RIPK1 kinase-dependent inflammation and cell death contribute to the pathogenesis of COPD.

BACKGROUND: Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD. METHODS: We assessed RIPK1 expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patients via immunohistochemistry. Next, we assessed the consequences of genetic and pharmacological inhibition of RIPK1 kinase activity in experimental COPD, using Ripk1 S25D/S25D kinase-deficient mice and the RIPK1 kinase inhibitor GSK'547. RESULTS: RIPK1 expression increased in alveolar type 1 (AT1), AT2, ciliated and neuroendocrine cells in human COPD. RIPK1 protein levels were significantly increased in airway epithelium of COPD patients compared with never-smokers and smokers without airflow limitation. In mice, exposure to cigarette smoke (CS) increased Ripk1 expression similarly in AT2 cells, and further in alveolar macrophages and T-cells. Genetic and/or pharmacological inhibition of RIPK1 kinase activity significantly attenuated airway inflammation upon acute and subacute CS exposure, as well as airway remodelling, emphysema, and apoptotic and necroptotic cell death upon chronic CS exposure. Similarly, pharmacological RIPK1 kinase inhibition significantly attenuated elastase-induced emphysema and lung function decline. Finally, RNA-seq on lung tissue of CS-exposed mice revealed downregulation of cell death and inflammatory pathways upon pharmacological RIPK1 kinase inhibition. CONCLUSIONS: RIPK1 kinase inhibition is protective in experimental models of COPD and may represent a novel promising therapeutic approach.

Necroptosis Signaling Promotes Inflammation, Airway Remodeling, and Emphysema in Chronic Obstructive Pulmonary Disease.

Rationale: Necroptosis, mediated by RIPK3 (receptor-interacting protein kinase 3) and MLKL (mixed lineage kinase domain-like), is a form of regulated necrosis that can drive tissue inflammation and destruction; however, its contribution to chronic obstructive pulmonary disease (COPD) pathogenesis is poorly understood. Objectives: To determine the role of necroptosis in COPD. Methods: Total and active (phosphorylated) RIPK3 and MLKL were measured in the lung tissue of patients with COPD and control subjects without COPD. Necroptosis-related mRNA and proteins as well as cell death were examined in lungs and pulmonary macrophages of mice with cigarette smoke (CS)-induced experimental COPD. The responses of Ripk3-/- and Mlkl-/- mice to acute and chronic CS exposure were compared with those of wild-type mice. The combined inhibition of apoptosis (with the pan-caspase inhibitor quinoline-Val-Asp-difluorophenoxymethylketone [qVD-OPh]) and necroptosis (with deletion of Mlkl in mice) was assessed. Measurements and Main Results: The total MLKL protein in the epithelium and macrophages and the pRIPK3 and pMLKL in lung tissue were increased in patients with severe COPD compared with never-smokers or smoker control subjects without COPD. Necroptosis-related mRNA and protein levels were increased in the lungs and macrophages in CS-exposed mice and experimental COPD. Ripk3 or Mlkl deletion prevented airway inflammation upon acute CS exposure. Ripk3 deficiency reduced airway inflammation and remodeling as well as the development of emphysematous pathology after chronic CS exposure. Mlkl deletion and qVD-OPh treatment reduced chronic CS-induced airway inflammation, but only Mlkl deletion prevented airway remodeling and emphysema. Ripk3 or Mlkl deletion and qVD-OPh treatment reduced CS-induced lung-cell death. Conclusions: Necroptosis is induced by CS exposure and is increased in the lungs of patients with COPD and in experimental COPD. Inhibiting necroptosis attenuates CS-induced airway inflammation, airway remodeling, and emphysema. Targeted inhibition of necroptosis is a potential therapeutic strategy in COPD.

Innate lymphoid cells in isocyanate-induced asthma: role of microRNA-155.

BACKGROUND: Occupational asthma, induced by workplace exposures to low molecular weight agents such as toluene 2,4-diisocyanate (TDI), causes a significant burden to patients and society. Little is known about innate lymphoid cells (ILCs) in TDI-induced asthma. A critical regulator of ILC function is microRNA-155, a microRNA associated with asthma. OBJECTIVE: To determine whether TDI exposure modifies the number of ILCs in the lung and whether microRNA-155 contributes to TDI-induced airway inflammation and hyperresponsiveness. METHODS: C57BL/6 wild-type and microRNA-155 knockout mice were sensitised and challenged with TDI or vehicle. Intracellular cytokine expression in ILCs and T-cells was evaluated in bronchoalveolar lavage (BAL) fluid using flow cytometry. Peribronchial eosinophilia and goblet cells were evaluated on lung tissue, and airway hyperresponsiveness was measured using the forced oscillation technique. Putative type 2 ILCs (ILC2) were identified in bronchial biopsies of subjects with TDI-induced occupational asthma using immunohistochemistry. Human bronchial epithelial cells were exposed to TDI or vehicle. RESULTS: TDI-exposed mice had higher numbers of airway goblet cells, BAL eosinophils, CD4+ T-cells and ILCs, with a predominant type 2 response, and tended to have airway hyperresponsiveness. In TDI-exposed microRNA-155 knockout mice, inflammation and airway hyperresponsiveness were attenuated. TDI exposure induced IL-33 expression in human bronchial epithelial cells and in murine lungs, which was microRNA-155 dependent in mice. GATA3+CD3- cells, presumably ILC2, were present in bronchial biopsies. CONCLUSION: TDI exposure is associated with increased numbers of ILCs. The proinflammatory microRNA-155 is crucial in a murine model of TDI asthma, suggesting its involvement in the pathogenesis of occupational asthma due to low molecular weight agents.

The myokine GDF-15 is a potential biomarker for myositis and associates with the protein aggregates of sporadic inclusion body myositis.

BACKGROUND: The cytokine growth differentiation factor-15 (GDF-15) has been associated with inflammatory and mitochondrial disease, warranting exploration of its expression in myositis patients. METHODS: GDF-15 protein levels are evaluated in 35 idiopathic inflammatory myopathy (IIM) serum samples using enzyme-linked immunosorbent assays, comparing with levels in samples from healthy individuals and from patients with genetically confirmed hereditary muscular dystrophies and mitochondrial disorders. Muscle tissue expression of GDF-15 protein is evaluated using immunofluorescent staining and Western blotting. RESULTS: GDF-15 protein levels are significantly higher in IIM sera (625 ± 358 pg/ml) than in that of healthy controls (326 ± 204 pg/ml, p = 0.01). Western blotting confirms increased GDF-15 protein levels in IIM muscle. In skeletal muscle tissue of IIM patients, GDF-15 localizes mostly to small regenerating or denervated muscle fibres. In patients diagnosed with sporadic inclusion body myositis, GDF-15 co-localizes with the characteristic protein aggregates within affected muscle fibres. CONCLUSIONS: We describe for the first time that GDF-15 is a myokine upregulated in myositis and present the cytokine as a potential diagnostic serum biomarker.

GDF-15 in Pulmonary and Critical Care Medicine.

GDF-15 (growth differentiation factor 15) acts both as a stress-induced cytokine with diverse actions at different body sites and as a cell-autonomous regulator linked to cellular senescence and apoptosis. For multiple reasons, this divergent transforming growth factor-ß molecular superfamily member should be better known to pulmonary researchers and clinicians. In ambulatory individuals, GDF-15 concentrations in peripheral blood are an established predictive biomarker of all-cause mortality and of adverse cardiovascular events. Concentrations upon admission of critically ill patients (without or with sepsis) correlate with organ dysfunction and independently predict short- and long-term mortality risk. GDF-15 is a major downstream mediator of p53 activation, but it can also be induced independently of p53, notably by nonsteroidal antiinflammatory agents. GDF-15 blood concentrations are markedly elevated in adults and children with pulmonary hypertension. Concentrations are also increased in chronic obstructive pulmonary disease, in which they contribute to mucus hypersecretion, airway epithelial cell senescence, and impaired antiviral defenses, which together with murine data support a role for GDF-15 in chronic obstructive pulmonary disease pathogenesis and progression. This review summarizes biological and clinical data on GDF-15 relevant to pulmonary and critical care medicine. We highlight the recent discovery of a central nervous system receptor for GDF-15, GFRAL (glial cell line-derived neurotrophic factor family receptor-α-like), an important advance with potential for novel treatments for obesity and cachexia. We also describe limitations and controversies in the existing literature, and we delineate research questions that must be addressed to determine whether GDF-15 can be therapeutically manipulated in other clinical settings.

DPP4, the Middle East Respiratory Syndrome Coronavirus Receptor, is Upregulated in Lungs of Smokers and Chronic Obstructive Pulmonary Disease Patients.

Background: Middle East respiratory syndrome coronavirus (MERS-CoV) causes pneumonia with a relatively high case fatality rate in humans. Smokers and chronic obstructive pulmonary disease (COPD) patients have been reported to be more susceptible to MERS-CoV infection. Here, we determined the expression of MERS-CoV receptor, dipeptidyl peptidase IV (DPP4), in lung tissues of smokers without airflow limitation and COPD patients in comparison to nonsmoking individuals (never-smokers). Methods: DPP4 expression was measured in lung tissue of lung resection specimens of never-smokers, smokers without airflow limitation, COPD GOLD stage II patients and in lung explants of end-stage COPD patients. Both control subjects and COPD patients were well phenotyped and age-matched. The mRNA expression was determined using qRT-PCR and protein expression was quantified using immunohistochemistry. Results: In smokers and subjects with COPD, both DPP4 mRNA and protein expression were significantly higher compared to never-smokers. Additionally, we found that both DPP4 mRNA and protein expression were inversely correlated with lung function and diffusing capacity parameters. Conclusions: We provide evidence that DPP4 is upregulated in the lungs of smokers and COPD patients, which could partially explain why these individuals are more susceptible to MERS-CoV infection. These data also highlight a possible role of DPP4 in COPD pathogenesis.

Heritability and genome-wide association study of diffusing capacity of the lung.

Although several genome-wide association studies (GWAS) have investigated the genetics of pulmonary ventilatory function, little is known about the genetic factors that influence gas exchange. The aim of the study was to investigate the heritability of, and genetic variants associated with the diffusing capacity of the lung.GWAS was performed on diffusing capacity of the lung measured by carbon monoxide uptake (DLCO) and per alveolar volume (VA) using the single-breath technique, in 8372 individuals from two population-based cohort studies, the Rotterdam Study and the Framingham Heart Study. Heritability was estimated in related (n=6246) and unrelated (n=3286) individuals.Heritability of DLCO and DLCO/VA ranged between 23% and 28% in unrelated individuals and between 45% and 49% in related individuals. Meta-analysis identified a genetic variant in ADGRG6 that is significantly associated with DLCO/VA Gene expression analysis of ADGRG6 in human lung tissue revealed a decreased expression in patients with chronic obstructive pulmonary disease (COPD) and subjects with decreased DLCO/VADLCO and DLCO/VA are heritable traits, with a considerable proportion of variance explained by genetics. A functional variant in ADGRG6 gene region was significantly associated with DLCO/VA Pulmonary ADGRG6 expression was decreased in patients with COPD.

MicroRNA Profiling Reveals a Role for MicroRNA-218-5p in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

RATIONALE: Aberrant expression of microRNAs (miRNAs) can have a detrimental role in disease pathogenesis. OBJECTIVES: To identify dysregulated miRNAs in lung tissue of patients with chronic obstructive pulmonary disease (COPD). METHODS: We performed miRNA and mRNA profiling using high throughput stem-loop reverse-transcriptase quantitative polymerase chain reaction and mRNA microarray, respectively, on lung tissue of 30 patients (screening cohort) encompassing 8 never-smokers, 10 smokers without airflow limitation, and 12 smokers with COPD. Differential expression of miRNA-218-5p (miR-218-5p) was validated by reverse-transcriptase quantitative polymerase chain reaction in an independent cohort of 71 patients, an in vivo murine model of COPD, and primary human bronchial epithelial cells. Localization of miR-218-5p was assessed by in situ hybridization. In vitro and in vivo perturbation of miR-218-5p combined with RNA sequencing and gene set enrichment analysis was used to elucidate its functional role in COPD pathogenesis. MEASUREMENTS AND MAIN RESULTS: Several miRNAs were differentially expressed among the different patient groups. Interestingly, miR-218-5p was significantly down-regulated in smokers without airflow limitation and in patients with COPD compared with never-smokers. Decreased pulmonary expression of miR-218-5p was validated in an independent validation cohort, in cigarette smoke-exposed mice, and in human bronchial epithelial cells. Importantly, expression of miR-218-5p strongly correlated with airway obstruction. Furthermore, cellular localization of miR-218-5p in human and murine lung revealed highest expression of miR-218-5p in the bronchial airway epithelium. Perturbation experiments with a miR-218-5p mimic or inhibitor demonstrated a protective role of miR-218-5p in cigarette smoke-induced inflammation and COPD. CONCLUSIONS: We highlight a role for miR-218-5p in the pathogenesis of COPD.

Role of B Cell-Activating Factor in Chronic Obstructive Pulmonary Disease.

RATIONALE: B cell-activating factor (BAFF) plays a major role in activation of B cells and in adaptive humoral immune responses. In chronic obstructive pulmonary disease (COPD), lymphoid follicles have been associated with disease severity, and overexpression of BAFF has been demonstrated within lymphoid follicles of patients with severe COPD. OBJECTIVES: To investigate expression and localization of BAFF in the lungs of patients with COPD and to study the role of BAFF in COPD by antagonizing BAFF in a mouse model of chronic cigarette smoke (CS) exposure. METHODS: We quantified and localized BAFF expression in lungs of never-smokers, smokers without COPD, and patients with COPD and in lungs of air- or CS-exposed mice by reverse-transcriptase polymerase chain reaction, ELISA, immunohistochemistry, and confocal imaging. Next, to investigate the role of BAFF in COPD, we antagonized BAFF by prophylactic or therapeutic administration of a soluble fusion protein of the BAFF-receptor, BAFFR-Fc, in mice exposed to air or CS for 24 weeks and evaluated several hallmarks of COPD and polarization of lung macrophages. MEASUREMENTS AND MAIN RESULTS: BAFF expression was significantly increased in lungs of patients with COPD and CS-exposed mice. BAFF staining in lymphoid follicles was observed around B cells, CD4(+) cells, dendritic cells, follicular dendritic cells, and fibroblastic reticular cells. Prophylactic and therapeutic administration of BAFFR-Fc in mice reduced pulmonary B-cell numbers and prevented CS-induced formation of lymphoid follicles and increases in immunoglobulin levels. Interestingly, prophylactic BAFFR-Fc administration significantly attenuated pulmonary inflammation and destruction of alveolar walls. Moreover, antagonizing BAFF altered the phenotype of alveolar and interstitial macrophages. CONCLUSIONS: BAFF is significantly increased in lungs of patients with COPD and is present around both immune and stromal cells within lymphoid follicles. Antagonizing BAFF in CS-exposed mice attenuates pulmonary inflammation and alveolar destruction.

Transforming growth factor-ß superfamily in obstructive lung diseases. more suspects than TGF-ß alone.

Asthma and chronic obstructive pulmonary disease are respiratory disorders and a major global health problem with increasing incidence and severity. Genes originally associated with lung development could be relevant in the pathogenesis of chronic obstructive pulmonary disease/asthma, owing to either an early-life origin of adult complex diseases or their dysregulation in adulthood upon exposure to environmental stressors (e.g., smoking). The transforming growth factor (TGF)-ß superfamily is conserved through evolution and is involved in a range of biological processes, both during development and in adult tissue homeostasis. TGF-ß1 has emerged as an important regulator of lung and immune system development. However, considerable evidence has been presented for a role of many of the other ligands of the TGF-ß superfamily in lung pathology, including activins, bone morphogenetic proteins, and growth differentiation factors. In this review, we summarize the current knowledge on the mechanisms by which activin, bone morphogenetic protein, and growth differentiation factor signaling contribute to the pathogenesis of obstructive airway diseases.

Role of activin-A in cigarette smoke-induced inflammation and COPD.

Activin-A is a pleiotropic cytokine belonging to the transforming growth factor-ß superfamily and has been implicated in asthma and pulmonary fibrosis. However, the role of activin-A and its endogenous inhibitor, follistatin, in the pathogenesis of chronic obstructive pulmonary disease (COPD) is unknown. We first quantified activin-A and follistatin in the lungs of air- or cigarette smoke-exposed mice and in the lungs of patients with COPD by immunohistochemistry, ELISA and quantitative real-time PCR. We subsequently studied the effect of cigarette smoke on primary human bronchial epithelial cells in vitro. Next, activin-A signalling was antagonised in vivo by administration of follistatin in mice exposed to air or cigarette smoke for 4 weeks. Protein levels of activin-A were increased in the airway epithelium of patients with COPD compared with never-smokers and smokers. Cigarette smoke-exposed human bronchial epithelial cells expressed higher levels of activin-A and lower levels of follistatin. Both mRNA and protein levels of activin-A were increased in the lungs of cigarette smoke-exposed mice, whereas follistatin levels were reduced upon cigarette smoke exposure. Importantly, administration of follistatin attenuated the cigarette smoke-induced increase of inflammatory cells and mediators in the bronchoalveolar lavage fluid in mice. These results suggest that an imbalance between activin-A and follistatin contributes to the pathogenesis of cigarette smoke-induced inflammation and COPD.

Role of CXCL13 in cigarette smoke-induced lymphoid follicle formation and chronic obstructive pulmonary disease.

RATIONALE: The B cell-attracting chemokine CXCL13 is an important mediator in the formation of tertiary lymphoid organs (TLOs). Increased numbers of ectopic lymphoid follicles have been observed in lungs of patients with severe chronic obstructive pulmonary disease (COPD). However, the role of these TLOs in the pathogenesis of COPD remains unknown. OBJECTIVES: By neutralizing CXCL13 in a mouse model of chronic cigarette smoke (CS) exposure, we aimed at interrogating the link between lymphoid follicles and development of pulmonary inflammation, emphysema, and airway wall remodeling. METHODS: We first quantified and localized CXCL13 in lungs of air- or CS-exposed mice and in lungs of never smokers, smokers without airflow obstruction, and patients with COPD by reverse transcriptase-polymerase chain reaction, ELISA, and immunohistochemistry. Next, CXCL13 signaling was blocked by prophylactic or therapeutic administration of anti-CXCL13 antibodies in mice exposed to air or CS for 24 weeks, and several hallmarks of COPD were evaluated. MEASUREMENTS AND MAIN RESULTS: Both mRNA and protein levels of CXCL13 were increased in lungs of CS-exposed mice and patients with COPD. Importantly, expression of CXCL13 was observed within B-cell areas of lymphoid follicles. Prophylactic and therapeutic administration of anti-CXCL13 antibodies completely prevented the CS-induced formation of pulmonary lymphoid follicles in mice. Interestingly, absence of TLOs attenuated destruction of alveolar walls and inflammation in bronchoalveolar lavage but did not affect airway wall remodeling. CONCLUSIONS: CXCL13 is produced within lymphoid follicles of patients with COPD and is crucial for the formation of TLOs. Neutralization of CXCL13 partially protects mice against CS-induced inflammation in bronchoalveolar lavage and alveolar wall destruction.

Epigenome-wide association study on diffusing capacity of the lung.

BACKGROUND: Epigenetics may play an important role in the pathogenesis of lung diseases. However, little is known about the epigenetic factors that influence impaired gas exchange at the lung. AIM: To identify the epigenetic signatures of the diffusing capacity of the lung measured by carbon monoxide uptake (the diffusing capacity of the lung for carbon monoxide (D LCO)). METHODS: An epigenome-wide association study (EWAS) was performed on diffusing capacity, measured by carbon monoxide uptake (D LCO) and per alveolar volume (V A) (as D LCO/V A), using the single-breath technique in 2674 individuals from two population-based cohort studies. These were the Rotterdam Study (RS, the "discovery panel") and the Framingham Heart Study (FHS, the "replication panel"). We assessed the clinical relevance of our findings by investigating the identified sites in whole blood and by lung tissue specific gene expression. RESULTS: We identified and replicated two CpG sites (cg05575921 and cg05951221) that were significantly associated with D LCO/V A and one (cg05575921) suggestively associated with D LCO. Furthermore, we found a positive association between aryl hydrocarbon receptor repressor (AHRR) gene (cg05575921) hypomethylation and gene expression of exocyst complex component 3 (EXOC3) in whole blood. We confirmed that the expression of EXOC3 in lung tissue is positively associated with D LCO/V A and D LCO. CONCLUSIONS: We report on epigenome-wide associations with diffusing capacity in the general population. Our results suggest EXOC3 to be an excellent candidate, through which smoking-induced hypomethylation of AHRR might affect pulmonary gas exchange.

Non-canonical Wnt/PCP signalling regulates intestinal stem cell lineage priming towards enteroendocrine and Paneth cell fates.

A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required to treat chronic intestinal diseases. However, the different models of ISC lineage hierarchy1-6 and segregation7-12 are subject to debate. Here, we have discovered non-canonical Wnt/planar cell polarity (PCP)-activated ISCs that are primed towards the enteroendocrine or Paneth cell lineage. Strikingly, integration of time-resolved lineage labelling with single-cell gene expression analysis revealed that both lineages are directly recruited from ISCs via unipotent transition states, challenging the existence of formerly predicted bi- or multipotent secretory progenitors7-12. Transitory cells that mature into Paneth cells are quiescent and express both stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ label-retaining cells7. Finally, Wnt/PCP-activated Lgr5+ ISCs are molecularly indistinguishable from Wnt/ß-catenin-activated Lgr5+ ISCs, suggesting that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch from canonical to non-canonical Wnt/PCP signalling. Taken together, we redefine the mechanisms underlying ISC lineage hierarchy and identify the Wnt/PCP pathway as a new niche signal preceding lateral inhibition in ISC lineage priming and segregation.

Quantification and role of innate lymphoid cell subsets in Chronic Obstructive Pulmonary Disease.

OBJECTIVES: Innate lymphoid cells (ILCs) secrete cytokines, such as IFN-γ, IL-13 and IL-17, which are linked to chronic obstructive pulmonary disease (COPD). Here, we investigated the role of pulmonary ILCs in COPD pathogenesis. METHODS: Lung ILC subsets in COPD and control subjects were quantified using flow cytometry and associated with clinical parameters. Tissue localisation of ILC and T-cell subsets was determined by immunohistochemistry. Mice were exposed to air or cigarette smoke (CS) for 1, 4 or 24 weeks to investigate whether pulmonary ILC numbers and activation are altered and whether they contribute to CS-induced innate inflammatory responses. RESULTS: Quantification of lung ILC subsets demonstrated that ILC1 frequency in the total ILC population was elevated in COPD and was associated with smoking and severity of respiratory symptoms (COPD Assessment Test [CAT] score). All three ILC subsets localised near lymphoid aggregates in COPD. In the COPD mouse model, CS exposure in C57BL/6J mice increased ILC numbers at all time points, with relative increases in ILC1 in bronchoalveolar lavage (BAL) fluid. Importantly, CS exposure induced increases in neutrophils, monocytes and dendritic cells that remained elevated in Rag2/Il2rg-deficient mice that lack adaptive immune cells and ILCs. However, CS-induced CXCL1, IL-6, TNF-α and IFN-γ levels were reduced by ILC deficiency. CONCLUSION: The ILC1 subset is increased in COPD patients and correlates with smoking and severity of respiratory symptoms. ILCs also increase upon CS exposure in C57BL/6J mice. In the absence of adaptive immunity, ILCs contribute to CS-induced pro-inflammatory mediator release, but are redundant in CS-induced innate inflammation.

Association of innate defense proteins BPIFA1 and BPIFB1 with disease severity in COPD.

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal inflammatory response in the lungs caused by the inhalation of noxious particles and gases. The airway epithelium has a protective function against these harmful agents by maintaining a physical barrier and by secreting defensive proteins, such as bactericidal/permeability-increasing fold-containing (BPIF) proteins, BPIFA1 and BPIFB1. However, inconsistent data regarding BPIFA1 expression in smokers and COPD patients have been reported to date. Therefore, we investigated the expression of BPIFA1 and BPIFB1 in a large cohort of never-smokers and smokers with and without COPD, both on the messenger RNA (mRNA) level in lung tissue and on the protein level in airway epithelium. Furthermore, we examined the correlation between BPIFA1 and BPIFB1 levels, goblet cell hyperplasia, and lung function measurements. BPIFA1 and BPIFB1 mRNA expressions were significantly increased in stage III-IV COPD patients compared with stage II COPD patients and subjects without COPD. In addition, protein levels in COPD patients were significantly increased in comparison with subjects without COPD. BPIFA1 and BPIFB1 levels were inversely correlated with measurements of airflow limitation and positively correlated with goblet cell hyperplasia. In addition, by the use of immunofluorescence double staining, we demonstrated the expression of BPIFB1 in goblet cells. In conclusion, we show that BPIFA1 and BPIFB1 levels are elevated in COPD patients and correlate with disease severity.

Characterization and Quantification of Innate Lymphoid Cell Subsets in Human Lung.

BACKGROUND: Innate lymphoid cells (ILC) are a new family of innate immune cells that have emerged as important regulators of tissue homeostasis and inflammation. However, limited data are available concerning the relative abundance and characteristics of ILC in the human lung. METHODS: The aim of this study was to characterize and enumerate the different ILC subsets in human lung by multi-color flow cytometry. RESULTS: Within the CD45+ Lin- CD127+ pulmonary ILC population, we identified group 1 (ILC1), group 2 (ILC2) and group 3 (ILC3) innate lymphoid cells using specific surface markers (i.e. IL12Rß2, CRTH2 and CD117 respectively) and key transcription factors (i.e. T-bet, GATA-3 and RORγT respectively). Based on the presence of NKp44, ILC3 were further subdivided in natural cytotoxicity receptor (NCR)+ and NCR- ILC3. In addition, we demonstrated the production of signature cytokines IFN-γ, IL-5, IL-17A, IL-22 and GM-CSF in the pulmonary ILC population. Interestingly, we observed a tendency to a higher frequency of NCR- ILC3 in lungs of patients with chronic obstructive pulmonary disease (COPD) compared with controls. CONCLUSIONS: We show that the three main ILC subsets are present in human lung. Importantly, the relative abundance of ILC subsets tended to change in COPD patients in comparison to control individuals.

Airway Surface Dehydration Aggravates Cigarette Smoke-Induced Hallmarks of COPD in Mice.

INTRODUCTION: Airway surface dehydration, caused by an imbalance between secretion and absorption of ions and fluid across the epithelium and/or increased epithelial mucin secretion, impairs mucociliary clearance. Recent evidence suggests that this mechanism may be implicated in chronic obstructive pulmonary disease (COPD). However, the role of airway surface dehydration in the pathogenesis of cigarette smoke (CS)-induced COPD remains unknown. OBJECTIVE: We aimed to investigate in vivo the effect of airway surface dehydration on several CS-induced hallmarks of COPD in mice with airway-specific overexpression of the ß-subunit of the epithelial Na⁺ channel (ßENaC). METHODS: ßENaC-Tg mice and wild-type (WT) littermates were exposed to air or CS for 4 or 8 weeks. Pathological hallmarks of COPD, including goblet cell metaplasia, mucin expression, pulmonary inflammation, lymphoid follicles, emphysema and airway wall remodelling were determined and lung function was measured. RESULTS: Airway surface dehydration in ßENaC-Tg mice aggravated CS-induced airway inflammation, mucin expression and destruction of alveolar walls and accelerated the formation of pulmonary lymphoid follicles. Moreover, lung function measurements demonstrated an increased compliance and total lung capacity and a lower resistance and hysteresis in ßENaC-Tg mice, compared to WT mice. CS exposure further altered lung function measurements. CONCLUSIONS: We conclude that airway surface dehydration is a risk factor that aggravates CS-induced hallmarks of COPD.

Cigarette smoke extract induces a phenotypic shift in epithelial cells; involvement of HIF1α in mesenchymal transition.

In COPD, matrix remodeling contributes to airflow limitation. Recent evidence suggests that next to fibroblasts, the process of epithelial-mesenchymal transition can contribute to matrix remodeling. CSE has been shown to induce EMT in lung epithelial cells, but the signaling mechanisms involved are largely unknown and subject of this study. EMT was assessed in A549 and BEAS2B cells stimulated with CSE by qPCR, Western blotting and immunofluorescence for epithelial and mesenchymal markers, as were collagen production, cell adhesion and barrier integrity as functional endpoints. Involvement of TGF-ß and HIF1α signaling pathways were investigated. In addition, mouse models were used to examine the effects of CS on hypoxia signaling and of hypoxia per se on mesenchymal expression. CSE induced EMT characteristics in A549 and BEAS2B cells, evidenced by decreased expression of epithelial markers and a concomitant increase in mesenchymal marker expression after CSE exposure. Furthermore cells that underwent EMT showed increased production of collagen, decreased adhesion and disrupted barrier integrity. The induction of EMT was found to be independent of TGF-ß signaling. On the contrary, CS was able to induce hypoxic signaling in A549 and BEAS2B cells as well as in mice lung tissue. Importantly, HIF1α knock-down prevented induction of mesenchymal markers, increased collagen production and decreased adhesion after CSE exposure, data that are in line with the observed induction of mesenchymal marker expression by hypoxia in vitro and in vivo. Together these data provide evidence that both bronchial and alveolar epithelial cells undergo a functional phenotypic shift in response to CSE exposure which can contribute to increased collagen deposition in COPD lungs. Moreover, HIF1α signaling appears to play an important role in this process.