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 α 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.

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.

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.

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.

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.

Effects of cigarette smoking on SARS-CoV-2 receptor ACE2 expression in the respiratory epithelium†.

Due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, the world is currently facing high morbidity and mortality rates as well as severe disruption to normal societal and social structures. SARS-CoV-2 uses the ACE2 receptor for cellular entry. In a recent publication of The Journal of Pathology, Liu and coworkers highlight the effects of cigarette smoking on ACE2 expression in the respiratory epithelium. The authors studied the effects of acute cigarette smoke exposure in a murine model and confirmed their findings in human lung tissues and gene expression datasets. Their findings demonstrate that cigarette smoking increases ACE2 expression specifically at the apical surface of the airway epithelium. Smoking cessation resulted in lower ACE2 expression, with implications for attenuating the risk of transmission of the virus. The role of ACE2 expression in the development of COVID-19 symptoms is still under investigation, with conflicting results from experimental models on the role of ACE2 expression in SARS-CoV-2-induced lung injury. In this commentary, we highlight the implications and limitations of the study of Liu et al as well as future therapeutic strategies directed towards ACE2. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

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.

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.

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.

Soluble receptor for advanced glycation end products (sRAGE) as a biomarker of COPD.

BACKGROUND: Soluble receptor for advanced glycation end products (sRAGE) is a proposed emphysema and airflow obstruction biomarker; however, previous publications have shown inconsistent associations and only one study has investigate the association between sRAGE and emphysema. No cohorts have examined the association between sRAGE and progressive decline of lung function. There have also been no evaluation of assay compatibility, receiver operating characteristics, and little examination of the effect of genetic variability in non-white population. This manuscript addresses these deficiencies and introduces novel data from Pittsburgh COPD SCCOR and as well as novel work on airflow obstruction. A meta-analysis is used to quantify sRAGE associations with clinical phenotypes. METHODS: sRAGE was measured in four independent longitudinal cohorts on different analytic assays: COPDGene (n = 1443); SPIROMICS (n = 1623); ECLIPSE (n = 2349); Pittsburgh COPD SCCOR (n = 399). We constructed adjusted linear mixed models to determine associations of sRAGE with baseline and follow up forced expiratory volume at one second (FEV1) and emphysema by quantitative high-resolution CT lung density at the 15th percentile (adjusted for total lung capacity). RESULTS: Lower plasma or serum sRAGE values were associated with a COPD diagnosis (P < 0.001), reduced FEV1 (P < 0.001), and emphysema severity (P < 0.001). In an inverse-variance weighted meta-analysis, one SD lower log10-transformed sRAGE was associated with 105 ± 22 mL lower FEV1 and 4.14 ± 0.55 g/L lower adjusted lung density. After adjusting for covariates, lower sRAGE at baseline was associated with greater FEV1 decline and emphysema progression only in the ECLIPSE cohort. Non-Hispanic white subjects carrying the rs2070600 minor allele (A) and non-Hispanic African Americans carrying the rs2071288 minor allele (A) had lower sRAGE measurements compare to those with the major allele, but their emphysema-sRAGE regression slopes were similar. CONCLUSIONS: Lower blood sRAGE is associated with more severe airflow obstruction and emphysema, but associations with progression are inconsistent in the cohorts analyzed. In these cohorts, genotype influenced sRAGE measurements and strengthened variance modelling. Thus, genotype should be included in sRAGE evaluations.

RAGE and TLR4 differentially regulate airway hyperresponsiveness: Implications for COPD.

BACKGROUND: The receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4) is implicated in COPD. Although these receptors share common ligands and signalling pathways, it is not known whether they act in concert to drive pathological processes in COPD. We examined the impact of RAGE and/or TLR4 gene deficiency in a mouse model of COPD and also determined whether expression of these receptors correlates with airway neutrophilia and airway hyperresponsiveness (AHR) in COPD patients. METHODS: We measured airway inflammation and AHR in wild-type, RAGE-/- , TLR4-/- and TLR4-/- RAGE-/- mice following acute exposure to cigarette smoke (CS). We also examined the impact of smoking status on AGER (encodes RAGE) and TLR4 bronchial gene expression in patients with and without COPD. Finally, we determined whether expression of these receptors correlates with airway neutrophilia and AHR in COPD patients. RESULTS: RAGE-/- mice were protected against CS-induced neutrophilia and AHR. In contrast, TLR4-/- mice were not protected against CS-induced neutrophilia and had more severe CS-induced AHR. TLR4-/- RAGE-/- mice were not protected against CS-induced neutrophilia but were partially protected against CS-induced mediator release and AHR. Current smoking was associated with significantly lower AGER and TLR4 expression irrespective of COPD status, possibly reflecting negative feedback regulation. However, consistent with preclinical findings, AGER expression correlated with higher sputum neutrophil counts and more severe AHR in COPD patients. TLR4 expression did not correlate with neutrophilic inflammation or AHR. CONCLUSIONS: Inhibition of RAGE but not TLR4 signalling may protect against airway neutrophilia and AHR in COPD.

Acute cigarette smoke-induced eQTL affects formyl peptide receptor expression and lung function.

BACKGROUND AND OBJECTIVE: Cigarette smoking is one of the most prevalent causes of preventable deaths worldwide, leading to chronic diseases, including chronic obstructive pulmonary disease (COPD). Cigarette smoke is known to induce significant transcriptional modifications throughout the respiratory tract. However, it is largely unknown how genetic profiles influence the smoking-related transcriptional changes and how changes in gene expression translate into altered alveolar epithelial repair responses. METHODS: We performed a candidate-based acute cigarette smoke-induced eQTL study, investigating the association between SNP and differential gene expression of FPR family members in bronchial epithelial cells isolated 24 h after smoking and after 48 h without smoking. The effects FPR1 on lung epithelial integrity and repair upon damage in the presence and absence of cigarette smoke were studied in CRISPR-Cas9-generated lung epithelial knockout cells. RESULTS: One significant (FDR < 0.05) inducible eQTL (rs3212855) was identified that induced a >2-fold change in gene expression. The minor allele of rs3212855 was associated with significantly higher gene expression of FPR1, FPR2 and FPR3 upon smoking. Importantly, the minor allele of rs3212855 was also associated with lower lung function. Alveolar epithelial FPR1 knockout cells were protected against CSE-induced reduction in repair capacity upon wounding. CONCLUSION: We identified a novel smoking-related inducible eQTL that is associated with a smoke-induced increase in the expression of FPR1, FPR2 and FPR3, and with lowered lung function. in vitro FPR1 down-regulation protects against smoke-induced reduction in lung epithelial repair.

Mitochondria: at the crossroads of regulating lung epithelial cell function in chronic obstructive pulmonary disease.

Disturbances in mitochondrial structure and function in lung epithelial cells have been implicated in the pathogenesis of various lung diseases, including chronic obstructive pulmonary disease (COPD). Such disturbances affect not only cellular energy metabolism but also alter a range of indispensable cellular homeostatic functions in which mitochondria are known to be involved. These range from cellular differentiation, cell death pathways, and cellular remodeling to physical barrier function and innate immunity, all of which are known to be impacted by exposure to cigarette smoke and have been linked to COPD pathogenesis. Next to their well-established role as the first physical frontline against external insults, lung epithelial cells are immunologically active. Malfunctioning epithelial cells with defective mitochondria are unable to maintain homeostasis and respond adequately to further stress or injury, which may ultimately shape the phenotype of lung diseases. In this review, we provide a comprehensive overview of the impact of cigarette smoke on the development of mitochondrial dysfunction in the lung epithelium and highlight the consequences for cell function, innate immune responses, epithelial remodeling, and epithelial barrier function in COPD. We also discuss the applicability and potential therapeutic value of recently proposed strategies for the restoration of mitochondrial function in the treatment of COPD.

Regulation of cellular senescence by extracellular matrix during chronic fibrotic diseases.

The extracellular matrix (ECM) is a complex network of macromolecules surrounding cells providing structural support and stability to tissues. The understanding of the ECM and the diverse roles it plays in development, homoeostasis and injury have greatly advanced in the last three decades. The ECM is crucial for maintaining tissue homoeostasis but also many pathological conditions arise from aberrant matrix remodelling during ageing. Ageing is characterised as functional decline of tissue over time ultimately leading to tissue dysfunction, and is a risk factor in many diseases including cardiovascular disease, diabetes, cancer, dementia, glaucoma, chronic obstructive pulmonary disease (COPD) and fibrosis. ECM changes are recognised as a major driver of aberrant cell responses. Mesenchymal cells in aged tissue show signs of growth arrest and resistance to apoptosis, which are indicative of cellular senescence. It was recently postulated that cellular senescence contributes to the pathogenesis of chronic fibrotic diseases in the heart, kidney, liver and lung. Senescent cells negatively impact tissue regeneration while creating a pro-inflammatory environment as part of the senescence-associated secretory phenotype (SASP) favouring disease progression. In this review, we explore and summarise the current knowledge around how aberrant ECM potentially influences the senescent phenotype in chronic fibrotic diseases. Lastly, we will explore the possibility for interventions in the ECM-senescence regulatory pathways for therapeutic potential in chronic fibrotic diseases.

Effect of long-term corticosteroid treatment on microRNA and gene-expression profiles in COPD.

The aim was to investigate whether microRNA (miRNA) expression is modulated by inhaled corticosteroid (ICS) treatmentWe performed genome-wide miRNA analysis on bronchial biopsies of 69 moderate/severe chronic obstructive pulmonary disease (COPD) patients at baseline and after 6- and 30-month treatment with the ICS fluticasone propionate or placebo. The effect of ICS on miRNA expression was validated in differentiated primary bronchial epithelial cultures, and functional studies were conducted in BEAS-2B cells. MiRNAs affected by ICS and their predicted targets were compared to an independent miRNA dataset of bronchial brushings from COPD patients and healthy controls.Treatment with ICS for both 6 and 30 months significantly altered the expression of four miRNAs, including miR-320d, which was increased during ICS treatment compared with placebo. The ICS-induced increase of miR-320d was confirmed in primary airway epithelial cells. MiR-320d negatively correlated targets were enriched for pro-inflammatory genes and were increased in the bronchial brushes of patients with lower lung function in the independent dataset. Overexpression of miR-320d in BEAS-2B cells dampened cigarette smoke extract-induced pro-inflammatory activity via inhibition of nuclear factor-κB.Collectively, we identified miR-320d as a novel mediator of ICS, regulating the pro-inflammatory response of the airway epithelium.

AGER expression and alternative splicing in bronchial biopsies of smokers and never smokers.

Cigarette smoking is one of the major risk factors for the development of chronic obstructive pulmonary disease (COPD). Evidence is accumulating that Receptor for Advanced Glycation-End products (RAGE)-signaling is a key pathway in the pathophysiology of COPD. To date, it is unknown how smoking affects RAGE expression. In the current study, we investigated the effect of smoking on AGER, the gene encoding RAGE, expression and on alternative splicing of AGER. To this end, we conducted RNA-Seq on bronchial biopsies for asymptomatic smokers (n = 36) and never smokers (n = 40). Total AGER gene expression was accessed using DESeq2, while alternative splicing was investigated by measuring the number of specific split reads spanning exon-exon junctions and the total split reads. One of the major isoforms of RAGE is endogenous soluble (es) RAGE, an anti-inflammatory decoy receptor, making up for approximately 10% of the total amount of soluble (s)RAGE. We found that smokers show decreased total gene expression of AGER in bronchial biopsies, while the relative abundance of the esRAGE isoform is increased. Furthermore, no difference in the serum levels of total sRAGE were observed between smokers and non-smokers. Our data indicates that smoking initiates a protective anti-inflammatory mechanism with decreased expression of the pro-inflammatory gene AGER and increased relative abundance of the anti-inflammatory isoform esRAGE.

Galectin-9 Is a Possible Promoter of Immunopathology in Rheumatoid Arthritis by Activation of Peptidyl Arginine Deiminase 4 (PAD-4) in Granulocytes.

The aetiology of rheumatoid arthritis (RA) is unknown, but citrullination of proteins is thought to be an initiating event. In addition, it is increasingly evident that the lung can be a potential site for the generation of autoimmune triggers before the development of joint disease. Here, we identified that serum levels of galectin-9 (Gal-9), a pleiotropic immunomodulatory protein, are elevated in RA patients, and are even further increased in patients with comorbid bronchiectasis, a lung disease caused by chronic inflammation. The serum concentrations of Gal-9 correlate with C-reactive protein levels and DAS-28 score. Gal-9 activated polymorphonuclear leukocytes (granulocytes) in vitro, which was characterized by increased cytokine secretion, migration, and survival. Further, granulocytes treated with Gal-9 upregulated expression of peptidyl arginine deiminase 4 (PAD-4), a key enzyme required for RA-associated citrullination of proteins. Correspondingly, treatment with Gal-9 triggered citrullination of intracellular granulocyte proteins that are known contributors to RA pathogenesis (i.e., myeloperoxidase, alpha-enolase, MMP-9, lactoferrin). In conclusion, this study identifies for the first time an immunomodulatory protein, Gal-9, that triggers activation of granulocytes leading to increased PAD-4 expression and generation of citrullinated autoantigens. This pathway may represent a potentially important mechanism for development of RA.

Genetic variance is associated with susceptibility for cigarette smoke-induced DAMP release in mice.

Chronic obstructive pulmonary disease (COPD) is characterized by unresolved neutrophilic airway inflammation and is caused by chronic exposure to toxic gases, such as cigarette smoke (CS), in genetically susceptible individuals. Recent data indicate a role for damage-associated molecular patterns (DAMPs) in COPD. Here, we investigated the genetics of CS-induced DAMP release in 28 inbred mouse strains. Subsequently, in lung tissue from a subset of strains, the expression of the identified candidate genes was analyzed. We tested whether small interfering RNA-dependent knockdown of candidate genes altered the susceptibility of the human A549 cell line to CS-induced cell death and DAMP release. Furthermore, we tested whether these genes were differentially regulated by CS exposure in bronchial brushings obtained from individuals with a family history indicative of either the presence or absence of susceptibility for COPD. We observed that, of the four DAMPs tested, double-stranded DNA (dsDNA) showed the highest correlation with neutrophilic airway inflammation. Genetic analyses identified 11 candidate genes governing either CS-induced or basal dsDNA release in mice. Two candidate genes (Elac2 and Ppt1) showed differential expression in lung tissue on CS exposure between susceptible and nonsusceptible mouse strains. Knockdown of ELAC2 and PPT1 in A549 cells altered susceptibility to CS extract-induced cell death and DAMP release. In bronchial brushings, CS-induced expression of ENOX1 and ARGHGEF11 was significantly different between individuals susceptible or nonsusceptible for COPD. Our study shows that genetic variance in a mouse model is associated with CS-induced DAMP release, and that this might contribute to susceptibility for COPD.

Increased neutrophil expression of pattern recognition receptors during COPD exacerbations.

Previously, we observed increased serum levels of damage-associated molecular patterns (DAMPs) during COPD exacerbations. Here, gene expression of DAMP receptors was measured in peripheral blood neutrophils of COPD patients during stable disease and severe acute exacerbation. The expression of toll-like receptor (TLR)2, TLR4 and NLR family, pyrin domain-containing 3 (NLRP3) was significantly increased, while serum levels of the soluble form of the decoy receptor for advanced glycation end-product (sRAGE) were decreased during exacerbation. Together, these data indicate that increased DAMP signalling contributes to activation of neutrophils during COPD exacerbations.