Upregulation of IL-17A/F from human lung tissue explants with cigarette smoke exposure: implications for COPD

Background

Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder marked by relative resistance to steroids. The IL-17 superfamily, which mediates cross-talk between the adaptive and innate immune systems, has been associated with diminished responses to steroids. Increasing evidence supports elevated IL-17 expression in the lung of COPD subjects. However, whether cells of the immune system (systemic) and/or local lung cells are contributing to the elevated IL-17 remains unclear. To address this issue, we utilized a human parenchymal lung tissue explant culture system with cigarette smoke exposure to investigate the expression of IL-17 and the mechanisms involved.

Methods

Parenchymal lung tissue removed from 10 non-COPD and 8 COPD patients was sectioned and cultured with different concentrations of cigarette smoke extract (CSE) for 3 or 6 hours. Tissue viability was evaluated by LDH (lactate dehydrogenase) in culture supernatants. Western blot and real-time PCR were performed to evaluate IL-17A/F expression. To investigate the mechanisms, pharmacological inhibitors for MAPK p38, ERK1/2, NF-κB and PI3K pathways were added into the culture media.

Results

No tissue damage was observed after the cigarette smoke exposure for 3 h or 6 h compared with the control media. At the protein level, the expression of both IL-17A (2.4 ± 0.6 fold) and IL-17 F (3.7 ± 0.7 fold) in the tissue from non-COPD subjects was significantly increased by 5% of CSE at 3 h. For COPD subjects, IL-17A/F expression were significantly increased only at 6 h with 10% of CSE (IL-17A: 4.2 ± 0.8 fold; IL-17 F: 3.3 ± 0.8 fold). The increased expression of IL-17A/F is also regulated at the mRNA level. The inhibitors for NF-κB and PI3K pathways significantly inhibited CSE-induced IL-17A/F expression from lung tissue of non-COPD subjects.

Conclusions

We found the evidence that the expression of both IL-17A and IL-17 F is increased by the cigarette smoke exposure in explants from both non-COPD and COPD subjects, supporting that local lung cells contribute IL-17 production. The elevated IL-17A/F expression is dependent on NF-κB and PI3K pathways. These observations add to the growing evidence which suggests that Th17 cytokines play a significant role in COPD.     

Delay of airway epithelial wound repair in COPD is associated with airflow obstruction severity

Background

Airway epithelium integrity is essential to maintain its role of mechanical and functional barrier. Recurrent epithelial injuries require a complex mechanism of repair to restore its integrity. In chronic obstructive pulmonary disease (COPD), an abnormal airway epithelial repair may participate in airway remodeling. The objective was to determine if airway epithelial wound repair of airway epithelium is abnormal in COPD.

Methods

Patients scheduled for lung resection were prospectively recruited. Demographic, clinical data and pulmonary function tests results were recorded. Emphysema was visually scored and histological remodeling features were noted. Primary bronchial epithelial cells (BEC) were extracted and cultured for wound closure assay. We determined the mean speed of wound closure (MSWC) and cell proliferation index, matrix metalloprotease (MMP)-2, MMP-9 and cytokines levels in supernatants of BEC 18 hours after cell wounding. In a subset of patients, bronchiolar epithelial cells were also cultured for wound closure assay for MSWC analyze.

Results

13 COPD and 7 non COPD patients were included. The severity of airflow obstruction and the severity of emphysema were associated with a lower MSWC in BEC (p = 0.01, 95% CI [0.15-0.80]; p = 0.04, 95% CI [−0.77;-0.03] respectively). Cell proliferation index was decreased in COPD patients (19 ± 6% in COPD vs 27 ± 3% in non COPD, p = 0.04). The severity of COPD was associated with a lower level of MMP-2 (7.8 ± 2 10⁵ AU in COPD GOLD D vs 12.8 ± 0.13 10⁵ AU in COPD GOLD A, p = 0.04) and a lower level of IL-4 (p = 0.03, 95% CI [0.09;0.87]). Moreover, higher levels of IL-4 and IL-2 were associated with a higher MSWC (p = 0.01, 95% CI [0.17;0.89] and p = 0.02, 95% CI [0.09;0.87] respectively). Clinical characteristics and smoking history were not associated with MSWC, cell proliferation index or MMP and cytokines levels. Finally, we showed an association of the MSWC of bronchial and corresponding bronchiolar epithelial cells obtained from the same patients (p = 0.02, 95% CI [0.12;0.89]).

Conclusion

Our results showed an abnormal bronchial epithelial wound closure process in severe COPD. Further studies are needed to elucidate the contribution and the regulation of this mechanism in the complex pathophysiology of COPD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0151-9) contains supplementary material, which is available to authorized users.     

Comparative cytoprotective effects of carbocysteine and fluticasone propionate in cigarette smoke extract-stimulated bronchial epithelial cells

Cigarette smoke extracts (CSE) induce oxidative stress, an important feature in chronic obstructive pulmonary disease (COPD), and oxidative stress contributes to the poor clinical efficacy of corticosteroids in COPD patients. Carbocysteine, an antioxidant and mucolytic agent, is effective in reducing the severity and the rate of exacerbations in COPD patients. The effects of carbocysteine on CSE-induced oxidative stress in bronchial epithelial cells as well as the comparison of these antioxidant effects of carbocysteine with those of fluticasone propionate are unknown. The present study was aimed to assess the effects of carbocysteine (10⁻⁴ M) in cell survival and intracellular reactive oxygen species (ROS) production (by flow cytometry) as well as total glutathione (GSH), heme oxygenase-1 (HO-1), nuclear-related factor 2 (Nrf2) expression and histone deacetylase 2 (HDAC-2) expression/activation in CSE-stimulated bronchial epithelial cells (16-HBE) and to compare these effects with those of fluticasone propionate (10⁻⁸ M). CSE, carbocysteine or fluticasone propionate did not induce cell necrosis (propidium positive cells) or cell apoptosis (annexin V-positive/propidium-negative cells) in 16-HBE. CSE increased ROS production, nuclear Nrf2 and HO-1 in 16-HBE. Fluticasone propionate did not modify intracellular ROS production, GSH and HDCA-2 but reduced Nrf2 and HO-1 in CSE-stimulated 16-HBE. Carbocysteine reduced ROS production and increased GSH, HO-1, Nrf2 and HDAC-2 nuclear expression/activity in CSE-stimulated cells and was more effective than fluticasone propionate in modulating the CSE-mediated effects. In conclusion, the present study provides compelling evidences that the use of carbocysteine may be considered a promising strategy in diseases associated with corticosteroid resistance.     

Severe vitamin D deficiency is associated with frequent exacerbations and hospitalization in COPD patients

Background

Acute exacerbations of COPD (AECOPD) are common and strongly influence disease severity and relative healthcare costs. Vitamin D deficiency is frequent among COPD patients and its contributory role in disease exacerbations is widely debated. Our aim was to assess the relationship of serum vitamin D levels with COPD severity and AECOPD.

Methods

Serum vitamin D (25-hydroxyvitamin D) levels were measured in 97 COPD patients and related to lung function, comorbidities, FEV1 decline, AECOPD and hospital admission during the previous year.

Results

Most patients (96%) had vitamin D deficiency, which was severe in 35 (36%). No significant relationship was found between vitamin D and FEV1 or annual FEV1 decline. No difference between patients with and without severe vitamin D deficiency was found in age, gender, BMI, smoking history, lung function, and comorbidities, apart from osteoporosis (60.9% in severe deficiency vs 22.7%, p = 0.001). In multiple logistic regression models, severe deficiency was independently associated with AECOPD [adjusted odds ratios (aOR) of 30.5 (95% CI 5.55, 168), p < 0.001] and hospitalization [aOR 3.83 (95% CI 1.29, 11.4), p = 0.02]. The odds ratio of being a frequent exacerbator if having severe vitamin D deficiency was 18.1 (95% CI 4.98, 65.8) (p < 0.001), while that of hospitalization was 4.57 (95% CI 1.83, 11.4) (p = 0.001).

Conclusions

In COPD patients severe vitamin D deficiency was related to more frequent disease exacerbations and hospitalization during the year previous to the measurement of vitamin D. This association was independent of patients’ characteristics and comorbidities.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0131-0) contains supplementary material, which is available to authorized users.     

Pancreatitis severity in mice with impaired CFTR function but pancreatic sufficiency is mediated via ductal and inflammatory cells-Not acinar cells

Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) are an established risk factor for cystic fibrosis (CF) and chronic pancreatitis. Whereas patients with CF usually develop complete exocrine pancreatic insufficiency, pancreatitis patients with CFTR mutations have mostly preserved exocrine pancreatic function. We therefore used a strain of transgenic mice with significant residual CFTR function (CFTR^tm1HGU) to induce pancreatitis experimentally by serial caerulein injections. Protease activation and necrosis were investigated in isolated acini, disease severity over 24h, pancreatic function by MRI, isolated duct stimulation and faecal chymotrypsin, and leucocyte function by ex vivo lipopolysaccharide (LPS) stimulation. Pancreatic and lung injury were more severe in CFTR^tm1HGU but intrapancreatic trypsin and serum enzyme activities higher than in wild-type controls only at 8h, a time interval previously attributed to leucocyte infiltration. CCK-induced trypsin activation and necrosis in acini from CFTR^tm1HGU did not differ from controls. Fluid and bicarbonate secretion were greatly impaired, whereas faecal chymotrypsin remained unchanged. LPS stimulation of splenocytes from CFTR^tm1HGU resulted in increased INF-γ and IL-6, but decreased IL-10 secretion. CFTR mutations that preserve residual pancreatic function significantly increase the severity of experimental pancreatitis—mostly via impairing duct cell function and a shift towards a pro-inflammatory phenotype, not by rendering acinar cells more susceptible to pathological stimuli.     

The role of bronchial epithelial cells in the pathogenesis of COPD in Z-alpha-1 antitrypsin deficiency

Background

Alpha-1 antitrypsin is the main inhibitor of neutrophil elastase in the lung. Although it is principally synthesized by hepatocytes, alpha-1 antitrypsin is also secreted by bronchial epithelial cells. Gene mutations can lead to alpha-1 antitrypsin deficiency, with the Z variant being the most clinically relevant due to its propensity to polymerize. The ability of bronchial epithelial cells to produce Z-variant protein and its polymers is unknown.We investigated the expression, accumulation, and secretion of Z-alpha-1 antitrypsin and its polymers in cultures of transfected cells and in cells originating from alpha-1 antitrypsin-deficient patients.

Methods

Experiments using a conformation-specific antibody were carried out on M- and Z-variant–transfected 16HBE cells and on bronchial biopsies and ex vivo bronchial epithelial cells from Z and M homozygous patients. In addition, the effect of an inflammatory stimulus on Z-variant polymer formation, elicited by Oncostatin M, was investigated. Comparisons of groups were performed using t-test or ANOVA. Non-normally distributed data were assessed by Mann–Whitney U test or the Kruskal-Wallis test, where appropriate. A P value of < 0.05 was considered to be significant.

Results

Alpha-1 antitrypsin polymers were found at a higher concentration in the culture medium of ex vivo bronchial epithelial cells from Z-variant homozygotes, compared with M-variant homozygotes (P < 0.01), and detected in the bronchial epithelial cells and submucosa of patient biopsies. Oncostatin M significantly increased the expression of alpha-1 antitrypsin mRNA and protein (P < 0.05), and the presence of Z-variant polymers in ex vivo cells (P < 0.01).

Conclusions

Polymers of Z-alpha-1 antitrypsin form in bronchial epithelial cells, suggesting that these cells may be involved in the pathogenesis of lung emphysema and in bronchial epithelial cell dysfunction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0112-3) contains supplementary material, which is available to authorized users.     

Cigarette smoke-induced autophagy is regulated by SIRT1-PARP-1-dependent mechanism: Implication in pathogenesis of COPD

Autophagy is a fundamental cellular process that eliminates long-lived proteins and damaged organelles through lysosomal degradation pathway. Cigarette smoke (CS)-mediated oxidative stress induces cytotoxic responses in lung cells. However, the role of autophagy and its mechanism in CS-mediated cytotoxic responses is not known. We hypothesized that NAD⁺-dependent deacetylase, sirtuin 1 (SIRT1) plays an important role in regulating autophagy in response to CS. CS exposure resulted in induction of autophagy in lung epithelial cells, fibroblasts and macrophages. Pretreatment of cells with SIRT1 activator resveratrol attenuated CS-induced autophagy whereas SIRT1 inhibitor, sirtinol, augmented CS-induced autophagy. Elevated levels of autophagy were induced by CS in the lungs of SIRT1 deficient mice. Inhibition of poly(ADP-ribose)-polymerase-1 (PARP-1) attenuated CS-induced autophagy via SIRT1 activation. These data suggest that the SIRT1-PARP-1 axis plays a critical role in the regulation of CS-induced autophagy and have important implications in understanding the mechanisms of CS-induced cell death and senescence.     

Dynamic hyperinflation is associated with a poor cardiovascular response to exercise in COPD patients

Background

Pulmonary hyperinflation has the potential for significant adverse effects on cardiovascular function in COPD. The aim of this study was to investigate the relationship between dynamic hyperinflation and cardiovascular response to maximal exercise in COPD patients.

Methods

We studied 48 patients (16F; age 68 yrs ± 8; BMI 26 ± 4) with COPD. All patients performed spirometry, plethysmography, lung diffusion capacity for carbon monoxide (TLco) measurement, and symptom-limited cardiopulmonary exercise test (CPET). The end-expiratory lung volume (EELV) was evaluated during the CPET. Cardiovascular response was assessed by change during exercise in oxygen pulse (ΔO₂Pulse) and double product, i.e. the product of systolic blood pressure and heart rate (DP reserve), and by the oxygen uptake efficiency slope (OUES), i.e. the relation between oxygen uptake and ventilation.

Results

Patients with a peak exercise EELV (%TLC) ≥ 75% had a significantly lower resting FEV₁/VC, FEF₅₀/FIF₅₀ ratio and IC/TLC ratio, when compared to patients with a peak exercise EELV (%TLC) < 75%. Dynamic hyperinflation was strictly associated to a poor cardiovascular response to exercise: EELV (%TLC) showed a negative correlation with ΔO₂Pulse (r = - 0.476, p = 0.001), OUES (r = - 0.452, p = 0.001) and DP reserve (r = - 0.425, p = 0.004). Furthermore, according to the ROC curve method, ΔO₂Pulse and DP reserve cut-off points which maximized sensitivity and specificity, with respect to a EELV (% TLC) value ≥ 75% as a threshold value, were ≤ 5.5 mL/bpm (0.640 sensitivity and 0.696 specificity) and ≤ 10,000 Hg · bpm (0.720 sensitivity and 0.783 specificity), respectively.

Conclusion

The present study shows that COPD patients with dynamic hyperinflation have a poor cardiovascular response to exercise. This finding supports the view that in COPD patients, dynamic hyperinflation may affect exercise performance not only by affecting ventilation, but also cardiac function.     

Hydrogen peroxide-mediated necrosis induction in HUVECs is associated with an atypical pattern of caspase-3 cleavage

Oxidative stress, continuously exerted during chronic inflammation, has been implicated as a major causative agent of cellular dysfunction and cell death. In the present study, we investigated the impact of oxidative stress on the mode of cell death in HUVECs using H2O2 as a model reagent. We found that the predominant form of cell death was necrosis. Necrosis induction was accompanied by a distinct mode of caspase-3 cleavage, yielding a 29-kDa fragment. While inhibition of caspases could not prevent the generation of the 29-kDa fragment, general protease inhibitors, such as leupeptin and LLNL, proved to be effective in inhibiting the distinct processing pattern of caspase-3. These results suggest that caspases can act as substrates for non-caspase proteases in cells primed for necrosis induction. Thus, the pattern of caspase-3 cleavage might reflect the proteolytic system engaged in the cell death machinery in HUVECs.     

脉冲震荡肺功能在稳定期COPD治疗中的应用价值

目的:探讨脉冲震荡肺功能(Impulse oscillometry,IOS)在稳定期COPD患者中应用价值及其和常规肺功能检测指标的相关性。方法:62例重度稳定期COPD患者,同时选择健康对照组人群40例纳入研究。经噻托溴铵联合沙美特罗替卡松治疗,将重度COPD患者缓解至中度。检测治疗前后常规肺功能指标(FEV1/FVC、FEV1)和IOS指标(ZRS、Fres、R5、X5、R20),分析常规肺功能指标和IOS各指标的相关性。结果:COPD患者FEV1和FEV1/FVC和对照组相比明显降低,差异有统计学意义(P0.05),观察组治疗后FEV1和FEV1/FVC明显改善,差异有统计学意义(P0.05)。观察组ZRS、Fres、R5、R20各项指标明显高于对照组,差异有统计学意义(P0.05);治疗后ZRS、Fres、R5明显改善,差异有统计学意义(P0.05)。FEV1、FEV1/FVC和ZRS、Fres、R5呈负相关性(P0.05),和X5呈正相关性(P0.05)。结论:脉冲震荡肺功能多项指标和传统肺功能指标有良好的相关性,是一种简便、低配合度、准确的的肺功能新的检测技术手段。     

Destabilization of the VCP-Ufd1-Npl4 complex is associated with decreased levels of ERAD substrates

p97/VCP associated with Ufd1-Npl4 is considered a key player in ER-associated degradation (ERAD). RNA interference (RNAi) of one component of the Ufd1-Npl4 heterodimer destabilizes the VCP-Ufd1-Npl4 complex inducing proteasome-dependent degradation of the other component and releasing free VCP. In contrast to RNAi of VCP, RNAi of Ufd1 or Npl4 depleting approximately 90% of the VCP-Ufd1-Npl4 complexes does not induce unfolded protein response, indicating that the Ufd1-Npl4 dimer is not involved in the regulation of ER function by VCP. RNAi of Ufd1 or Npl4 is associated with a 2-fold increase in the levels of polyubiquitinated proteins, which form dispersed aggregates often associated with calnexin-positive structures. However, contrary to the effects of proteasome inhibition, RNAi of Ufd1 or Npl4 does not induce an accumulation of alpha-TCR and delta-CD3, two ERAD substrates overexpressed in HeLa cells. Instead, a 60-70% decrease in their levels is observed. The decrease in alpha-TCR levels is associated with a 50% decrease of its half-life. Upregulation of the putative channel forming protein, derlin-1, may contribute to the increased degradation of ERAD substrates. To explain our findings, we propose a model, where association of emerging ERAD substrates with VCP-Ufd1-Npl4 is not required for their degradation but has a regulatory role.     

Identification of apoptosis‐associated protein factors distinctly expressed in cigarette smoke condensate‐exposed airway bronchial epithelial cells

Smoking is associated with an increased risk of respiratory diseases, including lung cancer and asthma. However, the mechanisms or diagnostic markers for smoking‐related diseases remain largely unknown. Here we investigated the role of cigarette smoke condensate (CSC) in the regulation of human bronchial epithelial cell (BEAS‐2B) behavior. We found that exposure to CSC significantly inhibited BEAS‐2B cell viability, impaired cell morphology, induced cell apoptosis, triggered oxidative damage, and promoted inflammatory response, which suggests a deleterious effect of CSC on bronchial epithelial cells. In addition, CSC markedly altered the expression of apoptosis‐associated protein factors, including p21, soluble tumor necrosis factor receptor 1, and Fas ligand. In sum, our study identified a panel of novel protein factors that may mediate the actions of CSC on bronchial epithelial cells and have a predictive value for the development and progression of smoking‐related diseases, thus providing insights into the development of potential diagnostic and therapeutic strategies against these diseases.     

HLA-B18 is associated with decreased levels of isoproterenol-stimulated cAMP in lymphocytes.

One hundred fifty-nine individuals were typed for HLA-A and B antigens and levels of isoproterenol-stimulated, lymphocyte cAMP. No significant age, sex, or caffeine effects on the natural log of the lymphocyte cAMP variable (ln cAMP) were found. A comparison of mean ln cAMP levels between individuals who carried a particular antigen (homozygous or heterozygous) and individuals who did not carry the antigen identified a highly significant decrease in ln cAMP levels associated with the HLA-B18 antigen. We estimated that 18.9% of the variability in ln cAMP was attributable to the HLA-B18 antigen. In addition, 38% of the variability in ln cAMP was attributable to factors that aggregate in families that were independent of the HLA-B18 effect. A weaker association of A10 with lymphocyte cAMP might be due to linkage disequilibrium between A10 and B18.     

Alveolar macrophage cytotoxic activity is inhibited by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenic component of cigarette smoke

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic compound of cigarette smoke that generates electrophilic intermediates capable of damaging DNA. Recently, we have shown that NNK can modulate mediator production by alveolar macrophages (AM) and bronchial and alveolar epithelial cells, suggesting that cigarette smoke can alter lung immune response. Thus, we investigated the effect of NNK and cigarette smoke extract (CSE) on AM capacity to eliminate tumoral cells. Rat AM cell line, NR8383, was treated with NNK (500 μM) or CSE (3%) and stimulated with lipopolysaccharide (10 ng/ml). The release of cytotoxic mediators, tumor necrosis factor (TNF) and reactive oxygen species (ROS), was measured in cell-free supernatants using ELISA and superoxide anion production. TNF- and ROS-dependent cytotoxicity were studied using a ⁵¹Chromium-release assay and WEHI-164 and P-815 cell lines. Treatment of AM with NNK and CSE for 18 h significantly inhibited AM TNF release. CSE exposure resulted in a significant increase of ROS production, whereas NNK did not. TNF-dependent cytotoxic activity of NR8383 and freshly isolated rat AM was significantly inhibited after treatment with NNK and CSE. Interestingly, although ROS production was stimulated by CSE and not affected by NNK, CSE inhibited AM ROS-dependent cytotoxicity. These results suggest that NNK may be one of the cigarette smoke components responsible for the reduction of pulmonary cytotoxicity. Thus, NNK may have a double pro-carcinogenic effect by contributing to DNA adduct formation and inhibiting AM cytotoxicity against tumoral cells.     

Lymphocyte senescence in COPD is associated with loss of glucocorticoid receptor expression by pro-inflammatory/cytotoxic lymphocytes

Background

Glucocorticoid (GC) resistance is a major barrier in COPD treatment. We have shown increased expression of the drug efflux pump, Pgp1 in cytotoxic/pro-inflammatory lymphocytes in COPD. Loss of lymphocyte co-stimulatory molecule CD28 (lymphocyte senescence) was associated with a further increase in their pro-inflammatory/cytotoxic potential and resistance to GC. We hypothesized that lymphocyte senescence and increased Pgp1 are also associated with down-regulation of the GC receptor (GCR).

Methods

Blood was collected from 10 COPD and 10 healthy aged-matched controls. Flow cytometry was applied to assess intracellular pro-inflammatory cytokines, CD28, Pgp1, GCR, steroid binding and relative cytoplasm/nuclear GCR by CD28+ and CD28null T, NKT-like cells. GCR localization was confirmed by fluorescent microscopy.

Results

COPD was associated with increased numbers of CD28nullCD8+ T and NKT-like cells. Loss of CD28 was associated with an increased percentage of T and NKT-like cells producing IFNγ or TNFα and associated with a loss of GCR and Dex-Fluor staining but unchanged Pgp1. There was a significant loss of GCR in CD8 + CD28null compared with CD8 + CD28+ T and NKT-like cells from both COPD and controls (eg, mean ± SEM 8 ± 3% GCR + CD8 + CD28null T-cells vs 49 ± 5% GCR + CD8 + CD28+ T-cells in COPD). There was a significant negative correlation between GCR expression and IFNγ and TNFα production by T and NKT-like cells(eg, COPD: T-cell IFNγ R = −.615; ) and with FEV1 in COPD (R = −.777).

Conclusions

COPD is associated with loss of GCR in senescent CD28null and NKT-like cells suggesting alternative treatment options to GC are required to inhibit these pro-inflammatory/cytotoxic cells.     

Impact of smoking on dendritic cell phenotypes in the airway lumen of patients with COPD

Background

Myeloid dendritic cells (DCs) are increased in the airway wall of patients with chronic obstructive pulmonary disease (COPD), and postulated to play a crucial role in COPD. However, DC phenotypes in COPD are poorly understood.

Methods

Function-associated surface molecules on bronchoalveolar lavage fluid (BALF) DCs were analyzed using flow cytometry in current smokers with COPD, in former smokers with COPD and in never-smoking controls.

Results

Myeloid DCs of current smokers with COPD displayed a significantly increased expression of receptors for antigen recognition such as BDCA-1 or Langerin, as compared with never-smoking controls. In contrast, former smokers with COPD displayed a significantly decreased expression of these receptors, as compared with never-smoking controls. A significantly reduced expression of the maturation marker CD83 on myeloid DCs was found in current smokers with COPD, but not in former smokers with COPD. The chemokine receptor CCR5 on myeloid DCs, which is also important for the uptake and procession of microbial antigens, was strongly reduced in all patients with COPD, independently of the smoking status.

Conclusion

COPD is characterized by a strongly reduced CCR5 expression on myeloid DCs in the airway lumen, which might hamper DC interactions with microbial antigens. Further studies are needed to better understand the role of CCR5 in the pathophysiology and microbiology of COPD.     

Inactivation of p53 is associated with decreased levels of radiation-induced apoptosis in medulloblastoma cell lines

Radiation is the primary therapeutic modality for children with medulloblastoma, a pediatric brain tumour. We examined the response of four medulloblastoma cell lines to ionising radiation. Our evaluation utilising flow cytometry, morphological analysis and terminal deoxynucleotidyl transferase assays demonstrated that medulloblastoma cells undergo radiation-induced apoptosis. p53 mediates radiation-induced apoptosis in many cell types, and p53 mutations have been associated with increased resistance to ionising radiation. p53 mutations are rare in medulloblastoma. We found that wildtype p53 is required for high levels of apoptosis in medulloblastoma, and cell lines in which p53 had been inactivated by mutation had very low levels of apoptosis. Inactivation of endogenous wildtype p53 in medulloblastoma cells by introduction of a dominant negative mutant of p53 decreased the level of radiation-induced apoptosis. Our results suggest that the sensitivity of medulloblastoma to irradiation involves p53-mediated apoptosis and that p53 gene status may be a predictor of response to radiation therapy.     

Cigarette smoke-induced skeletal muscle atrophy is associated with up-regulation of USP-19 via p38 and ERK MAPKs

Ubiquitin-specific proteases (USPs) deubiquitinate ubiquitin-protein conjugates in the ubiquitin-proteasome system. Previous research shows that ubiquitin-specific protease-19 (USP-19) is up-regulated in mammalian skeletal muscle in some degradative conditions, such as including fasting, diabetes, dexamethasone treatment, and cancer, and its function is associated with muscle atrophy. However, it is still unclear whether USP-19 is involved in muscle atrophy induced by chronic obstructive pulmonary disease. Rats exposed to chronic cigarette smoke and L6 myotubes incubated with cigarette smoke extract (CSE) were studied here. Using western blot analysis and quantitative real-time polymerase chain reaction (qPCR), we observed over-expression of USP-19 and down-regulation of myosin heavy chain (MHC) in both models. Moreover, CSE exposure inhibited myogenic differentiation and myotube formation in L6 myotubes. To explore the mechanism underlying these effects, we investigated the levels of phosphorylated mitogen-activated protein kinases (MAPKs) and total MAPKs. Exposing myotubes to CSE resulted in the general activation of MAPKs such as p38, JNK, and ERK1/2. The ERK inhibitor PD98059 and the p38 inhibitor SB203580 significantly blocked the increase in USP-19 gene expression induced by CSE. Our findings suggest that USP-19 is associated with muscle atrophy in response to cigarette smoke and is a potential therapeutic target. CSE promotes myotube wasting in culture partly by inhibiting myogenic differentiation and acts via p38 and ERK MAPK to stimulate expression of USP-19 in vitro.     

MYC-induced apoptosis in mammary epithelial cells is associated with repression of lineage-specific gene signatures

Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppression. However, it is still unclear how supraphysiological MYC expression levels engage specific sets of target genes to promote apoptosis. Recently, we demonstrated that repression of SRF target genes by MYC/MIZ1 complexes limits AKT-dependent survival signaling and contributes to apoptosis induction. Here we report that supraphysiological levels of MYC repress gene sets that include markers of basal-like breast cancer cells, but not luminal cancer cells, in a MIZ1-dependent manner. Furthermore, repressed genes are part of a conserved gene signature characterizing the basal subpopulation of both murine and human mammary gland. These repressed genes play a role in epithelium and mammary gland development and overlap with genes mediating cell adhesion and extracellular matrix organization. Strikingly, acute activation of oncogenic MYC in basal mammary epithelial cells is sufficient to induce luminal cell identity markers. We propose that supraphysiological MYC expression impacts on mammary epithelial cell identity by repressing lineage-specific target genes. Such abrupt cell identity switch could interfere with adhesion-dependent survival signaling and thus promote apoptosis in pre-malignant epithelial tissue.