Dysbiotic tumor microbiota associates with head and neck squamous cell carcinoma outcomes.

BACKGROUND: The need for an effective tool to predict prognosis of head and neck squamous cell carcinoma (HNSCC) patients is critical and unmet. Microbiota has recently been found involved in tumor progression and response to immunotherapy. However, the association of microbiota with the prognosis of HNSCC patients remains obscure. This study aims to investigate the association between tumor microbiota and outcomes of HNSCC patients. METHODS: A retrospective study including 129 primary tumors of HNSCC was conducted. Using 16S rRNA sequencing, the profile and the composition of tumor microbiota were measured and their associations with overall survival (OS) and disease free survival (DFS) were examined. RESULTS: We observed a reduced richness and enriched abundances of genera Schlegelella and Methyloversatilis in tumor microbiota of HNSCC patients with poor prognosis. However, a richer tumor microbiota with greater abundances of genera Bacillus, and Lactobacillus and Sphingomonas was characterized in the patients with favorable prognosis.The ratio of these differentially abundant taxa, microbial dysbiosis index (MDI), was significantly associated with OS (hazard ratio [HR], 4.67, 95% confidence interval [CI], 2.51 to 8.69,P < 0.001) and DFS (HR, 2.89; 95% CI, 1.74 to 4.80, P < 0.001) independently of age, tumor size, lymph node metastasis, differentiation and p16 status. The risk score of multivariate Cox regression exhibited an excellent performance for estimating three-year OS (AUC of 0.826). We also found a richer tumor microbiota was correlated with moderate peritumoral inflammatory infiltration. CONCLUSION: These results indicate that tumor microbiota associates with outcomes of HNSCC patients.

Whole transcriptome analysis of trophoblasts under hypoxia.

INTRODUCTION: A physiological hypoxia environment exists at maternal-fetal interface during early pregnancy. In addition, there is a pathological hypoxic microenvironment in patients with preeclampsia. Therefore, investigating the hypoxic adaptation and the effects of hypoxia on trophoblasts transcriptome is helpful to better understand the function and regulatory mechanism of trophoblasts at the maternal-fetal interface. METHODS: Trophoblast cell line HTR-8/SVneo was cultured under normoxia and hypoxia for 24 h, the full transcriptome was analyzed via RNA-Seq. GO and KEGG enrichment were performed on differentially expressed mRNA, adjacent genes of differentially expressed lncRNA, host genes of differentially expressed circRNA and target genes of differential expressed miRNA. RESULTS: The results showed that hypoxia differentially regulated 373 mRNAs, 334 lncRNAs, 71 circRNAs and 33 miRNAs. GO and KEGG enrichment showed that hypoxia negatively regulated TLR3 and PI3K-Akt signaling pathways. Consistently, we found hypoxia significantly inhibited TLR3 agonist-induced cytokines expression and the phosphorylation of Akt and mTOR. DISCUSSION: Our study obtained the full transcriptome data and potential regulatory network of trophoblasts under hypoxia, providing supportive data for revealing the function of trophoblasts.

CD68+ Macrophage Infiltration Associates With Poor Outcome of HPV Negative Oral Squamous Carcinoma Patients Receiving Radiation: Poly(I:C) Enhances Radiosensitivity of CAL-27 Cells but Promotes Macrophage Recruitment Through HMGB1.

Patients with human papillomavirus (HPV) negative oral squamous cell carcinoma (OSCC) generally have poor clinical outcomes and worse responses to radiotherapy. It is urgent to explore the underlining mechanisms of the distinct prognoses between HPV negative and HPV positive OSCC and to develop effective therapy strategy to increase the survival rate of HPV negative OSCC patients. We conducted a retrospective cohort of 99 resected OSCC patients to evaluate the prognosis of HPV negative and HPV positive OSCC patients receiving radiation or not. We further addressed the association of CD68+ macrophage infiltration with HPV status and the effects on survival of OSCC patients. We also used the TCGA-OSCC cohort for further verification. Based on the cohort study, we applied a synthetic dsRNA polymer, polyriboinosinic-polyribocytidylic acid (poly(I:C)), on CAL-27 (HPV negative OSCC cells). We co-cultured its condition medium with THP-1 derived macrophage and examined the cytokines and macrophage migration. We found that high CD68+ macrophage infiltration associated with poor overall survival in HPV negative OSCC patients receiving radiation. In vitro, poly(I:C) could induce apoptosis and enhance the radiosensitivity, but increase macrophage recruitment. Targeting HMGB1 could inhibit IL-6 induction and macrophage recruitment. Our findings indicated that CD68+ macrophage might play an important role in the outcomes of HPV negative OSCC patients receiving radiation. Our findings also suggested that radiation combined poly(I:C) might be a potential therapy strategy to increase the radiation response and prognosis of HPV negative OSCC. Notably, HMGB1 should be targeted to inhibit macrophage recruitment and enhance overall therapy effects.

The presence of elevated circulating trimethylamine N-oxide exaggerates postoperative cognitive dysfunction in aged rats.

Surgical trauma can cause brain oxidative stress and neuroinflammation, leading to postoperative cognitive dysfunction (POCD), especially in the elderly. Additionally, the pre-existing risk factors may enhance POCD. Gut microbiota-derived metabolite trimethylamine N-oxide (TMAO) has recently been shown to contribute to the pathogenesis of many diseases by increasing oxidative stress and inflammation in the peripheral tissues. Here we examined whether the presence of elevated circulating TMAO would influence surgery-induced cognitive decline. Aged rats were treated with vehicle or TMAO for 3 weeks. After two weeks of treatment, these rats underwent sham-operation or laparotomy. One week after surgery, rats underwent laparotomy exhibited hippocampal-dependent cognitive dysfunction as evidenced by reduced contextual freezing time, which was associated with elevated plasma proinflammatory cytokine levels, increased microglia-mediated neuroinflammation and reactive oxygen species (ROS) production in the hippocampus. Treatment with TMAO, which elevated plasma TMAO before and 1 week after surgery, further increased microglia-mediated neuroinflammation and ROS production in the hippocampus, resulting in exaggerated cognitive dysfunction in laparotomy group but not in sham-operation group. Moreover, TMAO treatment decreased expression of antioxidant enzyme methionine sulfoxide reductase (Msr) A in both groups. The results suggest that the presence of elevated circulating TMAO downregulates antioxidant enzyme MsrA in the hippocampus, which may increase the susceptibility to surgery-induced oxidative stress, contributing to exaggerations of neuroinflammation and cognitive decline in aged rats following surgery. Interventions to reduce circulating TMAO in the perioperative period may be a novel strategy to prevent the exaggeration of cognitive decline in elderly patients with high circulating TMAO.

Impaired Myocardial MIF/AMPK Activation Aggravates Myocardial Ischemia Reperfusion Injury in High-Fat Diet-Induced Obesity.

BACKGROUND: Obese patients are more sensitive to myocardial ischemia, which has been linked with high mortality rates. The following study investigates the effects of impaired macrophage Migration Inhibitory Factor (MIF)/AMP-Activated Protein Kinase (AMPK) activation on increased susceptibility to myocardial ischemia/reperfusion (I/R) in high-fat diet-induced obesity. METHODS: Male C57BL/6J mice were fed with a normal diet (10% kcal as fat, lean group) or a high-fat diet (60kcal as fat, obese group) for 12 consecutive weeks. To detect the MIF expression and AMPK activation in response to I/R in isolated hearts from lean and obese mice, myocardial samples were collected from left ventricular areas at different time points. To determine whether MIF supplementation is protective against I/R injury, recombined MIF (10 ng/mL) was applied before ischemia. Myocardial infarct size was estimated by triphenyltetrazolium staining. Western blot was used to detect myocardial MIF expression, AMPK activation and membrane glucose transporter 4 (Glut4) expression. RESULTS: The expression of MIF was remarkably higher in obese group compared to lean group. Ischemia increased myocardial MIF expression and phosphorylation of AMPK in lean mice, whereas it had no significant effect on obese mice. Furthermore, administration of recombinant MIF increased ischemic AMPK activation and membrane Glut4 expression in both lean and obese mice, while it reduced the infarct size in lean mice only. CONCLUSION: An impaired MIF/AMPK activation response and consequent reduced membrane Glut4 expression may play an important role in increasing myocardial susceptibility to I/R in obesity.

Preoperative Tim­3 expression on peripheral NK cells is correlated with pathologic TNM staging in colorectal cancer.

Previous research has indicated that T cell immunoglobulin and mucin domain 3 (Tim-3) serves an important regulatory role in lymphocytes and in several cancers. However, the association between Tim­3 expression on various lymphocyte subsets and human colorectal cancer (CRC) has not been elucidated. The present study aimed to characterize Tim­3 expression on peripheral lymphocytes, including cluster of differentiation CD3+CD56­ T cells, CD3­CD56+ natural killer (NK) cells and CD3+CD56+ natural killer T (NKT) cells, in patients with CRC. The frequency of T cells, NK cells and NKT cells expressing Tim­3 was assessed by multicolor flow cytometry of peripheral blood collected from 36 preoperative CRC patients and 38 healthy donors. The expression of Tim­3 on lymphocyte subsets from 53 postoperative blood samples of CRC patients was also analyzed. There were fewer circulating NK cells in patients with CRC compared with healthy controls (P=0.0027); NK cell expression of Tim­3 was also significantly decreased (P=0.0239). The frequency of circulating NK cells and Tim­3+ NK cells was negatively correlated with clinical cancer stage, compared with healthy controls, but not with other clinicopathological parameters or serum concentrations of CRC biomarkers. Furthermore, the expression of Tim­3 in NK cells was higher in CRC patients without metastasis. Notably, NK cell Tim­3 expression in CRC patients was significantly restored following surgical resection of the primary tumor. In conclusion, the present study indicates the presence of an altered frequency and expression of Tim­3 in peripheral NK cells in CRC patients. Preoperative Tim­3 expression on peripheral NK cells is correlated with differential staging in colorectal cancer, and may be useful as a serum biomarker.

Fucoidan inhibits CCL22 production through NF-κB pathway in M2 macrophages: a potential therapeutic strategy for cancer.

In tumor microenvironment, macrophages as a polarized M2 population promote tumor progression via releasing multiple cytokines and chemokines. A brown seaweed fucose-rich polysaccharide, fucoidan has antitumor activity and immune modulation through affecting tumor cells and lymphocytes. Here, we focused on the effect of fucoidan on macrophages especially M2 subtype. Our results demonstrated that fucoidan down-regulated partial cytokines and chemokines, especially a M2-type chemokine CCL22. Furthermore, fucoidan inhibited tumor cells migration and CD4+ T lymphocytes, especially Treg cells, recruitment induced by M2 macrophages conditioned medium through suppression of CCL22. Mechanismly, fucoidan inhibited CCL22 via suppressing p65-NF-κB phosphorylation and nuclear translocation. In addition, p38-MAPK and PI3K-AKT also affected the expression of CCL22 through differential modulation of NF-κB transcriptional activity. Taken together, we reveal an interesting result that fucoidan can inhibit tumor cell migration and lymphocytes recruitment by suppressing CCL22 in M2 macrophages via NF-κB-dependent transcription, which may be a novel and promising mechanism for tumor 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. Spearman's 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). Spearman's rank correlation indicated that there was a linear correlation between galectin-9 expression and CD56+ NK cell infiltration (R(2) = 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.

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-ß1 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.

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-ß. 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.

Silencing Kif2a induces apoptosis in squamous cell carcinoma of the oral tongue through inhibition of the PI3K/Akt signaling pathway.

Previous studies have demonstrated that the overexpression of Kif2a is involved in the progression, invasion and metastasis of squamous cell carcinoma of the oral tongue (SCCOT). Few studies have reported the correlation between Kif2a and apoptosis of tumor cells and which signaling pathways Kif2a is involved in remains unclear. The phosphatidylinositol­3­kinase (PI3K)/protein kinase B (Akt) pathway is frequently activated in many types of human cancer. The aim of the present study was to investigate the effects of downregulation of Kif2 expression on the P13K/Akt pathway in Tca8113 cells to determine whether silencing of Kif2 inhibits the P13K/Akt pathway, resulting in cell apoptosis. siRNA vector was constructed, western blot analysis was used to determine RNA interference and flow cytometry was used to determine promotion of Tca8113 cell apoptosis. The results revealed that silencing Kif2a induces apoptosis and decreases the mRNA and protein level of PI3K, Akt and B­cell lymphoma 2 (Bcl­2) in Tca8113 cells. The PI3K-specific agonist insulin­like growth factor 1 (IGF­1) eliminated the upregulation of apoptosis of Tca8113­Kif2a cells by phosphorylation of Akt. The results suggest that silencing Kif2a induces tumor cell apoptosis, at least partially, through the PI3K/Akt signaling pathway.

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.

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.

Involvement of the mitochondrial pathway and Bim/Bcl-2 balance in dihydroartemisinin-induced apoptosis in human breast cancer in vitro.

Dihydroartemisinin (DHA), a semi-synthetic derivative and active metabolite of artemisinin, has been shown to have profound anticancer potential in addition to its strong anti-malarial activity. The purpose of the present study was to thoroughly investigate the anti-neoplastic effects induced by DHA and to provide a molecular basis for the use of DHA in the treatment of breast cancer. Our results demonstrated that DHA could significantly inhibit the cell proliferation of breast cancer in a dose- and time-dependent manner that was associated with induced apoptosis and G0/G1 cell cycle arrest, and the half maximal inhibitory concentrations (IC50) of DHA treatment were 60.03, 33.86 and 17.18 µM for 24, 48 and 72 h, respectively. Moreover, the DHA treatment dramatically increased the protein expression of caspase-8, cleaved caspase-9, activated Bid and induced the release of cytochrome c from mitochondria into the cytosol. In addition, the apoptotic action of DHA was associated with the increased expression of the pro-apoptotic gene Bim and a decreased expression of the anti-apoptotic gene Bcl-2. Therefore, the mitochondrial pathway is involved in the apoptosis of breast cancer cells induced by DHA and the imbalance of the Bim/Bcl-2 interaction may promote the beneficial effect against breast cancer cells. Overall, our study provides the scientific rationale for the clinical usage of DHA for breast cancer.

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.

Single-molecule-counting protein microarray assay with nanoliter samples and its application in the dynamic protein expression of living cells.

A novel ultra-sensitive single-molecule-counting microarray assay (SMCMA) with a 1.8-nL sample volume for quantification of proteins was provided using total internal reflection fluorescence microscopy coupled with quantum dot (QD)-labeling. In the SMCMA, the microarray consisting of ∼ 300 µm diameter microspots with the spot-to-spot pitch distance of 500 µm was fabricated by spotting 1.8 nL of solutions containing the target protein onto the substrate which was modified with primary antibody of the protein and blocked with ethanolamine and BSA using a pin-tool type microarraying robot. Then, biotinylated secondary antibody of the protein was bound to the protein to form sandwich immunocomplexes. After labeling with streptavidin-coated QDs, the whole image of the microarray was acquired using a homemade single-molecule microarray reader. The target protein was quantified based on the number of bright dots from the QDs corresponding to single target protein molecules on the microarray. Using the SMCMA, an amount as low as 1.5 × 10(-21) mole (904 molecules) for proteins could be detected. The SMCMA was applied to measure dynamic expression of osteopontin in living cells.

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-alpha by hypoxic mDCs and elevate the amount of Th1 cytokine IFN-gamma 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-1 alpha 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.