Meixiang Yang

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.

Hypoxia inhibits the migratory capacity of human monocyte-derived dendritic cells

Hypoxia, a prominent characteristic of inflammatory tissue lesions and solid tumour microenvironments, is a crucial stimulus capable of modulating the expression of specific genes involved in leucocyte recruitment. Although studies have shown that hypoxia can affect leucocyte migration by influencing the expression of migration‐related genes, such as matrix metalloproteinases (MMP) and their endogenous tissue inhibitors of matrix metalloproteinases (TIMP), it remains unclear whether hypoxia can affect the migration of dendritic cells (DC). In this study, we showed that human monocyte‐derived DC under hypoxic conditions in a transwell system have significantly reduced migratory capacity compared to normoxic controls. A moderate phenotypic change of hypoxic DC was observed. In hypoxic DC, we detected a twofold increase in TIMP‐1 transcript levels, and downregulated expression of MMP‐9 and membrane type 1‐MMP genes by threefold and 17‐fold, respectively. Our results suggest that hypoxia may inhibit DC migratory activity by regulating the balance between MMP and TIMP gene expression.

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.

Hypoxia induces T-cell apoptosis by inhibiting chemokine C receptor 7 expression: the role of adenosine receptor A(2).

Hypoxia is a major characteristic of the tumor microenvironment, and its effects on immune cells are proposed to be important factors for the process of tumor immune escape. It has been reported that hypoxia affects the function of dendritic cells and the antitumor function of T cells. Here we discuss the effects of hypoxia on T-cell survival. Our results showed that hypoxia induced apoptosis of T cells. Adenosine and adenosine receptors (AR) are important to the hypoxia-related signaling pathway. Using AR agonists and antagonists, we demonstrated that hypoxia-induced apoptosis of T cells was mediated by A2a and A2b receptors. Furthermore, we are the first, to our knowledge, to report that hypoxia significantly inhibited the expression of chemokine C receptor 7 (CCR7) of T cells via the A2R signal pathway, perhaps representing a mechanism of hypoxia-induced apoptosis of T cells. Collectively, our research demonstrated that hypoxia induces T-cell apoptosis by the A2R signaling pathway partly by suppressing CCR7. Blocking the A2R signaling pathway and/or activation of CCR7 can increase the anti-apoptosis function of T cells and may become a new strategy to improve antitumor potential.

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.

Osteopontin splice variants expressed by breast tumors regulate monocyte activation via MCP-1 and TGF-β1

Osteopontin (OPN), a multifunctional glycoprotein, has three transcripts that have distinct roles in tumors in vitro. Whether OPN transcripts have different functions in tumor processes in vivo is unclear. It has been reported that immune cell-derived OPN can promote tumor formation. We propose a hypothesis that tumor-derived OPN may facilitate tumor immune escape by affecting immune cell differentiation and function. In this study, we constructed lentiviral expression vectors of OPN transcripts and transfected them into the MCF-7 cell line. MCF-7 cells transfected with OPN transcripts were injected into the armpit of nude mice, and tumor growth was monitored. The results showed that all OPN transcripts promoted local tumor formation, but that there was no significant difference among transcripts. We also investigated the effect of the OPN expressed by tumor cells on monocyte differentiation by coculturing monocytes with tumor supernatant. We found OPN-c upregulated CD163 levels compared with OPN-a and OPN-b; however, none of the transcripts affected HLA-DR and CD206 levels. All OPN transcripts significantly inhibited TNF-α and enhanced IL-10 production by monocytes. Furthermore, we found that the overexpression of OPN transcripts significantly upregulated TGF-β1 and MCP-1 production by tumor cells. Using neutralizing antibody and recombinant cytokines, we found that OPN overexpressed by tumor cells regulates the production of TNF-α and IL-10 by monocytes partly via MCP-1 and TGF-β1, respectively. Collectively, our results show that OPN transcripts have no distinct role in breast cancer formation in vivo. We also demonstrate that OPN regulates the alternative activation of monocytes via TGF-β1 and MCP-1, which may represent an additional mechanism for tumor immune escape.

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.