Wanli Ma

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.

Cell Origins and Diagnostic Accuracy of Interleukin 27 in Pleural Effusions

The objective of the present study was to investigate the presence of interleukin (IL)-27 in pleural effusions and to evaluate the diagnostic significance of pleural IL-27. The concentrations of IL-27 were determined in pleural fluids and sera from 68 patients with tuberculous pleural effusion, 63 malignant pleural effusion, 22 infectious pleural effusion, and 21 transudative pleural effusion. Flow cytometry was used to identify which pleural cell types expressed IL-27. It was found that the concentrations of pleural IL-27 in tuberculous group were significantly higher than those in malignant, infectious, and transudative groups, respectively. Pleural CD4+ T cells, CD8+ T cells, NK cells, NKT cells, B cells, monocytes, macrophages, and mesothelial cells might be the cell sources for IL-27. IL-27 levels could be used for diagnostic purpose for tuberculous pleural effusion, with the cut off value of 1,007 ng/L, IL-27 had a sensitivity of 92.7% and specificity of 99.1% for differential diagnosing tuberculous pleural effusion from non-tuberculous pleural effusions. Therefore, compared to non-tuberculous pleural effusions, IL-27 appeared to be increased in tuberculous pleural effusion. IL-27 in pleural fluid is a sensitive and specific biomarker for the differential diagnosing tuberculous pleural effusion from pleural effusions with the other causes.

Bleomycin induced epithelial-mesenchymal transition (EMT) in pleural mesothelial cells.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis. Recent studies revealed that pleural mesothelial cells (PMCs) undergo epithelial-mesenchymal transition (EMT) and play a pivotal role in IPF. In animal model, bleomycin induces pulmonary fibrosis exhibiting subpleural fibrosis similar to what is seen in human IPF. It is not known yet whether bleomycin induces EMT in PMCs. In the present study, PMCs were cultured and treated with bleomycin. The protein levels of collagen-I, mesenchymal phenotypic markers (vimentin and α-smooth muscle actin), and epithelial phenotypic markers (cytokeratin-8 and E-cadherin) were measured by Western blot. PMC migration was evaluated using wound-healing assay of culture PMCs in vitro, and in vivo by monitoring the localization of PMC marker, calretinin, in the lung sections of bleomycin-induced lung fibrosis. The results showed that bleomycin induced increases in collagen-I synthesis in PMC. Bleomycin induced significant increases in mesenchymal phenotypic markers and decreases in epithelial phenotypic markers in PMC, and promoted PMC migration in vitro and in vivo. Moreover, TGF-β1-Smad2/3 signaling pathway involved in the EMT of PMC was demonstrated. Taken together, our results indicate that bleomycin induces characteristic changes of EMT in PMC and the latter contributes to subpleural fibrosis.

Interplay of Th1 and Th17 Cells in Murine Models of Malignant Pleural Effusion

RATIONALE IFN-γ-producing CD4(+) T (Th1) cells and IL-17-producing CD4(+) T (Th17) cells have been found to be involved in multiple malignancies; however, the reciprocal relationship between Th1 and Th17 cells in malignant pleural effusion (MPE) remains to be elucidated. OBJECTIVES To explore the differentiation and immune regulation of Th1 and Th17 cells in the development of MPE in murine models. METHODS The distribution and differentiation of Th1 and Th17 cells in MPE were investigated in IFN-γ(-/-), IL-17(-/-), and wild-type mice. The effects of Th1 and Th17 cells on the development of MPE and the survival of mice bearing MPE were also investigated. MEASUREMENTS AND MAIN RESULTS We have demonstrated that increased Th1 and Th17 cells could be found in MPE as compared with blood and spleen. Compared with wild-type mice, Th17 cells were markedly augmented in MPE from IFN-γ(-/-) mice, and improved survival could be seen in IFN-γ(-/-) mice. Th1 cell numbers were elevated in MPE from IL-17(-/-) mice, and decreased survival could be seen in IL-17(-/-) mice. The in vitro experiments showed that IFN-γ deficiency promoted Th17-cell differentiation by suppressing the STAT3 pathway and that IL-17 deficiency promoted Th1-cell differentiation by suppressing the STAT1 pathway. CONCLUSIONS In mouse models of MPE, IFN-γ inhibited Th17-cell differentiation, whereas IL-17 inhibited Th1-cell differentiation. IL-17 inhibited the formation of MPE and improved the survival of mice bearing MPE; in contrast, IFN-γ promoted MPE formation and mouse death.

Crosstalk between calpain activation and TGF-β1 augments collagen-I synthesis in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease of unknown cause that typically leads to respiratory failure and death within 3-5years of diagnosis. TGF-β1 is considered a major profibrotic factor. However, TGF-β1 is necessary but not sufficient to the pathogenesis of fibrotic lesion of the lungs. Recent observations have revealed that calpain, a calcium dependent protease, plays a pivotal role in tissue remodeling and fibrosis. However, the mechanism of calpain mediating pulmonary fibrosis is not understood. Calpain conditional knockout (ER-Cre(+/-)capns1(flox/flox)) mice and primary human lung fibroblasts (HLFs) were used here to investigate the relationship between calpain and TGF-β1. Calpain knockout mice were protected from fibrotic effects of bleomycin. Bleomycin induced increases in TGF-β1 via calpain activation in HLFs. Moreover, TGF-β1 also activated calpain. This crosstalk between calpain activation and TGF-β1 triggered the downstream signaling pathway including TGF-β1 Smad2/3 and non-Smad (Akt) pathways, as well as collagen-I synthesis. Taken together, our data indicate that the crosstalk between calpain activation and TGF-β1 augments collagen-I synthesis in HLFs and in pulmonary fibrosis. Intervention in the crosstalk between calpain activation and TGF-β1 is a novel potential strategy to prevent pulmonary fibrosis.

Transforming growth factor-β1 suppresses the up-regulation of matrix metalloproteinase-2 by lung fibroblasts in response to tumor necrosis factor-α.

Exposed to inflammatory factors or cytokines, fibroblasts appear to play additional roles beyond the deposition of extracellular matrix. It has been reported that tumor necrosis factor‐α (TNF‐α) induces the production of matrix metalloproteinase‐2 (MMP‐2) and transforming growth factor‐β1 (TGF‐β1) in fibroblasts. In this study, we demonstrated that the active MMP‐2 secreted by lung fibroblasts reached the peak level at 12 hours after TNF‐α treatment, whereas, by adding anti‐TGF‐β1 antibody in the culture medium, the MMP‐2 production in response to TNF‐α was maintained at high levels after 24 hours of treatment. We also confirmed that TNF‐α induced up‐regulation of active TGF‐β1 and exogenous TGF‐β1 induced down‐regulation of MMP‐2 synthesis in lung fibroblasts. Moreover, an increased MMP‐2 level was observed in a rat model with pulmonary inflammation and fibrosis induced by bleomycin‐A5. This revealed that MMP‐2 in the lung reached the peak level when TNF‐α reached the peak level at the 7th day, and then MMP‐2 decreased along with an increase in the TGF‐β1 level. Taken together, our results demonstrate that TNF‐α induced an increase of MMP‐2 and TGF‐β1 in lung fibroblasts, and the TGF‐β1 attenuated the up‐regulation of MMP‐2. This suggests that MMP‐2 secreted from fibroblasts modulated by TNF‐α/TGF‐β1 might play an important role in pulmonary inflammation and fibrosis.

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.

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.

Activation of calpain by renin-angiotensin system in pleural mesothelial cells mediates tuberculous pleural fibrosis

Pleural fibrosis is defined as an excessive deposition of extracellular matrix (ECM) components that results in destruction of the normal pleural tissue architecture. It can result from diverse inflammatory conditions, especially tuberculous pleurisy. Pleural mesothelial cells (PMCs) play a pivotal role in pleural fibrosis. Calpain is a family of calcium-dependent endopeptidases, which plays an important role in ECM remodeling. However, the role of calpain in pleural fibrosis remains unknown. In the present study, we found that tuberculous pleural effusion (TPE) induced calpain activation in PMCs and that inhibition of calpain prevented TPE-induced collagen-I synthesis and cell proliferation of PMCs. Moreover, our data revealed that the levels of angiotensin (ANG)-converting enzyme (ACE) were significantly higher in pleural fluid of patients with TPE than those with malignant pleural effusion, and ACE-ANG II in TPE resulted in activation of calpain and subsequent triggering of the phosphatidylinositol 3-kinase (PI3K)/Akt/NF-κB signaling pathway in PMCs. Finally, calpain activation in PMCs and collagen depositions were confirmed in pleural biopsy specimens from patients with tuberculous pleurisy. Together, these studies demonstrated that calpain is activated by renin-angiotensin system in pleural fibrosis and mediates TPE-induced collagen-I synthesis and proliferation of PMCs via the PI3K/Akt/NF-κB signaling pathway. Calpain in PMCs might be a novel target for intervention in tuberculous pleural fibrosis.

Regulation of CD4(+) T cells by pleural mesothelial cells via adhesion molecule-dependent mechanisms in tuberculous pleurisy.

Background Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) have been demonstrated to be expressed on pleural mesothelial cells (PMCs), and to mediate leukocyte adhesion and migration; however, little is known about whether adhesion molecule-dependent mechanisms are involved in the regulation of CD4+ T cells by PMCs in tuberculous pleural effusion (TPE). Methods Expressions of ICAM-1 and VCAM-1 on PMCs, as well as expressions of CD11a and CD29, the counter-receptors for ICAM-1 and VCAM-1, respectively, expressed on CD4+ T cells in TPE were determined using flow cytometry. The immune regulations on adhesion, proliferation, activation, selective expansion of CD4+ helper T cell subgroups exerted by PMCs via adhesion molecule-dependent mechanisms were explored. Results Percentages of ICAM-1-positive and VCAM-1‒positive PMCs in TPE were increased compared with PMC line. Interferon-γ enhanced fluorescence intensity of ICAM-1, while IL-4 promoted VCAM-1 expression on PMCs. Percentages of CD11ahighCD4+ and CD29highCD4+ T cells in TPE significantly increased as compared with peripheral blood. Prestimulation of PMCs with anti‒ICAM-1 or ‒VCAM-1 mAb significantly inhibited adhesion, activation, as well as effector regulatory T cell expansion induced by PMCs. Conclusions Our current data showed that adhesion molecule pathways on PMCs regulated adhesion and activation of CD4+ T cells, and selectively promoted the expansion of effector regulatory T cells.