Helicobacter pylori and Its Role in Gastric Cancer.

Gastric cancer is a challenging public health concern worldwide and remains a leading cause of cancer-related mortality. The primary risk factor implicated in gastric cancer development is infection with Helicobacter pylori. H. pylori induces chronic inflammation affecting the gastric epithelium, which can lead to DNA damage and the promotion of precancerous lesions. Disease manifestations associated with H. pylori are attributed to virulence factors with multiple activities, and its capacity to subvert host immunity. One of the most significant H. pylori virulence determinants is the cagPAI gene cluster, which encodes a type IV secretion system and the CagA toxin. This secretion system allows H. pylori to inject the CagA oncoprotein into host cells, causing multiple cellular perturbations. Despite the high prevalence of H. pylori infection, only a small percentage of affected individuals develop significant clinical outcomes, while most remain asymptomatic. Therefore, understanding how H. pylori triggers carcinogenesis and its immune evasion mechanisms is critical in preventing gastric cancer and mitigating the burden of this life-threatening disease. This review aims to provide an overview of our current understanding of H. pylori infection, its association with gastric cancer and other gastric diseases, and how it subverts the host immune system to establish persistent infection.

Helicobacter pylori Immune Response in Children Versus Adults.

H. pylori is perhaps the most prevalent human pathogen worldwide and infects almost half of the world's population. Despite the decreasing prevalence of infection overall, it is significant in developing countries. Most infections are acquired in childhood and persist for a lifetime unless treated. Children are often asymptomatic and often develop a tolerogenic immune response that includes T regulatory cells and their products, immunosuppressive cytokines, such as interleukin (IL)-10, and transforming growth factor-ß (TGF-ß). This contrasts to the gastric immune response seen in H. pylori-infected adults, where the response is mainly inflammatory, with predominant Th1 and Th17 cells, as well as, inflammatory cytokines, such as TNF-α, IFN-γ, IL-1, IL-6, IL-8, and IL-17. Therefore, compared to adults, infected children generally have limited gastric inflammation and peptic ulcer disease. H. pylori surreptitiously subverts immune defenses to persist in the human gastric mucosa for decades. The chronic infection might result in clinically significant diseases in adults, such as peptic ulcer disease, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. This review compares the infection in children and adults and highlights the H. pylori virulence mechanisms responsible for the pathogenesis and immune evasion.

Helicobacter pylori infection downregulates the DNA glycosylase NEIL2, resulting in increased genome damage and inflammation in gastric epithelial cells.

Infection with the Gram-negative, microaerophilic bacterium Helicobacter pylori induces an inflammatory response and oxidative DNA damage in gastric epithelial cells that can lead to gastric cancer (GC). However, the underlying pathogenic mechanism is largely unclear. Here, we report that the suppression of Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase that specifically removes oxidized bases, is one mechanism through which H. pylori infection may fuel the accumulation of DNA damage leading to GC. Using cultured cell lines, gastric biopsy specimens, primary cells, and human enteroid-derived monolayers from healthy human stomach, we show that H. pylori infection greatly reduces NEIL2 expression. The H. pylori infection-induced downregulation of NEIL2 was specific, as Campylobacter jejuni had no such effect. Using gastric organoids isolated from the murine stomach in coculture experiments with live bacteria mimicking the infected stomach lining, we found that H. pylori infection is associated with the production of various inflammatory cytokines. This response was more pronounced in Neil2 knockout (KO) mouse cells than in WT cells, suggesting that NEIL2 suppresses inflammation under physiological conditions. Notably, the H. pylori-infected Neil2-KO murine stomach exhibited more DNA damage than the WT. Furthermore, H. pylori-infected Neil2-KO mice had greater inflammation and more epithelial cell damage. Computational analysis of gene expression profiles of DNA glycosylases in gastric specimens linked the reduced Neil2 level to GC progression. Our results suggest that NEIL2 downregulation is a plausible mechanism by which H. pylori infection impairs DNA damage repair, amplifies the inflammatory response, and initiates GC.

Helicobacter pylori Deregulates T and B Cell Signaling to Trigger Immune Evasion.

Helicobacter pylori is a prevalent human pathogen that successfully establishes chronic infection, which leads to clinically significant gastric diseases including chronic gastritis, peptic ulcer disease (PUD), and gastric cancer (GC). H. pylori is able to produce a persistent infection due in large part to its ability to hijack the host immune response. The host adaptive immune response is activated to strategically and specifically attack pathogens and normally clears them from the infected host. Since B and T lymphocytes are central mediators of adaptive immunity, in this chapter we review their development and the fundamental mechanisms regulating their activation in order to understand how some of the normal processes are subverted by H. pylori. In this review, we place particular emphasis on the CD4+ T cell responses, their subtypes, and regulatory mechanisms because of the expanding literature in this area related to H. pylori. T lymphocyte differentiation and function are finely orchestrated through a series of cell-cell interactions, which include immune checkpoint receptors. Among the immune checkpoint receptor family, there are some with inhibitory properties that are exploited by tumor cells to facilitate their immune evasion. Gastric epithelial cells (GECs), which act as antigen-presenting cells (APCs) in the gastric mucosa, are induced by H. pylori to express immune checkpoint receptors known to sway T lymphocyte function and thus circumvent effective T effector lymphocyte responses. This chapter reviews these and other mechanisms used by H. pylori to interfere with host immunity in order to persist.

Helicobacter pylori elicits B7H3 expression on gastric epithelial cells: Implications in local T cell regulation and subset development during infection.

Helicobacter pylori (H. pylori) is a gram negative bacterium that infects more than 50% of humanity and is associated with gastritis, peptic ulcer and gastric cancer. Although CD4+ T cells are recruited to the gastric mucosa, the host is unable to clear the bacteria. Previously, we demonstrated that H. pylori infection upregulates the expression of the T cell co-inhibitory molecule B7-H1 while simultaneously downregulating the expression of T cell co-stimulatory molecule B7-H2 on gastric epithelial cells (GEC), which together affect the Treg and Th17 cell balance and foster bacterial persistence. Because B7-H3, another member of the B7 family of co-inhibitory receptors, has been found to have important immunoregulatory roles and in cancer, in this study we examined the expression of B7-H3 molecules on GEC and how the expression is regulated by H. pylori during infection. Our study showed that both human and murine GEC constitutively express B7-H3 molecules, but their expression levels increased during H. pylori infection. We further demonstrated that H. pylori uses its type 4 secretion system (T4SS) components CagA and cell wall peptidoglycan (PG) fragment to upregulate B7-H3. Th17 cells and Treg cells which are increased during H. pylori infection also had an effect on B7-H3 induction. The underlying cell signaling pathway involves modulation of p38MAPK pathway. Since B7-H3 were shown to up-regulate Th2 responses, the phenotype of T cell subpopulations in mice infected with H. pylori PMSS1 or SS1 strains were characterized. A mixed Th1/Th2 response in H. pylori infected mice was observed. Consistent with previous findings, increased Treg cells and decreased Th17 cells in MLN of PMSS1 infected mice compared to SS1 infected mice was observed. Human biopsy samples collected from gastritis biopsies and gastric tumors showed a strong association between increased B7-H3 and Th2 responses in H. pylori strains associated with gastritis. T cell: GEC co-cultures and anti-B7-H3 blocking Ab confirmed that the induction of Th2 is mediated by B7-H3 and associated exclusively with an H. pylori gastritis strain not cancer or ulcer strains. In conclusion, these studies revealed a novel regulatory mechanism employed by H. pylori to influence the type of T cell response that develops within the infected gastric mucosa.

Expression of Programmed Death-Ligand 1 by Human Colonic CD90+ Stromal Cells Differs Between Ulcerative Colitis and Crohn's Disease and Determines Their Capacity to Suppress Th1 Cells.

Background and Aims: The role of programmed cell death protein 1 (PD-1) and its ligands in the dysregulation of T helper immune responses observed in the inflammatory bowel disease (IBD) is unclear. Recently, a novel concept emerged that CD90+ colonic (myo)fibroblasts (CMFs), also known as stromal cells, act as immunosuppressors, and are among the key regulators of acute and chronic inflammation. The objective of this study was to determine if the level of the PD-1 ligands is changed in the IBD inflamed colonic mucosa and to test the hypothesis that changes in IBD-CMF-mediated PD-1 ligand-linked immunosuppression is a mechanism promoting the dysregulation of Th1 cell responses. Methods: Tissues and cells derived from Crohn's disease (CD), ulcerative colitis (UC), and healthy individuals (N) were studied in situ, ex vivo, and in culture. Results: A significant increase in programmed death-ligand 1 (PD-L1) was observed in the inflamed UC colonic mucosa when compared to the non-inflamed matched tissue samples, CD, and healthy controls. UC-CMFs were among the major populations in the colonic mucosa contributing to the enhanced PD-L1 expression. In contrast, PD-L1 expression was decreased in CD-CMFs. When compared to CD-CMFs and N-CMFs, UC-CMFs demonstrated stronger suppression of IL-2, Th1 transcriptional factor Tbet, and IFN-γ expression by CD3/CD28-activated CD4+ T cells, and this process was PD-L1 dependent. Similar observations were made when differentiated Th1 cells were cocultured with UC-CMFs. In contrast, CD-CMFs showed reduced capacity to suppress Th1 cell activity and addition of recombinant PD-L1 Fc to CD-CMF:T cell cocultures partially restored the suppression of the Th1 type responses. Conclusion: We present evidence showing that increased PD-L1 expression suppresses Th1 cell activity in UC. In contrast, loss of PD-L1 expression observed in CD contributes to the persistence of the Th1 inflammatory milieu in CD. Our data suggest that dysregulation of the Th1 responses in the inflamed colonic mucosa of IBD patients is promoted by the alterations in PD-L1 expression in the mucosal mesenchymal stromal cell compartment.

Helicobacter pylori cag pathogenicity island's role in B7-H1 induction and immune evasion.

During Helicobacter pylori (H. pylori) infection CD4+ T cells in the gastric lamina propria are hyporesponsive and polarized by Th1/Th17 cell responses controlled by Treg cells. We have previously shown that H. pylori upregulates B7-H1 expression on GEC, which, in turn, suppress T cell proliferation, effector function, and induce Treg cells in vitro. In this study, we investigated the underlying mechanisms and the functional relevance of B7-H1 induction by H. pylori infection to chronic infection. Using H. pylori wild type (WT), cag pathogenicity island (cag PAI-) and cagA- isogenic mutant strains we demonstrated that H. pylori requires its type 4 secretion system (T4SS) as well as its effector protein CagA and peptidoglycan (PG) fragments for B7-H1 upregulation on GEC. Our study also showed that H. pylori uses the p38 MAPK pathway to upregulate B7-H1 expression in GEC. In vivo confirmation was obtained when infection of C57BL/6 mice with H. pylori PMSS1 strain, which has a functional T4SS delivery system, but not with H. pylori SS1 strain lacking a functional T4SS, led to a strong upregulation of B7-H1 expression in the gastric mucosa, increased bacterial load, induction of Treg cells in the stomach, increased IL-10 in the serum. Interestingly, B7-H1-/- mice showed less Treg cells and reduced bacterial loads after infection. These studies demonstrate how H. pylori T4SS components activate the p38 MAPK pathway, upregulate B7-H1 expression by GEC, and cause Treg cell induction; thus, contribute to establishing a persistent infection characteristic of H. pylori.

Immune evasion strategies used by Helicobacter pylori.

Helicobacter pylori (H. pylori) is perhaps the most ubiquitous and successful human pathogen, since it colonizes the stomach of more than half of humankind. Infection with this bacterium is commonly acquired during childhood. Once infected, people carry the bacteria for decades or even for life, if not treated. Persistent infection with this pathogen causes gastritis, peptic ulcer disease and is also strongly associated with the development of gastric cancer. Despite induction of innate and adaptive immune responses in the infected individual, the host is unable to clear the bacteria. One widely accepted hallmark of H. pylori is that it successfully and stealthily evades host defense mechanisms. Though the gastric mucosa is well protected against infection, H. pylori is able to reside under the mucus, attach to gastric epithelial cells and cause persistent infection by evading immune responses mediated by host. In this review, we discuss how H. pylori avoids innate and acquired immune response elements, uses gastric epithelial cells as mediators to manipulate host T cell responses and uses virulence factors to avoid adaptive immune responses by T cells to establish a persistent infection. We also discuss in this review how the genetic diversity of this pathogen helps for its survival.

Effect of Helicobacter pylori on gastric epithelial cells.

The gastrointestinal epithelium has cells with features that make them a powerful line of defense in innate mucosal immunity. Features that allow gastrointestinal epithelial cells to contribute in innate defense include cell barrier integrity, cell turnover, autophagy, and innate immune responses. Helicobacter pylori (H. pylori) is a spiral shape gram negative bacterium that selectively colonizes the gastric epithelium of more than half of the world's population. The infection invariably becomes persistent due to highly specialized mechanisms that facilitate H. pylori's avoidance of this initial line of host defense as well as adaptive immune mechanisms. The host response is thus unsuccessful in clearing the infection and as a result becomes established as a persistent infection promoting chronic inflammation. In some individuals the associated inflammation contributes to ulcerogenesis or neoplasia. H. pylori has an array of different strategies to interact intimately with epithelial cells and manipulate their cellular processes and functions. Among the multiple aspects that H. pylori affects in gastric epithelial cells are their distribution of epithelial junctions, DNA damage, apoptosis, proliferation, stimulation of cytokine production, and cell transformation. Some of these processes are initiated as a result of the activation of signaling mechanisms activated on binding of H. pylori to cell surface receptors or via soluble virulence factors that gain access to the epithelium. The multiple responses by the epithelium to the infection contribute to pathogenesis associated with H. pylori.

Diagnostics for Statistical Variable Selection Methods for Prediction of Peptic Ulcer Disease in Helicobacter pylori Infection.

BACKGROUND: The development of accurate classification models depends upon the methods used to identify the most relevant variables. The aim of this article is to evaluate variable selection methods to identify important variables in predicting a binary response using nonlinear statistical models. Our goals in model selection include producing non-overfitting stable models that are interpretable, that generate accurate predictions and have minimum bias. This work was motivated by data on clinical and laboratory features of Helicobacter pylori infections obtained from 60 individuals enrolled in a prospective observational study. RESULTS: We carried out a comprehensive performance comparison of several nonlinear classification models over the H. pylori data set. We compared variable selection results by Multivariate Adaptive Regression Splines (MARS), Logistic Regression with regularization, Generalized Additive Models (GAMs) and Bayesian Variable Selection in GAMs. We found that the MARS model approach has the highest predictive power because the nonlinearity assumptions of candidate predictors are strongly satisfied, a finding demonstrated via deviance chi-square testing procedures in GAMs. CONCLUSIONS: Our results suggest that the physiological free amino acids citrulline, histidine, lysine and arginine are the major features for predicting H. pylori peptic ulcer disease on the basis of amino acid profiling.

TLR4 activation enhances the PD-L1-mediated tolerogenic capacity of colonic CD90+ stromal cells.

Signaling via programmed death ligand-1 (PD-L1) and PD-L2 is crucial for maintaining peripheral tolerance. CD90(+) myofibroblasts/fibroblasts (CMFs) are major programmed cell death-1 (PD-1) ligand-expressing cells in normal human colonic mucosa. CMFs suppress activated CD4(+) T cell proliferation via PD-1 ligands. It is not known whether signaling through TLRs contribute to the regulation PD-1 ligands on CMFs upon colonic mucosal tolerance. In this study, we demonstrated that stimulation of TLR4 on human CMFs upregulates PD-L1, but not PD-L2, and reinforces CMF-mediated suppression of CD4(+) T cell proliferation and IFN-γ production. TLR4-mediated upregulation of PD-L1 on CMFs involved NF-κB pathways and was JAK2 and MyD88 dependent. MyD88-dependent stimulation of TLR1/2 and TLR5 also upregulated PD-L1 expression on CMFs in culture. PD-L1 expression was drastically decreased in vivo in the colonic mucosa of mice devoid of MyD88. Induction of MyD88 deficiency in CMFs in fibroblast-specific MyD88 conditional knockout mice resulted in a strong increase in a mucosal IFN-γ expression concomitantly with the abrogation of PD-L1 expression in CMFs under homeostasis and epithelial injury induced by dextran sodium sulfate. Together, these data suggest that MyD88-dependent TLR stimulation of CMFs in the normal colonic mucosa may reinforce these cells' anti-inflammatory capacity and thus contribute to the maintenance of mucosal tolerance.

Amniotic fluid-borne hepatocyte growth factor protects rat pups against experimental necrotizing enterocolitis.

Fetal swallowing of amniotic fluid, which contains numerous cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid can provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (HGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC6 intestinal epithelial cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Amniotic fluid supplementation in oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by HGF and blocked by anti-HGF antibody or a PI3K inhibitor. HGF transactivated several RTKs in IEC6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, recombinant HGF also reduced the frequency and severity of NEC-like injury in rat pups. Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF.

CagA-dependent downregulation of B7-H2 expression on gastric mucosa and inhibition of Th17 responses during Helicobacter pylori infection.

Gastric epithelial cells (GECs) are the primary target for Helicobacter pylori infection and may act as APCs regulating local T cell responses. We previously reported that H. pylori infection of GECs induces the expression of the T cell coinhibitory molecule B7-H1 on GECs. This process contributes to the hyporesponsiveness of CD4(+) effector T cells and accumulation of regulatory T cells. In the present study, we investigated the impact of H. pylori cytotoxin-associated gene A (CagA) on the modulation of the expression of the T cell costimulator B7-H2 by GECs. B7-H2 is involved in promoting Th17 type responses. H. pylori infection downregulates B7-H2 expression by GECs in a CagA-dependent manner. IFN-γ, which is increased in the H. pylori-infected gastric mucosa, synergizes with H. pylori in downregulating B7-H2 expression by GECs. CagA-mediated modulation of B7-H2 on GECs involves p70 S6 kinase phosphorylation. The CagA-dependent B7-H2 downregulation in GECs correlates with a decrease in Th17 type responses in vitro and in vivo. Furthermore, CagA-dependent modulation of Th17 responses was inversely correlated with the H. pylori colonization levels in vivo. Our data suggest that CagA contributes to the ability of H. pylori to evade Th17-mediated clearance by modulating expression of B7-H2 and, thus, to the establishment of the H. pylori chronic infection.

Role of gastric epithelial cell-derived transforming growth factor beta in reduced CD4+ T cell proliferation and development of regulatory T cells during Helicobacter pylori infection.

Gastric epithelial cells (GECs) express the class II major histocompatibility complex (MHC) and costimulatory molecules, enabling them to act as antigen-presenting cells (APCs) and affect local T cell responses. During Helicobacter pylori infection, GECs respond by releasing proinflammatory cytokines and by increasing the surface expression of immunologically relevant receptors, including class II MHC. The CD4(+) T cell response during H. pylori infection is skewed toward a Th1 response, but these cells remain hyporesponsive. Activated T cells show decreased proliferation during H. pylori infection, and CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) are present at the site of infection. In this study, we examined the mechanisms surrounding the CD4(+) T cell responses during H. pylori infection and found that transforming growth factor ß (TGF-ß) plays a major role in these responses. GECs produced TGF-ß1 and TGF-ß2 in response to infection. Activated CD4(+) T cells in culture with H. pylori-treated GECs were decreased in proliferation but increased upon neutralization of TGF-ß. Naïve CD4(+) T cell development into Tregs was also enhanced in the presence of GEC-derived TGF-ß. Herein, we demonstrate a role for GEC-produced TGF-ß in the inhibition of CD4(+) T cell responses seen during H. pylori infection.

Human colonic myofibroblasts promote expansion of CD4+ CD25high Foxp3+ regulatory T cells.

BACKGROUND & AIMS: Regulatory T (Treg) cells (CD4+ CD25high FoxP3+) regulate mucosal tolerance; their adoptive transfer prevents or reduces symptoms of colitis in mouse models of inflammatory bowel disease. Colonic CD90+ mesenchymal myofibroblasts and fibroblasts (CMFs) are abundant, nonprofessional antigen-presenting cells in the normal human colonic mucosa that suppress proliferation of activated CD4+ effector T cells. We studied CMF suppressive capacity and evaluated the ability of CMF to induce Treg cells. METHODS: Allogeneic cocultures of CD4+ T cells and CMFs, derived from normal mucosa of patients undergoing colectomy for colon cancer or inflamed colonic tissues from patients with ulcerative colitis or Crohn's disease, were used to assess activation of the Treg cells. RESULTS: Coculture of normal CMF with resting or naïve CD4+ T cells led to development of cells with a Treg phenotype; it also induced proliferation of a CD25+ CD127- FoxP3+ T cells, which expressed CTLA-4, interleukin-10, and transforming growth factor-ß and had suppressive activities. In contrast to dendritic cells, normal CMFs required exogenous interleukin-2 to induce proliferation of naturally occurring Treg cells. Induction of Treg cells by normal CMFs required major histocompatibility complex class II and prostaglandin E2. CMFs from patients with inflammatory bowel diseases had reduced capacity to induce active Treg cells and increased capacity to transiently generate CD4+CD25+/- CD127+ T cells that express low levels of FoxP3. CONCLUSIONS: CMFs suppress the immune response in normal colon tissue and might therefore help maintain colonic mucosal tolerance. Alterations in CMF-mediated induction of Treg cells might promote pathogenesis of inflammatory bowel diseases.

Staphylococcal enterotoxins.

Staphylococcus aureus (S. aureus) is a Gram positive bacterium that is carried by about one third of the general population and is responsible for common and serious diseases. These diseases include food poisoning and toxic shock syndrome, which are caused by exotoxins produced by S. aureus. Of the more than 20 Staphylococcal enterotoxins, SEA and SEB are the best characterized and are also regarded as superantigens because of their ability to bind to class II MHC molecules on antigen presenting cells and stimulate large populations of T cells that share variable regions on the ß chain of the T cell receptor. The result of this massive T cell activation is a cytokine bolus leading to an acute toxic shock. These proteins are highly resistant to denaturation, which allows them to remain intact in contaminated food and trigger disease outbreaks. A recognized problem is the emergence of multi-drug resistant strains of S. aureus and these are a concern in the clinical setting as they are a common cause of antibiotic-associated diarrhea in hospitalized patients. In this review, we provide an overview of the current understanding of these proteins.

CD74 in antigen presentation, inflammation, and cancers of the gastrointestinal tract.

CD74 is a protein whose initial role in antigen presentation was recognized two decades ago. Recent studies have revealed that it has additional functions as a receptor for macrophage migration inhibitory factor and as a receptor for an important human pathogen, Helicobacter pylori (H pylori). The role of CD74 as a receptor is important because after binding of migration inhibitory factor or H pylori, NF-kappaB and Erk1/2 activation occurs, along with the induction of proinflammatory cytokine secretion. This review provides an up-to-date account of the functions of CD74 and how it might be involved in inflammation and cancer within the gastrointestinal tract.

PD-1 ligand expression by human colonic myofibroblasts/fibroblasts regulates CD4+ T-cell activity.

BACKGROUND & AIMS: A prominent role for inhibitory molecules PD-L1 and PD-L2 in peripheral tolerance has been proposed. However, the phenotype and function of PD-L-expressing cells in human gut remains unclear. Recent studies suggest that colonic myofibroblasts (CMFs) and fibroblasts are important in the switch from acute inflammation to adaptive immunity. In the normal human colon, CMFs represent a distinct population of major histocompatibility complex class II(+) cells involved in the regulation of mucosal CD4(+) T-cell responses. METHODS: PD-L1 and PD-L2 expression on human CMFs was determined using Western blot, fluorescence-activated cell sorter analysis and confocal microscopy. Lymphoproliferation assays and cytokine enzyme-linked immunosorbent assays were used to evaluate the role of B7 costimulators expressed by CMFs with regard to the regulation of preactivated T-helper cell responses. RESULTS: We demonstrate here the expression of PD-L1/2 molecules by normal human CMF and fibroblasts in situ and in culture. Both molecules support suppressive functions of CMFs in the regulation of activated CD4(+) T-helper cell proliferative responses; blocking this interaction reverses the suppressive effect of CMFs on T-cell proliferation and leads to increased production of the major T-cell growth factor, interleukin (IL)-2. PD-L1/2-mediated CMF suppressive functions are mainly due to the inhibition of IL-2 production, because supplementation of the coculture media with exogenous IL-2 led to partial recovery of activated T-cell proliferation. CONCLUSIONS: Our data suggest that stromal myofibroblasts and fibroblasts may limit T-helper cell proliferative activity in the gut and, thus, might play a prominent role in mucosal intestinal tolerance.

Macrophage migration inhibitory factor and interleukin-8 produced by gastric epithelial cells during Helicobacter pylori exposure induce expression and activation of the epidermal growth factor receptor.

While a link between Helicobacter pylori exposure and gastric cancer has been established, the underlying mechanisms remain unclear. H. pylori induces a chronic inflammatory response in infected individuals. A link between chronic inflammation and carcinogenesis has long been suggested but never elucidated. Epidermal growth factor receptor (EGFR) signaling plays an important role in both proinflammatory and procarcinogenic mechanisms and is upregulated on gastric epithelial cells (GECs) during H. pylori exposure. The aim of this study was to examine the effects of two important proinflammatory cytokines released during H. pylori infection, macrophage migration inhibitory factor (MIF) and interleukin-8 (IL-8), on the expression and transactivation of EGFR and on the proliferation of GECs during H. pylori exposure. The expression of EGFR by GECs was increased by exposure to either H. pylori, recombinant MIF, or recombinant IL-8. However, cag pathogenicity island knockout strains of H. pylori had very little effect on expression. MIF and IL-8 also induced phosphorylation of EGFR, signaling events, and proliferation during H. pylori exposure, all of which were decreased when they were neutralized by these cytokines or were blocked from their receptors. The overall role of EGFR in these responses to H. pylori exposure was assessed by knocking down EGFR expression by small interfering RNA.

Expression of the programmed death ligand 1, B7-H1, on gastric epithelial cells after Helicobacter pylori exposure promotes development of CD4+ CD25+ FoxP3+ regulatory T cells.

During Helicobacter pylori infection, T cells are recruited to the gastric mucosa, but the host T-cell response is not sufficient to clear the infection. Some of the recruited T cells respond in a polarized manner to a Th1 response, while others become anergic. We have previously shown that T-cell anergy may be induced during infection by the interaction of T cells with B7-H1, which is up-regulated on the gastric epithelium during H. pylori infection. Recently, regulatory T (Treg) cells with a CD4(+) CD25(high) FoxP3(+) phenotype were found at an increased frequency in the gastric mucosa of biopsy specimens from H. pylori-infected patients. While Treg cells are important in maintaining tolerance, they can also suppress immune responses during infection. In this study, we examined the induction of the Treg phenotype when naïve T cells were incubated with gastric epithelial cells exposed to H. pylori. The frequency of this phenotype was markedly decreased when B7-H1 was blocked with monoclonal antibodies or its expression was blocked with small interfering RNA. The functional role of these Treg cells was assessed in proliferation assays when the cells were cocultured with activated T cells, which effectively decreased proliferation of the cells.