Qianqian Shao

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.

Fucoidan stimulation induces a functional maturation of human monocyte-derived dendritic cells.

Fucoidan is a complex sulfated polysaccharide with a wide variety of biological activities for modulating immune cell functions. However, the effects of fucoidan on maturation process and activation of human monocyte-derived dendritic cells (DCs) remain to be elucidated. The present study demonstrated that the level of special marks and polarization phenotype of DCs was altered by fucoidan. Human monocytes were cultured with GM-CSF and IL-4 for 5 days followed by another 2 days in the presence of fucoidan or LPS. Then DCs were harvested on day 7 and were examined using functional assays. We demonstrated that fucoidan up-regulated the expression of HLA-DR and co-stimulatory molecules of DCs. However the endocytic activity was impaired markedly. Fucoidan induces their Th1-promoting tumor necrosis factor alpha (TNF-alpha) and interleukin-12 (IL-12) secretion, and enhances their allostimulatory capacity. In an allogeneic MLR assay, DCs treated with fucoidan were potent in the secretion of IL-12p70, TNF-alpha and IFN-gamma. Naive T cells stimulated by fucoidan-treated DCs differentiated towards a helper T cell type 1 (Th1) response depending on IL-12 secretion. These results suggest that fucoidan may induce immature DCs maturation and drive their differentiation towards a Th1-polarizing phenotype. Moreover, our data suggest that DCs appear to be a potential target for the immunomodulatory capacity of fucoidan and fucoidan may be used on DC-based vaccines for cancer immunotherapy.

Hypoxia skews dendritic cells to a T helper type 2-stimulating phenotype and promotes tumour cell migration by dendritic cell-derived osteopontin

It is well recognized that tissue microenvironments are involved in regulating the development and function of dendritic cells (DC). Oxygen supply, which varies in different tissues, has been accepted as an important microenvironmental factor in regulating the biological functions of several immune cells and as being involved in tumour progression and metastasis. However, little is known about the effect of hypoxia on the biological functions of DC and the effect of these hypoxia‐conditioned DC on tumour metastasis. In this study, we analysed the transcriptional profiles of human monocyte‐derived immature DC (imDC) and mature DC (mDC) cultured under normoxia and hypoxia by microarray, and found a body of potential targets regulating the functions of DC during hypoxia. In addition, the phagocytic ability of hypoxic imDC markedly decreased compared with that of normoxic imDC. Importantly, hypoxic DC poorly induced the proliferation of allogeneic T cells, but polarized allogeneic CD4+ naive T cells into a T helper type 2 (Th2) response. Moreover, hypoxic DC secreted large amounts of osteopontin, which were responsible for the enhanced migration of tumour cells. Therefore, our study provides new insights into the biological functions of DC under hypoxic conditions and one of mechanisms underlying tumour immune escape during hypoxia.

Synergy between the ectoenzymes CD39 and CD73 contributes to adenosinergic immunosuppression in human malignant gliomas

BACKGROUND The importance of ectoenzymes CD39 and CD73 in mediating adenosinergic immunosuppression has been recognized, but their roles in human malignant glioma-associated immunosuppression remain largely unknown. METHODS In this study, the ectoenzyme characteristics of malignant glioma cells and infiltrating CD4(+) T lymphocytes isolated from newly diagnosed malignant glioma patients were investigated. The ectoenzyme activities of both cell populations were determined by nucleotide hydrolysis assay. The immunosuppressive property of the CD39-CD73 synergic effect was evaluated via responder T-cell proliferation assay. RESULTS We observed that CD39(-)CD73(+) glioma cells and infiltrating CD4(+)CD39(high)CD73(low) T lymphocytes exhibited 2 distinct but complementary ectoenzyme phenotypes, which were further verified by enzyme activity assay. The nucleotide hydrolysis cascade was incomplete unless CD39 derived from T lymphocytes and CD73 collaborated synergistically. We demonstrated that increased suppression of responder CD4(+) T-cell proliferation suppression was induced by CD4(+)CD39(+) T cells in the presence of CD73(+) glioma cells, which could be alleviated by the CD39 inhibitor ARL67156, the CD73 inhibitor APCP, or the adenosine receptor A2aR antagonist SCH58261. In addition, survival analysis suggested that CD73 downregulation was a positive prognostic factor related to the extended disease-free survival of glioblastoma patients. CONCLUSIONS Our data indicate that glioma-derived CD73 contributes to local adenosine-mediated immunosuppression in synergy with CD39 from infiltrating CD4(+)CD39(+) T lymphocytes, which could become a potential therapeutic target for treatment of malignant glioma and other immunosuppressive diseases.

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.

Glioma cells escaped from cytotoxicity of temozolomide and vincristine by communicating with human astrocytes.

Resistance to chemotherapeutic drugs remains a great obstacle to successful treatment of gliomas. Understanding the mechanism of glioma chemoresistance is conducive to develop effective strategies to overcome resistance. Astrocytes are the major stromal cells in the brain and have been demonstrated to play a key role in the malignant phenotype of gliomas. However, little is known regarding its role in glioma chemoresistance. In our study, we established a co-culture system of human astrocytes and glioma in vitro to simulate tumor microenvironment. Our results showed that astrocytes significantly reduced glioma cell apoptosis induced by the chemotherapeutic drugs temozolomide and vincristine. This protective effect was dependent on direct contact between astrocytes and glioma cells through Cx43-GJC. Moreover, in human glioma specimens, we found astrocytes infiltrating around the tumor, with a reactive appearance, suggesting that these astrocytes would play the same chemoprotective effect on gliomas in vivo. Our results expand the understanding of the interaction between astrocytes and glioma cells and provide a possible explanation for unsatisfactory clinical outcomes of chemotherapeutic drugs. Cx43-GJC between astrocytes and glioma cells may be a potential target for overcoming chemoresistance in gliomas clinically.

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.

Membrane type 1-matrix metalloproteinase is involved in the migration of human monocyte-derived dendritic cells

Dendritic cells (DC) are highly mobile APC. The trafficking of both immature and mature DC is crucial for their functions, which depends mainly on chemotactic attraction and matrix metalloproteinases (MMP) activity. MMP that are in a transmembrane form belong to membrane type (MT)‐MMP, among which MT1‐MMP has been shown to possess strong proteolytic activity that is capable of degrading extracellular matrix molecules. Although it is well established that MMP are zinc‐dependent endopeptidases that collectively degrade most components of the extracellular matrix, relatively little is known about MT‐MMP‐mediated matrix degradation during DC migration. In this study, we showed that MT1‐MMP was expressed in human monocyte‐derived immature and mature DC by semi‐quantitative reverse transcription PCR and western blotting analyses. Moreover, immunofluorescence microscopic studies showed that MT1‐MMP was expressed on the membrane surface of DC. Blocking of MT1‐MMP activity greatly reduced the invasion capacity of immature DC in Matrigel, whereas mature DC mobility was not affected. Taken together, our results show a novel functional link between MT1‐MMP and DC motility and suggest that MT1‐MMP may play an important role in modulating the migration of immature DC.

Leptin-Promoted Human Extravillous Trophoblast Invasion Is MMP14 Dependent and Requires the Cross Talk Between Notch1 and PI3K/Akt Signaling

ABSTRACT The overexpression of leptin is a crucial feature for the maintenance of pregnancy. The effects of leptin on trophoblast invasion are important to its reproductive function, but the underlying mechanisms remain poorly understood. MMP14 is a member of matrix metalloproteinase (MMP) family that is closely involved in the invasion process. Here, we characterized the importance of MMP14 in the proinvasion effect of leptin on EVT cells and elucidated its molecular mechanisms. Transwell assay revealed that leptin promoted invasion of the immortalized EVT cell line HTR-8/SVneo in a dose- and time-related fashion. Further studies suggested that leptin enhanced HTR-8/SVneo cell invasion by up-regulating MMP14 expression and that knockdown of MMP14 by small interference RNA (siRNA) blocked the proinvasion effect of leptin. Notably, leptin promoted the expression of Notch1 receptor and activated its signaling in HTR-8/SVneo cells, and blocking this pathway by siRNA inhibited both leptin-enhanced MMP14 expression and invasiveness of HTR-8/SVneo cells. Such effects of Notch1 signaling were related with the activation of the PI3K/Akt pathway, which was significantly activated after leptin stimulation and was interfered by Notch1 signaling perturbation. Taken together, our observations suggest that leptin is an effective regulator of MMP14 expression, which consequently plays critical roles in invasion of EVT cells. The promoting effects of leptin on MMP14 require the cross talk between Notch1 and PI3K/Akt signaling pathways.