IL-33 Expression Is Lower in Current Smokers at both Transcriptomic and Protein Levels.

Rationale: IL-33 is a proinflammatory cytokine thought to play a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). A recent clinical trial using an anti-IL-33 antibody showed a reduction in exacerbation and improved lung function in ex-smokers but not current smokers with COPD. Objectives: This study aimed to understand the effects of smoking status on IL-33. Methods: We investigated the association of smoking status with the level of gene expression of IL-33 in the airways in eight independent transcriptomic studies of lung airways. Additionally, we performed Western blot analysis and immunohistochemistry for IL-33 in lung tissue to assess protein levels. Measurements and Main Results: Across the bulk RNA-sequencing datasets, IL-33 gene expression and its signaling pathway were significantly lower in current versus former or never-smokers and increased upon smoking cessation (P < 0.05). Single-cell sequencing showed that IL-33 is predominantly expressed in resting basal epithelial cells and decreases during the differentiation process triggered by smoke exposure. We also found a higher transitioning of this cellular subpopulation into a more differentiated cell type during chronic smoking, potentially driving the reduction of IL-33. Protein analysis demonstrated lower IL-33 levels in lung tissue from current versus former smokers with COPD and a lower proportion of IL-33-positive basal cells in current versus ex-smoking controls. Conclusions: We provide strong evidence that cigarette smoke leads to an overall reduction in IL-33 expression in transcriptomic and protein level, and this may be due to the decrease in resting basal cells. Together, these findings may explain the clinical observation that a recent antibody-based anti-IL-33 treatment is more effective in former than current smokers with COPD.

The development, validation, and in vivo testing of a high-precision bronchial epithelial lining fluid sampling device.

Introduction: Analysis of respiratory biomarkers or pharmaceutical drug concentrations in bronchial epithelial lining fluid (bELF) using a high-precision sampling method is crucial for effective clinical respiratory diagnostics and research. Here, we utilized a cellulose matrix as an absorptive probe for bELF sampling, subsequently testing the design of a device and sampling technique in vivo. Methods: The absorptive matrix [Whatman® qualitative filter paper (Grade CF-12)] was first tested through tissue-contact experiments on porcine airway tissue. The absorption and elution capacity of the matrix, as well as the laboratory processing and analysis method, was validated with a range of Interleukin-8 (CXCL8) and C-Reactive protein (CRP) stock solutions. Subsequently, the device's design was optimized for universal in-house production and both, safe and efficient sampling. The airway sampling method was then tested in a group of 10 patients with Chronic Obstructive Pulmonary Disease (COPD). For each patient, a bELF sample was obtained using the newly developed bELF probe, as well as a reference 20 mL saline bronchial wash sample. Supernatants were assessed, using an immunoassay, for levels of the pro-inflammatory markers CXCL8, Myeloperoxidase (MPO), and CRP. The bELF samples were compared to bronchial wash. Results: The Whatman® qualitative filter paper (Grade CF-12) bELF probes adhered to porcine airway tissue, softening slightly upon wetting. The material maintained architectural integrity following the removal of the probes, leaving no residual fibers on the porcine airway mucosa. The bELF probe design was optimized for bronchoscopic delivery and in-house production. On average, a fully saturated bELF probe carried 32 µL of protein-rich fluid. The mean return of CXCL8 and CRP from samples collected from a serial dilution series (1, 5, 10, 20 ng/mL) was 69% (range 48%-87%). The bELF probe detected, on average, 7 (MPO), 14 (CRP), and 59 (CXCL8) times higher equivalent inflammatory protein concentrations in the collected bELF probe samples compared to the bronchial wash. Conclusion: The bELF probe is an effective absorptive technology for high-precision bELF sampling without dilution. With a simple in-house production procedure and bronchoscopic sampling technique, this method can be introduced in any bronchoscopic center for a consistent sampling of bELF.

Precision Medicine for More Oxygen (P4O2)-Study Design and First Results of the Long COVID-19 Extension.

Introduction: The coronavirus disease 2019 (COVID-19) pandemic has led to the death of almost 7 million people, however, with a cumulative incidence of 0.76 billion, most people survive COVID-19. Several studies indicate that the acute phase of COVID-19 may be followed by persistent symptoms including fatigue, dyspnea, headache, musculoskeletal symptoms, and pulmonary functional-and radiological abnormalities. However, the impact of COVID-19 on long-term health outcomes remains to be elucidated. Aims: The Precision Medicine for more Oxygen (P4O2) consortium COVID-19 extension aims to identify long COVID patients that are at risk for developing chronic lung disease and furthermore, to identify treatable traits and innovative personalized therapeutic strategies for prevention and treatment. This study aims to describe the study design and first results of the P4O2 COVID-19 cohort. Methods: The P4O2 COVID-19 study is a prospective multicenter cohort study that includes nested personalized counseling intervention trial. Patients, aged 40-65 years, were recruited from outpatient post-COVID clinics from five hospitals in The Netherlands. During study visits at 3-6 and 12-18 months post-COVID-19, data from medical records, pulmonary function tests, chest computed tomography scans and biological samples were collected and questionnaires were administered. Furthermore, exposome data was collected at the patient's home and state-of-the-art imaging techniques as well as multi-omics analyses will be performed on collected data. Results: 95 long COVID patients were enrolled between May 2021 and September 2022. The current study showed persistence of clinical symptoms and signs of pulmonary function test/radiological abnormalities in post-COVID patients at 3-6 months post-COVID. The most commonly reported symptoms included respiratory symptoms (78.9%), neurological symptoms (68.4%) and fatigue (67.4%). Female sex and infection with the Delta, compared with the Beta, SARS-CoV-2 variant were significantly associated with more persisting symptom categories. Conclusions: The P4O2 COVID-19 study contributes to our understanding of the long-term health impacts of COVID-19. Furthermore, P4O2 COVID-19 can lead to the identification of different phenotypes of long COVID patients, for example those that are at risk for developing chronic lung disease. Understanding the mechanisms behind the different phenotypes and identifying these patients at an early stage can help to develop and optimize prevention and treatment strategies.

Host-device interactions: exposure of lung epithelial cells and fibroblasts to nickel, titanium, or nitinol affect proliferation, reactive oxygen species production, and cellular signaling.

Endoscopic implantation of medical devices for the treatment of lung diseases, including airway stents, unidirectional valves and coils, is readily used to treat central airway disease and emphysema. However, granulation and fibrotic tissue formation impairs treatment effectiveness. To date little is known about the interaction between implanted devices, often made from metals, such as nickel, titanium or nitinol, and cells in the airways. Here, we study the response of lung epithelial cells and fibroblasts to implant device materials. The adhesion and proliferation of bronchial epithelial cells and lung fibroblasts upon exposure to 10 × 3 × 1 mm pieces of nickel, titanium or nitinol is examined using light and scanning electron microscopy. Pro-inflammatory cytokine mRNA expression and release, signaling kinase activity and intracellular free radical production are assessed. Nitinol, and to a lesser extent nickel and titanium, surfaces support the attachment and growth of lung epithelial cells. Nitinol induces a rapid and significant alteration of kinase activity. Cells directly exposed to nickel or titanium produce free radicals, but those exposed to nitinol do not. The response of lung epithelial cells and fibroblasts depends on the metal type to which they are exposed. Nitinol induces cellular surface growth and the induction of kinase activity, while exposure of lung epithelial cells to nickel and titanium induces free radical production, but nitinol does not.

Smoking increases expression of the SARS-CoV-2 spike protein-binding long ACE2 isoform in bronchial epithelium.

After more than two years the COVID-19 pandemic, that is caused by infection with the respiratory SARS-CoV-2 virus, is still ongoing. The risk to develop severe COVID-19 upon SARS-CoV-2 infection is increased in individuals with a high age, high body mass index, and who are smoking. The SARS-CoV-2 virus infects cells of the upper respiratory tract by entering these cells upon binding to the Angiotensin-converting enzyme 2 (ACE2) receptor. ACE2 is expressed in various cell types in the lung but the expression is especially high in goblet and ciliated cells. Recently, it was shown that next to its full-length isoform, ACE2 also has a short isoform. The short isoform is unable to bind SARS-CoV-2 and does not facilitate viral entry. In the current study we investigated whether active cigarette smoking increases the expression of the long or the short ACE2 isoform. We showed that in active smokers the expression of the long, active isoform, but not the short isoform of ACE2 is higher compared to never smokers. Additionally, it was shown that the expression of especially the long, active isoform of ACE2 was associated with secretory, club and goblet epithelial cells. This study increases our understanding of why current smokers are more susceptible to SARS-CoV-2 infection, in addition to the already established increased risk to develop severe COVID-19.

Endobronchial Valve Treatment Does Not Cause Significant Nickel Deposition in Lung Tissue.

Bronchoscopic lung volume reduction using endobronchial valves (EBVs) is a treatment option for patients with severe emphysema. These EBVs are made out of a nitinol mesh covered by a silicone layer. Nitinol is an alloy of nickel and titanium and is commonly used in implantable medical devices because of its biocompatibility and memory-shape properties. However, there are some concerns that nickel ions can be released from nitinol-containing devices which might cause adverse health effects, especially in patients with a known nickel hypersensitivity. In vitro, it was found that EBV release significant amounts of nickel in the first hours. Our aim was to assess the nickel concentration in lung tissue from a patient who previously underwent EBV treatment but, due to treatment failure, underwent lung volume reduction surgery and to compare this to a reference sample. We found no significant difference in the median nickel concentration between the EBV-treated patient and the non-EBV-treated patient (0.270 vs. 0.328 µg/g, respectively, p = 0.693) and these concentrations were also comparable to previously published nickel concentrations in human lung tissue samples not having any medically implanted devices in the lung. Our results suggest that there is no significant long-term nickel deposition in lung tissue after EBV treatment.

Airway wall splice quantitative trait locus analysis reveals novel downstream mechanisms for known asthma single-nucleotide polymorphisms.

Studying the effects of asthma SNPs on alternative splicing can lead to new insights into asthma pathophysiology. More specifically, a 17q12 SNP is associated to alternative splicing of GSDMB. https://bit.ly/3W49oTs.

Highway to heal: Influence of altered extracellular matrix on infiltrating immune cells during acute and chronic lung diseases.

Environmental insults including respiratory infections, in combination with genetic predisposition, may lead to lung diseases such as chronic obstructive pulmonary disease, lung fibrosis, asthma, and acute respiratory distress syndrome. Common characteristics of these diseases are infiltration and activation of inflammatory cells and abnormal extracellular matrix (ECM) turnover, leading to tissue damage and impairments in lung function. The ECM provides three-dimensional (3D) architectural support to the lung and crucial biochemical and biophysical cues to the cells, directing cellular processes. As immune cells travel to reach any site of injury, they encounter the composition and various mechanical features of the ECM. Emerging evidence demonstrates the crucial role played by the local environment in recruiting immune cells and their function in lung diseases. Moreover, recent developments in the field have elucidated considerable differences in responses of immune cells in two-dimensional versus 3D modeling systems. Examining the effect of individual parameters of the ECM to study their effect independently and collectively in a 3D microenvironment will help in better understanding disease pathobiology. In this article, we discuss the importance of investigating cellular migration and recent advances in this field. Moreover, we summarize changes in the ECM in lung diseases and the potential impacts on infiltrating immune cell migration in these diseases. There has been compelling progress in this field that encourages further developments, such as advanced in vitro 3D modeling using native ECM-based models, patient-derived materials, and bioprinting. We conclude with an overview of these state-of-the-art methodologies, followed by a discussion on developing novel and innovative models and the practical challenges envisaged in implementing and utilizing these systems.

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

In fibrosis remodelling of ECM leads to changes in composition and stiffness. Such changes can have a major impact on cell functions including proliferation, secretory profile and differentiation. Several studies have reported that fibrosis is characterised by increased senescence and accumulating evidence suggests that changes to the ECM including altered composition and increased stiffness may contribute to premature cellular senescence. This study investigated if increased stiffness could modulate markers of senescence and/or fibrosis in primary human lung fibroblasts. Using hydrogels representing stiffnesses that fall within healthy and fibrotic ranges, we cultured primary fibroblasts from non-diseased lung tissue on top of these hydrogels for up to 7 days before assessing senescence and fibrosis markers. Fibroblasts cultured on stiffer (±15 kPa) 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-ß1 (TGF-ß1) or connective tissue growth factor (CTGF) expression and higher decorin protein deposition on stiffer matrices. With respect to genes associated with fibrosis, fibroblasts on stiffer hydrogels compared to soft had higher expression of smooth muscle alpha (α)-2 actin (ACTA2), collagen (COL) 1A1 and fibulin-1 (Fbln1) and higher Fbln1 protein deposition after 7 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 phenotype (SASP). This overlap may contribute to the creation of a feedback loop whereby fibroblasts create a perpetuating cycle reinforcing progression of a fibrotic response.

The Decrease in Serum sRAGE Levels Upon Smoking is Associated with Activated Neutrophils.

The serum level of the soluble Receptor for Advanced Glycation End-products (sRAGE) is a promising blood biomarker for the development, severity, and progression of chronic obstructive pulmonary disease (COPD). However, cigarette smoking causes a nearly instant drop in circulating sRAGE levels, strongly impacting on the variability in sRAGE levels. In the current study, we investigated the possible mechanism behind the sudden drop in sRAGE upon smoking. We showed that the number of activated neutrophils in blood significantly increases within two hours upon smoking three cigarettes within one hour. Furthermore, an increased expression of the leukocyte activation marker CD11b, which is a known ligand for RAGE, was observed upon smoking. Additionally, the in vitro activation of neutrophils increased their capacity to bind sRAGE. Together, these data indicate that smoking activates neutrophils in the circulation with concomitant upregulation of the RAGE ligand CD11b, leading to reduced levels of sRAGE in serum.

Plasma sRAGE levels strongly associate with centrilobular emphysema assessed by HRCT scans.

BACKGROUND: There is a strong need for biomarkers to better characterize individuals with COPD and to take into account the heterogeneity of COPD. The blood protein sRAGE has been put forward as promising biomarker for COPD in general and emphysema in particular. Here, we measured plasma sRAGE levels using quantitative LC-MS and assessed whether the plasma sRAGE levels associate with (changes in) lung function, radiological emphysema parameters, and radiological subtypes of emphysema. METHODS: Three hundred and twenty-four COPD patients (mean FEV1: 63%predicted) and 185 healthy controls from the COPDGene study were selected. Plasma sRAGE was measured by immunoprecipitation in 96-well plate methodology to enrich sRAGE, followed by targeted quantitative liquid chromatography-mass spectrometry. Spirometry and HRCT scans (inspiration and expiration) with a 5-year follow-up were used; both subjected to high quality control standards. RESULTS: Lower sRAGE values significantly associated with the presence of COPD, the severity of airflow obstruction, the severity of emphysema on HRCT, the heterogeneous distribution of emphysema, centrilobular emphysema, and 5-year progression of emphysema. However, sRAGE values did not associate with airway wall thickness or paraseptal emphysema. CONCLUSIONS: Rather than being a general COPD biomarker, sRAGE is especially a promising biomarker for centrilobular emphysema. Follow-up studies should elucidate whether sRAGE can be used as a biomarker for other COPD phenotypes as well.

Identification of damage associated molecular patterns and extracellular matrix proteins as major constituents of the surface proteome of lung implantable silicone/nitinol devices.

Lung implantable devices have been widely adopted as mechanical interventions for a wide variety of pulmonary pathologies. Despite successful initial treatment, long-term efficacy can often be impacted by fibrotic or granulation tissue formation at the implant sites. This study aimed to explore the lung-device interface by identifying the adhered proteome on lung devices explanted from patients with severe emphysema. In this study, scanning electron microscopy is used to visualize the adhesion of cells and proteins to silicone and nitinol surfaces of explanted endobronchial valves. By applying high-resolution mass-spectrometry, the surface proteome of eight explanted valves is characterized, identifying 263 unique protein species to be mutually adsorbed on the valves. This subset is subjected to gene enrichment analysis, matched with known databases and further validated using immunohistochemistry. Enrichment analyses reveal dominant clusters of functionally-related ontology terms associated with coagulation, pattern recognition receptor signaling, immune responses, cytoskeleton organization, cell adhesion and migration. Matching results show that extracellular matrix proteins and damage-associated molecular patterns are cardinal in the formation of the surface proteome. This is the first study investigating the composition of the adhered proteome on explanted lung devices, setting the groundwork for hypothesis generation and further exploration. STATEMENT OF SIGNIFICANCE: This is the first study investigating the composition of the adhered proteome on explanted lung devices. Lung implantable devices have been widely adopted as mechanical interventions for pulmonary pathologies. Despite successful initial treatment, long-term efficacy can often be impacted by fibrotic or granulation tissue formation around the implant sites. We identified the adhered proteome on explanted lung devices using several techniques. We identified 263 unique protein species to be mutually adsorbed on explanted lung devices. Pathway analyses revealed that these proteins are associated with coagulation, pattern recognition receptor signaling, immune responses, cytoskeleton organization, cell adhesion and migration. Furthermore, we identified that especially extracellular matrix proteins and damage-associated molecular patterns were cardinal in the formation of the surface proteome.

Crosslink bio-adhesives for bronchoscopic lung volume reduction: current status and future direction.

Several bronchoscopic lung volume reduction (BLVR) treatments have been developed to reduce hyperinflation in emphysema patients. Lung bio-adhesives are among the most promising new BLVR treatment options, as they potentially provide a permanent solution for emphysematous patients after only a single application. To date, bio-adhesives have mainly been used as haemostats and tissue sealants, while their application in permanently contracting and sealing hyperinflated lung tissue has recently been identified as a novel and enticing opportunity. However, a major drawback of the current adhesive technology is the induction of severe inflammatory responses and adverse events upon administration. In our review, we distinguish between and discuss various natural, semi-synthetic and synthetic tissue haemostats and sealants that have been used for pulmonary applications such as sealing air/fluid leaks. Furthermore, we present an overview of the different materials including AeriSeal and autologous blood that have been used to achieve lung volume reduction and discuss their respective advantages and drawbacks. In conclusion, we describe the key biological (therapeutic benefit and biocompatibility) and biomechanical (degradability, adhesive strength, stiffness, viscoelasticity, tunability and self-healing capacity) characteristics that are essential for an ideal lung bio-adhesive material with the potential to overcome the concerns related to current adhesives.

Paracrine Regulation of Alveolar Epithelial Damage and Repair Responses by Human Lung-Resident Mesenchymal Stromal Cells.

COPD is characterized by irreversible lung tissue damage. We hypothesized that lung-derived mesenchymal stromal cells (LMSCs) reduce alveolar epithelial damage via paracrine processes, and may thus be suitable for cell-based strategies in COPD. We aimed to assess whether COPD-derived LMSCs display abnormalities. LMSCs were isolated from lung tissue of severe COPD patients and non-COPD controls. Effects of LMSC conditioned-medium (CM) on H2O2-induced, electric field- and scratch-injury were studied in A549 and NCI-H441 epithelial cells. In organoid models, LMSCs were co-cultured with NCI-H441 or primary lung cells. Organoid number, size and expression of alveolar type II markers were assessed. Pre-treatment with LMSC-CM significantly attenuated oxidative stress-induced necrosis and accelerated wound repair in A549. Co-culture with LMSCs supported organoid formation in NCI-H441 and primary epithelial cells, resulting in significantly larger organoids with lower type II-marker positivity in the presence of COPD-derived versus control LMSCs. Similar abnormalities developed in organoids from COPD compared to control-derived lung cells, with significantly larger organoids. Collectively, this indicates that LMSCs' secretome attenuates alveolar epithelial injury and supports epithelial repair. Additionally, LMSCs promote generation of alveolar organoids, with abnormalities in the supportive effects of COPD-derived LMCS, reflective of impaired regenerative responses of COPD distal lung cells.

Adsorptive Microtiter Plates As Solid Supports in Affinity Purification Workflows.

Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biological samples. These ligands are generally immobilized onto solid supports which facilitate the separation of a captured protein from the sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochemical detection (e.g., immunoassays) but hardly ever prior to liquid chromatography-mass spectrometry (LC-MS-)-based detection. Here, we describe the use of adsorptive microtiter plates for protein enrichment prior to LC-MS detection, and we discuss opportunities and challenges of corresponding workflows, based on examples of targeted (i.e., soluble receptor for advanced glycation end-products (sRAGE) in human serum) and discovery-based workflows (i.e., transcription factor p65 (NF-κB) in lysed murine RAW 264.7 macrophages and peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells). Thereby, we aim to highlight the potential usefulness of adsorptive microtiter plates in affinity purification workflows prior to LC-MS detection, which could increase their usage in mass spectrometry-based protein research.

Airway granulation response to lung-implantable medical devices: a concise overview.

Increasing numbers of endoscopically implantable devices are implanted in the airways, such as airway stents, one-way valves and coils, to treat both malignant and benign diseases. They significantly improve patient outcomes, but their long-term effectiveness and sustainability is hampered by the reaction of the formation of granulation tissue. Factors including procedural-related tissue injury; micro-organism presence; device-related factors, such as the material, design and sizing in relation to the airway; and patient-related factors, including genetic susceptibility, comorbidities and medication use, might all effect the severity of the tissue response and the subsequent degree of granulation tissue formation. However, research into the underlying mechanism and risk factors is scarce and therefore our knowledge is limited. Joint efforts from the scientific community, both pre-clinical and clinical, are needed to gain a deeper understanding and eventually improve the long-term treatment effectiveness of lung-implantable devices.

LL-37 and HMGB1 induce alveolar damage and reduce lung tissue regeneration via RAGE.

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, it is still unknown whether RAGE directly contributes to alveolar epithelial damage and abnormal repair responses. We hypothesize that RAGE activation not only induces lung tissue damage but also hampers alveolar epithelial repair responses. The effects of the RAGE ligands LL-37 and HMGB1 were examined on airway inflammation and alveolar tissue damage in wild-type and RAGE-deficient mice and on lung damage and repair responses using murine precision cut lung slices (PCLS) and organoids. In addition, their effects were studied on the repair response of human alveolar epithelial A549 cells, using siRNA knockdown of RAGE and treatment with the RAGE inhibitor FPS-ZM1. We observed that intranasal installation of LL-37 and HMGB1 induces RAGE-dependent inflammation and severe alveolar tissue damage in mice within 6 h, with stronger effects in a mouse strain susceptible for emphysema compared with a nonsusceptible strain. In PCLS, RAGE inhibition reduced the recovery from elastase-induced alveolar tissue damage. In organoids, RAGE ligands reduced the organoid-forming efficiency and epithelial differentiation into pneumocyte-organoids. Finally, in A549 cells, we confirmed the role of RAGE in impaired repair responses upon exposure to LL-37. Together, our data indicate that activation of RAGE by its ligands LL-37 and HMGB1 induces acute lung tissue damage and that this impedes alveolar epithelial repair, illustrating the therapeutic potential of RAGE inhibitors for lung tissue repair in emphysema.