Bingfeng Song

HIF-dependent induction of adenosine receptor A2b skews human dendritic cells to a Th2-stimulating phenotype under hypoxia

Hypoxia is a common characteristic of many pathological and physiological conditions that can markedly change cellular metabolism and cause the accumulation of extracellular adenosine. Recent studies have shown that adenosine can modulate the function of certain immune cell types through binding with different adenosine receptors. Our previous studies have shown that hypoxia has an effect on the biological activity of dendritic cells (DCs) by inducing their differentiation towards a Th2 polarising phenotype. However, the mechanisms underlying this suppression remain unclear. In this study, we have demonstrated that hypoxic mDCs predominantly express adenosine receptor A2b. The A2b receptor antagonist MRS1754 was able to increase the production of IL‐12p70 and TNF‐α by hypoxic mDCs and elevate the amount of Th1 cytokine IFN‐γ production in a mDCs‐T‐cell co‐culture system. We also found that the effect of hypoxia on IL‐12p70 production was mediated via increased intracellular cAMP levels through the up‐regulation of A2b adenosine receptor and the preferential expression of adenosine A2b receptors in hypoxic mDCs was HIF‐1α dependent. Therefore, the hypoxic mDCs could provide a useful tool for researching the function of A2bR in human DCs. Our results offer new insights into understanding the molecular mechanisms underlying the biological activities of DCs in local‐tissue hypoxic microenvironments.

Hypoxia suppresses the production of MMP-9 by human monocyte-derived dendritic cells and requires activation of adenosine receptor A2b via cAMP/PKA signaling pathway.

The migration of dendritic cells (DCs) from the site of antigen-encounter to regional lymphoid organs is crucial for DCs to function as potent antigen-presenting cells. Matrix metalloproteinase-9 (MMP-9) is critically for DCs migration across extracellular matrix (ECM). We verified in previous studies that hypoxia diminished the production of MMP-9 in human monocyte-derived DCs via an unknown mechanism. In this study, we found, for the first time to our knowledge, that hypoxia altered the expression of adenosine receptors on matured DCs (mDCs) toward the predominant expression of adenosine receptor A(2b). MRS1754 (an A(2b)-receptor specific antagonist) was able to counteract the inhibition of hypoxia on MMP-9 by mDCs. We also found that forskolin (a direct adenylate cyclase activator) can mimic the action of hypoxia on the production of MMP-9 by DCs, whereas the adenylate cyclase inhibitor SQ22536 and the PKA inhibitor H89 can abrogate the inhibition of MMP-9 produce by mDCs under hypoxia. The results herein provide initial evidence that the inhibitory effect of hypoxia on MMP-9 by mDCs requires the activation of A(2b) in a cAMP/PKA-dependent pathway. These data offer new insights into our understanding of the molecular mechanisms underlying the migratory function of DCs in local-tissue hypoxic microenvironments.

Human placental trophoblasts express the immunosuppressive cytokine IL-35

Studies of maternal-fetal tolerance focus on defining mechanisms for establishment of immunological privilege within the uterus during pregnancy. Fetal trophoblasts play a key role in maternal tolerance, in part through cytokines production. As a novel inhibitory cytokine, IL-35 is produced by Foxp3(+) regulatory T cells (Tregs) and mediates maximal suppression of Tregs. The purpose of the study is to analyze the expression of IL-35 in first-trimester human placental trophoblasts. IL-35 expression was detected at both protein and mRNA levels by immunohistochemical staining and quantitative real-time PCR method, respectively and secretion of IL-35 was measured by ELISA assay. Our results demonstrated that human trophoblasts constitutively expressed IL-35. Ebi3 and p35 (two subunits of IL-35) mRNA was shown to be co-expressed in trophoblast cells. Moreover, large amounts of secreted IL-35 were detected in the supernatants of trophoblast cells. But we did not detect the constitutive expression of IL-35 in decidual stromal cells. Our findings confirmed for the first time that first-trimester human trophoblast cells expressed and secreted IL-35, which might contribute to their suppressive capacity to maternal immune cells. Therefore, IL-35 may be an important factor of the cytokine network regulating local immune responses during human pregnancy.

Regulation of Th1/Th2 polarization by tissue inhibitor of metalloproteinase-3 via modulating dendritic cells

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is one of a family of proteins inhibiting matrix metalloproteinases, which has also been identified as a mediator for checking inflammation. Meanwhile, it is well known that inflammation causes the activation of the immune response. However, it is not clear whether TIMP-3 plays a role in the immune system. In the present study, we demonstrated a novel function of TIMP-3 in Th1/Th2 polarization through its influence on the antigen-presenting cells. First, TIMP-3 was found strikingly up-regulated by IL-4 during the differentiation of human dendritic cells via the p38MAPK pathway. Second, the expression of costimulatory molecule-CD86 was repressed by TIMP-3. Besides, the induction of IL-12 in matured dendritic cells was significantly inhibited in a PI3K-dependent manner. Furthermore, dendritic cells matured in the presence of TIMP-3 could stimulate allogeneic naive T helper (Th) cells to display a prominent Th2 polarization. Importantly, in an autoimmune disorder-primary immune thrombocytopenia, TIMP-3 showed a statistically positive correlation with IL-4 and platelet count, but a negative correlation with IFN-γ in patient blood samples. Collectively, these in vitro and in vivo data clearly suggested a novel role of TIMP-3 in Th1/Th2 balance in humans.

RhoBTB2 (DBC2) functions as tumor suppressor via inhibiting proliferation, preventing colony formation and inducing apoptosis in breast cancer cells

RhoBTB2 was isolated recently as a tumor suppressor gene from sporadic breast cancer. Although RhoBTB2 was found to be frequently lost in breast cancer and a variety of cancers, its antitumor effect, however, remains unclear. In this study, we constructed a recombinant expression vector pEGFP-N1-RhoBTB2 and transfected it into RhoBTB2-negative breast tumor cell line T-47D. Stable transformanted cells were identified by fluorescence microscope, RT-PCR and Western blot. Cell viability was measured by MTT assay. Colony forming efficiency of breast tumor cells was detected by colony formation assay. Morphological change of apoptotic cells was observed by hematoxylin-eosin staining. Apoptotic ratio was determined by flow cytometry. Cell invasion and migration ability assay were performed using transwell system. Overexpression of RhoBTB2 in breast tumor cells significantly inhibited the proliferation and colony formation of tumor cells. In addition, RhoBTB2 also elevated the apoptotic ratio and caused typical changes of apoptotic morphology in breast tumor cells of RhoBTB2 overexpression. But RhoBTB2 did not influence the invasion and migration ability of breast tumor cells. Therefore, RhoBTB2 is an important tumor suppressor gene related with breast cancer and may play antitumor roles by inhibiting proliferation, preventing colony formation and promoting the apoptosis of tumor cells. However, the precise mechanism behind the antitumor effects of RhoBTB2 needs to be investigated further.

Response gene to complement 32 (RGC-32) expression on M2-polarized and tumor-associated macrophages is M-CSF-dependent and enhanced by tumor-derived IL-4.

Response gene to complement 32 (RGC-32) is a cell cycle regulator involved in the proliferation, differentiation and migration of cells and has also been implicated in angiogenesis. Here we show that RGC-32 expression in macrophages is induced by IL-4 and reduced by LPS, indicating a link between RGC-32 expression and M2 polarization. We demonstrated that the increased expression of RGC-32 is characteristic of alternatively activated macrophages, in which this protein suppresses the production of pro-inflammatory cytokine IL-6 and promotes the production of the anti-inflammatory mediator TGF-β. Consistent with in vitro data, tumor-associated macrophages (TAMs) express high levels of RGC-32, and this expression is induced by tumor-derived ascitic fluid in an M-CSF- and/or IL-4-dependent manner. Collectively, these results establish RGC-32 as a marker for M2 macrophage polarization and indicate that this protein is a potential target for cancer immunotherapy, targeting tumor-associated macrophages.

Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway

NK cells accumulate at the maternal-fetal interface (MFI) and play essential roles in maintaining immune tolerance during pregnancy. The mechanisms that facilitate NK cells tolerance to fetal tissue are largely unknown. T cell Ig and mucin domain-containing protein 3 (Tim-3) is a newly defined molecule with essential immunological function in many physiological and pathological processes. Recent study showed that Tim-3 was involved in the regulation of immune tolerance at MFI. However, whether Tim-3 regulates NK cells cytotoxicity toward trophoblasts is unclear. Here, we showed Tim-3 was mainly expressed by decidual NK cells (dNK) and Tim-3 level in dNK was higher than peripheral NK cells (pNK). Tim-3+ dNK expressed more levels of mature markers CD94 and CD69 than Tim-3- dNK cells and blocking Tim-3 significantly inhibited dNK IFN-γ and TNF-α secretion. Furthermore, we found TGF-β1 may contribute to such up-regulation of Tim-3 in NK cells. Interestingly, blocking Tim-3 enhanced NK cytotoxicity toward trophoblast cell line HTR-8 but not K562. We found HTR-8 expressed Tim-3 ligand Galectin-9, in contrast K562 did not. Small interfering RNA-mediated silencing of Galectin-9 expression enhanced NK cytotoxicity toward HTR-8. We further showed Tim-3/Galecin-9 inhibited NK cytotoxicity toward trophoblast partially via impairing the degranulation process. In addition, clinical data showed that abnormal Tim-3 level on pNK might be associated with recurrent spontaneous abortion (RSA). Thus, our data demonstrate Tim-3/Galectin-9 pathway maintains local tolerance by suppressing NK cytotoxicity toward trophoblasts which may represent a new immunologic tolerance mechanism at MFI.

Effects and mechanisms of curcumin and basil polysaccharide on the invasion of SKOV3 cells and dendritic cells

In the present study, a polysaccharide extract was obtained from Ocimum basilicum (basil polysaccharide, BPS) and the effects of curcumin and BPS on the invasion activity of the SKOV3 ovarian cancer cells and human monocyte-derived dendritic cells (DCs) were investigated. SKOV3 cells and immature or mature DCs were treated with 50 µM curcumin or 100 µg/ml BPS. A transwell invasion assay demonstrated that curcumin and BPS differentially regulate the invasion of SKOV3 cells and DCs. Curcumin significantly decreased the invasion of SKOV3 cells and immature and mature DCs, while BPS only decreased SKOV3 cell invasion. Osteopontin (OPN) mRNA and protein expression were significantly reduced in curcumin and BPS-treated SKOV3 cells and curcumin-treated DCs. Furthermore, flow cytometry showed that curcumin significantly inhibited the surface expression of CD44 in SKOV3 cells and DCs, while BPS had a minimal effect on CD44 expression. Matrix metallopeptidase-9 (MMP-9) mRNA and protein expression were also reduced in all curcumin-treated cells and BPS-treated SKOV3 cells. The results indicated that curcumin and BPS regulated invasion of SKOV3 cells and DCs by distinctly downregulating OPN, CD44 and MMP-9 expression. Therefore, Curcumin and BPS may be suitable candidates for DC-based vaccines for ovarian cancer immunotherapy.

Reduced Expression of Galectin-9 Contributes to a Poor Outcome in Colon Cancer by Inhibiting NK Cell Chemotaxis Partially through the Rho/ROCK1 Signaling Pathway

Galectin-9 is a widely expressed protein that is involved in immune regulation and tumorpathogenesis and serves as a marker of a poor prognosis in various types of cancers. However, the clinical impact and the precise mechanism by which this protein contributes to colon tumor progression are unclear. In the present study, we detected the expression of galectin-9 and CD56 cells using immunohistochemistry. Spearmans rank correlation was used to clarify the association between galectin-9 expression and natural killer (NK) cell infiltration. The influence of galectin-9 on NK-92 cell migration was evaluated in vitro using transwell chemotaxis assays. The role of rh-galectin-9 in F-actin polarization in NK-92 cells was investigated using laser scanning confocal microscopy. We showed that galectin-9 was expressed in 101 (78.91%) colon tumor tissues and that was expressed at lower levels in these tissues than in para-tumor tissues. Low levels of galectin-9 expression were positively correlated with a poor histological grade and lymph node metastasis (P<0.05). A Kaplan-Meier method and Cox proportional hazards regression analysis showed that overall survival was longer in patients with high galectin-9 expression in an 8-year follow-up (P<0.05). Spearmans rank correlation indicated that there was a linear correlation between galectin-9 expression and CD56+ NK cell infiltration (R2 = 0.658; P<0.0001). Galectin-9 stimulated migration in human NK-92 cells by affecting F-actin polarization through the Rho/ROCK1 signaling pathway. These results suggest that galectin-9 expression potentially represents a novel mechanism for tumors to escape immune surveillance in colon tumors.

Expression of RGC32 in human normal and preeclamptic placentas and its role in trophoblast cell invasion and migration

INTRODUCTION Preeclampsia (PE) is a pregnancy-specific complication and it is related to insufficient extravillous trophoblast invasion. To date, the pathophysiology of PE has not yet been fully elucidated. Response gene to complement 32 (RGC32) is a novel cellular protein, and it plays important roles in the regulation of cell differentiation, angiogenesis, migration, and invasion. This study aimed to determine the RGC32 expression and function in human placentas and to explore the underlying mechanisms. METHODS RGC32 expression in term placentas collected after cesarean section from pregnant women with PE and normal pregnant women was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. The effects of RGC32 expression on trophoblast invasion, migration, and the underlying mechanisms were studied in HTR8/SVneo cells. RESULTS The messenger RNA (mRNA) and protein levels of RGC32 were significantly downregulated in preeclamptic placentas compared with normal controls (P < 0.05). RGC32 silencing significantly inhibited HTR8/SVneo cell migration and invasion (P < 0.001, respectively). These effects were associated with decreased activities and expression of matrix metalloproteinase (MMP)-2/9, and with the reduced phosphorylation level of Akt. DISCUSSION RGC32 may play important roles in the pathophysiology of PE by directly affecting the invasion/migration of trophoblast.