A 3D Epithelial-Mesenchymal Co-Culture Model of the Airway Wall Using Native Lung Extracellular Matrix.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by ongoing inflammation, impaired tissue repair, and aberrant interplay between airway epithelium and fibroblasts, resulting in an altered extracellular matrix (ECM) composition. The ECM is the three-dimensional (3D) scaffold that provides mechanical support and biochemical signals to cells, now recognized not only as a consequence but as a potential driver of disease progression. To elucidate how the ECM influences pathophysiological changes occurring in COPD, in vitro models are needed that incorporate the ECM. ECM hydrogels are a novel experimental tool for incorporating the ECM in experimental setups. We developed an airway wall model by combining lung-derived ECM hydrogels with a co-culture of primary human fibroblasts and epithelial cells at an air-liquid interface. Collagen IV and a mixture of collagen I, fibronectin, and bovine serum albumin were used as basement membrane-mimicking coatings. The model was initially assembled using porcine lung-derived ECM hydrogels and subsequently with COPD and non-COPD human lung-derived ECM hydrogels. The resulting 3D construct exhibited considerable contraction and supported co-culture, resulting in a differentiated epithelial layer. This multi-component 3D model allows the investigation of remodelling mechanisms, exploring ECM involvement in cellular crosstalk, and holds promise as a model for drug discovery studies exploring ECM involvement in cellular interactions.

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), collagen type VI α chain 1 (COL6A1); collagen type VI α chain 2 (COL6A2), collagen type XIV α chain 1 (COL14A1), fibulin 2 and 5 (FBLN2 and FBLN5), latent transforming growth factor ß binding protein 4 (LTBP4), lumican (LUM), versican (VCAN), decorin (DCN), and elastin (ELN) using image analysis and statistical modeling. 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 forced expiratory volume in 1 s (FEV1) measurements, indicating a relationship with disease severity. Furthermore, we identified six unique ECM signatures where LUM and COL6A1 in parenchyma and COL1A1, FBLN5, DCN, and VCAN in airway walls appear essential in reflecting the presence and severity of COPD. These signatures emphasize the need to examine groups of proteins to represent an overall difference in the ECM landscape in COPD that are more likely to be related to functional effects than individual proteins. Our study revealed differences in the lung ECM landscape between control and COPD and between SEO and moderate COPD signifying distinct pathological processes in the different subgroups.NEW & NOTEWORTHY Our study identified chronic obstructive pulmonary disease (COPD)-associated differences in the lung extracellular matrix (ECM) composition. We highlight the compartmental differences in the ECM landscape in different subtypes of COPD. The most prominent differences were observed for severe-early onset COPD. Moreover, we identified unique ECM signatures that describe airway walls and parenchyma providing insight into the intertwined nature and complexity of ECM changes in COPD that together drive ECM remodeling and may contribute to disease pathogenesis.

Systemic oxidative stress may be associated with reduced IgG antibody titers against SARS-CoV-2 in vaccinated kidney transplant recipients: A post-hoc analysis of the RECOVAC-IR observational study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) poses an increased risk for severe illness and suboptimal vaccination responses in patients with kidney disease, in which oxidative stress may be involved. Oxidative stress can be reliably measured by determining circulating free thiols (R-SH, sulfhydryl groups), since R-SH are rapidly oxidized by reactive species. In this study, we aimed to examine the association between serum free thiols and the ability to mount a humoral immune response to SARS-CoV-2 vaccination in kidney patients. METHODS: Serum free thiol concentrations were measured in patients with chronic kidney disease stages 4/5 (CKD G4/5) (n = 46), on dialysis (n = 43), kidney transplant recipients (KTR) (n = 73), and controls (n = 50). Baseline serum free thiol and interferon-γ-induced protein-10 (IP-10) - a biomarker of the interferon response - were analyzed for associations with seroconversion rates and SARS-CoV-2 spike (S1)-specific IgG concentrations after two doses of the mRNA-1273 vaccine. RESULTS: Albumin-adjusted serum free thiol concentrations were significantly lower in patients with CKD G4/5 (P < 0.001), on dialysis (P < 0.001), and KTR (P < 0.001), as compared to controls. Seroconversion rates after full vaccination were markedly reduced in KTR (52.1%) and were significantly associated with albumin-adjusted free thiols (OR = 1.76, P = 0.033). After adjustment for MMF use, hemoglobin, and eGFR, this significance was not sustained (OR = 1.49, P = 0.241). CONCLUSIONS: KTR show suboptimal serological responses to SARS-CoV-2 vaccination, which is inversely associated with serum R-SH, reflecting systemic oxidative stress. Albeit this association was not robust to relevant confounding factors, it may at least partially be involved in the inability of KTR to generate a positive serological response after SARS-CoV-2 vaccination.

Targeted epigenetic silencing of UCHL1 expression suppresses collagen-1 production in human lung epithelial cells.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is highly expressed in smokers, but little is known about the molecular mechanism of UCHL1 in airway epithelium and its possible role in affecting extracellular matrix (ECM) remodelling in the underlying submucosa. Since cigarette smoking is a major cause of lung diseases, we studied its effect on UCHL1 expression and DNA methylation patterns in human bronchial epithelial cells, obtained after laser capture micro-dissection (LCM) or isolated from residual tracheal/main stem bronchial tissue. Targeted regulation of UCHL1 expression via CRISPR/dCas9 based-epigenetic editing was used to explore the function of UCHL1 in lung epithelium. Our results show that cigarette smoke extract (CSE) stimulated the expression of UCHL1 in vitro. The methylation status of the UCHL1 gene was negatively associated with UCHL1 transcription in LCM-obtained airway epithelium at specific sites. Treatment with a UCHL1 inhibitor showed that the TGF-ß1-induced upregulation of the ECM gene COL1A1 can be prevented by the inhibition of UCHL1 activity in cell lines. Furthermore, upon downregulation of UCHL1 by epigenetic editing using CRISPR/dCas-EZH2, mRNA expression of COL1A1 and fibronectin was reduced. In conclusion, we confirmed higher UCHL1 expression in current smokers compared to non- and ex-smokers, and induced downregulation of UCHL1 by epigenetic editing. The subsequent repression of genes encoding ECM proteins suggest a role for UCHL1 as a therapeutic target in fibrosis-related disease.

Postnatal Smoke Exposure Further Increases the Hepatic Nicotine Metabolism in Prenatally Smoke Exposed Male Offspring and Is Linked with Aberrant Cyp2a5 Methylation.

Prenatal smoke exposure (PreSE) is a risk factor for nicotine dependence, which is further enhanced by postnatal smoke exposure (PostSE). One susceptibility gene to nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine in the liver. Higher CYP2A6 activity is associated with nicotine dependence and could be regulated through DNA methylation. In this study we investigated whether PostSE further impaired PreSE-induced effects on nicotine metabolism, along with Cyp2a5, orthologue of CYP2A6, mRNA expression and DNA methylation. Using a mouse model where prenatally smoke-exposed adult offspring were exposed to cigarette smoke for 3 months, enzyme activity, mRNA levels, and promoter methylation of hepatic Cyp2a5 were evaluated. We found that in male offspring, PostSE increased PreSE-induced cotinine levels and Cyp2a5 mRNA expression. In addition, both PostSE and PreSE changed Cyp2a5 DNA methylation in male groups. PreSE however decreased cotinine levels whereas it had no effect on Cyp2a5 mRNA expression or methylation. These adverse outcomes of PreSE and PostSE were most prominent in males. When considered in the context of the human health aspects, the combined effect of prenatal and adolescent smoke exposure could lead to an accelerated risk for nicotine dependence later in life.

Prenatal smoke exposure dysregulates lung epithelial cell differentiation in mouse offspring: role for AREG-induced EGFR signaling.

Prenatal smoke exposure is a risk factor for impaired lung development in children. Recent studies have indicated that amphiregulin (AREG), which is a ligand of the epidermal growth factor receptor (EGFR), has a regulatory role in airway epithelial cell differentiation. In this study, we investigated the effect of prenatal smoke exposure on lung epithelial cell differentiation and linked this with AREG-EGFR signaling in 1-day-old mouse offspring. Bronchial and alveolar epithelial cell differentiations were assessed by immunohistochemistry. Areg, epidermal growth factor (Egf), and mRNA expressions of specific markers for bronchial and alveolar epithelial cells were assessed by RT-qPCR. The results in neonatal lungs were validated in an AREG-treated three-dimensional mouse lung organoid model. We found that prenatal smoke exposure reduced the number of ciliated cells and the expression of the cilia-related transcription factor Foxj1, whereas it resulted in higher expression of mucus-related transcription factors Spdef and Foxm1 in the lung. Moreover, prenatally smoke-exposed offspring had higher numbers of alveolar epithelial type II cells (AECII) and lower expression of the AECI-related Pdpn and Gramd2 markers. This was accompanied by higher expression of Areg and lower expression of Egf in prenatally smoke-exposed offspring. In bronchial organoids, AREG treatment resulted in fewer ciliated cells and more basal cells when compared with non-treated bronchiolar organoids. In alveolar organoids, AREG treatment led to more AECII cells than non-treated AECII cells. Taken together, the observed impaired bronchial and alveolar cell development in prenatally smoke-exposed neonatal offspring may be induced by increased AREG-EGFR signaling.

Prenatal smoke exposure induces persistent Cyp2a5 methylation and increases nicotine metabolism in the liver of neonatal and adult male offspring.

Prenatal smoke exposure (PSE) is a risk factor for nicotine dependence. One susceptibility gene for nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine and nicotine clearance in the liver. Higher activity of the CYP2A6 enzyme is associated with nicotine dependence, but no research has addressed the PSE effects on the CYP2A6 gene or its mouse homologue Cyp2a5. We hypothesized that PSE affects Cyp2a5 promoter methylation, Cyp2a5 mRNA levels, and nicotine metabolism in offspring. We used a smoke-exposed pregnant mouse model. RNA, DNA, and microsomal protein were isolated from liver tissue of foetal, neonatal, and adult offspring. Enzyme activity, Cyp2a5 mRNA levels, and Cyp2a5 methylation status of six CpG sites within the promoter region were analysed via HPLC, RT-PCR, and bisulphite pyrosequencing. Our data show that PSE induced higher cotinine levels in livers of male neonatal and adult offspring compared to controls. PSE-induced cotinine levels in neonates correlated with Cyp2a5 mRNA expression and promoter methylation at CpG-7 and CpG+45. PSE increased methylation in almost all CpG sites in foetal offspring, and this effect persisted at CpG-74 in male neonatal and adult offspring. Our results indicate that male offspring of mothers which were exposed to cigarette smoke during pregnancy have a higher hepatic nicotine metabolism, which could be regulated by DNA methylation. Given the detected persistence into adulthood, extrapolation to the human situation suggests that sons born from smoking mothers could be more susceptible to nicotine dependence later in life.

Prenatal smoke effect on mouse offspring Igf1 promoter methylation from fetal stage to adulthood is organ and sex specific.

Prenatal smoke exposure (PSE) is associated with reduced birth weight, impaired fetal development, and increased risk for diseases later in life. Changes in DNA methylation may be involved, as multiple large-scale epigenome-wide association studies showed that PSE is robustly associated with DNA methylation changes in blood among offspring in early life. Insulin-like growth factor-1 (IGF1) is important in growth, differentiation, and repair processes after injury. However, no studies investigated the organ-specific persistence of PSE-induced methylation change of Igf1 into adulthood. Based on our previous studies on the PSE effect on Igf1 promoter methylation in fetal and neonatal mouse offspring, we now have extended our studies to adulthood. Our data show that basal Igf1 promoter methylation generally increased in the lung but decreased in the liver (except for 2 persistent CpG sites in both organs) across three different developmental stages. PSE changed Igf1 promoter methylation in all three developmental stages, which was organ and sex specific. The PSE effect was less pronounced in adult offspring compared with the fetal and neonatal stages. In addition, the PSE effect in the adult stage was more pronounced in the lung compared with the liver. For most CpG sites, an inverse correlation was found for promoter methylation and mRNA expression when the data of all three stages were combined. This was more prominent in the liver. Our findings provide additional evidence for sex- and organ-dependent prenatal programming, which supports the developmental origins of health and disease (DOHaD) hypothesis.

Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution.

The human bronchial epithelium is composed of multiple distinct cell types that cooperate to defend against environmental insults. While studies have shown that smoking alters bronchial epithelial function and morphology, its precise effects on specific cell types and overall tissue composition are unclear. We used single-cell RNA sequencing to profile bronchial epithelial cells from six never and six current smokers. Unsupervised analyses led to the characterization of a set of toxin metabolism genes that localized to smoker ciliated cells, tissue remodeling associated with a loss of club cells and extensive goblet cell hyperplasia, and a previously unidentified peri-goblet epithelial subpopulation in smokers who expressed a marker of bronchial premalignant lesions. Our data demonstrate that smoke exposure drives a complex landscape of cellular alterations that may prime the human bronchial epithelium for disease.

A cellular census of human lungs identifies novel cell states in health and in asthma.

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.

Atopy and Inhaled Corticosteroid Use Associate with Fewer IL-17+ Cells in Asthmatic Airways.

BACKGROUND: Interleukin (IL)-17 plays a critical role in numerous immune and inflammatory responses and was recently suggested to contribute to the pathogenesis of nonatopic (non-eosinophil/neutrophil-dominant) asthma. We aimed to compare expression of IL-17 in bronchial airways between atopic and nonatopic asthmatics, with/without inhaled corticosteroid (ICS) use and to identify its major cellular source. METHODS: Bronchial biopsies from 114 patients with mild-to-moderate asthma were investigated: 33 nonatopic, 63 non-corticosteroid users, 90 nonsmokers. IL-17 expression was correlated with atopy and inflammatory cell counts (EPX, NP57, CD3, CD4, CD8, CD20, CD68), taking ICS use and smoking into account. Multiple linear regression analyses were used to determine the independent factors as well as the most relevant inflammatory cells contributing to IL-17 expression. Double immunostainings were performed to confirm the major cellular source of IL-17. RESULTS: In non-ICS users, nonatopic asthmatics had more IL-17+ cells in the airway wall than atopic asthmatics. In both atopic and nonatopic asthmatics, ICS use was associated with lower numbers of IL-17+ cells, independent of smoking. The number of IL-17+ cells was associated with the number of neutrophils (B: 0.26, 95% CI: 0.17-0.35) and eosinophils (B: 0.18, 95% CI: 0.07-0.29). The majority of IL-17+ cells were neutrophils, as confirmed by double immunostaining. CONCLUSIONS: We show for the first time that atopy and ICS use are associated with lower numbers of IL-17+ cells in asthmatic airways. Importantly, IL-17+ cells were mostly neutrophils which conflicts with the paradigm that lymphocytes (Th17) are the main source of IL-17.

Distinct macrophage phenotypes in allergic and nonallergic lung inflammation.

Chronic exposure to farm environments is a risk factor for nonallergic lung disease. In contrast to allergic asthma, in which type 2 helper T cell (Th2) activation is dominant, exposure to farm dust extracts (FDE) induces Th1/Th17 lung inflammation, associated with neutrophil infiltration. Macrophage influx is a common feature of both types of lung inflammation, allergic and nonallergic. However, macrophage functions and phenotypes may vary according to their polarized state, which is dependent on the cytokine environment. In this study, we aimed to characterize and quantify the lung macrophage populations in two established murine models of allergic and nonallergic lung inflammation by means of fluorescence-activated cell sorting and immunohistochemistry. We demonstrated that, whereas in allergic asthma M2-dominant macrophages predominated in the lungs, in nonallergic inflammation M1-dominant macrophages were more prevalent. This was confirmed in vitro using a macrophage cell line, where FDE exerted a direct effect on macrophages, inducing M1-dominant polarization. The polarization of macrophages diverged depending on the exposure and inflammatory status of the tissue. Interfering with this polarization could be a target for treatment of different types of lung inflammation.

Mouse protocadherin-1 gene expression is regulated by cigarette smoke exposure in vivo.

Protocadherin-1 (PCDH1) is a novel susceptibility gene for airway hyperresponsiveness, first identified in families exposed to cigarette smoke and is expressed in bronchial epithelial cells. Here, we asked how mouse Pcdh1 expression is regulated in lung structural cells in vivo under physiological conditions, and in both short-term cigarette smoke exposure models characterized by airway inflammation and hyperresponsiveness and chronic cigarette smoke exposure models. Pcdh1 gene-structure was investigated by Rapid Amplification of cDNA Ends. Pcdh1 mRNA and protein expression was investigated by qRT-PCR, western blotting using isoform-specific antibodies. We observed 87% conservation of the Pcdh1 nucleotide sequence, and 96% conservation of the Pcdh1 protein sequence between men and mice. We identified a novel Pcdh1 isoform encoding only the intracellular signalling motifs. Cigarette smoke exposure for 4 consecutive days markedly reduced Pcdh1 mRNA expression in lung tissue (3 to 4-fold), while neutrophilia and airway hyperresponsiveness was induced. Moreover, Pcdh1 mRNA expression in lung tissue was reduced already 6 hours after an acute cigarette-smoke exposure in mice. Chronic exposure to cigarette smoke induced loss of Pcdh1 protein in lung tissue after 2 months, while Pcdh1 protein levels were no longer reduced after 9 months of cigarette smoke exposure. We conclude that Pcdh1 is highly homologous to human PCDH1, encodes two transmembrane proteins and one intracellular protein, and is regulated by cigarette smoke exposure in vivo.

Susceptibility to chronic mucus hypersecretion, a genome wide association study.

BACKGROUND: Chronic mucus hypersecretion (CMH) is associated with an increased frequency of respiratory infections, excess lung function decline, and increased hospitalisation and mortality rates in the general population. It is associated with smoking, but it is unknown why only a minority of smokers develops CMH. A plausible explanation for this phenomenon is a predisposing genetic constitution. Therefore, we performed a genome wide association (GWA) study of CMH in Caucasian populations. METHODS: GWA analysis was performed in the NELSON-study using the Illumina 610 array, followed by replication and meta-analysis in 11 additional cohorts. In total 2,704 subjects with, and 7,624 subjects without CMH were included, all current or former heavy smokers (≥20 pack-years). Additional studies were performed to test the functional relevance of the most significant single nucleotide polymorphism (SNP). RESULTS: A strong association with CMH, consistent across all cohorts, was observed with rs6577641 (p = 4.25×10(-6), OR = 1.17), located in intron 9 of the special AT-rich sequence-binding protein 1 locus (SATB1) on chromosome 3. The risk allele (G) was associated with higher mRNA expression of SATB1 (4.3×10(-9)) in lung tissue. Presence of CMH was associated with increased SATB1 mRNA expression in bronchial biopsies from COPD patients. SATB1 expression was induced during differentiation of primary human bronchial epithelial cells in culture. CONCLUSIONS: Our findings, that SNP rs6577641 is associated with CMH in multiple cohorts and is a cis-eQTL for SATB1, together with our additional observation that SATB1 expression increases during epithelial differentiation provide suggestive evidence that SATB1 is a gene that affects CMH.

Association of mast cells with lung function in chronic obstructive pulmonary disease.

BACKGROUND: In asthma, higher chymase positive mast cell (MC-C) numbers are associated with less airway obstruction. In COPD, the distribution of MC-C and tryptase positive mast cells (MC-T) in central and peripheral airways, and their relation with lung function, is unknown. We compared MC-T and MC-C distributions in COPD and controls without airflow limitation, and determined their relation with lung function. METHODS: Lung tissue sections from 19 COPD patients (median [interquartile range] FEV1% predicted 56 [23-75]) and 10 controls were stained for tryptase and chymase. Numbers of MC-T and MC-C were determined in different regions of central and peripheral airways and percentage of degranulation was determined. RESULTS: COPD patients had lower MC-T numbers in the subepithelial area of central airways than controls. In COPD, MC-T numbers in the airway wall and more specifically in the epithelium and subepithelial area of peripheral airways correlated positively with FEV1/VC (Spearman's rho (rs) 0.47, p = 0.05 and rs 0.48, p = 0.05, respectively); MC-C numbers in airway smooth muscle of peripheral airways correlated positively with FEV1% predicted (rs 0.57, p = 0.02). Both in COPD patients and controls the percentage of degranulated MC-T and MC-C mast cells was higher in peripheral than in central airways (all p < 0.05), but this was not different between the groups. CONCLUSION: More MC-T and MC-C in peripheral airways correlate with better lung function in COPD patients. It is yet to determine whether this reflects a protective association of mast cells with COPD pathogenesis, or that other explanations are to be considered.

Heme oxygenase-1 prevents smoke induced B-cell infiltrates: a role for regulatory T cells?

BACKGROUND: Smoking is the most important cause for the development of COPD. Since not all smokers develop COPD, it is obvious that other factors must be involved in disease development. We hypothesize that heme oxygenase-1 (HO-1), a protective enzyme against oxidative stress and inflammation, is insufficiently upregulated in COPD. The effects of HO-1 modulation on cigarette smoke induced inflammation and emphysema were tested in a smoking mouse model. METHODS: Mice were either exposed or sham exposed to cigarette smoke exposure for 20 weeks. Cobalt protoporphyrin or tin protoporphyrin was injected during this period to induce or inhibit HO-1 activity, respectively. Afterwards, emphysema development, levels of inflammatory cells and cytokines, and the presence of B-cell infiltrates in lung tissue were analyzed. RESULTS: Smoke exposure induced emphysema and increased the numbers of inflammatory cells and numbers of B-cell infiltrates, as well as the levels of inflammatory cytokines in lung tissue. HO-1 modulation had no effects on smoke induced emphysema development, or the increases in neutrophils and macrophages and inflammatory cytokines. Interestingly, HO-1 induction prevented the development of smoke induced B-cell infiltrates and increased the levels of CD4+CD25+ T cells and Foxp3 positive cells in the lungs. Additionally, the CD4+CD25+ T cells correlated positively with the number of Foxp3 positive cells in lung tissue, indicating that these cells were regulatory T cells. CONCLUSION: These results support the concept that HO-1 expression influences regulatory T cells and indicates that this mechanism is involved in the suppression of smoke induced B-cell infiltrates. The translation of this interaction to human COPD should now be pursued.

Nitrogen dioxide exposure attenuates cigarette smoke-induced cytokine production in mice.

Cigarette smoke is the most important cause for the development of chronic obstructive pulmonary disease (COPD). Since only a minority of smokers and some nonsmokers develop COPD, other factors must be involved as well. NO2 is an important air pollutant associated with respiratory symptoms in humans and emphysema development in animal models. We hypothesized that combined exposure to NO2 and cigarette smoke will enhance pulmonary inflammation and emphysema development. Mice were exposed to 20 ppm NO2 for 17 h/day, to 24 puffs of cigarette smoke 2 times per day, to their combination, or to control air for 5 days/wk during 4 wk. Following the last NO2 exposure and within 24 h after the last smoke exposure the mice were sacrificed. Lungs were removed and analyzed for several inflammatory parameters and emphysema. Cigarette smoke exposure increased eosinophil numbers and levels of tumor necrosis factor (TNF)-alpha, KC, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6. NO2 exposure increased goblet cells, eosinophils, and the levels of IL-6, while it decreased the levels of IL-10. Four weeks of NO2, cigarette smoke, or their combination was not sufficient to induce significant emphysema, nor did it lead to increased numbers of lymphocytes, neutrophils, or macrophages in lung tissue. Instead, NO2 exposure attenuated the smoke-induced increases in levels of TNF-alpha, KC, and MCP-1. These dampening effects of NO2 may be due to modulating effects of NO2 on cytokine production by macrophages and epithelial cells, which have been reported earlier. The next step is to translate these findings of combined, controlled exposure in animals to the human situation.

Proteomics analysis of Hodgkin lymphoma: identification of new players involved in the cross-talk between HRS cells and infiltrating lymphocytes.

Hodgkin and Reed-Sternberg (HRS) cells in Hodgkin lymphoma (HL) secrete factors that interact with inflammatory background cells and may serve as biomarkers for disease activity. To detect new proteins related to pathogenesis, we analyzed the secretome of HRS cells. Proteins in cell culture supernatant of 4 HL cell lines were identified using 1DGE followed by in-gel trypsin digestion and LC-MS/MS. In total, 1290 proteins, including 368 secreted proteins, were identified. Functional grouping of secreted proteins revealed 37 proteins involved in immune response. Sixteen of the 37 proteins (ie, ALCAM, Cathepsin C, Cathepsin S, CD100, CD150, CD26, CD44, CD63, CD71, Fractal-kine, IL1R2, IL25, IP-10, MIF, RANTES, and TARC) were validated in HL cell lines and patient material using immunohistochemistry and/or ELISA. Expression of all 16 proteins was confirmed in HL cell lines, and 15 were also confirmed in HL tissues. Seven proteins (ALCAM, cathepsin S, CD26, CD44, IL1R2, MIF, and TARC) revealed significantly elevated levels in patient plasma compared with healthy controls. Proteomics analyses of HL cell line supernatant allowed detection of new secreted proteins, which may add to our insights in the interaction between HRS cells and infiltrating lymphocytes and in some instances might serve as biomarkers.

Cigarette smoke-induced emphysema: A role for the B cell?

RATIONALE: Little is known about what drives the inflammatory reaction in the development of chronic obstructive lung disease. B cells have been found. OBJECTIVE: To study the involvement of B cells in the development of emphysema. METHODS: The presence of B-cell follicles and their interaction with other cells were investigated in lungs of patients with chronic obstructive pulmonary disease and of smoking mice. B cells were isolated from lymphoid follicles by laser microdissection and analyzed for the presence of immunoglobulin rearrangements and somatic mutations. MAIN RESULTS: Lymphoid follicles consisting of B cells and follicular dendritic cells with adjacent T cells were demonstrated both in the parenchyma and in bronchial walls of patients with emphysema. A clonal process was observed in all follicles and the presence of ongoing somatic mutations was observed in 75% of the follicles, indicating oligoclonal, antigen-specific proliferation. Similar lymphoid follicles were detected in mice that had developed pulmonary inflammation and progressive alveolar airspace enlargement after smoking. The increase in the number of B-cell follicles was progressive with time and correlated with the increase in mean linear intercept. Specific bacterial or viral nucleic acids could not be detected. CONCLUSIONS: B-cell follicles with an oligoclonal, antigen-specific reaction were found in men and mice with emphysema. In mice, the development was progressive with time and correlated with the increase in airspace enlargement. We hypothesize that these B cells contribute to the inflammatory process and/or the development and perpetuation of emphysema by producing antibodies against either tobacco smoke residues or extracellular matrix components.