Comparison of gastric inflammation and metaplasia induced by Helicobacter pylori or Helicobacter felis colonization in mice.

Gastric cancer is the fifth most diagnosed cancer in the world. Infection by the bacteria Helicobacter pylori (HP) is associated with approximately 75% of gastric cancer cases. HP infection induces chronic gastric inflammation, damaging the stomach and fostering carcinogenesis. Most mechanistic studies on gastric cancer initiation are performed in mice and utilize either mouse-adapted strains of HP or the natural mouse pathogen Helicobacter felis (HF). Here, we identified the differences in gastric inflammation, atrophy, and metaplasia associated with HP and HF infection in mice. PMSS1 HP strain or the CS1 HF strain were co-cultured with mouse peritoneal macrophages to assess their immunostimulatory effects. HP and HF induced similar cytokine production from cultured mouse peritoneal macrophages revealing that both bacteria exhibit similar immunostimulatory effects in vitro. Next, C57BL/6J mice were infected with HP or HF and were assessed 2 months post-infection. HP-infected mice caused modest inflammation within both the gastric corpus and antrum, and did not induce significant atrophy within the gastric corpus. In contrast, HF induced significant inflammation throughout the gastric corpus and antrum. Moreover, HF infection was associated with significant atrophy of the chief and parietal cell compartments and induced the expression of pyloric metaplasia (PM) markers. HP is poorly immunogenic compared to HF. HF induces dramatic CD4+ T cell activation, which is associated with increased gastric cancer risk in humans. Thus, HP studies in mice are better suited for studies on colonization, while HF is more strongly suited for studies on the effects of gastric inflammation on tumorigenesis. . IMPORTANCE: Mouse infection models with Helicobacter species are widely used to study Helicobacter pathogenesis and gastric cancer initiation. However, Helicobacter pylori is not a natural mouse pathogen, and mouse-adapted H. pylori strains are poorly immunogenic. In contrast, Helicobacter felis is a natural mouse pathogen that induces robust gastric inflammation and is often used in mice to investigate gastric cancer initiation. Although both bacterial strains are widely used, their disease pathogenesis in mice differs dramatically. However, few studies have directly compared the pathogenesis of these bacterial species in mice, and the contrasting features of these two models are not clearly defined. This study directly compares the gastric inflammation, atrophy, and metaplasia development triggered by the widely used PMSS1 H. pylori and CS1 H. felis strains in mice. It serves as a useful resource for researchers to select the experimental model best suited for their studies.

The Cxcr2+ subset of the S100a8+ gastric granylocytic myeloid-derived suppressor cell population (G-MDSC) regulates gastric pathology.

Introduction: Gastric myeloid-derived suppressor cells (MDSCs) are a prominent population that expands during gastric pre-neoplastic and neoplastic development in humans and mice. However, the heterogeneity of this population has circumvented the ability to study these cells or understand their functions. Aside from Schlafen-4+ (Slfn-4+) MDSCs in mouse studies, which constitute a subset of this population, limitations exist in characterizing the heterogeneity of the gastric CD11b+Ly6G+ population and targeting its different subsets. Here we identify S100a8 as a pan-specific marker for this population and utilize it to study the role of the S100a8+Cxcr2+ subset. Methods: We profiled gastric CD11b+Ly6G+ versus CD11b+Ly6G- myeloid cells by transcriptomic and single-cell RNA sequencing. We identified S100a8 as a pan-specific marker of the gastric granulocytic MDSC (G-MDSC) population, and generated S100a8CreCxcr2flox/flox to study the effects of Cxcr2 knockdown. Results: Following 6-months of Helicobacter felis infection, gastric CD11b+Ly6G+ G-MDSCs were highly enriched for the expression of S100a8, S100a9, Slfn4, Cxcr2, Irg1, Il1f9, Hcar2, Retnlg, Wfdc21, Trem1, Csf3R, Nlrp3, and Il1b. The expression of these distinct genes following 6mo H. felis infection marked heterogeneous subpopulations, but they all represented a subset of S100a8+ cells. S100a8 was identified as a pan-marker for CD11b+Ly6G+ cells arising in chronic inflammation, but not neutrophils recruited during acute gut infection. 6mo Helicobacter felis-infected S100a8CreCxcr2flox/flox mice exhibited worsened gastric metaplastic pathology than Cxcr2flox/flox mice, which was associated with dysregulated lipid metabolism and peroxidation. Conclusion: S100a8 is a pan-specific marker that can be used to target gastric G-MDSC subpopulations, of which the Cxcr2+ subset regulates gastric immunopathology and associates with the regulation of lipid peroxidation.

Schlafen4+-MDSC in Helicobacter-induced gastric metaplasia reveals role for GTPases.

Introduction: MDSCs express SCHLAFEN 4 (SLFN4) in Helicobacter-infected stomachs coincident with spasmolytic polypeptide-expressing metaplasia (SPEM), a precursor of gastric cancer. We aimed to characterize SLFN4+ cell identity and the role of Slfn4 in these cells. Methods: Single-cell RNA sequencing was performed on immune cells sorted from PBMCs and stomachs prepared from uninfected and 6-month H. felis-infected mice. Knockdown of Slfn4 by siRNA or PDE5/6 inhibition by sildenafil were performed in vitro. Intracellular ATP/GTP levels and GTPase activity of immunoprecipitated Slfn4 complexes were measured using the GTPase-Glo assay kit. The intracellular level of ROS was quantified by the DCF-DA fluorescent staining, and apoptosis was determined by cleaved Caspase-3 and Annexin V expression. Gli1CreERT2 x Slfn4 fl/fl mice were generated and infected with H. felis. Sildenafil was administered twice over 2 weeks by gavaging H. felis infected mice ~4 months after inoculation once SPEM had developed. Results: Slfn4 was highly induced in both monocytic and granulocytic MDSCs from infected stomachs. Both Slfn4 +-MDSC populations exhibited strong transcriptional signatures for type-I interferon responsive GTPases and exhibited T cell suppressor function. SLFN4-containing protein complexes immunoprecipitated from myeloid cell cultures treated with IFNa exhibited GTPase activity. Knocking down Slfn4 or PDE5/6 inhibition with sildenafil blocked IFNa induction of GTP, SLFN4 and NOS2. Moreover, IFNa induction of Slfn +-MDSC function was inhibited by inducing their reactive oxygen species (ROS) production and apoptosis through protein kinase G activation. Accordingly, in vivo disruption of Slfn4 in Gli1CreERT2 x Slfn4 fl/fl mice or pharmacologic inhibition by sildenafil after Helicobacter infection also suppressed SLFN4 and NOS2, reversed T cell suppression and mitigated SPEM development. Conclusion: Taken together, SLFN4 regulates the activity of the GTPase pathway in MDSCs and precludes these cells from succumbing to the massive ROS generation when they acquire MDSC function.

CD4+ CD8αα+ T cells in the gastric epithelium mediate chronic inflammation induced by Helicobacter felis.

CD4+ CD8αα+ double-positive intraepithelial T lymphocytes (DP T cells), a newly characterized subset of intraepithelial T cells, are reported to contribute to local immunosuppression. However, the presence of DP T cells in Helicobacter. pylori -induced gastritis and their relationship with disease prognosis has yet to be elucidated. In this study, a chronic gastritis model was established by infecting mice with Helicobacter felis. Gastric-infiltrating lymphocytes were isolated from these mice and analyzed by flow cytometry. The frequency of DP T cells in H. felis-induced gastritis mice was higher than that in uninfected mice. The gastric DP T cells were derived from lamina propria cells but were predominantly distributed in the gastric epithelial layer. These gastric DP T cells also exhibited anti-inflammatory functions, and they inhibited the maturation of dendritic cells and proliferation of CD4+ T lymphocytes in vitro. Elimination of DP T cells simultaneously resulted in severe gastritis and a reduction of H. felis load in vivo. Finally, vaccine mixed with different adjuvants was used to explore the relationship between vaccine efficacy and DP cells. Silk fibroin as the vaccine delivery system enhanced vaccine efficacy by reducing the number of DP T cells. This study demonstrated that DP T cells perform an immunosuppressive role in Helicobacter felis-induced gastritis, and consequently, DP T cells may affect disease prognosis and vaccine efficacy.

Helicobacter pylori-induced gastric cancer is orchestrated by MRCKß-mediated Siah2 phosphorylation.

BACKGROUND: Helicobacter pylori-mediated gastric carcinogenesis is initiated by a plethora of signaling events in the infected gastric epithelial cells (GECs). The E3 ubiquitin ligase seven in absentia homolog 2 (Siah2) is induced in GECs in response to H. pylori infection. Posttranslational modifications of Siah2 orchestrate its function as well as stability. The aim of this study was to evaluate Siah2 phosphorylation status under the influence of H. pylori infection and its impact in gastric cancer progression. METHODS: H. pylori-infected various GECs, gastric tissues from H. pylori-infected GC patients and H. felis-infected C57BL/6 mice were evaluated for Siah2 phosphorylation by western blotting or immunofluorescence microscopy. Coimmunoprecipitation assay followed by mass spectrometry were performed to identify the kinases interacting with Siah2. Phosphorylation sites of Siah2 were identified by using various plasmid constructs generated by site-directed mutagenesis. Proteasome inhibitor MG132 was used to investigate proteasome degradation events. The importance of Siah2 phosphorylation on tumorigenicity of infected cells were detected by using phosphorylation-null mutant and wild type Siah2 stably-transfected cells followed by clonogenicity assay, cell proliferation assay, anchorage-independent growth and transwell invasion assay. RESULTS: Siah2 was phosphorylated in H. pylori-infected GECs as well as in metastatic GC tissues at residues serine6 (Ser6) and threonine279 (Thr279). Phosphorylation of Siah2 was mediated by MRCKß, a Ser/Thr protein kinase. MRCKß was consistently expressed in uninfected GECs and noncancer gastric tissues but its level decreased in infected GECs as well as in metastatic tissues which had enhanced Siah2 expression. Infected murine gastric tissues showed similar results. MRCKß could phosphorylate Siah2 but itself got ubiquitinated from this interaction leading to the proteasomal degradation of MRCKß and use of proteasomal inhibitor MG132 could rescue MRCKß from Siah2-mediated degradation. Ser6 and Thr279 phosphorylated-Siah2 was more stable and tumorigenic than its non-phosphorylated counterpart as revealed by the proliferation, invasion, migration abilities and anchorage-independent growth of stable-transfected cells. CONCLUSIONS: Increased level of Ser6 and Thr279-phosphorylated-Siah2 and downregulated MRCKß were prominent histological characteristics of Helicobacter-infected gastric epithelium and metastatic human GC. MRCKß-dependent Siah2 phosphorylation stabilized Siah2 which promoted anchorage-independent survival and proliferative potential of GECs. Phospho-null mutants of Siah2 (S6A and T279A) showed abated tumorigenicity.

Early detection of tumor cells in bone marrow and peripheral blood in a fast­progressing gastric cancer model.

Helicobacter pylori (H. pylori) infection is a major risk factor for the development of gastric cancer. The authors previously demonstrated that in mice deficient in myeloid differentiation primary response 88 (Myd88­/­), infection with Helicobacter felis (H. felis) a close relative of H. pylori, subsequently rapidly progressed to neoplasia. The present study examined circulating tumor cells (CTCs) by measuring the expression of cytokeratins, epithelial­to­mesenchymal transition (EMT)­related markers and cancer stem cell (CSC) markers in bone marrow and peripheral blood from Myd88­/­ and wild­type (WT) mice. Cytokeratins CK8/18 were detected as early as 4 months post­infection in Myd88­/­ mice. By contrast, cytokeratins were not detected in WT mice even after 7 months post­infection. The expression of Mucin­1 (MUC1) was observed in both bone marrow and peripheral blood at different time points, suggesting its role in gastric cancer metastasis. Snail, Twist and ZEB were expressed at different levels in bone marrow and peripheral blood. The expression of these EMT­related markers suggests the manifestation of cancer metastasis in the early stages of disease development. LGR5, CD44 and CD133 were the most prominent CSC markers detected. The detection of CSC and EMT markers along with cytokeratins does reinforce their use as biomarkers for gastric cancer metastasis. This early detection of markers suggests that CTCs leave primary site even before cancer is well established. Thus, cytokeratins, EMT, and CSCs could be used as biomarkers to detect aggressive forms of gastric cancers. This information may prove to be of significance in stratifying patients for treatment prior to the onset of severe disease­related characteristics.

PD-1 Signaling Promotes Tumor-Infiltrating Myeloid-Derived Suppressor Cells and Gastric Tumorigenesis in Mice.

BACKGROUND & AIMS: Immune checkpoint inhibitors have limited efficacy in many tumors. We investigated mechanisms of tumor resistance to inhibitors of programmed cell death-1 (PDCD1, also called PD-1) in mice with gastric cancer, and the role of its ligand, PD-L1. METHODS: Gastrin-deficient mice were given N-methyl-N-nitrosourea (MNU) in drinking water along with Helicobacter felis to induce gastric tumor formation; we also performed studies with H/K-ATPase-hIL1B mice, which develop spontaneous gastric tumors at the antral-corpus junction and have parietal cells that constitutively secrete interleukin 1B. Mice were given injections of an antibody against PD-1 or an isotype control before tumors developed, or anti-PD-1 and 5-fluorouracil and oxaliplatin, or an antibody against lymphocyte antigen 6 complex locus G (also called Gr-1), which depletes myeloid-derived suppressor cells [MDSCs]), after tumors developed. We generated knock-in mice that express PD-L1 specifically in the gastric epithelium or myeloid lineage. RESULTS: When given to gastrin-deficient mice before tumors grew, anti-PD-1 significantly reduced tumor size and increased tumor infiltration by T cells. However, anti-PD-1 alone did not have significant effects on established tumors in these mice. Neither early nor late anti-PD-1 administration reduced tumor growth in the presence of MDSCs in H/K-ATPase-hIL-1ß mice. The combination of 5-fluorouracil and oxaliplatin reduced MDSCs, increased numbers of intra-tumor CD8+ T cells, and increased the response of tumors to anti-PD-1; however, this resulted in increased tumor expression of PD-L1. Expression of PD-L1 by tumor or immune cells increased gastric tumorigenesis in mice given MNU. Mice with gastric epithelial cells that expressed PD-L1 did not develop spontaneous tumors, but they developed more and larger tumors after administration of MNU and H felis, with accumulation of MDSCs. CONCLUSIONS: In mouse models of gastric cancer, 5-fluorouracil and oxaliplatin reduce numbers of MDSCs to increase the effects of anti-PD-1, which promotes tumor infiltration by CD8+ T cells. However, these chemotherapeutic agents also induce expression of PD-L1 by tumor cells. Expression of PD-L1 by gastric epithelial cells increases tumorigenesis in response to MNU and H felis, and accumulation of MDSCs, which promote tumor progression. The timing and site of PD-L1 expression is therefore important in gastric tumorigenesis and should be considered in design of therapeutic regimens.

Silk fibroin hydrogel as mucosal vaccine carrier: induction of gastric CD4+TRM cells mediated by inflammatory response induces optimal immune protection against Helicobacter felis.

Tissue-resident memory T (TRM) cells, located in the epithelium of most peripheral tissues, constitute the first-line defense against pathogen infections. Our previous study reported that gastric subserous layer (GSL) vaccination induced a "pool" of protective tissue-resident memory CD4+T (CD4+TRM) cells in the gastric epithelium. However, the mechanistic details how CD4+TRM cells form in the gastric epithelium are unknown. Here, our results suggested that the vaccine containing CCF in combination with Silk fibroin hydrogel (SF) broadened the distribution of gastric intraepithelial CD4+TRM cells. It was revealed that the gastric intraepithelial TRM cells were even more important than circulating memory T cells against infection by Helicobacter felis. It was also shown that gastric-infiltrating neutrophils were involved as indispensable mediators which secreted CXCL10 to chemoattract CXCR3+CD4+T cells into the gastric epithelium. Blocking of CXCR3 or neutrophils significantly decreased the number of gastric intraepithelial CD4+TRM cells due to reduced recruitment of CD4+T cells. This study demonstrated the protective efficacy of gastric CD4+TRM cells against H. felis infection, and highlighted the influence of neutrophils on gastric intraepithelial CD4+TRM cells formation.

TET repression and increased DNMT activity synergistically induce aberrant DNA methylation.

Chronic inflammation is deeply involved in various human disorders, such as cancer, neurodegenerative disorders, and metabolic disorders. Induction of epigenetic alterations, especially aberrant DNA methylation, is one of the major mechanisms, but how it is induced is still unclear. Here, we found that expression of TET genes, methylation erasers, was downregulated in inflamed mouse and human tissues, and that this was caused by upregulation of TET-targeting miRNAs such as MIR20A, MIR26B, and MIR29C, likely due to activation of NF-κB signaling downstream of IL-1ß and TNF-α. However, TET knockdown induced only mild aberrant methylation. Nitric oxide (NO), produced by NOS2, enhanced enzymatic activity of DNA methyltransferases (DNMTs), methylation writers, and NO exposure induced minimal aberrant methylation. In contrast, a combination of TET knockdown and NO exposure synergistically induced aberrant methylation, involving genomic regions not methylated by either alone. The results showed that a vicious combination of TET repression, due to NF-κB activation, and DNMT activation, due to NO production, is responsible for aberrant methylation induction in human tissues.

Innate Immune Molecule NLRC5 Protects Mice From Helicobacter-induced Formation of Gastric Lymphoid Tissue.

BACKGROUND & AIMS: Helicobacter pylori induces strong inflammatory responses that are directed at clearing the infection, but if not controlled, these responses can be harmful to the host. We investigated the immune-regulatory effects of the innate immune molecule, nucleotide-binding oligomerization domain-like receptors (NLR) family CARD domain-containing 5 (NLRC5), in patients and mice with Helicobacter infection. METHODS: We obtained gastric biopsies from 30 patients in Australia. We performed studies with mice that lack NLRC5 in the myeloid linage (Nlrc5møKO) and mice without Nlrc5 gene disruption (controls). Some mice were gavaged with H pylori SS1 or Helicobacter felis; 3 months later, stomachs, spleens, and sera were collected, along with macrophages derived from bone marrow. Human and mouse gastric tissues and mouse macrophages were analyzed by histology, immunohistochemistry, immunoblots, and quantitative polymerase chain reaction. THP-1 cells (human macrophages, controls) and NLRC5-/- THP-1 cells (generated by CRISPR-Cas9 gene editing) were incubated with Helicobacter and gene expression and production of cytokines were analyzed. RESULTS: Levels of NLRC5 messenger RNA were significantly increased in gastric tissues from patients with H pylori infection, compared with patients without infection (P < .01), and correlated with gastritis severity (P < .05). H pylori bacteria induced significantly higher levels of chemokine and cytokine production by NLRC5-/- THP-1 macrophages than by control THP-1 cells (P < .05). After 3 months of infection with H felis, Nlrc5mø-KO mice developed gastric hyperplasia (P < .0001), splenomegaly (P < .0001), and increased serum antibody titers (P < .01), whereas control mice did not. Nlrc5mø-KO mice with chronic H felis infection had increased numbers of gastric B-cell follicles expressing CD19 (P < .0001); these follicles had features of mucosa-associated lymphoid tissue lymphoma. We identified B-cell-activating factor as a protein that promoted B-cell hyperproliferation in Nlrc5mø-KO mice. CONCLUSIONS: NLRC5 is a negative regulator of gastric inflammation and mucosal lymphoid formation in response to Helicobacter infection. Aberrant NLRC5 signaling in macrophages can promote B-cell lymphomagenesis during chronic Helicobacter infection.

Alzheimer's Disease and Helicobacter pylori Infection: Inflammation from Stomach to Brain?

Despite extensive research, the origin of Alzheimer's disease (AD) remains unknown. The role of infectious pathogens has recently emerged. Epidemiological studies have shown that Helicobacter pylori infection increases the risk of developing AD. We hypothesized that H. pylori-induced gastritis may be associated with a systemic inflammation and finally neuroinflammation. C57BL/6 mice were infected with H. pylori (n = 15) or Helicobacter felis (n = 13) or left uninfected (n = 9) during 18 months. Gastritis, amyloid deposition, astroglial and microglial cell area, and systemic and brain cytokines were assessed. The infection (H. felis> H. pylori) induced a severe gastritis and an increased neuroinflammation but without brain amyloid deposition or systemic inflammation.

Increased LIGHT expression and activation of non-canonical NF-κB are observed in gastric lesions of MyD88-deficient mice upon Helicobacter felis infection.

Helicobacter pylori infection induces a number of pro-inflammatory signaling pathways contributing to gastric inflammation and carcinogenesis. Among those, NF-κB signaling plays a pivotal role during infection and malignant transformation of the gastric epithelium. However, deficiency of the adaptor molecule myeloid differentiation primary response 88 (MyD88), which signals through NF-κB, led to an accelerated development of gastric pathology upon H. felis infection, but the mechanisms leading to this phenotype remained elusive. Non-canonical NF-κB signaling was shown to aggravate H. pylori-induced gastric inflammation via activation of the lymphotoxin ß receptor (LTßR). In the present study, we explored whether the exacerbated pathology observed in MyD88-deficient (Myd88-/-) mice was associated with aberrant activation of non-canonical NF-κB. Our results indicate that, in the absence of MyD88, H. felis infection enhances the activation of non-canonical NF-κB that is associated with increase in Cxcl9 and Icam1 gene expression and CD3+ lymphocyte recruitment. In addition, activation of signal transducer and activator of transcription 3 (STAT3) signaling was higher in Myd88-/- compared to wild type (WT) mice, indicating a link between MyD88 deficiency and STAT3 activation in response to H. felis infection. Thereby, MyD88 deficiency results in accelerated and aggravated gastric pathology induced by Helicobacter through activation of non-canonical NF-κB.

Myeloid-Derived Suppressor Cells and γδT17 Cells Contribute to the Development of Gastric MALT Lymphoma in H. felis-Infected Mice.

Helicobacter-induced chronic inflammation and immune disorders are closely associated with the development of gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Myeloid-derived suppressor cells (MDSCs) exhibit strong immunosuppressive properties and promote the growth of various solid tumors. However, the role of MDSCs in the development of MALT lymphoma has not been elucidated so far. We detected significant infiltration and enrichment of MDSCs in patients with MALT lymphoma, as well in Helicobacter felis-infected mouse model of gastric MALT lymphoma. In addition, the expression of arginase-1 and inducible nitric oxide synthase was significantly elevated both in gastric MALT lymphoma tissues and H. felis-infected stomach. Persistent H. felis infection closely reproduced the development of gastric MALT lymphoma and was accompanied by increased numbers of γδT17 cells. Accumulation of γδT17 cells was also validated in the human gastric MALT lymphoma tissues. Furthermore, the elevated cytokines interleukin-23 and interleukin-1ß, as well as chemokines CCL20/CCR6, may be involved in the accumulation of γδT17 cells and the subsequent immunosuppression. These findings highlight the role of MDSCs and γδT17 cells in immune dysregulation during gastric MALT lymphoma development and their potential as therapeutic targets.

An atypical presentation of an acute gastric Helicobacter felis infection.

Helicobacter pylori is a Gram negative bacterium that has been associated with a wide variety of gastric pathologies in humans. Besides this well studied gastric pathogen, other Helicobacter spp. have been detected in a minority of patients with gastric disease. These species, also referred to as "H. heilmanii sensu lato" or "non Helicobacter pylori Helicobacter spp. (NHPH)", have a very fastidious nature which makes their in vitro isolation difficult. This group compromises several different Helicobacter species which naturally colonize the stomach of animals. In this article we present a case of a patient with severe gastritis in which H. felis was identified. The necrotic lesions observed at gastroscopy differ from the less active and less severe lesions generally associated with NHPH infections in human patients. The patient was successfully treated with a combination of amoxicillin, clarithromycin and pantoprazole. Infections with NHPH should be included in the differential diagnosis of gastritis when anatomopathological findings show an atypically shaped helicobacter.

Bacterially activated B-cells drive T cell differentiation towards Tr1 through PD-1/PD-L1 expression.

Regulatory B cells (Bregs) play a crucial role in immunological tolerance primarily through the production of IL-10 in many diseases including autoimmune disorders, allergy, infectious diseases, and cancer. To date, various Breg subsets with overlapping phenotypes have been identified. However, the roles of Bregs in Helicobacter infection are largely unknown. In the present study, we investigate the phenotype and function of Helicobacter -stimulated B cells. Our results demonstrate that Helicobacter felis -stimulated IL-10- producing B cells (Hfstim- IL-10+ B) are composed of B10 and Transitional 2 Marginal Zone Precursor (T2-MZP) cells with expression of CD9, Tim-1, and programmed death 1 (PD-1). On the other hand, Helicobacter felis -stimulated IL-10- nonproducing B (Hfstim- IL-10- B) cells are mainly marginal zone (MZ) B cells that express PD-L1 and secrete TGF-ß, IL-6, and TNF-α, and IgM and IgG2b. Furthermore, we show that both Hfstim- IL-10+ B cells and Hfstim- IL-10- B cells induce CD49b+LAG-3+ Tr1 cells. Here, we describe a novel mechanism for PD-1/PD-L1- driven B cell-dependent Tr1 cell differentiation. Finally, we explore the capability of Hfstim- IL-10- B cells to induce Th17 cell differentiation, which we find to be dependent on TGF-ß. Taken together, the current study demonstrates that Hfstim- B cells induce Tr1 cells through the PD-1/PD-L1 axis and Th17 cells by secreting TGF-ß.

Helicobacter-induced gastric inflammation alters the properties of gastric tissue stem/progenitor cells.

BACKGROUND: Although Helicobacter-induced gastric inflammation is the major predisposing factor for gastric carcinogenesis, the precise mechanism by which chronic gastritis causes gastric cancer remains unclear. Intestinal and spasmolytic polypeptide-expressing metaplasia (SPEM) is considered as precancerous lesions, changes in epithelial tissue stem/progenitor cells after chronic inflammation has not been clarified yet. In this study, we utilized three-dimensional gastric epithelial cell culture systems that could form organoids, mimicking gastric epithelial layer, and characterized the changes in epithelial cells after chronic Helicobacter felis infection. METHODS: We used three mice model; 1) long-term H. felis infection, 2) H. felis eradication, and 3) MNU chemical carcinogenesis model. We performed cRNA microarray analysis after organoid culture, and analyzed the effects of chronic gastric inflammation on tissue stem cells, by the size of organoid, mRNA expression profile and immunohistochemical analysis. RESULTS: The number of organoids cultured from gastric epithelial cells was significantly higher in organoids isolated from H. felis-infected mice compared with those from uninfected gastric mucosa. Based on the mRNA expression profile, we found that possible stem cell markers such as Cd44, Dclk1, and genes associated with the intestinal phenotype, such as Villin, were increased in organoids isolated from H. felis-infected mucosa compared with the control. The upregulation of these genes were cancelled after H. felis eradication. In a xenograft model, tumors were generated only from organoids cultured from carcinogen-treated gastric mucosa, not from H. felis infected mucosa or control organoids. CONCLUSIONS: Our results suggested that, as a possible mechanism of gastric carcinogenesis, chronic inflammation induced by H. felis infection increased the number of tissue stem/progenitor cells and the expression of stem cell markers. These findings suggest that chronic inflammation may alter the direction of differentiation toward undifferentiated state and that drawbacks may enable cells to redifferentiate to intestinal metaplasia or neoplasia.

Mice lacking NF-κB1 exhibit marked DNA damage responses and more severe gastric pathology in response to intraperitoneal tamoxifen administration.

Tamoxifen (TAM) has recently been shown to cause acute gastric atrophy and metaplasia in mice. We have previously demonstrated that the outcome of Helicobacter felis infection, which induces similar gastric lesions in mice, is altered by deletion of specific NF-κB subunits. Nfkb1-/- mice developed more severe gastric atrophy than wild-type (WT) mice 6 weeks after H. felis infection. In contrast, Nfkb2-/- mice were protected from this pathology. We therefore hypothesized that gastric lesions induced by TAM may be similarly regulated by signaling via NF-κB subunits. Groups of five female C57BL/6 (WT), Nfkb1-/-, Nfkb2-/- and c-Rel-/- mice were administered 150 mg/kg TAM by IP injection. Seventy-two hours later, gastric corpus tissues were taken for quantitative histological assessment. In addition, groups of six female WT and Nfkb1-/- mice were exposed to 12 Gy γ-irradiation. Gastric epithelial apoptosis was quantified 6 and 48 h after irradiation. TAM induced gastric epithelial lesions in all strains of mice, but this was more severe in Nfkb1-/- mice than in WT mice. Nfkb1-/- mice exhibited more severe parietal cell loss than WT mice, had increased gastric epithelial expression of Ki67 and had an exaggerated gastric epithelial DNA damage response as quantified by γH2AX. To investigate whether the difference in gastric epithelial DNA damage response of Nfkb1-/- mice was unique to TAM-induced DNA damage or a generic consequence of DNA damage, we also assessed gastric epithelial apoptosis following γ-irradiation. Six hours after γ-irradiation, gastric epithelial apoptosis was increased in the gastric corpus and antrum of Nfkb1-/- mice. NF-κB1-mediated signaling regulates the development of gastric mucosal pathology following TAM administration. This is associated with an exaggerated gastric epithelial DNA damage response. This aberrant response appears to reflect a more generic sensitization of the gastric mucosa of Nfkb1-/- mice to DNA damage.

Nardilysin regulates inflammation, metaplasia, and tumors in murine stomach.

Chronic inflammation contributes to a wide variety of human disorders. In the stomach, longstanding gastritis often results in structural alterations in the gastric mucosa, including metaplastic changes and gastric cancers. Therefore, it is important to elucidate factors that are involved in gastric inflammation. Nardilysin (N-arginine dibasic convertase; Nrdc) is a metalloendopeptidase of the M16 family that promotes ectodomain shedding of the precursor forms of various growth factors and cytokines by enhancing the protease activities of a disintegrin and metalloproteinase (ADAM) proteins. Here, we have demonstrated that Nrdc crucially regulates gastric inflammation caused by Helicobacter felis infection or forced expression of prostaglandin E2 in K19-C2mE mice. Metaplastic changes following gastric inflammation were suppressed by the deletion of Nrdc. Furthremore, the deletion of Nrdc significantly suppressed N-methyl-N-nitrosourea (MNU)-induced gastric tumorigenesis in the murine stomach. These data may lead to a global therapeutic approach against various gastric disorders by targeting Nrdc.

A Gain-Of-Function Mutation in the Plcg2 Gene Protects Mice from Helicobacter felis-Induced Gastric MALT Lymphoma.

Gastric mucosa-associated lymphoid tissue (MALT) lymphomas develop from a chronic Helicobacter infection. Phospholipase C gamma 2 (PLCG2) is important for B-cell survival and proliferation. We used BALB/c mice with a gain-of-function mutation in the Plcg2 gene (Ali5) to analyze its role in the development of gastric MALT lymphoma. Heterozygous BALB/c Plcg2Ali5/+ and wildtype (WT) mice were infected with Helicobacter felis (H. felis) and observed up to 16 months for development of gastric MALT lymphomas. In contrast to our initial hypothesis, Plcg2Ali5/+ mice developed MALT lymphomas less frequently than their WT littermates after long-term infection of 16 months. Infected Plcg2Ali5/+ mice showed downregulation of proinflammatory cytokines and decreased H. felis-specific IgG1 and IgG2a antibody responses. These results suggested a blunted immune response of Plcg2Ali5/+ mice towards H. felis infection. Intriguingly, Plcg2Ali5/+ mice harboured higher numbers of CD73 expressing regulatory T cells (Tregs), possibly responsible for impaired immune response towards Helicobacter infection. We suggest that Plcg2Ali5/+ mice may be protected from developing gastric MALT lymphomas as a result of elevated Treg numbers, reduced response to H. felis and decrease of proinflammatory cytokines.