Deyu Chen

ANRIL inhibits p15INK4b through the TGFβ1 signaling pathway in human esophageal squamous cell carcinoma

The INK4b-ARF-INK4a gene cluster encodes three tumor suppressors: p15(INK4b), p14(ARF), and p16(INK4a). Antisense non-coding RNA in the INK4 locus (ANRIL) is transcribed in the opposite direction from this gene cluster. Recent studies suggest that ANRIL represses the expression of p15(INK4b), p14(ARF), and p16(INK4a); however, the underlying mechanism is unclear. In this study, the expressions of ANRIL in human esophageal squamous cell carcinoma (ESCC) tissues and matched adjacent non-tumor tissues were examined by quantitative real-time polymerase chain reaction. Compared with matched adjacent non-tumor tissues, the expression levels of ANRIL in ESCC tissues were significantly increased. Furthermore, inhibition of ANRIL was found to increase the expression of p15(INK4b) and transforming growth factor β1 (TGFβ1) and depletion of ANRIL in ESCC cell lines may inhibit cellular proliferation. Thus, our findings suggest a significant role of ANRIL in the occurrence and development of ESCC through TGFβ1 signaling pathways.

Polarization of ILC2s in Peripheral Blood Might Contribute to Immunosuppressive Microenvironment in Patients with Gastric Cancer

Newly identified nuocytes or group 2 innate lymphoid cells (ILC2s) play an important role in Th2 cell mediated immunity such as protective immune responses to helminth parasites, allergic asthma, and chronic rhinosinusitis. However, the contributions of ILC2s in the occurrence and development of cancer remain unknown. Our previous study found that there was a predominant Th2 phenotype in patients with gastric cancer. In this study, the ILC2s related genes or molecules in PBMC from patients with gastric cancer were measured, and the potential correlation between them was analyzed. The expression levels of RORα, GATA3, T1/ST2, IL-17RB, CRTH2, IL-33, IL-5, and IL-4 mRNA were significantly increased in patients, but no significant changes were found in ICOS, CD45, and IL-13 expression, and there was a positive correlation between RORα or IL-13 and other related factors, such as ICOS and CD45. The increased frequency of ILC2s was also found in PBMC of patients by flow cytometry. In addition, the mRNA of Arg1 and iNOS were also significantly increased in patients. These results suggested that there are polarized ILC2s in gastric cancer patients which might contribute to immunosuppressive microenvironment and closely related to the upregulation of MDSCs and M2 macrophages.

Correlation between circulating myeloid-derived suppressor cells and Th17 cells in esophageal cancer.

AIM To perform a comprehensive investigation into the potential correlation between circulating myeloid-derived suppressor cells (MDSCs) and Th17 cells in esophageal cancer (ECA). METHODS A total of 31 patients newly diagnosed with ECA and 26 healthy subjects were included in the current study. The frequencies of MDSCs and Th17 cells in peripheral blood were determined by flow cytometry. The mRNA expression of cytokines, arginase 1 (Arg1) and inducible NO synthase (iNOS) in peripheral blood mononuclear cells (PBMCs) and plasma Arg1 were assessed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS There was an increased prevalence of MDSCs in the peripheral blood from ECA patients (15.21% ± 2.25%) when compared with healthy control (HC) (1.10% ± 0.12%, P < 0.0001). The plasma levels of Arg1 in ECA patients were significantly higher than those in HC (28.28 ± 4.10 ng/mL vs 9.57 ± 1.51 ng/mL, P = 0.0003). iNOS mRNA levels in the peripheral blood of ECA patients also showed a threefold increase compared with HC (P = 0.0162). The frequencies of Th17 cells (CD4⁺IL-17A⁺) were significantly elevated in ECA patients versus HC (3.50% ± 0.33% vs 1.82% ± 0.19%, P = 0.0001). Increased mRNA expression of IL-17 and ROR-γt was also observed in ECA patients compared with HC (P = 0.0041 and P = 0.0004, respectively), while the mRNA expression of IL-6 and tumor necrosis factor-α (TNF-α) showed significant decreases (P = 0.0049 and P < 0.0001, respectively). No obvious correlations were found between the frequencies of MDSCs and Th17 cells in the peripheral blood from ECA patients(r = -0.1725, P = 0.3534). Arg1 mRNA levels were positively correlated with levels of IL-6 (r = 0.6404, P = 0.0031) and TNF-α (r = 0.7646, P = 0.0001). Similarly, iNOS mRNA levels were also positively correlated with levels of IL-6 (r = 0.6782, P = 0.0007) and TNF-α (r = 0.7633, P < 0.0001). CONCLUSION This study reveals the relationship between circulating MDSCs and Th17 cells, which may lead to new immunotherapy approaches for ECA based on the associated metabolites and cytokines.

Interleukin-23 promotes the epithelial-mesenchymal transition of oesophageal carcinoma cells via the Wnt/β-catenin pathway

As the eighth most common malignant tumour worldwide, oesophageal cancer (OC) is often diagnosed during the metastasis of its advanced stage. Interleukin (IL)-23 is an immunomodulatory cytokine that has recently been identified as a cancer-associated factor. However, the role of IL-23 in the evolution of OC remains unclear. In the present study, we found that IL-23 was significantly expressed in the tumours of OC patients suffering metastasis and demonstrated that IL-23 contributed to epithelial-mesenchymal transition (EMT) through the Wnt/β-catenin pathway, promoting the migration and invasion of OC cells. In conclusion, IL-23 plays a pivotal role in the development of OC via EMT.

Chemokine/chemokine receptor interactions contribute to the accumulation of Th17 cells in patients with esophageal squamous cell carcinoma.

Chemokine/chemokine receptor interactions play a critical role in lymphocyte infiltration of tumors. Recent studies suggest that Th17 cells accumulate within many types of tumors, although the mechanisms that control this are unclear. We studied the distribution and phenotypic features of Th17 cells chemokine receptors, as well as the mRNA levels of CCL2, CCL17, CCL20, and CCL22 in tumors of patients with esophageal squamous cell carcinoma. We found that Th17 cells accumulated in tumors, and high expressions of CCR4, CCR6 were detected in Th17 cells. Levels of the chemokines CCL17, CCL20, and CCL22 in tumors were significantly higher than in tumor-free tissues, and were positively correlated with the distribution of Th17 cells in tumors. Furthermore, an in vitro migration assay showed that CCL17, CCL20 and CCL22 had chemotactic effects on tumor-derived Th17 cells. In conclusion, the CCR4-CCL17/22 and CCR6-CCL20 axis might play an important role in Th17 cell infiltration of tumors.

Tumor-activated TCRγδ⁺ T cells from gastric cancer patients induce the antitumor immune response of TCRαβ⁺ T cells via their antigen-presenting cell-like effects.

Human γδ T cells display the principal characteristics of professional antigen-presenting cells (APCs), in addition to playing a vital role in immunity through cytokine secretion and their cytotoxic activity. However, it is not clear whether γδ T cells perform APC-like functions under pathological conditions. In this study, we showed that, in contrast to peripheral-derived γδ T cells directly isolated from PBMCs of gastric cancer patients, tumor-activated γδ T cells not only killed tumor cells efficiently but also strongly induced primary CD4+ and CD8+   αβ T cells proliferation and differentiation. More importantly, they abrogated the immunosuppression induced by CD4+CD25+ Treg cells and induced the cytotoxic function of CD8+   αβ T cells from patients with gastric cancer. In conclusion, tumor-activated γδ T cells can induce adaptive immune responses through their APC-like functions, and these cells may be a potentially useful tool in the development of tumor vaccines and immunotherapy.

Schistosoma japonicum HSP60-derived peptide SJMHE1 suppresses delayed-type hypersensitivity in a murine model

BackgroundParasite-derived molecules with immunomodulatory properties, which have been optimised during host-parasite co-evolution, exhibit potential applications as novel immunotherapeutics. We have previously demonstrated that Schistosoma japonicum HSP60-derived peptide SJMHE1 induces CD4+CD25+ regulatory T-cells (Tregs) and that adoptively transferred SJMHE1-induced CD4+CD25+ Tregs inhibit delayed-type hypersensitivity (DTH) in mice. However, multiple concerns regarding this method render this treatment unsuitable. To gain further insights into the potential effects of SJMHE1, we used ovalbumin (OVA)-induced DTH and evaluated the effect of SJMHE1 on DTH mice.MethodsBALB/c mice were sensitised with OVA alone or combined with SJMHE1 and then challenged with OVA to induce DTH. We first analysed the potential effects of SJMHE1 by measuring DTH responses, T-cell responses, cytokine secretion, and Treg proportions. We then evaluated the expression levels of IL-10 and TGF-β1 in CD4+CD25+ T-cells during DTH and Treg generation to identify the mechanism by which SJMHE1 suppresses DTH.ResultsSJMHE1 modulated the effector response against OVA-induced DTH and stimulated the production of the anti-inflammatory cytokines IL-10 and TGF-β1 in immunised mice through a mechanism involving CD4+CD25+ Tregs. SJMHE1-induced CD4+CD25+ Tregs expressed high levels of CTLA-4, IL-10, and TGF-β1, which substantially contributed to the suppressive activity during DTH. The administration of SJMHE1 to DTH in mice led to the expansion of CD4+CD25+ Tregs from CD4+CD25− T-cells in the periphery, which inhibited DTH responses.ConclusionsOur study proves that the parasite-driven peptide suppresses DTH in mice, which may confer a new option for inflammation treatment.

The Regulatory Effect of UL-16 Binding Protein-3 Expression on the Cytotoxicity of NK Cells in Cancer Patients

The activating immunoreceptor NKG2D (natural killer group 2, member D) and its ligands play important roles in the innate and adaptive immune responses. UL16-binding protein 3 (ULBP3), an NKG2D ligand, is overexpressed on certain epithelial tumor cells. In this study, we investigated the effect of ULBP3 expression on the cytotoxic activity of natural killer (NK) cells. ULBP3 were measured by flow cytometry analysis, immunohistochemistry, and time-resolved fluoroimmunoassay. The cytotoxicity of NK cells was determined with the lactate dehydrogenase release assay. We found that ULBP3 was overexpressed on tumor cell lines and tumor tissues. Serum from cancer patients, but not from healthy donors, contained elevated levels of soluble ULBP3 (sULBP3). Importantly, high expression of ULBP3 on the cell surface of tumor cells augmented NKG2D-mediated NK cell cytotoxicity. However, low levels of sULBP3 (<15 ng/ml) weakened the cytotoxicity of NK cells by decreasing NKG2D expression on NK cells. Further analysis showed that serum samples from most cancer patients (>70%) contained the low level of sULBP3. Our results demonstrate that tumor cells express surface and soluble ULBP3, which regulate NK cell activity. Thus, ULBP3 is a potential therapeutic target for improving the immune response against cancer.

A monoclonal antibody against human UL16-binding protein 3.

The UL16-binding proteins (ULBPs) are a novel family of human MHC class I-related, cell surface proteins that function as ligands for NKG2D. In this study, the gene encoding human ULBP3 was cloned into prokaryotic expression vector pQE30, resulting in a recombinant plasmid pQE30-ULBP3. The pQE30-ULBP3 was transformed into Escherichia coli M15 and induced the expression of recombinant protein ULBP3 (rec-ULBP3). The purified rec-ULBP3 as an antigen was used to immunize BALB/c mice. Through cell fusion, sub-cloning, and screening approach, three hybridoma cell clones expressing monoclonal antibodies (MAb) were acquired. The results from Western blot analysis, flow cytometry, and enzyme-linked immunosorbent assay showed that the hybridoma clones B2-F1-F1 and B4-C5-D11, and not G2-A4-A12, reacted with rec-ULBP3 and nature ULBP3 expressed on the cell surface of the tumor cells. In conclusion, the new MAb described here provides a valuable tool for further investigating ULBP3 function and clinical application.

Inhibition of cytokine response to TLR stimulation and alleviation of collagen-induced arthritis in mice by Schistosoma japonicum peptide SJMHE1

Helminth‐derived products have recently been shown to prevent the development of inflammatory diseases in mouse models. However, most identified immunomodulators from helminthes are mixtures or macromolecules with potentially immunogenic side effects. We previously identified an immunomodulatory peptide called SJMHE1 from the HSP60 protein of Schistosoma japonicum. In this study, we assessed the ability of SJMHE1 to affect murine splenocytes and human peripheral blood mononuclear cells (PBMCs) stimulated by toll‐like receptor (TLR) ligands in vitro and its treatment effect on mice with collagen‐induced arthritis (CIA). We show that SJMHE1 not only modulates the cytokine production of murine macrophage (MΦ) and dendritic cell but also affects cytokine production upon coculturing with allogeneic CD4+ T cell. SJMHE1 potently inhibits the cytokine response to TLR ligands lipopolysaccharide (LPS), CpG oligodeoxynucleotides (CpG) or resiquimod (R848) from mouse splenocytes, and human PBMCs stimulated by LPS. Furthermore, SJMHE1 suppressed clinical signs of CIA in mice and blocked joint erosion progression. This effect was mediated by downregulation of key cytokines involved in the pathogenesis of CIA, such as interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐6, IL‐17, and IL‐22 and up‐regulation of the inhibitory cytokine IL‐10, Tgf‐β1 mRNA, and CD4+CD25+Foxp3+ Tregs. This study provides new evidence that the peptide from S. japonicum, which is the ‘safe’ selective generation of small molecule peptide that has evolved during host–parasite interactions, is of great value in the search for novel anti‐inflammatory agents and therapeutic targets for autoimmune diseases.