Helicobacter pylori-Induced Angiopoietin-Like 4 Promotes Gastric Bacterial Colonization and Gastritis.

Helicobacter pylori infection is characterized as progressive processes of bacterial persistence and chronic gastritis with features of infiltration of mononuclear cells more than granulocytes in gastric mucosa. Angiopoietin-like 4 (ANGPTL4) is considered a double-edged sword in inflammation-associated diseases, but its function and clinical relevance in H. pylori-associated pathology are unknown. Here, we demonstrate both pro-colonization and pro-inflammation roles of ANGPTL4 in H. pylori infection. Increased ANGPTL4 in the infected gastric mucosa was produced from gastric epithelial cells (GECs) synergistically induced by H. pylori and IL-17A in a cagA-dependent manner. Human gastric ANGPTL4 correlated with H. pylori colonization and the severity of gastritis, and mouse ANGPTL4 from non-bone marrow-derived cells promoted bacteria colonization and inflammation. Importantly, H. pylori colonization and inflammation were attenuated in Il17a -/-, Angptl4 -/-, and Il17a -/- Angptl4 -/- mice. Mechanistically, ANGPTL4 bound to integrin αV (ITGAV) on GECs to suppress CXCL1 production by inhibiting ERK, leading to decreased gastric influx of neutrophils, thereby promoting H. pylori colonization; ANGPTL4 also bound to ITGAV on monocytes to promote CCL5 production by activating PI3K-AKT-NF-κB, resulting in increased gastric influx of regulatory CD4+ T cells (Tregs) via CCL5-CCR4-dependent migration. In turn, ANGPTL4 induced Treg proliferation by binding to ITGAV to activate PI3K-AKT-NF-κB, promoting H. pylori-associated gastritis. Overall, we propose a model in which ANGPTL4 collectively ensures H. pylori persistence and promotes gastritis. Efforts to inhibit ANGPTL4-associated pathway may prove valuable strategies in treating H. pylori infection.

Helicobacter pylori-induced REDD1 modulates Th17 cell responses that contribute to gastritis.

OBJECTIVE: Regulated in development and DNA damage responses-1 (REDD1) is a conserved and ubiquitous protein, which is induced in response to multiple stimuli. However, the regulation, function and clinical relevance of REDD1 in Helicobacter pylori-associated gastritis are presently unknown. APPROACH: Immunohistochemistry, real-time PCR and Western blot analyses were performed to examine the levels of REDD1 in gastric samples from H. pylori-infected patients and mice. Gastric tissues from Redd1-/- and wildtype (WT, control) mice were examined for inflammation. Gastric epithelial cells (GECs), monocytes and T cells were isolated, stimulated and/or cultured for REDD1 regulation and functional assays. RESULTS: REDD1 was increased in gastric mucosa of H. pylori-infected patients and mice. H. pylori induced GECs to express REDD1 via the phosphorylated cytotoxin associated gene A (cagA) that activated MAPKp38 pathway to mediate NF-κB directly binding to REDD1 promoter. Human gastric REDD1 increased with the severity of gastritis, and mouse REDD1 from non-marrow chimera-derived cells promoted gastric inflammation that was characterized by the influx of MHCII+ monocytes. Importantly, gastric inflammation, MHCII+ monocyte infiltration, IL-23 and IL-17A were attenuated in Redd1-/- mice. Mechanistically, REDD1 in GECs regulated CXCL1 production, which attracted MHCII+ monocytes migration by CXCL1-CXCR2 axis. Then H. pylori induced MHCII+ monocytes to secrete IL-23, which favored IL-17A-producing CD4+ cell (Th17 cell) polarization, thereby contributing to the development of H. pylori-associated gastritis. CONCLUSIONS: The present study identifies a novel regulatory network involving REDD1, which collectively exert a pro-inflammatory effect within gastric microenvironment. Efforts to inhibit this REDD1-dependent pathway may prove valuable strategies in treating of H. pylori-associated gastritis.

L-Plastin Promotes Gastric Cancer Growth and Metastasis in a Helicobacter pylori cagA-ERK-SP1-Dependent Manner.

Actin cytoskeleton dynamic rearrangement is required for tumor cell metastasis and is a key characteristic of Helicobacter pylori (H. pylori)-infected host cells. Actin cytoskeleton modulation is coordinated by multiple actin-binding proteins (ABP). Through Kyoto encyclopedia of gene and genomes database, GEPIA website, and real-time PCR data, we found that H. pylori infection significantly induced L-plastin, a key ABP, in gastric cancer cells. We further explored the regulation and function of L-plastin in H. pylori-associated gastric cancer and found that, mechanistically, H. pylori infection induced gastric cancer cells to express L-plastin via cagA-activated ERK signaling pathway to mediate SP1 binding to L-plastin promoter. Moreover, this increased L-plastin promoted gastric cancer cell proliferation and migration in vitro and facilitated the growth and metastasis of gastric cancer in vivo. Finally, we detected the expression pattern of L-plastin in gastric cancer tissues, and found that L-plastin was increased in gastric cancer tissues and that this increase of L-plastin positively correlated with cagA + H. pylori infection status. Overall, our results elucidate a novel mechanism of L-plastin expression induced by H. pylori, and a new function of L-plastin-facilitated growth and metastasis of gastric cancer, and thereby implicating L-plastin as a potential therapeutic target against gastric cancer. IMPLICATIONS: Our results elucidate a novel mechanism of L-plastin expression induced by H. pylori in gastric cancer, and a new function of L-plastin-facilitated gastric cancer growth and metastasis, implicating L-plastin as a potential therapeutic target against gastric cancer.

Helicobacter pylori-Induced Rev-erbα Fosters Gastric Bacteria Colonization by Impairing Host Innate and Adaptive Defense.

BACKGROUND & AIMS: Rev-erbα represents a powerful transcriptional repressor involved in immunity. However, the regulation, function, and clinical relevance of Rev-erbα in Helicobacter pylori infection are presently unknown. METHODS: Rev-erbα was examined in gastric samples from H pylori-infected patients and mice. Gastric epithelial cells (GECs) were isolated and infected with H pylori for Rev-erbα regulation assays. Gastric tissues from Rev-erbα-/- and wild-type (littermate control) mice or these mice adoptively transferred with CD4+ T cells from IFN-γ-/- and wild-type mice, bone marrow chimera mice and mice with in vivo pharmacological activation or inhibition of Rev-erbα were examined for bacteria colonization. GECs, CD45+CD11c-Ly6G-CD11b+CD68- myeloid cells and CD4+ T cells were isolated, stimulated and/or cultured for Rev-erbα function assays. RESULTS: Rev-erbα was increased in gastric mucosa of H pylori-infected patients and mice. H pylori induced GECs to express Rev-erbα via the phosphorylated cagA that activated ERK signaling pathway to mediate NF-κB directly binding to Rev-erbα promoter, which resulted in increased bacteria colonization within gastric mucosa. Mechanistically, Rev-erbα in GECs not only directly suppressed Reg3b and ß-defensin-1 expression, which resulted in impaired bactericidal effects against H pylori of these antibacterial proteins in vitro and in vivo; but also directly inhibited chemokine CCL21 expression, which led to decreased gastric influx of CD45+CD11c-Ly6G-CD11b+CD68- myeloid cells by CCL21-CCR7-dependent migration and, as a direct consequence, reduced bacterial clearing capacity of H pylori-specific Th1 cell response. CONCLUSIONS: Overall, this study identifies a model involving Rev-erbα, which collectively ensures gastric bacterial persistence by suppressing host gene expression required for local innate and adaptive defense against H pylori.

Arrestin domain containing 3 promotes Helicobacter pylori-associated gastritis by regulating protease-activated receptor 1.

Arrestin domain containing 3 (ARRDC3) represents a newly discovered α-arrestin involved in obesity, inflammation, and cancer. Here, we demonstrate a proinflammation role of ARRDC3 in Helicobacter pylori-associated gastritis. Increased ARRDC3 was detected in gastric mucosa of patients and mice infected with H. pylori. ARRDC3 in gastric epithelial cells (GECs) was induced by H. pylori, regulated by ERK and PI3K-AKT pathways in a cagA-dependent manner. Human gastric ARRDC3 correlated with the severity of gastritis, and mouse ARRDC3 from non-BM-derived cells promoted gastric inflammation. This inflammation was characterized by the CXCR2-dependent influx of CD45+CD11b+Ly6C-Ly6G+ neutrophils, whose migration was induced via the ARRDC3-dependent production of CXCL2 by GECs. Importantly, gastric inflammation was attenuated in Arrdc3-/- mice but increased in protease-activated receptor 1-/- (Par1-/-) mice. Mechanistically, ARRDC3 in GECs directly interacted with PAR1 and negatively regulated PAR1 via ARRDC3-mediated lysosomal degradation, which abrogated the suppression of CXCL2 production and following neutrophil chemotaxis by PAR1, thereby contributing to the development of H. pylori-associated gastritis. This study identifies a regulatory network involving H. pylori, GECs, ARRDC3, PAR1, and neutrophils, which collectively exert a proinflammatory effect within the gastric microenvironment. Efforts to inhibit this ARRDC3-dependent pathway may provide valuable strategies in treating of H. pylori-associated gastritis.

Helicobacter pylori-induced adrenomedullin modulates IFN-γ-producing T-cell responses and contributes to gastritis.

Adrenomedullin (ADM) is a multifunctional peptide that is expressed by many surface epithelial cells, but its relevance to Helicobacter pylori (H. pylori)-induced gastritis is unknown. Here, we found that gastric ADM expression was elevated in gastric mucosa of H. pylori-infected patients and mice. In H. pylori-infected human gastric mucosa, ADM expression was positively correlated with the degree of gastritis; accordingly, blockade of ADM resulted in decreased inflammation within the gastric mucosa of H. pylori-infected mice. During H. pylori infection, ADM production was promoted via PI3K-AKT signaling pathway activation by gastric epithelial cells in a cagA-dependent manner, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterized by the increased IFN-γ-producing T cells, whose differentiation was induced via the phosphorylation of AKT and STAT3 by ADM derived from gastric epithelial cells. ADM also induced macrophages to produce IL-12, which promoted the IFN-γ-producing T-cell responses, thereby contributing to the development of H. pylori-associated gastritis. Accordingly, blockade of IFN-γ or knockout of IFN-γ decreased inflammation within the gastric mucosa of H. pylori-infected mice. This study identifies a novel regulatory network involving H. pylori, gastric epithelial cells, ADM, macrophages, T cells, and IFN-γ, which collectively exert a pro-inflammatory effect within the gastric microenvironment.

Upexpression of BHLHE40 in gastric epithelial cells increases CXCL12 production through interaction with p-STAT3 in Helicobacter pylori-associated gastritis.

BHLHE40, a member of the basic helix-loop-helix transcription factor family, has been reported to play an important role in inflammatory diseases. However, the regulation and function of BHLHE40 in Helicobacter pylori (H pylori)-associated gastritis is unknown. We observed that gastric BHLHE40 was significantly elevated in patients and mice with H pylori infection. Then, we demonstrate that H pylori-infected GECs express BHLHE40 via cagA-ERK pathway. BHLHE40 translocates to cell nucleus, and then binds to cagA protein-activated p-STAT3 (Tyr705). The complex increases chemotactic factor CXCL12 expression (production). Release of CXCL12 from GECs fosters CD4+ T cell infiltration in the gastric mucosa. Our results identify the cagA-BHLHE40-CXCL12 axis that contributes to inflammatory response in gastric mucosa during H pylori infection.

Helicobacter pylori-induced matrix metallopeptidase-10 promotes gastric bacterial colonization and gastritis.

The interaction between gastric epithelium and immune response plays key roles in H. pylori-associated pathology. We demonstrated a procolonization and proinflammation role of MMP-10 in H. pylori infection. MMP-10 is elevated in gastric mucosa and is produced by gastric epithelial cells synergistically induced by H. pylori and IL-22 via the ERK pathway. Human gastric MMP-10 was correlated with H. pylori colonization and the severity of gastritis, and mouse MMP-10 from non-BM-derived cells promoted bacteria colonization and inflammation. H. pylori colonization and inflammation were attenuated in IL-22-/-, MMP-10-/-, and IL-22-/-MMP-10-/- mice. MMP-10-associated inflammation is characterized by the influx of CD8+ T cells, whose migration is induced via MMP-10-CXCL16 axis by gastric epithelial cells. Under the influence of MMP-10, Reg3a, E-cadherin, and zonula occludens-1 proteins decrease, resulting in impaired host defense and increased H. pylori colonization. Our results suggest that MMP-10 facilitates H. pylori persistence and promotes gastritis.

Decreased IL-17RB expression impairs CD11b+CD11c- myeloid cell accumulation in gastric mucosa and host defense during the early-phase of Helicobacter pylori infection.

Interleukin-17 receptor B (IL-17RB), a member of the IL-17 receptor family activated by IL-17B/IL-17E, has been shown to be involved in inflammatory diseases. However, the regulation and function of IL-17RB in Helicobacter pylori (H. pylori) infection, especially in the early-phase is still unknown. Here, we found that gastric IL-17RB mRNA and protein were decreased in gastric mucosa of both patients and mice infected with H. pylori. In vitro experiments show that IL-17RB expression was down regulated via PI3K/AKT pathway on gastric epithelial cells (GECs) stimulated with H. pylori in a cagA-involved manner, while in vivo studies showed that the effect was partially dependent on cagA expression. IL-17E was also decreased during the early-phase of H. pylori infection, and provision of exogenous IL-17E resulted in increased CD11b+CD11c- myeloid cells accumulation and decreased bacteria colonization within the gastric mucosa. In the early-phase of H. pylori infection, IL-17E-IL-17RB promoted gastric epithelial cell-derived CXCL1/2/5/6 to attract CD11b+CD11c- myeloid cells, and also contributed to host defense by promoting the production of antibacterial protein Reg3a. This study defines a negative regulatory network involving IL-17E, GECs, IL-17RB, CD11b+CD11c- myeloid cells, and Reg3a in the early-phase of H. pylori infection, which results in an impaired host defense within the gastric microenvironment, suggesting IL-17RB as a potential early intervening target in H. pylori infection.

Abrogation of cathepsin C by Helicobacter pylori impairs neutrophil activation to promote gastric infection.

Cathepsin C (CtsC) functions as a central coordinator for activation of many serine proteases in immune cells. However, CtsC expression in gastric epithelial cells and its role in Helicobacter pylori infection remain unclear. Real-time PCR, Western blot, and immunohistochemistry analyses identified that CtsC was decreased in gastric mucosa of H. pylori-infected patients and mice. Isolated gastric epithelial cells and cell lines were stimulated with H. pylori and/or TGF-ß1 showed that down-regulation of CtsC in gastric epithelial cells largely depended on H. pylori cagA via Src/ERK and Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathways, and the effect could be synergistically augmented by TGF-ß1 in an autocrine manner. In human gastric mucosa, CtsC expression was negatively correlated with bacteria colonization; accordingly, provision of exogenous active CtsC overwhelmed H. pylori persistence in gastric mucosa of mice. In the presence of active CtsC, isolated human neutrophils activated via NF-κB pathway with augmented bactericidal capacity in vitro. We also found that neutrophils activated and cleared bacteria in active CtsC-injected mice and that there was no bactericidal capacity in mice that were simultaneously neutrophil-depleted by Ly6G antibody. Our findings identified a mechanism that H. pylori abrogate CtsC to impair neutrophil activation and to ensure persistence in gastric mucosa. Efforts to enable and boost this neutrophil activation pathway by active CtsC may therefore become valuable strategies in treating H. pylori infection.-Liu, Y. G., Teng, Y. S., Cheng, P., Kong, H., Lv, Y. P., Mao, F. Y., Wu, X. L., Hao, C. J., Chen, W., Yang, S. M., Zhang, J. Y., Peng, L. S., Wang, T. T., Han, B., Ma, Q., Zou, Q. M., Zhuang, Y. Abrogation of cathepsin C by Helicobacter pylori impairs neutrophil activation to promote gastric infection.

Degranulation of mast cells induced by gastric cancer-derived adrenomedullin prompts gastric cancer progression.

Mast cells are prominent components of solid tumors and exhibit distinct phenotypes in different tumor microenvironments. However, their precise mechanism of communication in gastric cancer remains largely unclear. Here, we found that patients with GC showed a significantly higher mast cell infiltration in tumors. Mast cell levels increased with tumor progression and independently predicted reduced overall survival. Tumor-derived adrenomedullin (ADM) induced mast cell degranulation via PI3K-AKT signaling pathway, which effectively promoted the proliferation and inhibited the apoptosis of GC cells in vitro and contributed to the growth and progression of GC tumors in vivo, and the effect could be reversed by blocking interleukin (IL)-17A production from these mast cells. Our results illuminate a novel protumorigenic role and associated mechanism of mast cells in GC, and also provide functional evidence for these mast cells to prevent, and to treat this immunopathogenesis feature of GC.

Altered NKp30, NKp46, NKG2D, and DNAM-1 Expression on Circulating NK Cells Is Associated with Tumor Progression in Human Gastric Cancer.

Natural killer (NK) cell activity is influenced by a complex integration of signaling pathways activated downstream of both activating and inhibitory surface receptors. The tumor microenvironment can suppress NK cell activity, and there is a great clinical interest in understanding whether modulating tumor-mediated NK cell suppression and/or boosting preexisting NK cell numbers in cancer patients is therapeutically viable. To this light, we characterized the surface receptor phenotypes of peripheral blood NK cells and examined their clinical relevance to human gastric cancer (GC). We found that the proportion of peripheral blood NK cells which expressed the activating receptors NKp30, NKp46, NKG2D, and DNAM-1 was significantly decreased in GC patients compared to healthy donors, and that this decrease was positively associated with tumor progression. At the same time, plasma TGF-ß1 concentrations were significantly increased in GC patients and negatively correlated with the proportion of NKp30, NKp46, NKG2D, and DNAM-1 expressing NK cells. Furthermore, TGF-ß1 significantly downregulated the expression of NKp30, NKp46, NKG2D, and DNAM-1 on NK cells in vitro, and the addition of galunisertib, an inhibitor of the TGF-ß receptor subunit I, reversed this downregulation. Altogether, our data suggest that the decreased expression of activating receptors NKp30, NKp46, NKG2D, and DNAM-1 on peripheral blood NK cells is positively associated with GC progression, and that TGF-ß1-mediated NK cell suppression may be a therapeutically targetable characteristic of GC.

CD45+CD33lowCD11bdim myeloid-derived suppressor cells suppress CD8+ T cell activity via the IL-6/IL-8-arginase I axis in human gastric cancer.

Myeloid-derived suppressor cells (MDSCs) are a prominent component of the pro-tumoral response. The phenotype of and mechanisms used by MDSCs is heterogeneous and requires more precise characterization in gastric cancer (GC) patients. Here, we have identified a novel subset of CD45+CD33lowCD11bdim MDSCs in the peripheral blood of GC patients compared to healthy individuals. CD45+CD33lowCD11bdim MDSCs morphologically resembled neutrophils and expressed high levels of the neutrophil marker CD66b. Circulating CD45+CD33lowCD11bdim MDSCs effectively suppressed CD8+ T cells activity through the inhibition of CD8+ T cell proliferation and interferon-γ (IFN-γ) and granzyme B (GrB) production. The proportion of CD45+CD33lowCD11bdim MDSCs also negatively correlated with the proportion of IFN-γ+CD8+ T cell in the peripheral blood of GC patients. GC patient serum-derived IL-6 and IL-8 activated and induced CD45+CD33lowCD11bdim MDSCs to express arginase I via the PI3K-AKT signaling pathway. This pathway contributed to CD8+ T cell suppression as it was partially rescued by the blockade of the IL-6/IL-8-arginase I axis. Peripheral blood CD45+CD33lowCD11bdim MDSCs, as well as IL-6, IL-8, and arginase I serum levels, positively correlated with GC progression and negatively correlated with overall patient survival. Altogether, our results highlight that a subset of neutrophilic CD45+CD33lowCD11bdim MDSCs is functionally immunosuppressive and activated via the IL-6/IL-8-arginase I axis in GC patients.

Modulation of CD8+ memory stem T cell activity and glycogen synthase kinase 3ß inhibition enhances anti-tumoral immunity in gastric cancer.

The potential contributions of CD8+ memory stem T cells to anti-tumor immunity and immunotherapy responses in gastric cancer has not been demonstrated. We found that CD8+ memory stem T cell frequencies were increased in the peripheral blood of gastric cancer patients compared to healthy donors and declined in frequency with disease progression. Despite minimal in vitro cytotoxic activity, the adoptive transfer of CD8+ memory stem T cells into Rag1-/- tumor bearing mice enhanced tumor regression compared to CD8+ central or effector memory T cell counterparts. This effect was associated with an increase in splenic, draining lymph node and tumor infiltrating CD8+ T cell numbers and the development of an altered CD8+ T cell phenotype not seen during homeostasis. GSK-3ß inhibition is known to promote memory stem T cell accumulation by arresting effector T cell differentiation in vivo. Surprisingly however, GSK-3ß inhibition conversely increased the cytotoxic capacity of CD8+ memory stem T cells in vitro, and this was associated with the induction of effector T cell-associated effector proteins including FasL. Finally, FasL neutralization following GSK-3ß inhibition directly attenuated the anti-tumoral capacity of CD8+ memory stem T cells both in vitro and in vivo. Altogether, our findings identify the therapeutic potential of modulating CD8+ memory stem T cells for improved anti-tumoral responses against gastric cancer.

Helicobacter pylori-induced IL-33 modulates mast cell responses, benefits bacterial growth, and contributes to gastritis.

Interleukin (IL)-induced inflammatory responses are critical for the pathogenesis of Helicobacter pylori (H. pylori)-induced gastritis. IL-33 represents a recently discovered proinflammatory cytokine involved in inflammatory diseases, but its relevance to H. pylori-induced gastritis is unknown. Here, we found that gastric IL-33 mRNA and protein expression were elevated in gastric mucosa of both patients and mice infected with H. pylori, which is positively correlated with bacterial load and the degree of gastritis. IL-33 production was promoted via extracellular regulated protein kinases (ERK) signaling pathway activation by gastric epithelial cells in a cagA-dependent manner during H. pylori infection, and resulted in increased inflammation and bacteria burden within the gastric mucosa. Gastric epithelial cell-derived IL-33 promoted TNF-α production from mast cells in vitro, and IL-33 increased TNF-α production in vivo. Increased TNF-α inhibited gastric epithelial cell proliferation, conducing to the progress of H. pylori-associated gastritis and bacteria colonization. This study defined a patent regulatory networks involving H. pylori, gastric epithelial cell, IL-33, mast cell, and TNF-α, which jointly play a pathological effect within the gastric circumstances. It may be a valuable strategy to restrain this IL-33-dependent pathway in the treatment of H. pylori-associated gastritis.

Increased tumor-infiltrating CD45RA-CCR7- regulatory T-cell subset with immunosuppressive properties foster gastric cancer progress.

Regulatory T cells (Tregs) are major components of tumor-infiltrating immune cells with potent immunosuppressive properties in gastric cancer (GC) microenvironment. However, different subsets of the Tregs and their relevance to GC are unknown. Here, we found that patients with GC showed a significantly higher Tregs infiltration in tumors, and CD45RA-CCR7- Treg subset constituted most tumor-infiltrating Tregs. Tumor-infiltrating CD45RA-CCR7- Treg subset with an effector/memory phenotype accumulated in tumors and expressed low level of HLA-DR. Gastric tumor-derived TNF-α induced CD45RA-CCR7- Treg subset with similar phenotype to their status in tumors and inhibited their HLA-DR expression via activating STAT3 phosphorylation. These tumor-associated CD45RA-CCR7- Treg subset exerted superior immunosuppressive properties to effectively suppress CD8+ T cells' anti-tumor function including CD8+ T-cell IFN-γ and granzyme B (GrB) production as well as CD8+ T-cell proliferation in vitro, and also contributed to the growth and progression of human gastric tumors in vivo, via IL-10 secretion and cell-cell contact mechanisms. Moreover, increased tumor-infiltrating CD45RA-CCR7- Treg subset as well as higher intratumoral CD45RA-CCR7- Treg/CD8+ T-cell ratio was associated with advanced disease progression and reduced GC patient survival. This study therefore identifies a novel immunosuppressive pathway involving CD45RA-CCR7- Treg subset development within the GC microenvironment. Efforts to inhibit this pathway may therefore prove a valuable strategy to prevent, and to treat this immune suppressive of GC.

Tumour-activated neutrophils in gastric cancer foster immune suppression and disease progression through GM-CSF-PD-L1 pathway.

OBJECTIVE: Neutrophils are prominent components of solid tumours and exhibit distinct phenotypes in different tumour microenvironments. However, the nature, regulation, function and clinical relevance of neutrophils in human gastric cancer (GC) are presently unknown. DESIGN: Flow cytometry analyses were performed to examine levels and phenotype of neutrophils in samples from 105 patients with GC. Kaplan-Meier plots for overall survival were performed using the log-rank test. Neutrophils and T cells were isolated, stimulated and/or cultured for in vitro and in vivo regulation and function assays. RESULTS: Patients with GC showed a significantly higher neutrophil infiltration in tumours. These tumour-infiltrating neutrophils showed an activated CD54+ phenotype and expressed high level immunosuppressive molecule programmed death-ligand 1 (PD-L1). Neutrophils activated by tumours prolonged their lifespan and strongly expressed PD-L1 proteins with similar phenotype to their status in GC, and significant correlations were found between the levels of PD-L1 and CD54 on tumour-infiltrating neutrophils. Moreover, these PD-L1+ neutrophils in tumours were associated with disease progression and reduced GC patient survival. Tumour-derived GM-CSF activated neutrophils and induced neutrophil PD-L1 expression via Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signalling pathway. The activated PD-L1+ neutrophils effectively suppressed normal T-cell immunity in vitro and contributed to the growth and progression of human GC in vivo; the effect could be reversed by blocking PD-L1 on these neutrophils. CONCLUSIONS: Our results illuminate a novel mechanism of PD-L1 expression on tumour-activated neutrophils in GC, and also provide functional evidence for these novel GM-CSF-PD-L1 pathways to prevent, and to treat this immune tolerance feature of GC.

Tumor-Associated Monocytes/Macrophages Impair NK-Cell Function via TGFß1 in Human Gastric Cancer.

Natural killer (NK) cells are a major component of the host antitumor immune response in human cancer. However, the nature, functional regulation, and clinical relevance of NK cells in gastric cancer remain largely unknown. In this study, we showed that the percentages of NK cells in tumors were significantly decreased, and low percentages of tumor-infiltrating NK cells were positively correlated with poor survival and disease progression. Although the expression of activating and inhibitory receptors on NK cells was shown to be not different between tumor and nontumor tissues, NK cells in tumors had impaired effector functions, characterized by decreased IFNγ, TNFα, and Ki-67 expression. We found that tumor-infiltrating monocytes/macrophages were physically close to NK cells, and their percentages negatively correlated with IFNγ+ and TNFα+ NK-cell percentages. Ex vivo study showed that isolated tumor-associated monocytes/macrophages could impair NK-cell expression of IFNγ, TNFα, and Ki-67. Blockade of TGFß1 attenuated such monocytes/macrophages-mediated impairment of NK-cell function. Our data suggest that human NK-cell function was impaired by tumor-associated monocytes/macrophages, and that restoring NK-cell function may be an important therapeutic strategy to prevent tumor immune escape in gastric cancer. Cancer Immunol Res; 5(3); 248-56. ©2017 AACR.

Altered phenotypic and functional characteristics of CD3+CD56+ NKT-like cells in human gastric cancer.

CD3+CD56+ natural killer T (NKT)-like cells are a group of CD3+ T cells sharing characteristics of NK and T cells and constitute a major component of host anti-tumor immune response in human cancer. However, the nature, function and clinical relevance of CD3+CD56+ NKT-like cells in human gastric cancer (GC) remain unclear. In this study, we showed that the frequencies of CD3+CD56+NKT-like cells in GC tumors were significantly decreased and low levels of tumor-infiltrating CD3+CD56+ NKT-like cells were positively correlated with poor survival and disease progression. Most CD3+CD56+NKT-like cells in GC tumors were CD45RA-CD27+/- central/effector-memory cells with decreased activity and lower expression levels of CD69, NKG2D and DNAM-1 than those in non-tumor tissues. We further observed that tumor-infiltrating CD3+CD56+ NKT-like cells had impaired effector function as shown by decreased IFN-γ, TNF-α, granzyme B and Ki-67 expression. Moreover, in vitro studies showed that soluble factors released from GC tumors could induce the functional impairment of CD3+CD56+ NKT-like cells. Collectively, our data indicate that decreased tumor-infiltrating CD3+CD56+ NKT-like cells with impaired effector function are associated with tumor progression and poor survival of GC patients, which may contribute to immune escape of GC.

A pro-inflammatory role for Th22 cells in Helicobacter pylori-associated gastritis.

OBJECTIVE: Helper T (Th) cell responses are critical for the pathogenesis of Helicobacter pylori-induced gastritis. Th22 cells represent a newly discovered Th cell subset, but their relevance to H. pylori-induced gastritis is unknown. DESIGN: Flow cytometry, real-time PCR and ELISA analyses were performed to examine cell, protein and transcript levels in gastric samples from patients and mice infected with H. pylori. Gastric tissues from interleukin (IL)-22-deficient and wild-type (control) mice were also examined. Tissue inflammation was determined for pro-inflammatory cell infiltration and pro-inflammatory protein production. Gastric epithelial cells and myeloid-derived suppressor cells (MDSC) were isolated, stimulated and/or cultured for Th22 cell function assays. RESULTS: Th22 cells accumulated in gastric mucosa of both patients and mice infected with H. pylori. Th22 cell polarisation was promoted via the production of IL-23 by dendritic cells (DC) during H. pylori infection, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterised by the CXCR2-dependent influx of MDSCs, whose migration was induced via the IL-22-dependent production of CXCL2 by gastric epithelial cells. Under the influence of IL-22, MDSCs, in turn, produced pro-inflammatory proteins, such as S100A8 and S100A9, and suppressed Th1 cell responses, thereby contributing to the development of H. pylori-associated gastritis. CONCLUSIONS: This study, therefore, identifies a novel regulatory network involving H. pylori, DCs, Th22 cells, gastric epithelial cells and MDSCs, which collectively exert a pro-inflammatory effect within the gastric microenvironment. Efforts to inhibit this Th22-dependent pathway may therefore prove a valuable strategy in the therapy of H. pylori-associated gastritis.