Helicobacter pylori infection is associated with an altered gastric microbiota in children.

The intestinal microbiome in early life influences development of the mucosal immune system and predisposition to certain diseases. Because less is known about the microbiome in the stomach and its relationship to disease, we characterized the microbiota in the stomachs of 86 children and adults and the impact of Helicobacter pylori infection on the bacterial communities. The overall composition of the gastric microbiota in children and adults without H. pylori infection was similar, with minor differences in only low abundance taxa. However, the gastric microbiota in H. pylori-infected children, but not infected adults, differed significantly in the proportions of multiple high abundance taxa compared with their non-infected peers. The stomachs of H. pylori-infected children also harbored more diverse microbiota, smaller abundance of Firmicutes, and larger abundance of non-Helicobacter Proteobacteria and several lower taxonomic groups than stomachs of H. pylori-infected adults. Children with restructured gastric microbiota had higher levels of FOXP3, IL10, and TGFß expression, consistent with increased T-regulatory cell responses, compared with non-infected children and H. pylori-infected adults. The gastric commensal bacteria in children are altered during H. pylori infection in parallel with more tolerogenic gastric mucosae, potentially contributing to the reduced gastric disease characteristic of H. pylori-infected children.

Human gastric epithelial cells contribute to gastric immune regulation by providing retinoic acid to dendritic cells.

Despite the high prevalence of chronic gastritis caused by Helicobacter pylori, the gastric mucosa has received little investigative attention as a unique immune environment. Here, we analyzed whether retinoic acid (RA), an important homeostatic factor in the small intestinal mucosa, also contributes to gastric immune regulation. We report that human gastric tissue contains high levels of the RA precursor molecule retinol (ROL), and that gastric epithelial cells express both RA biosynthesis genes and RA response genes, indicative of active RA biosynthesis. Moreover, primary gastric epithelial cells cultured in the presence of ROL synthesized RA in vitro and induced RA biosynthesis in co-cultured monocytes through an RA-dependent mechanism, suggesting that gastric epithelial cells may also confer the ability to generate RA on gastric dendritic cells (DCs). Indeed, DCs purified from gastric mucosa had similar levels of aldehyde dehydrogenase activity and RA biosynthesis gene expression as small intestinal DCs, although gastric DCs lacked CD103. In H. pylori-infected gastric mucosa, gastric RA biosynthesis gene expression was severely disrupted, which may lead to reduced RA signaling and thus contribute to disease progression. Collectively, our results support a critical role for RA in human gastric immune regulation.

Extracellular matrix-associated cytokines regulate CD4+ effector T-cell responses in the human intestinal mucosa.

Extracellular matrix (stroma) regulation of mucosal T-cell function is incompletely understood. In this study, we uncovered a role for intestinal stromal products in the innate regulation of effector T cells. Stroma-conditioned media (S-CM) derived from the normal human intestinal stroma (transforming growth factor-ß (TGF-ß)(hi)/interleukin (IL)-6(lo)/IL-1ß(lo)) significantly downregulated T-cell proliferation and interferon-γ (IFN-γ) production compared with S-CM derived from the inflamed Crohn's mucosa (TGF-ß(hi)/IL-6(hi)/IL-1ß(hi)). Antibody neutralization studies showed that TGF-ß in normal S-CM inhibited T-cell proliferation and IFN-γ production, whereas IL-6 plus IL-1ß in Crohn's S-CM promoted T-cell proliferation, and IL-1ß alone promoted IFN-γ and IL-17 release. Importantly, normal S-CM inhibited T-bet expression, whereas Crohn's S-CM activated signal transducer and activator of transcription 3, suggesting that discordant T-cell responses are regulated at the transcription factor and signaling levels. These findings implicate stromal TGF-ß in the downregulation of T-cell 2 responses in the normal intestinal mucosa, and stromal IL-6 and IL-1ß in the promotion of Th1 and Th17 responses in the inflamed Crohn's mucosa, suggesting an innate regulatory function for the intestinal extracellular matrix.

Intestinal macrophages and response to microbial encroachment.

Macrophages in the gastrointestinal mucosa represent the largest pool of tissue macrophages in the body. In order to maintain mucosal homeostasis, resident intestinal macrophages uniquely do not express the lipopolysaccharide (LPS) co-receptor CD14 or the IgA (CD89) and IgG (CD16, 32, and 64) receptors, yet prominently display Toll-like receptors (TLRs) 3-9. Remarkably, intestinal macrophages also do not produce proinflammatory cytokines in response to TLR ligands, likely because of extracellular matrix (stromal) transforming growth factor-ß (TGF-ß) dysregulation of nuclear factor (NF)-κB signal proteins and, via Smad signaling, expression of IκBα, thereby inhibiting NF-κB-mediated activities. Thus, in noninflamed mucosa, resident macrophages are inflammation anergic but retain avid scavenger and host defense function, an ideal profile for macrophages in close proximity to gut microbiota. In the event of impaired epithelial integrity during intestinal infection or inflammation, however, blood monocytes also accumulate in the lamina propria and actively pursue invading microorganisms through uptake and degradation of the organism and release of inflammatory mediators. Consequently, resident intestinal macrophages are inflammation adverse, but when the need arises, they receive assistance from newly recruited circulating monocytes.

Human primary gastric dendritic cells induce a Th1 response to H. pylori.

Adaptive CD4 T-cell responses are important in the pathogenesis of chronic Helicobacter pylori gastritis. However, the gastric antigen-presenting cells that induce these responses have not yet been identified. Here we show that dendritic cells (DCs) are present in the gastric mucosa of healthy subjects and are more prevalent and more activated in the gastric mucosa of H. pylori-infected subjects. H. pylori induced gastric DCs isolated from noninfected subjects to express increased levels of CD11c, CD86 and CD83, and to secrete proinflammatory cytokines, particularly interleukin (IL)-6 and IL-8. Importantly, gastric DCs pulsed with live H. pylori, but not control DCs, mediated T-cell secretion of interferon-gamma. The ability of H. pylori to induce gastric DC maturation and stimulate gastric DC activation of Th1 cells implicates gastric DCs as initiators of the immune response to H. pylori.

Intestinal macrophages lack CD14 and CD89 and consequently are down-regulated for LPS- and IgA-mediated activities.

The intestinal mucosa normally displays minimal inflammation despite the close proximity between mucosal macrophages and lumenal bacteria. Macrophages interact with bacteria and their products through CD14, a surface receptor involved in the response to LPS, and CD89, the receptor for IgA (FcalphaR). Here we show that resident macrophages isolated from normal human intestine lack CD14 and CD89. The absence of CD14 and CD89 was not due to the isolation procedure or mucosal cell products, but was evident at the transcriptional level, as the macrophages expressed neither CD14- nor CD89-specific mRNAs, but did express Toll-like receptor 2 and 4 transcripts. Consistent with their CD14(-) phenotype, lamina propria macrophages displayed markedly reduced LPS-induced cytokine production and LPS-enhanced phagocytosis. In addition, IgA-enhanced phagocytosis was sharply reduced in lamina propria macrophages. Thus, the absence of CD14 and CD89 on resident intestinal macrophages, due to down-regulated gene transcription, causes down-modulated LPS- and IgA-mediated functions and probably contributes to the low level of inflammation in normal human intestinal mucosa.

Quantitative analysis of Helicobacter pylori infection in a mouse model.

Progress in elucidating the pathogenesis of Helicobacter pylori gastric infection and in developing an H. pylori vaccine will be aided by an animal model in which H. pylori can be reliably detected. To validate the use of the mouse model of H. pylori infection, we determined the susceptibility of three inbred strains of mice (C57BL/6J, C57BL/10J and BALB/c) to two VacA+/CagA+ isolates of H. pylori (SPM326 and M1.16) and determined the effectiveness of microbiological, histological and molecular assays for H. pylori detection. For the detection of H. pylori in inoculated mice, reverse transcriptase-polymerase chain reaction was the most sensitive assay (82%), histological evaluation the next most sensitive (66%) and microbiological evaluation the least sensitive (38%); the assays were equally specific (100%). Of the two H. pylori isolates, M1.16 showed the highest rate of colonization, but SPM326 displayed the highest rate of persistent infection. Among the three mouse strains, C57BL/6J mice showed the highest level of both susceptibility to colonization and persistent infection. Anti-H. pylori antibody responses were induced in all inoculated mice and persisted for up to 8 weeks after H. pylori clearance. These results indicate that inbred mice experimentally infected with H. pylori is a reliable model for human infection, but host susceptibility to colonization and persistence of infection are dependent on the H. pylori isolate and the mouse strain.

Helicobacter pylori-induced mucosal inflammation is Th1 mediated and exacerbated in IL-4, but not IFN-gamma, gene-deficient mice.

To elucidate the pathogenesis of Helicobacter pylori-associated gastritis, we studied immune responses of C57BL/6J wild-type (WT), SCID, and gene deficient (IFN-gamma-/- and IL-4-/-) mice following infection with a pathogenic isolate of H. pylori (SPM326). During early infection in WT mice, mononuclear and polymorphonuclear cells accumulated in the gastric lamina propria, and the numbers of cells in the inflamed mucosa expressing IFN-gamma, but not IL-4, mRNA rose significantly (p < 0.005), consistent with a local Th1 response. Splenic T cells from the same infected WT mice produced high levels of IFN-gamma, no detectable IL-4, and low amounts of IL-10 following in vitro H. pylori urease stimulation, reflecting a systemic Th1 response. Infected C57BL/6J SCID mice did not develop gastric inflammation despite colonization by many bacteria. Infected C57BL/10J and BALB/c mice also did not develop gastric inflammation and displayed a mixed Th1/Th2 splenic cytokine profile. These data imply a major role for the Th1 cytokine IFN-gamma in H. pylori-associated gastric inflammation in C57BL/6J mice. Compared with WT animals, infected IL-4-/- animals had more severe gastritis and higher levels of IFN-gamma production by urease-stimulated splenocytes (p < 0.01), whereas IFN-gamma-/- mice exhibited no gastric inflammation and higher levels of IL-4 production by stimulated splenocytes. These findings establish C57BL/6J mice as an important model for H. pylori infection and demonstrate that up-regulated production of IFN-gamma, in the absence of the opposing effects of IL-4 (and possibly IL-10), plays a pivotal role in promoting H. pylori-induced mucosal inflammation.

Inflammatory cytokine mRNA expression during early and persistent Helicobacter pylori infection in nonhuman primates.

The role of mononuclear phagocytes in orchestrating the host responses to Helicobacter pylori is inadequately understood. Therefore, gene expression for the monocyte/macrophage-derived cytokines interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha was determined before and during H. pylori infection of rhesus monkeys by use of a highly sensitive quantitative reverse transcriptase polymerase chain reaction. The numbers of molecules of IL-1beta, IL-6, and TNF-alpha mRNA in gastric tissue during early infection (7 weeks) significantly exceeded the preinfection numbers (P<.03). Moreover, the numbers of IL-1beta, IL-6, and TNF-alpha mRNA molecules in persistently infected animals (6 years) also were elevated compared with preinfection numbers (P<.02, P=.03, P=.16, respectively). Cytokine gene expression coincided with progressive H. pylori gastritis, confirmed by increased gastritis scores over preinfection scores (P<.005). These findings provide quantitative evidence that H. pylori induces local gene expression of monocyte/macrophage-derived inflammatory cytokines and evokes an innate response in gastric tissue of nonhuman primates.

Poliovirus replicons encoding the B subunit of Helicobacter pylori urease elicit a Th1 associated immune response.

The development of a vaccine for Helicobacter pylori is a key strategy for reducing the worldwide prevalence of H. pylori infection. Although immunization with recombinant B subunit of H. pylori urease (ureB) has yielded promising results, for the most part, these studies relied on the use of strong adjuvant, cholera toxin, precluding the use in humans. Thus, the development of new vaccine strategies for H. pylori is essential. Previous studies from our laboratory have described a vaccine vector based on poliovirus in which foreign genes are substituted for the poliovirus capsid genes. The genomes encoding foreign proteins (replicons) are encapsidated into authentic poliovirions by providing the capsids in trans. To test the utility of replicons as a vaccine vector for H. pylori, a replicon was constructed which encodes ureB. Expression of ureB in cells from the replicon was demonstrated by metabolic labeling followed by immunoprecipitation with anti-urease antibodies. To investigate the immunogenicity of the replicons, mice containing the transgene for the receptor for poliovirus were immunized via the intramuscular route. Mice given three doses of replicons did not develop substantial antibodies to ureB as determined by Western blot analysis using lysates from H. pylori. In contrast, mice given two doses of replicon followed by a single injection of recombinant ureB developed serum antibodies to ureB which were predominately IgG2a. Splenic lymphocytes from mice immunized with replicons alone, or replicons plus recombinant ureB produced abundant interferon-gamma and no detectable interleukin-4 upon stimulation with recombinant ureB. These results establish that poliovirus replicons encoding H. pylori ureB are immunogenic and induce primarily a T helper 1 associated immune response.

African trypanosome infections in mice that lack the interferon-gamma receptor gene: nitric oxide-dependent and -independent suppression of T-cell proliferative responses and the development of anaemia.

Infection of mice with African trypanosomes leads to a severe immunosuppression, mediated by suppressor macrophages. Using ex vivo macrophage culture and in vivo cell transfer, it has been shown that nitric oxide (NO) is a potent effector product of these cells and causes both lymphocyte unresponsiveness and dyserythropoiesis. We explored the role of NO in vivo during trypanosome infection using mice with a disrupted interferon-gamma-receptor gene, which were unable to respond with macrophage activation and NO synthesis. These mice were less effective at controlling parasitaemia than the wild types, but showed an improved splenic T-cell responsiveness and reduced anaemia during the early stages of infection. The data indicate that, in the mouse, NO is a significant mediator of immunosuppression only in early infection. Beyond day 10 of infection, NO-independent mechanisms are of primary significance and the control of parasitaemia and T-cell responsiveness are not directly related.

Nitric oxide produced in the lungs of mice immunized with the radiation-attenuated schistosome vaccine is not the major agent causing challenge parasite elimination.

Mice vaccinated with radiation-attenuated cercariae of Schistosoma mansoni exhibit high levels of protection against a challenge with normal larvae. The immune effector mechanism, which operates against schistosomula in the lungs, requires CD4+ T cells capable of producing interferon-gamma (IFN-gamma). This cytokine can stimulate production of nitric oxide (NO), via its ability to up-regulate inducible nitric oxide synthase (iNOS). We have therefore evaluated the potential role of NO in the effector mechanism operating in vaccinated mice. Evidence for the production of NO in the lungs of such animals was obtained from assays on antigen-stimulated airway cell cultures. Enhanced levels of NO, compared with those in cultures from control mice, were detected both after vaccination and after challenge; elevated levels of iNOS mRNA were also present in whole lung after challenge. However, administration of an iNOS inhibitor to vaccinated mice after percutaneous challenge did not significantly increase the worm burden. Furthermore, when mice with a disrupted iNOS gene were vaccinated they showed a highly significant level of protection. Although NO from activated macrophages can mediate cytotoxic killing of newly transformed schistosomula in vitro, we have demonstrated that the addition of erythrocytes to these larvicidal assays abolishes its effects. We interpret this to mean that once migrating schistosomula enter the bloodstream they will be protected against the cytotoxic actions of NO. Our data thus provide little evidence to implicate NO as a major component of the pulmonary effector response to S. mansoni in vaccinated mice.

Kinetics and mechanism of effector focus formation in the lungs of mice vaccinated with irradiated cercariae of Schistosoma mansoni.

The sequence of events involved in effector focus formation around challenge schistosomula in the lungs of mice vaccinated with radiation-attenuated cercariae of Schistosoma mansoni has been characterized following intravenous administration of lung stage larvae. Histopathological analysis of the lungs of vaccinated animals revealed that infiltrating cells were present around larvae within 24 h. The main increment in cell recruitment occurred between 2 and 4 days, with foci reaching maximal diameter on day 8. No additional infiltration of the airways was detected by bronchoalveolar sampling before day 4 when the maximum number of cells, predominantly lymphocytes, was recovered. In contrast, responses in challenge control animals were relatively slight prior to day 12. IFN gamma was the major cytokine in airway cultures from vaccinated mice, the greatest increment in production coinciding with peak cell recruitment. A similar pattern of IFN gamma mRNA expression was observed in whole lung extracts, highlighting the dominance of Th1 responses in the effector mechanism. The slow start to focus formation may be due to the need for antigen, released by the intravascular parasite, to be translocated across the endothelium, processed by accessory cells and presented to the helper T cells which orchestrate the effector mechanism. The delay is of the same order as the period of development which the parasite must undergo in the lung, to facilitate further migration. This similarity in the timing may explain why some larvae are able to avoid the consequences of the pulmonary effector response.

Impaired immunity and altered pulmonary responses in mice with a disrupted interferon-gamma receptor gene exposed to the irradiated Schistosoma mansoni vaccine.

A high level of protection against Schistosoma mansoni is elicited in mice by the irradiated cercaria vaccine and interferon-gamma (IFN-gamma) is a key cytokine in the pulmonary effector response. The role of this cytokine has been investigated in mice with a targeted disruption of the IFN-gamma receptor gene (IFN-gamma R-/- mice). The level of protection was impaired relative to that elicited in C57BL/6 and 129 wild-type (WT) animals. These two groups developed compact effector foci, of largely mononuclear cell composition, around individual challenge parasites migrating through the lungs. In contrast the IFN-gamma R-/- mice showed a massive and generalized leucocytic infiltration of the airways and interstitium in which eosinophils were a prominent feature. Cultures of airway leucocytes from C57BL/6 mice produced abundant IFN-gamma whilst those from IFN-gamma R-/- mice produced interleukin-4 (IL-4), IL-5 and IL-10, indicating default to the Th2 pathway; the WT animals showed an intermediate response. The pattern of cytokine gene transcripts in whole lung tissue agreed remarkably well with the level of cytokine protein detected in leucocyte cultures, with the exception of substantial IL-4 mRNA but negligible protein in C57BL/6 mice. The loose but intense infiltrate of leucocytes in the lungs of IFN-gamma R-/- mice was clearly ineffective in eliminating challenge parasites, whereas the level of IFN-gamma protein and mRNA in the lungs of C57BL/6 and WT mice correlated with the size and compactness of effector foci. On the basis of these and earlier observations, we suggest that a primary role for IFN-gamma is to promote intercellular adhesion between the leucocytes in an effector focus, promoting its ability to block parasite migration.

Immunity to Schistosoma mansoni in mice vaccinated with irradiated cercariae: cytokine interactions in the pulmonary protective response.

In C57BL/6 strain mice vaccinated with attenuated cercariae of Schistosoma mansoni, the major site of immune elimination of challenge parasites is the lungs. We have monitored pulmonary events after both vaccination and challenge by bronchoalveolar lavage, and examined the profile of cytokines released by recovered cells upon stimulation with larval antigens in vitro. From 14 days post-vaccination, lavage samples contain infiltrating lymphocytes which produce abundant interferon-gamma (IFN-gamma) and interleukin-3 (IL-3). We suggest that the lymphocytes recruited to the lungs are effector/memory cells of the Th1 subset. Challenge of vaccinated mice results in a second influx of IFN-gamma- and IL-3-secreting cells into the airways, earlier than after vaccination alone, or in appropriate controls. Ablation studies reveal that CD4+ T cells are the source of the IFN-gamma. The timing of cytokine production after both vaccination and challenge coincides with phases of macrophage activation already recorded, and with the presence of parasites in the lungs. Administration of monoclonal antibody directed against IFN-gamma, over the period of challenge elimination, almost completely abrogates protection in vaccinated mice, but does not affect the ratio of Th1:Th2 cells in the lungs. Immunity in this model is not, however, affected by inhibition of nitric oxide production, or neutralization of TNF. We suggest that the effector mechanism may operate by blocking parasite migration, and that loss of protection following neutralization of IFN-gamma may be attributed to changes in composition, density and cohesiveness of pulmonary foci.

Phenotypic and functional properties of Th lines and clones recognizing larval antigens of Schistosoma mansoni.

Five T cell clones and two lines were derived from the lymph nodes (LN) of C57BL/6 mice immunized with radiation-attenuated lung-stage larvae of Schistosoma mansoni. All seven clones/lines were CD4+, CD8- and expressed high levels of CD44 and CD45RB surface markers. After prolonged maintenance in-vitro, with soluble antigen from 18 h schistosomula (SSP), five retained the ability to proliferate readily and release IFNg in response to concanavalin A (Con-A) and to SSP and/or soluble adult worm antigen (SWAP). These Th clones/lines induced significant footpad DTH reactions when injected with SWAP, but were unable to confer protective immunity after transfer to naïve recipient mice. This result could be explained by the antigen specificity of the clones/lines, since they were not able to release IFNg when cultured in-vitro with living lung-stage larvae. A second possibility is that the high level of CD45RB expression, which is not seen on the surface of pulmonary CD4+ memory/effector cells isolated directly from protectively-vaccinated mice, alters the ability of the clones/lines to release IFNg and to induce a DTH response in the lungs when they encounter antigen released from migrating schistosomula.

Monoclonal antibody to IFN-gamma modifies pulmonary inflammatory responses and abrogates immunity to Schistosoma mansoni in mice vaccinated with attenuated cercariae.

In C57Bl/6 mice vaccinated with a single dose of attenuated cercariae of Schistosoma mansoni, the major site of immune elimination of intact challenge parasites is the lungs. The effector mechanism involves the formation of focal inflammatory responses throughout the pulmonary tissues. These foci are rich in CD4+ T cells, believed to be memory:effector cells of the Th1 type. To investigate the role of IFN-gamma in these inflammatory responses, vaccinated mice were treated with neutralizing mAb. Administration on days 4, 8, 12, and 16 post-challenge, the period over which elimination of challenge parasites takes place in the lungs, gave an average 89.5% abrogation of protective immunity. Analysis of pulmonary cell populations recovered by bronchoalveolar lavage from treated nonimmune mice at day 14 post-challenge revealed a sharp increase in pulmonary eosinophilia, relative to intact vaccinated and challenged animals. The inverse relationship between eosinophilia and protection suggests that eosinophils do not play a vital role in the immune effector mechanism in this model. Pulmonary foci of treated mice were larger, less compact, and of different cellular composition from those of control groups. They contained increased numbers of eosinophils, together with numerous multinucleated giant cells. The effects observed in the anti-IFN-gamma mAb-treated mice, together with the maintenance of MHC class II expression on alveolar macrophages in these animals, could all be explained by the production of IL-4 and other Th2 cytokines. Thus, neutralization of IFN-gamma during challenge responses may shift the Th balance towards domination by the Th2 subset.

T cell-derived cytokines associated with pulmonary immune mechanisms in mice vaccinated with irradiated cercariae of Schistosoma mansoni.

In C57Bl/6 strain mice vaccinated with attenuated cercariae of Schistosoma mansoni, the major site of immune elimination of normal challenge parasites is the lungs. The immune effector mechanism involves formation of focal inflammatory responses; the abundance of CD4+ T cells and the activation of alveolar macrophages suggests a role for inflammatory cytokines. We report the profile of cytokines produced by cultures of leukocytes recovered by bronchoalveolar lavage (BAL) from the lungs of vaccinated and challenged mice. From 14 days after vaccination, BAL cultures contained infiltrating lymphocytes that produced abundant quantities of IFN-gamma and IL-3 on stimulation with larval Ag. Production declined from day 21 although the infiltrate of lymphocytes persisted. Challenge of vaccinated mice resulted in a second influx of IFN-gamma and IL-3-producing cells, earlier than after vaccination or in the appropriate controls. Ablation studies revealed that CD4+ T cells were essential for the production of IFN-gamma. The timing of cytokine production after vaccination, and challenge was coincident with the phases of macrophage activation previously reported. At no time could lymphocytes in BAL cultures be stimulated to proliferate with either larval Ag or mitogen, in contrast to splenocytes from the same mice. Furthermore, T cell growth factor activity was not detected in BAL cultures stimulated with Ag. We suggest that the lymphocytes recruited to the lungs are memory/effector cells. When Ag released from challenge schistosomula is presented to these cells, they respond by secreting cytokines that mediate the formation of cellular aggregates around the parasites, blocking their onward migration.