2023

Cellular Senescence Affects ECM Regulation in COPD Lung Tissue

IntroductionHigher levels of cellular senescence have been demonstrated in COPD patients, including severe early-onset (SEO)-COPD. Recently, we demonstrated that senescence induces extracellular matrix (ECM) dysregulation in lung fibroblasts. However, this in vitro observation has not been demonstrated in vivo yet. Therefore, we investigated whether cellular senescence can contribute to COPD-associated ECM changes in parenchymal lung tissue. MethodsTranscriptomic and proteomic analyses were performed on parenchymal lung tissue from 60 COPD patients (including 18 SEO-COPD patients) and 32 controls. Differential expression of ECM-related genes and proteins was compared between (SEO-)COPD and controls, followed by correlations with six senescence markers and four senescence signature scores. Significant ECM-senescence correlations were verified using histology and primary lung fibroblasts. ResultsWe identified 12 COPD- and 57 SEO-COPD-associated ECM genes and 4 COPD- and 9 SEO-COPD-associated ECM proteins of which the majority, 45 genes and 5 proteins, correlated with senescence. The correlations for COL6A1, COL6A2 and FBLN5 were confirmed in situ and correlations for 21 ECM genes were confirmed in primary lung fibroblasts at baseline. Four genes were successfully functionally validated in our senescence-induced lung fibroblast model, including increased protein levels of ADAMST1 and a non-functional FBLN5 protein. ConclusionsWe confirm a strong link between (SEO-)COPD-associated ECM changes and senescence in vivo in peripheral lung tissue from COPD patients. The strongest and most consistent senescence-associated ECM components include proteases, elastogenesis genes, and collagens 6. These results indicate a contributing role for senescence in disturbed ECM and elastic fiber organization, and protease-antiprotease imbalance in COPD.

pathology↗

bioRxiv2023

Childhood-onset asthma is characterized by airway epithelial hillock-to-squamous differentiation in early life

Childhood-onset asthma is characterized by Type 2-inflammation and airway wall remodeling, but mechanisms of asthma development in the first years of life remain unclear. Here, we investigate transcriptional changes in airway wall biopsies of 22 symptomatic one year old children and relate these to asthma at school age. We demonstrate that pre-asthmatic children (n = 10) overexpressed a gene signature characteristic for an airway epithelial differentiation trajectory via hillock cells towards squamous cells (adjusted p-value 8.06e-16), whilst there was no association with gene signatures of Type 2-inflammation or eosinophil activation. Genes expressed along this trajectory are linked to an altered epithelial barrier function, innate immune activation and extracellular matrix remodeling. Functional GWAS analysis supports a causal link between childhood-onset, but not adult-onset asthma, and the hillock-squamous cell differentiation trajectory. Next, we confirmed the presence of hillock-like cells at the RNA and protein level in pediatric upper and lower airway samples. These findings identify a novel mechanism by which an aberrant airway epithelial differentiation trajectory may contribute to a pre-asthmatic state, highlighting the difference between the early origins of childhood-onset asthma and adult asthma, and point to possible new targets for the early diagnosis and treatment of asthma in the first two years of life. One Sentence SummaryRNA sequencing in bronchial biopsies from wheezing infants and children < 2 years shows evidence for an airway epithelial hillock-to-squamous differentiation pathway that marks the development of asthma.

genomics↗

bioRxiv2022

IL-11 disrupts alveolar epithelial progenitor function

IL-11 is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF), since IL-11 induces myofibroblast differentiation and stimulates their excessive collagen deposition in the lung. The alveolar architecture is disrupted in IPF, yet the effect of IL-11 on dysregulated alveolar repair associated with IPF remains to be elucidated. We hypothesized that epithelial-fibroblast communication associated with lung repair is disrupted by IL-11. Thus, we studied whether IL-11 affects the repair responses of alveolar lung epithelium using mouse lung organoids and precision cut lung slices (PCLS). Additionally, we assessed the anatomical distribution of IL-11 and IL-11 receptor in human control and IPF lungs using immunohistochemistry. IL-11 protein was observed in human control lungs in airway epithelium, macrophages and in IPF lungs, in areas of AT2 cell hyperplasia. IL-11R staining was predominantly present in smooth muscle and macrophages. In mouse organoid co-cultures of epithelial cells with lung fibroblasts, IL-11 decreased organoid number and reduced the fraction of pro-SPC expressing organoids, indicating dysfunctional regeneration initiated by epithelial progenitors. In mouse PCLS alveolar marker gene expression declined, whereas airway markers were increased. The response of primary human fibroblasts to IL-11 on gene expression level was minimal, though bulk RNA-sequencing revealed IL-11 modulated a number of processes which may play a role in IPF, including unfolded protein response, glycolysis and Notch signaling. In conclusion, IL-11 disrupts alveolar epithelial regeneration by inhibiting progenitor activation and suppressing the formation of mature alveolar epithelial cells. The contribution of dysregulated fibroblast - epithelial communication to this process appears to be limited.

cell biology↗

bioRxiv2022

Age-associated Differences in the Human Lung Extracellular Matrix

IntroductionExtracellular matrix (ECM) remodelling has been associated with chronic lung diseases. However, information about specific age-associated differences in lung ECM is currently limited. In this study we aimed to identify and localize age-associated ECM differences in human lung using comprehensive transcriptomic, proteomic and immunohistochemical analyses. MethodsOur previously identified age-associated gene expression signature of the lung was re-analysed limiting it to an aging signature based on 270 control patients (37-80 years) and focused on the Matrisome core geneset using geneset enrichment analysis. To validate the age-associated transcriptomic differences on protein level, we compared the age-associated ECM genes (F <0.05) with a profile of age-associated proteins identified from a lung tissue proteomics dataset from 9 control patients (49-76 years) (FDR<0.05). Extensive immunohistochemical analysis was used to localize the age-associated ECM differences in lung tissues from control patients (9-82 years). ResultsComparative analysis of transcriptomic and proteomic data identified 7 ECM proteins with higher expression with age at both gene and protein level: COL1A1, COL6A1, COL6A2, COL14A1, FBLN2, LTBP4 and LUM. With immunohistochemistry we demonstrated higher protein expression with age for COL6A2 in whole tissue, parenchyma, airway wall and blood vessel, for COL14A1 in bronchial epithelium and blood vessel, and for FBLN2 and COL1A1 in lung parenchyma. ConclusionOur study revealed that higher age is associated with lung ECM remodelling, with specific differences occurring in defined regions within the lung. These differences may affect lung structure and physiology with aging and as such may increase susceptibility for developing chronic lung diseases. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSsummarise the state of scientific knowledge on this subject before you did your study and why this study needed to be done. In animal models, it has been demonstrated that aging alters the composition of the lung ECM, with more deposition of collagen and degradation of elastin. Similar ECM differences have been observed in age-associated chronic lung diseases, including COPD; moreover, we observed in lung tissue that several ECM genes associate differently with age in COPD patients compared to non-COPD controls(1). Detailed knowledge on age-associated changes in specific ECM proteins as well as regional differences within the lung is lacking. What this study addssummarise what we now know as a result of this study that we did not know before. We identified 7 age-associated ECM proteins i.e. COL1A1, COL6A1, COL6A2 COL14A1, FBLN2, LTBP4 and LUM with higher transcript and protein levels in human lung tissue with age. Extensive immunohistochemical analysis revealed significant age-associated differences for 3 of these ECM proteins in specific compartments of the lung, with the most notable differences in the blood vessels and parenchyma. How this study might affect research, practice, or policysummarise the implications of this study. The identification of age-associated differences in specific human lung ECM proteins lays a new foundation for the investigation of ECM differences in age-associated chronic lung diseases. Additionally, examining the function of these age-associated ECM proteins and their cellular interactions in lung injury and repair responses may provide novel insight in mechanisms underlying chronic lung diseases.

molecular biology↗

bioRxiv2020

Osteoprotegerin is elevated in pulmonary fibrosis and associates with IPF progression

Osteoprotegerin (OPG), a decoy receptor for receptor activator of NF-kB ligand (RANKL), is used as a biomarker for assessing severity of liver fibrosis. However, its expression and role in pulmonary fibrosis are unknown. We hypothesized that OPG also has a role in pulmonary fibrosis. Human and mouse control and fibrotic lung tissue were used to examine OPG expression, and mouse precision-cut lung slices to study OPG regulation in pulmonary fibrosis. Serum from idiopathic pulmonary fibrosis (IPF) patients and controls was analysed to investigate whether OPG levels correlate with disease status as measured by lung function. OPG-protein levels were significantly higher in mouse and human fibrotic lung tissue compared to control. OPG-mRNA and protein production were induced in mouse precision-cut-lung slices upon TGF{beta} stimulation and could be inhibited with galunisertib, a TGF{beta} receptor kinase inhibitor. OPG-protein levels in fibrotic mouse lung tissue correlated with degree of fibrosis. Isolated lung fibroblasts from IPF patients had higher OPG-protein levels than control fibroblasts. Serum OPG levels in IPF patients, at first presentation, negatively correlated with diffusing capacity to carbon monoxide. Finally, serum OPG levels higher than 1234 pg/ml at first presentation were associated with progression of disease in IPF patients. In conclusion, OPG is produced in lung tissue, associates with fibrosis, and may be a potential prognostic biomarker for IPF disease progression. Validation in a larger cohort is warranted to further explore the role of OPG in pulmonary fibrosis and its potential for assessing the prognosis of fibrotic lung disease in individual patients. Take home messageOsteoprotegerin is present in fibrotic lung tissue and high serum levels correlate with low lung function and IPF disease progression in this small study, indicating osteoprotegerin may have value as a biomarker to predict IPF progression

pathology↗

bioRxiv2020

Gene Expression Network Analysis Provides Potential Targets Against SARS-CoV-2

ABSTRACTBACKGROUND Cell entry of SARS-CoV-2, the novel coronavirus causing COVID-19, is facilitated by host cell angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We aimed to identify and characterize genes that are co-expressed with ACE2 and TMPRSS2, and to further explore their biological functions and potential as druggable targets.METHODS Using the gene expression profiles of 1,038 lung tissue samples, we performed a weighted gene correlation network analysis (WGCNA) to identify modules of co-expressed genes. We explored the biology of co-expressed genes using bioinformatics databases, and identified known drug-gene interactions.RESULTS ACE2 was in a module of 681 co-expressed genes; 12 genes with moderate-high correlation with ACE2 (r>0.3, FDR<0.05) had known interactions with existing drug compounds. TMPRSS2 was in a module of 1,086 co-expressed genes; 15 of these genes were enriched in the gene ontology biologic process ‘Entry into host cell’, and 53 TMPRSS2-correlated genes had known interactions with drug compounds.CONCLUSION Dozens of genes are co-expressed with ACE2 and TMPRSS2, many of which have plausible links to COVID-19 pathophysiology. Many of the co-expressed genes are potentially targetable with existing drugs, which may help to fast-track the development of COVID-19 therapeutics.Competing Interest StatementS.M. reports personal fees from Novartis and Boehringer-Ingelheim, outside the submitted work. W.T. reports fees to Institution from Roche-Ventana, AbbVie, Merck-Sharp-Dohme and Bristol-Myers-Squibb, outside the submitted work. M.B. reports research grants paid to University from Astra Zeneca, Novartis, outside the submitted work. D.D.S. reports research funding from AstraZeneca and received honoraria for speaking engagements from Boehringer Ingelheim and AstraZeneca over the past 36 months, outside of the submitted work.View Full Text

genomics↗

bioRxiv2020

Novel Type 2-high gene clusters associated with corticosteroid sensitivity in COPD

RationaleSevere asthma and COPD share common pathophysiologic traits such as relative corticosteroid insensitivity. We recently published three transcriptome-associated clusters (TACs) using hierarchical analysis of the sputum transcriptome in asthmatics from the U-BIOPRED cohort with one Type 2-high signature (TAC1) and 2 Type 2-low signatures. ObjectiveWe examined whether gene-expression signatures obtained in asthma can be used to identify the subgroup of COPD patients with steroid sensitivity. MethodsUsing gene set variation analysis (GSVA), we examined the distribution and enrichment scores (ES) of the 3 TACs in the transcriptome of bronchial biopsies from 46 patients who participated in the GLUCOLD COPD study that received 30 months of treatment with inhaled corticosteroids (ICS) with and without an added long-acting {beta}-agonist (LABA). The identified signatures were then associated with longitudinal clinical variables after treatment. Measurements and main resultsBronchial biopsies in COPD patients at baseline showed a wide range of expression of the 3 TACs. After ICS{+/-}LABA treatment, the ES of TAC1 was significantly reduced at 30 months, but those of TAC2 and TAC3 were unaffected. A corticosteroid-sensitive TAC1 (sub)signature was developed from the TAC1 ICS-responsive genes. This signature consisted of mast cell-specific genes identified by single-cell RNA-seq and positively correlated with bronchial biopsy mast cell numbers following ICS{+/-}LABA. Baseline levels of gene transcription predicted change in FEV1 %predicted following 30-month ICS{+/-}LABA. ConclusionsSputum-derived transcriptomic signatures from an asthma cohort can be recapitulated in bronchial biopsies of COPD patients and identified airway wall mast cells as a predictor of those COPD patients who will benefit from inhaled corticosteroids.

bioinformatics↗