Jian-Bao Xin

Generation and Differentiation of IL-17–Producing CD4+ T Cells in Malignant Pleural Effusion

IL-17–producing CD4+ T (Th17) cells have been found to be increased in some human cancers; however, the possible implication of Th17 cells in regulating antitumor responses in malignant pleural effusion (MPE) remains to be elucidated. In the current study, distribution and phenotypic features of Th17 cells in both MPE and peripheral blood from patients with lung cancer were determined by flow cytometry or double immunofluorescence staining. The impacts of cytokines on Th17 cell generation and differentiation were explored. The chemoattractant activity of chemokines CCL20 and CCL22 for Th17 cells in vitro was also observed. It was found that the increased Th17 cells could be found in MPE compared with blood. The in vitro experiments showed that IL-1β, IL-6, IL-23, or their various combinations could promote Th17 cell generation and differentiation from naive CD4+ T cells. MPE was chemotactic for Th17 cells, and this activity was partly blocked by anti-CCL20 and/or CCL22 Abs. Our data also showed that the accumulation of Th17 cells in MPE predicted improved patient survival. It could be concluded that the overrepresentation of Th17 cells in MPE might be due to Th17 cell differentiation and expansion stimulated by pleural proinflammatory cytokines and to recruitment of Th17 cells from peripheral blood induced by pleural chemokines CCL20 and CCL22. Furthermore, the accumulation of Th17 cells in MPE predicted improved patient survival. These data provide the basis for developing immune-boosting strategies based on ridding the cancer patient of this cell population.

Diagnostic accuracy of T-cell interferon-γ release assays in tuberculous pleurisy: a meta-analysis.

Background and objective:  The diagnosis of tuberculous pleurisy by analysis of pleural fluid using standard diagnostic tools is difficult. Recently, T‐cell interferon‐γ release assays (IGRA) have been introduced for the diagnosis of tuberculous pleurisy. The aim of the present meta‐analysis was to establish the overall diagnostic accuracy of IGRA on both pleural fluid and peripheral blood, for diagnosing tuberculous pleurisy.

Interleukin 22-producing CD4+ T cells in malignant pleural effusion

Th22 cells have been reported to be involved in human cancers. However, differentiation and immune regulation of Th22 cells in malignant pleural effusion (MPE) remain unknown. We noted that Th22 cell numbers were increased in MPE, and that IL-22 substantially promoted the proliferation and migratory activity of A549 cells. Moreover, IL-22 could strongly facilitate intercellular adhesion of A549 cells to pleural mesothelial cell monolayers. Our data revealed that the increase in Th22 cells in MPE was due to pleural cytokines and chemokines, and that Th22 exerted an important immune regulation on cancer cells in human pleural malignant environment.

CD39+Regulatory T cells suppress generation and differentiation of Th17 cells in human malignant pleural effusion via a LAP-dependent mechanism

BackgroundBoth regulatory T cells (Tregs) and T helper IL-17-producing cells (Th17 cells) have been found to be involved in human malignancies, however, the possible implication of Tregs in regulating generation and differentiation of Th17 cells in malignant pleural effusion remains to be elucidated.MethodsThe numbers of both CD39+Tregs and Th17 cells in malignant pleural effusion and peripheral blood from patients with lung cancer were determined by flow cytometry. The regulation and mechanism of Tregs on generation and differentiation of Th17 cells were explored.ResultsBoth CD39+Tregs and Th17 cells were increased in malignant pleural effusion when compared with blood, and the numbers of CD39+Tregs were correlated negatively with those of Th17 cells. It was also noted that high levels of IL-1β, IL-6, and TGF-β1 could be observed in malignant pleural effusion when compared the corresponding serum, and that pleural CD39+Tregs could express latency-associated peptide on their surface. When naïve CD4+ T cells were cocultured with CD39+Tregs, Th17 cell numbers decreased as CD39+Treg numbers increased, addition of the anti-latency-associated peptide mAb to the coculture reverted the inhibitory effect exerted by CD39+Tregs.ConclusionsTherefore, the above results indicate that CD39+Tregs inhibit generation and differentiation of Th17 cells via a latency-associated peptide-dependent mechanism.

Treg/IL-17 Ratio and Treg Differentiation in Patients with COPD

Background Chronic obstructive pulmonary disease (COPD) is characterized by chronic pulmonary and systematic inflammation. An abnormal adaptive immune response leads to an imbalance between pro- and anti-inflammatory processes. T-helper (Th), T-cytotoxic (Tc) and T-regulatory (Treg) cells may play important roles in immune and inflammatory responses. This study was conducted to clarify the changes and imbalance of cytokines and T lymphocyte subsets in patients with COPD, especially during acute exacerbations (AECOPD). Methods Twenty-three patients with stable COPD (SCOPD) and 21 patients with AECOPD were enrolled in the present study. In addition, 20 age-, sex- and weight-matched non-smoking healthy volunteers were included as controls. The serum levels of selected cytokines (TGF-β, IL-10, TNF-α, IL-17 and IL-9) were measured by enzyme-linked immunosorbent assay (ELISA) kits. Furthermore, the T lymphocyte subsets collected from peripheral blood samples were evaluated by flow cytometry after staining with anti-CD3-APC, anti-CD4-PerCP, anti-CD8- PerCP, anti-CD25-FITC and anti-FoxP3-PE monoclonal antibodies. Importantly, to remove the confounding effects of inflammatory factors, the authors introduced a concept of “inflammation adjustment” and corrected each measured value using representative inflammatory markers, such as TNF-α and IL-17. Results Unlike the other cytokines, serum TGF-β levels were considerably higher in patients with AECOPD relative to the control group regardless of adjustment. There were no significant differences in the percentages of either CD4+ or CD8+ T cells among the three groups. Although Tregs were relatively upregulated during acute exacerbations, their capacities of generation and differentiation were far from sufficient. Finally, the authors noted that the ratios of Treg/IL-17 were similar among groups. Conclusions These observations suggest that in patients with COPD, especially during acute exacerbations, both pro-inflammatory and anti-inflammatory reactions are strengthened, with the pro-inflammatory reactions dominating. Although the Treg/IL-17 ratios were normal, the regulatory T cells were still insufficient to suppress the accompanying increases in inflammation. All of these changes suggest a complicated mechanism of pro- and anti-inflammatory imbalance which needs to be further investigated.

Cigarette Smoking Promotes Inflammation in Patients with COPD by Affecting the Polarization and Survival of Th/Tregs through Up-Regulation of Muscarinic Receptor 3 and 5 Expression

Background CD4+ T cells in the lung are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD), although CD4+ T cell subsets and the direct effect of smoking on these cells, especially the expression of MRs, have not been comprehensively examined. Methods First, circulating CD4+ T cell subsets in healthy nonsmokers, patients with SCOPD and patients with AECOPD were evaluated by flow cytometry. Then, differentiation experiments were carried out using RT-PCR, and Ki-67/Annexin V antibodies were used to measure proliferation and apoptosis. We also explored the impact of CSE on the differentiation and survival of CD4+Th/Tregs and examined the expression of MRs in healthy nonsmokers and patients with SCOPD. Results We found the percentages of circulating Th1 and Th17 cells were increased in patients with AECOPD, while the percentage of Th2 cells was decreased in patients with SCOPD. The percentages of Th10 cells were decreased in both patients with SCOPD and patients with AECOPD, while the percentages of Tregs were increased. In addition, the percentages of CD4+α-7+ T cells were decreased in patients with SCOPD and patients with AECOPD. However, only the decrease observed in patients with AECOPD was significant. In vitro studies also revealed MR expression affected the polarization of T cells, with different CD4+ T cell subtypes acquiring different MR expression profiles. The addition of CSE facilitated CD4+ T cell polarization towards pro-inflammatory subsets (Th1 and Th17) and affected the survival of CD4+ T cells and Treg cells by up-regulating the expression of MR3 and 5, resulting in an imbalance of CD4+ T cell subsets. Conclusions Our findings suggest an imbalance of circulating CD4+ T cell subsets is involved in COPD pathogenesis in smokers. Cigarette smoking may contribute to this imbalance by affecting the polarization and survival of Th/Tregs through the up-regulation of MR3 and MR5.

Egr-1 mediates Si0(2)-driven transcription of membrane type I matrix metalloproteinase in macrophages.

The up-regulation mechanism of membrane type I matrix metalloproteinase (MT1-MMP) in macrophages stimulated by silica in vitro and the contribution of early growth response 1 (Egr-1) transcription factor in the gene expression pathway were investigated. Macrophages stimulated by silica were treated with Egr-1 antibody or Egr-1 decoy oligodeoxynucleotides (ODN). The levels of MT1-MMP proteins were determined by Western blot and the expression of MT1-MMP mRNAs was detected by RT-PCR. The results showed as compared with control macrophages, silica-stimulated group showed up-regulated gene expression of MT1-MMP via Egr-1 (P<0.01). Compared with silica-stimulated macrophages untreated with antibody, the cells treated with 5 μg/mL Egr-1 antibody were associated with reduced expression of MT1-MMP protein (P<0.01) and mRNA (P<0.01). Compared with silica-stimulated untransfected group, the Egr-1 “decoy” ODN group was associated with reduction in the expression of MT1-MMP protein and mRNA (P<0.01). It was concluded gene expression of MT1-MMP which may play a critical role in silicosis was up-regulated by silica in macrophages. Egr-1 participated in the expression of MT1-MMP and positively regulated the expression. Both Egr-1 antibody and Egr-1 decoy ODN suppressed the expression of MT1-MMP through the Egr-1 pathway and may become a potential therapeutic tool in the management of silicosis in the future.

Cigarette Smoke Disturbs the Survival of CD8+ Tc/Tregs Partially through Muscarinic Receptors-Dependent Mechanisms in Chronic Obstructive Pulmonary Disease.

Background CD8+ T cells (Cytotoxic T cells, Tc) are known to play a critical role in the pathogenesis of smoking related airway inflammation including chronic obstructive pulmonary disease (COPD). However, how cigarette smoke directly impacts systematic CD8+ T cell and regulatory T cell (Treg) subsets, especially by modulating muscarinic acetylcholine receptors (MRs), has yet to be well elucidated. Methods Circulating CD8+ Tc/Tregs in healthy nonsmokers (n = 15), healthy smokers (n = 15) and COPD patients (n = 18) were evaluated by flow cytometry after incubating with anti-CD3, anti-CD8, anti-CD25, anti-Foxp3 antibodies. Peripheral blood T cells (PBT cells) from healthy nonsmokers were cultured in the presence of cigarette smoke extract (CSE) alone or combined with MRs agonist/antagonist for 5 days. Proliferation and apoptosis were evaluated by flow cytometry using Ki-67/Annexin-V antibodies to measure the effects of CSE on the survival of CD8+ Tc/Tregs. Results While COPD patients have elevated circulating percentage of CD8+ T cells, healthy smokers have higher frequency of CD8+ Tregs. Elevated percentages of CD8+ T cells correlated inversely with declined FEV1 in COPD. CSE promoted the proliferation and inhibited the apoptosis of CD8+ T cells, while facilitated both the proliferation and apoptosis of CD8+ Tregs. Notably, the effects of CSE on CD8+ Tc/Tregs can be mostly simulated or attenuated by muscarine and atropine, the MR agonist and antagonist, respectively. However, neither muscarine nor atropine influenced the apoptosis of CD8+ Tregs. Conclusion The results imply that cigarette smoking likely facilitates a proinflammatory state in smokers, which is partially mediated by MR dysfunction. The MR antagonist may be a beneficial drug candidate for cigarette smoke-induced chronic airway inflammation.

Effect of tiotropium bromide on expression of CD8 +CD25 +FoxP3 + regulatory T cells in patients with stable chronic obstructive pulmonary disease

SummaryThe expression of CD8+CD25+FoxP3+ regulatory T cells (CD8+Tregs) in the peripheral blood of patients with stable chronic obstructive pulmonary disease (COPD), and the effect of muscarinic cholinergic receptor antagonist tiotropium bromide on the expression of CD8+Tregs were investigated. Twenty-three patients with moderate to severe stable COPD were enrolled in this study. All patients inhaled tiotropium bromide (18 μg daily) for 3 months. Before and after inhalation of tiotropium bromide, peripheral blood samples were collected from the patients, and T cells were labeled by three-color labeled monoclonal antibodies. Flow cytometry was used to detect the quantity and percentage of CD8+T cells, CD8+CD25+T cells, CD8+Tregs, CD4+T cells, CD4+CD25+T cells and CD4+CD25+FoxP3+ regulatory T cells (CD4+Tregs) respectively. The percentage of CD4+T cells was increased from (27.82±2.18)% to (35.53±1.3)% (t=3.20, P=0.004) in the peripheral blood of patients with stable COPD after inhalation of tiotropium bromide for 3 months, that of CD4+CD25+T cells was decreased from (10.03 ±1.42)% to (4.21 ±0.65)% (t=3.78, P=0.001), and that of CD8+Tregs was increased from (8.41 ±1.68)% to (21.34 ±4.20)% (t=2.72, P=0.013). At baseline, CD8+T cells, CD8+CD25+T cells and CD4+Tregs were detectable in the peripheral blood, but no significant changes were observed after treatment. Linear correlation analysis revealed that the difference before and after treatment in CD4+T cells and CD4+CD25+T cells was negatively correlated with the ratio of change in CD8+Tregs before and after treatment (r=−0.61, P=0.013; r=-0.72, P=0.001 respectively). In the peripheral blood of patients with stable COPD, there was the expression of CD8+Tregs and CD4+Tregs. Muscarinic receptor antagonist, tiotropium bromide, can promote the amplification of CD4+T cells, inhibit the expression of CD25+T cells, and enhance the expression of CD8+Tregs. CD8+Tregs and CD4+Tregs can be used as new indicators to understand the immune status of patients. They are helpful in judging the treatment efficacy and disease immunophenotype.The expression of CD8 +CD25 +FoxP3 + regulatory T cells (CD8 +Tregs) in the peripheral blood of patients with stable chronic obstructive pulmonary disease (COPD), and the effect of muscarinic cholinergic receptor antagonist tiotropium bromide on the expression of CD8 +Tregs were investigated. Twenty-three patients with moderate to severe stable COPD were enrolled in this study. All patients inhaled tiotropium bromide (18 μg daily) for 3 months. Before and after inhalation of tiotropium bromide, peripheral blood samples were collected from the patients, and T cells were labeled by three-color labeled monoclonal antibodies. Flow cytometry was used to detect the quantity and percentage of CD8 +T cells, CD8 +CD25 +T cells, CD8 +Tregs, CD4 +T cells, CD4 +CD25 +T cells and CD4 +CD25 +FoxP3 + regulatory T cells (CD4 +Tregs) respectively. The percentage of CD4 +T cells was increased from (27.82±2.18)% to (35.53±1.3)% (t=3.20, P=0.004) in the peripheral blood of patients with stable COPD after inhalation of tiotropium bromide for 3 months, that of CD4 +CD25 +T cells was decreased from (10.03 ±1.42)% to (4.21 ±0.65)% (t=3.78, P=0.001), and that of CD8 +Tregs was increased from (8.41 ±1.68)% to (21.34 ±4.20)% (t=2.72, P=0.013). At baseline, CD8 +T cells, CD8 +CD25 +T cells and CD4 +Tregs were detectable in the peripheral blood, but no significant changes were observed after treatment. Linear correlation analysis revealed that the difference before and after treatment in CD4 +T cells and CD4 +CD25 +T cells was negatively correlated with the ratio of change in CD8 +Tregs before and after treatment (r=−0.61, P=0.013; r=-0.72, P=0.001 respectively). In the peripheral blood of patients with stable COPD, there was the expression of CD8 +Tregs and CD4 +Tregs. Muscarinic receptor antagonist, tiotropium bromide, can promote the amplification of CD4 +T cells, inhibit the expression of CD25 +T cells, and enhance the expression of CD8 +Tregs. CD8 +Tregs and CD4 +Tregs can be used as new indicators to understand the immune status of patients. They are helpful in judging the treatment efficacy and disease immunophenotype.

Diagnostic and Prognostic Value of Plasma Adrenomedullin in COPD Exacerbation

BACKGROUND: Adrenomedullin (ADM) is a regulatory peptide with many biological actions, but little is known about its role in patients with COPD exacerbation. The purpose of this study was to evaluate the diagnostic and prognostic value of plasma ADM levels on hospital admission in patients with COPD exacerbation. METHODS: Consecutive subjects admitted to the hospital for COPD exacerbation were included and were followed up for 1 y; in addition, subjects with stable COPD from an out-patient clinic and healthy volunteers were recruited as controls. RESULTS: Compared with healthy subjects (145 pg/mL [interquartile range {IQR} 103–290 pg/mL]), plasma ADM levels were significantly higher in subjects with COPD exacerbation (270 pg/mL [IQR 170–510 pg/mL], P = .001) and in subjects with stable COPD (400 pg/mL [IQR 210–525 pg/mL], P < .001). In subjects with COPD exacerbation, ADM levels were significantly elevated during exacerbation (560 pg/mL [IQR 495–630 pg/mL]) compared with the recovery phase (470 pg/mL [IQR 393–553 pg/mL], P = .01) and the stable phase (200 pg/mL [IQR 143–308 pg/mL], P < .001). In receiver operating characteristic analysis, in subjects with COPD exacerbation, ADM had high diagnostic accuracy in differentiating between exacerbation and the stable phase (area under the curve 0.97, 95% CI 0.93–1.02, P < .001). In Cox regression analysis, plasma ADM was not independently associated with 1-y survival (P = .97), but it could accurately predicted the need for ICU care (hazard ratio 1.37, 95% CI 1.09–1.72, P = .008). CONCLUSIONS: Plasma ADM is a valuable biomarker to confirm COPD exacerbation; furthermore, plasma ADM independently predicts the need of ICU care, although it is not associated with long-term mortality in patients with COPD exacerbation.