Tumor-associated macrophages (TAMs) are enriched in gliomas and help create a tumor-immunosuppressive microenvironment. A distinct M2-skewed type of macrophages makes up the majority of glioma TAMs, and these cells exhibit pro-tumor functions. Gliomas contain large hypoxic areas, and the presence of a correlation between the density of M2-polarized TAMs and hypoxic areas suggests that hypoxia plays a supportive role during TAM recruitment and induction. Here, we investigated the effects of hypoxia on human macrophage recruitment and M2 polarization. We also investigated the influence of the HIF inhibitor acriflavine (ACF) on M2 TAM infiltration and tumor progression in vivo. We found that hypoxia increased periostin (POSTN) expression in glioma cells and promoted the recruitment of macrophages. Hypoxia-inducible POSTN expression was increased by TGF-α via the RTK/PI3K pathway, and this effect was blocked by treating hypoxic cells with ACF. We also demonstrated that both a hypoxic environment and hypoxia-treated glioma cell supernatants were capable of polarizing macrophages toward a M2 phenotype. ACF partially reversed the M2 polarization of macrophages by inhibiting the upregulation of M-CSFR in macrophages and TGF-β in glioma cells under hypoxic conditions. Administering ACF also ablated tumor progression in vivo. Our findings reveal a mechanism that underlies hypoxia-induced TAM enrichment and M2 polarization and suggest that pharmacologically inhibiting HIFs may reduce M2-polarized TAM infiltration and glioma progression.

Hypoxia induces protective autophagy in glioblastoma cells and new therapeutic avenues that target this process may improve the outcome for glioblastoma patients. Recent studies have suggested that the autophagic process is upregulated in glioblastomas in response to extensive hypoxia. Hypoxia also induces the upregulation of a specific set of proteins and microRNAs (miRNAs) in a variety of cell types. IL6 (interleukin 6), an inflammatory autocrine and paracrine cytokine that is overexpressed in glioblastoma, has been reported to be a biomarker for poor prognosis because of its tumor-promoting effects. Here, we describe a novel tumor-promoting mechanism of IL6, whereby hypoxia-induced IL6 acts as a potent initiator of autophagy in glioblastoma via the phosphorylated (p)-STAT3-MIR155-3p pathway. IL6 and p-STAT3 levels correlated with the abundance of autophagic cells and HIF1A levels in human glioma tissues and with the grade of human glioma, whereas inhibition of exogenous or endogenous IL6 repressed autophagy in glioblastoma cells in vitro. Knockdown of endogenous MIR155-3p inhibited IL6-induced autophagy, and enforced expression of MIR155-3p restored the anti-autophagic activity of IL6 inhibitors. We show that the hypoxia-IL6-p-STAT3-MIR155-3p-CREBRF-CREB3-ATG5 pathway plays a central role in malignant glioma progression, with blockade of the IL6 receptor by tocilizumab demonstrating a certain level of therapeutic efficacy in a xenograft model in vivo, especially in combination with temozolomide. Moreover, tocilizumab inhibits autophagy by promoting tumor apoptosis. Collectively, our findings provide new insight into the molecular mechanisms underlying hypoxia-induced glioma cell autophagy and point toward a possible efficacious adjuvant therapy for glioblastoma patients.

SummaryIt 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.Keywords: cancer; dendritic cells; hypoxia; T helper type 2Please cite this article in press as: Yang M. et al. Hypoxia skews dendritic cells to a T helper type 2-stimulating phenotype and promotes tumour cell migration by dendritic cell-derived osteopontin, Immunology (2009)

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 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-a by hypoxic mDCs and elevate the amount of Th1 cytokine IFN-c 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-1a 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.

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.

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.

CTLA-4 is a negative regulator of the proliferation and the effector function of T-cells. Therefore, it might be important to determine its expression on tumor cells and T-lymphocytes from cancer patients, to investigate its role in initiating and maintaining the neoplastic pathogenesis. CTLA-4 expression was detected in breast tissue by immunohistochemical staining and RT-PCR in 60 patients with breast cancer and 30 normal controls. The levels of CTLA-4 on T lymphocytes in 33 of the patients and 27 of the control group were determined by flow cytometry. Isolated peripheral blood mononuclear cells (PBMCs) were stimulated with phytohaemagglutinin (PHA). Stimulation index and IL-2 level in the cell culture supernatant were measured by MTT assay and ELISA method, respectively. Patients showed strong expression of CTLA-4 in the tumor cells of all specimens at both the protein and mRNA level, but only weakly positive or negative expression in normal breast tissue. Patients with higher mRNA level of CTLA-4 had obvious axillary lymph nodes metastases and higher clinical stage. Spontaneous expression of CD3 CTLA-4 on PBMCs of tumor patients was also significantly higher than that of the controls. Moreover, PBMCs derived from patients with high expression of CD3 CTLA-4 T-cells showed poor responsiveness to PHA stimulation and lower IL-2 production. Therefore, abnormal expression and dysregulation of CTLA-4 could partly explain the mechanism of evasion of anti-tumor immune responses in breast cancer patients and therefore highlight its importance in the development and progression of breast cancer.