Biology subjects

Melgert, B. N.

Publications and source records attributed to Melgert, B. N..

14 recordsLinked to original sources

bioRxiv2026

Immune-competent apical out airway organoids reveal distinct antiviral strategies of macrophages, neutrophils, and monocytes during influenza infection

The respiratory mucosa is the primary entry site for influenza virus and early antiviral immunity is governed there by interactions between epithelial cells and innate immune cells. However, mechanistic insight into these interactions in a human context is limited. Here, we established an immune-competent apical-out human airway organoid model that enables direct epithelial infection and controlled integration of innate immune cells. The organoids recapitulate key features of the human upper airway epithelium and support productive influenza virus replication. Using defined co-cultures, we uncovered distinct innate immune functions: macrophages suppress viral replication and restrict epithelial spread via production of type I and III interferons; neutrophils reduce extracellular virus levels without limiting epithelial infection, consistent with antiviral clearance mechanisms independent of interferon signaling; and monocytes exert modest, transient antiviral effects. Together, we use a human-relevant platform to define how distinct innate immune cells shape mucosal immunity at the respiratory epithelium, with direct implications for antiviral and vaccine development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/729766v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@612404org.highwire.dtl.DTLVardef@183aff0org.highwire.dtl.DTLVardef@93df5forg.highwire.dtl.DTLVardef@2de4a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2026

Differential collagen crosslinking and network organization creates distinct tissue remodeling patterns in fibrosis and COPD

Collagens are key components of the extracellular matrix (ECM) that play a crucial role in maintaining structure, strength, and function of the lungs. Fibrillar collagens are crosslinked by enzymes such as lysyl oxidases and transglutaminases and organized into networks by proteoglycans and glycoproteins. Collagens are the main load-bearing components and along with elastin may impart a non-linear strain hardening behavior to the lung. In disease, collagen crosslinking and organization can be disrupted, possibly due to abnormal levels of enzymes or ECM components. Few studies have examined collagen crosslinking and organization in healthy and diseased human lungs. In this study, alterations in collagen crosslinking and organization were investigated in human lung control, fibrotic and chronic obstructive pulmonary disease (COPD) tissue sections. Ultra-performance liquid chromatography and second harmonic generation microscopy measured pyridinoline crosslinks and the distribution of mature and immature collagens within the decellularized scaffolds, respectively. Fibrotic scaffolds had higher total collagen but less crosslinking per mole of collagen compared with COPD donors. Image analysis by second harmonic generation microscopy showed mature collagens populated airway or blood vessel walls in all three groups and in the parenchyma of fibrotic scaffolds. Immature collagens, on the other hand, were mainly localized to parenchymal regions in control and COPD scaffolds, with fewer immature collagens in fibrotic parenchyma. Additionally, quantification of the mature to immature collagen ratio in defined regions of control and diseased scaffolds showed increased organized collagen in fibrotic tissue. Our study shows that collagen crosslinking and organization are disrupted in fibrotic and COPD lungs and these changes may be compartment specific and can contribute to aberrant mechanical properties of diseased lungs. Our findings highlight that along with total collagen content, collagen crosslinking and organization are equally important while investigating collagen-mediated pathological changes in lung tissue. These changes may have implications for developing ECM-based therapeutics for patients with lung diseases.

molecular biology↗

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↗

bioRxiv2025

Contrasting responses of control and fibrotic lung fibroblasts to fibrotic stimuli: the role of osteoprotegerin in extracellular matrix remodeling

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by dysregulated wound healing, leading to excessive production of extracellular matrix (ECM) proteins, particularly collagen, by activated fibroblasts. Osteoprotegerin (OPG), a soluble protein associated with ECM remodeling in bone, has been found in higher levels in serum and lung tissue of IPF patients compared to controls. However, whether OPG is associated with ECM remodeling by fibroblasts in lung fibrosis remains poorly understood. In this study we assessed differences in OPG levels in ECM-derived hydrogels from non-disease controls and patients with IPF encapsulated with primary human lung fibroblasts from control and patients with IPF (7 days). OPG levels were assessed in a collected culture medium and stained lung tissue sections, and related to biomechanical properties. Fibrotic human lung fibroblasts secreted and deposited more OPG compared to controls, which was observed in both non-diseased and IPF ECM hydrogels. OPG levels correlated significantly with matrix stiffness and collagen fiber structural properties, but only in IPF hydrogels, independent of fibroblast source. Strikingly, we also observed opposite correlations for control and fibrotic fibroblasts in fibrotic hydrogels. Control fibroblasts within a fibrotic microenvironment, secreted less OPG when hydrogel stiffness increased and deposited more OPG when collagen fibers were more aligned. Interestingly, fibrotic fibroblasts secreted more OPG when collagen fiber length increased and when collagen fiber curvature increased. Overall, our findings suggest that control and fibrotic lung fibroblasts exhibited opposite behavior with respect to OPG and ECM remodeling under fibrotic conditions and this may have consequences for matrix stiffness

cell biology↗

bioRxiv2025

It takes two: Aberrant repair and low-grade inflammation characterizes bronchiolitis obliterans syndrome after lung transplantation in serum proteomic analysis

AimThe obstructive phenotype of chronic lung allograft dysfunction, bronchiolitis obliterans syndrome (BOS), is diagnosed after lung transplantation (LTx) when irreversible airway obstruction is already present. This study aimed to investigate the fibrotic response and inflammation signals in serum of BOS patients. MethodsLTx patients transplanted at the University Medical Center Groningen between 2004 and 2017 were screened. Nineteen patients with BOS were selected and matched to 19 non-BOS patients. Only patients for whom lung function and longitudinal serum samples post-LTx were available were included. Enzyme-linked immunosorbent assays were performed for neoepitopes of collagen types I, III, and VI and osteoprotegerin (OPG) in serum. Additionally, serum samples were analyzed by label free liquid chromatography with tandem mass spectrometry proteomics analysis. ResultsCollagen neoepitopes did not differ significantly between BOS and non-BOS patients at any timepoint. OPG was significantly higher in non-BOS compared to BOS six months before BOS onset (p<0.04). In proteomics analysis, proteins indicating cell repair and proliferation, namely human type II keratin-6 and centromere protein F (both FDR<0.1), were significantly lower three months before BOS onset in BOS compared to non-BOS patients. C-reactive protein (FDR<0.05) and SERPINA3 (FDR<0.05) amongst others, were higher in end-stage BOS compared to non-BOS patients. ConclusionDifferences in expression of proteins that reflect the complex interplay between fibrosis and inflammation in BOS were identified. These proteins should be investigated and validated in larger cohorts and may aid in expanding knowledge about the development of BOS.

bioinformatics↗

bioRxiv2024

Interaction of osteoprotegerin with fibulin-1 is essential for extracellular matrix deposition in fibrotic lung tissue

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease with an unclear cause and no cure. Osteoprotegerin (OPG), a well-known regulator of bone extracellular matrix (ECM), is increased in pulmonary fibrosis and this is associated with lower lung function and IPF progression. Fibulin-1, a key ECM protein, is increased in lung fibroblasts and lung tissues from patients with IPF and high levels are predictive of a poor prognosis for these patients. We found a positive correlation between levels of OPG and fibulin-1 in serum from patients with IPF, but it is unknown if these proteins interact. This study aimed at investigating interactions between OPG and fibulin-1 in lung fibrosis, hypothesizing that OPG may form a complex with fibulin-1 thereby possibly influencing lung fibrosis. We investigated intracellular interactions between OPG and fibulin-1 in human lung fibroblasts, showing neither OPG nor fibulin-1 expression were dependent on each other. However, OPG protein deposition was significantly decreased in lung tissues of fibulin-1 knockout mice (fibulin-1-/-)compared to controls suggesting extracellular interactions. Subsequently, we detected colocalization of both proteins in both IPF and control lung tissues using immunofluorescence, with both of them also colocalizing with latent TGF{beta} binding protein 1 (LTBP1). Proximity ligation analyses confirmed close proximity of OPG to fibulin-1 and fibulin-1 to LTBP1, particularly in the interstitial regions of both control and IPF lung tissue, but not OPG and LTBP1. In conclusion, we found that OPG, fibulin-1 and LTBP1 seem to form a complex, with fibulin-1 playing a bridging role between OPG and LTBP1, in the extracellular environment. This complex may play a regulatory role in deposition of extracellular matrix in lung tissue.

pathology↗

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

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↗

bioRxiv2022

Substrate stiffness engineered to replicate disease conditions influence senescence and fibrotic responses in primary lung fibroblasts

In idiopathic pulmonary fibrosis (IPF) there is excessive ECM deposition, increased stiffness and ultimately destruction of lung parenchyma. IPF presents mainly in the elderly, implying that senescence, a hallmark of ageing, contributes to disease progression. Several studies have reported that IPF is characterised by increased senescence and accumulating evidence suggests that structural changes, such as increased stiffness may contribute to senescence. This study therefore investigated if increased tissue stiffness could modulate markers of senescence and/or fibrosis in primary lung fibroblasts. Using hydrogels representing healthy and fibrotic stiffnesses, we cultured primary fibroblasts from non-diseased lung tissue on top of these hydrogels for up to seven days before assessing senescence and fibrosis markers. Fibroblasts cultured on stiff ({+/-}15kPa) hydrogels showed higher Yes-associated protein-1 (YAP) nuclear translocation compared to soft hydrogels. When looking at senescence-associated proteins we also found higher secretion of receptor activator of nuclear factor kappa-B ligand (RANKL) but no change in transforming growth factor-{beta}1 (TGF-{beta}1) or connective tissue growth factor (CTGF) expression and higher decorin protein deposition on stiff matrices. With respect to genes associated with fibrosis, fibroblasts on stiff hydrogels compared to soft had higher expression of smooth muscle alpha ()-2 actin (ACTA2), collagen (COL) 1A1 and fibulin-1 (Fbln1) and higher Fbln1 protein deposition after seven days. Our results show that exposure of lung fibroblasts to fibrotic stiffness activates genes and secreted factors that are part of fibrotic responses and part of the senescence-associated secretory profile (SASP). This overlap may contribute to the creation of a feedback loop whereby fibroblasts create a perpetuating cycle reinforcing disease progression in IPF.

cell biology↗

bioRxiv2021

Inhalable textile microplastic fibers impair airway epithelial growth

Synthetic textiles shed fibers that accumulate indoors and this results in continuous exposure when indoors. High exposure to microplastic fibers in nylon flock workers has been linked to the development of airway and interstitial lung disease, but the exact health effects of microplastic fibers on the lungs are unknown. Here we determined effects of polyester and nylon textile microplastic fibers on airway and alveolar epithelial cells using human and murine lung organoids. We observed that particularly nylon microfibers had a negative impact on the growth and development of airway organoids. We demonstrated that this effect was mediated by components leaking from nylon. Moreover, our data suggested that microplastic textile fibers may especially harm the developing airways or airways undergoing repair. Our results call for a need to assess exposure and inhalation levels in indoor environments to accurately determine the actual risk of these fibers to human health. TeaserAirborne fibers shed from synthetic textiles, in particular nylon, can inhibit repair of the cells coating the airways

pharmacology and toxicology↗

bioRxiv2020

Osteoprotegerin expression in liver is induced by IL-13 through TGF-β

1.BackgroundsOsteoprotegerin (OPG) is a profibrotic mediator produced by myofibroblasts under influence of transforming growth factor {beta} (TGF{beta}). Its expression in experimental models of liver fibrosis correlates well with disease severity and treatment responses. The regulation of OPG in liver tissue is largely unknown and we therefore set out to elucidate which growth factors/interleukins associated with fibrosis induce OPG and through which pathways. MethodsPrecision-cut liver slices of wild type and STAT6-deficient mice and 3T3 fibroblasts were used to investigate the effects of TGF{beta}, interleukin (IL) 13 (IL13), IL1{beta}, and platelet-derived growth factor BB (PDGF-BB) on expression of OPG. ResultsIn addition to TGF{beta}, only IL13 and not PDGF-BB or IL1{beta} could induce OPG expression in 3T3 fibroblasts and liver slices. This IL13-dependent induction was not shown in liver slices of STAT6-deficient mice and when wild type slices were cotreated with TGF{beta} receptor 1 kinase inhibitor galunisertib, STAT6 inhibitor AS1517499, or AP1 inhibitor T5224. This suggests that the OPG-inducing effect of IL13 is mediated through IL13 receptor 1-activation and subsequent STAT6-dependent upregulation of IL13 receptor 2, which in turn activates AP1 and induces production of TGF{beta} and subsequent production of OPG. ConclusionWe have shown that IL13 induces OPG release by liver tissue through a TGF{beta}-dependent pathway involving both the 1 and the 2 receptor of IL13 and transcription factors STAT6 and AP1. OPG may therefore be a novel target for the treatment liver fibrosis as it is mechanistically linked to two important regulators of fibrosis in liver, namely IL13 and TGF{beta}1.

pathology↗

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↗