SEARCH

SEARCH BY CITATION

Keywords:

  • Anergy;
  • Helicobacter pylori;
  • Inflammation;
  • Mouse;
  • Treg cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

The gastric pathogen Helicobacter pylori infects over half the world's population. The lifelong infection induces gastric inflammation but the host fails to generate protective immunity. To study the lack of protective H. pylori immunity, CD4+CD25+ Treg cells were investigated for their ability to down-regulate H. pylori-specific CD4+CD25 cells in a murine model. CD25 lymphocytes from infected mice were hyporesponsive to antigenic stimulation in vitro even in the absence of CD25+ Treg cells unless treated with high-dose IL-2. Transfer of CD45RBhi naïve CD25 cells from infected mice into rag1−/− mice challenged with H. pylori resulted in severe gastritis and reduced bacterial loads, whereas transfer of CD45RBlo memory CD25 cells from H. pylori-infected mice resulted in only mild gastritis and persistent infection. CD25 cells stimulated in the absence of CD25+ cells in rag1−/− mice promoted bacterial clearance, but lost this ability when subsequently transferred to WT mice harboring CD25+ cells. These results demonstrate that CD25+ cells induce anergy in CD25 cells in response to H. pylori infection but are not required to maintain hyporesponsiveness. In addition, CD25+ cells are able to suppress previously activated CD25 cells when responding to H. pylori challenge in vivo.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

CD4+CD25+ Treg cells have been identified in the peripheral tissue of mice and humans where they help prevent the development of autoimmunity by down-regulation of self-reactive T cells that escape thymic education 1. Although the level of regulatory CD25hi T cells in human blood ranges from 2 to 4%, a distinct population of regulatory CD25+ cells makes up approximately 10% of the mouse peripheral T-cell pool 2–4. Treg cells are further characterized by the expression of the transcription factor Foxp3 and by their anergic response to in vitro stimulation 5, 6. The suppressive activity of these cells is contact-dependent and requires TCR activation 7. It has recently been shown that Treg cells are also necessary for the maintenance of tolerance to food antigens and commensal flora of the gut 8, 9 as transfer of CD4+ T cells depleted of Treg cells into immunodeficient mice results in the spontaneous development of colitis and wasting disease 10, 11. Treg cells were shown to prevent the development of colitis when simultaneously transferred with a CD25+ Treg cell-depleted population 9, 12.

Treg cells have been identified in the host response to specific pathogens 13–16. Reduced and non-protective immune responses have been described for the parasites Plasmodium yoelii and Leishmania major and the fungus Pneumocystis carinii, which contributes to the ability of these microorganisms to establish persistent infection of the host 14–16. Recently, CD25+ Treg cells have also been identified in the gastric mucosa of Helicobacter pylori-infected patients 17–19. H. pylori is a gram-negative bacterium that infects the stomach of more than half the world's population and in many developing nations its prevalence exceeds 80% of the population 20, 21. Infection persists for the life of the host and is accompanied by active-chronic inflammation and an adaptive immune response that fails to generate protective immunity. Several early studies on H. pylori immunity indicated that infection might induce T-cell hyporesponsiveness as both peripheral blood lymphocytes and gastric lymphocytes from H. pylori-positive patients were shown to respond to in vitro stimulation by H. pylori antigen with low cytokine secretion and proliferation relative to H. pylori-negative patients 22, 23. In mice, the in vitro H. pylori-specific recall response of T cells from experimentally infected animals is significantly weaker than mice immunized with H. pylori antigens 24, 25 and limiting the contribution of CD25+ cells can result in increased gastritis and reduced bacterial loads 26, 27.

In this study, we investigated the extent of the regulatory activity of CD4+CD25+ Treg cells during H. pylori infection in mice. We demonstrate here that the presence of Treg cells at the time of T-cell activation in the gastric mucosa results in the generation of a population of CD25H. pylori-specific anergic T cells. Although several reports have suggested that these H. pylori-specific CD25 T cells become responsive when CD25+ Treg cells are removed 26–28, our in vitro and in vivo data suggest that once stimulated in the presence of Treg cells, this hyporesponsive CD25 population remains anergic even in the absence of CD25+ Treg cells. In addition, we demonstrate that Treg cells are not only capable of inducing H. pylori-specific anergy in naïve CD25 cells but capable of down-regulating anti-H. pylori immunity in previously activated CD25 cells as well.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Foxp3 expression is increased in the gastric mucosa during H. pylori infection

Prior to investigating the role of CD25+ Treg cells during H. pylori infection, we confirmed the recruitment of these cells to the gastric mucosa in our model of H. pylori infection by measuring Foxp3 mRNA levels in gastric biopsies by quantitative PCR. Foxp3+ Treg cells have been previously reported to reside in the gastric mucosa after H. pylori infection as has been demonstrated in both human and murine tissues 17, 26. In our model, Foxp3 expression was increased almost threefold compared with naïve control mice 4 wk after challenge (data not shown). Although the increase was not statistically significant, we observed a comparable increase in two independent experiments.

Hyporesponsive CD25 cells are activated by high-dose IL-2 and antigen

We have previously demonstrated H. pylori-specific anergy in bulk spleen cells from H. pylori-infected mice that could be induced to produce IFN-γ in the presence of H. pylori and high-dose IL-2 (1000 U/mL) 24. To control for the possibility that other cell types in the spleen cell population might contribute to the responsiveness of the T cells, purified CD4+ cells were cultured in the presence or absence of high-dose IL-2. Figure 1 shows that the CD4+ cells from infected mice displayed activity comparable to naïve mice when stimulated by antigen alone. This activity was significantly less than observed for antigen-stimulated CD4+ cells from immune mice (p<0.001). The addition of IL-2 resulted in IFN-γ production by cells from infected mice comparable to the response of the cells from immune mice.

thumbnail image

Figure 1. H. pylori-specific hyporesponsiveness of CD4+ cells from infected mice is reversed in the presence of high-dose IL-2. CD4+ cells were isolated from the spleens of naïve, infected, or immunized mice and stimulated in vitro for 24 h with 10 μg/mL of H. pylori lysate (Hp), 1000 U/mL of rhIL-2, or both. T-cell activation was determined by production of IFN-γ as quantified by ELISA. *p<0.01 compared with immunized mice similarly treated. Each value shown is the mean of each group±the standard deviation.

Download figure to PowerPoint

We then assessed the role of CD25+ cells in preventing the T cells from responding to antigen exposure in vitro. Spleen cell populations from infected mice in which the CD25+ cells had been depleted by either complement-mediated killing or by fluorescence-activated cell sorting remained anergic when stimulated with H. pylori lysate antigen. However, activity could be recovered only when cells were simultaneously exposed to antigen and high-dose IL-2 (data not shown).

The cellular response of the infected mice was further analyzed by specifically measuring the activity of the CD4+ cells in the presence or absence of CD25+ cells. Initially, CD25+ cells were depleted from bulk spleen cells by complement-mediated killing and the CD25 cells were stimulated in vitro with H. pylori lysate antigen. Removal of the CD25+ cells did not result in the ability of the CD25 cells to respond to antigen (Fig. 2A). Flow cytometric assessment of cell populations depleted of CD25+ cells by complement-mediated lysis showed that less than 1% of the cells continued to stain positively for CD25 compared with approximately 11% for untreated cells (data not shown). We then purified CD4+ cells from spleens by positive selection and then further fractionated the cells into CD25+ and CD25 populations by affinity column isolation. The direct stimulation of CD4+ cells from naïve mice with H. pylori antigen generated low levels of activity (Fig. 1). Similar observations have been made by others 29. Therefore, we stimulated the fractionated cells with antigen-pulsed macrophages in which soluble antigen was removed by washing prior to co-culture with the fractionated spleen cells. CD4+CD25 cells failed to respond to H. pylori antigen but were induced to produce significantly greater quantities of IFN-γ when co-stimulated with high-dose IL-2 (p=0.008) (Fig. 2B).

thumbnail image

Figure 2. H. pylori-specific CD25 T cells remain hyporesponsive in the absence of CD25+ cells but are activated by the addition of exogenous high-dose IL-2. (A) Splenocytes isolated from H. pylori-infected mice were depleted of CD25+ cells by complement-mediated killing and stimulated with 10 μg/mL of H. pylori lysate antigen. (B) CD4+ cells from infected mice fractionated into CD25 or CD25+ T-cell populations by flow cytometry were stimulated with antigen pulsed macrophages in the presence or absence of high-dose (1000 U/mL) rhIL-2. IFN-γ was measured as a marker of T-cell activation. CD4+ cells from naïve mice were unfractionated. *p<0.01 compared with CD25 cells stimulated in a comparable manner. Each value shown is the mean of each group±the standard deviation.

Download figure to PowerPoint

CD4+CD25 cells from infected mice promote gastritis and reduce bacterial load

Since CD25 T cells from H. pylori-infected mice were hyporesponsive in vitro, we transferred these cells into immunodeficient rag1−/− mice prior to challenge with live H. pylori to measure their responsiveness in vivo in the absence of Treg cells. Since mice deficient in the rag1 enzyme lack mature T cells and B cells, the CD25 cells from the donor mice would be stimulated in the absence of any recipient mouse lymphocytes. We observed significant reductions in bacterial load in rag1−/− mice reconstituted with CD25 cells from naïve donors (Fig. 3). These mice had the lowest average number of bacteria and displayed the most severe inflammation, significantly greater than experimentally infected WT control mice (Fig. 4, p<0.006). Transfer of bulk CD4+ cells from H. pylori-infected mice into rag1−/− recipients resulted in a significant decrease in bacterial load compared with infected WT control mice (Fig. 3, p<0.05) and mild gastric inflammation (Fig. 4). Contrary to our expectations, we also observed decreased bacterial load in rag1−/− mice receiving CD4+CD25 cells from H. pylori-infected mice that was statistically equivalent to groups transferred with CD4+CD25 cells from naïve donors. Histologic examination of the gastric mucosa demonstrated that rag1−/− mice reconstituted with CD4+CD25 cells from H. pylori-infected mice responded with mild inflammation (Fig. 4).

thumbnail image

Figure 3. Adoptive transfer of bulk CD4+ or CD4+CD25 cells from H. pylori-infected donors results in decreased bacterial colonization. Bulk CD4+ or purified CD4+CD25 splenocytes isolated from naïve or H. pylori-infected mice were adoptively transferred into immunodeficient recipient mice on day 0. All mice were then challenged with live H. pylori on days 1 and 2. Mice were sacrificed on day 28 and the level of bacterial colonization was compared with infected WT control mice. Gray bars represent the median for each group. N=7 mice per group. *p<0.05 for all three transfer groups compared with infected WT control.

Download figure to PowerPoint

thumbnail image

Figure 4. Adoptive transfer of bulk CD4+ or CD4+CD25 cells from H. pylori-infected donors results in mild gastric inflammation. Bulk CD4+ or purified CD4+CD25 splenocytes isolated from naïve or H. pylori-infected mice were adoptively transferred into immunodeficient recipient mice on day 0. All mice were then challenged with live H. pylori on days 1 and 2. Mice were sacrificed on day 28 and the level of gastric inflammation was compared with infected WT control mice. N=7 mice per group.

Download figure to PowerPoint

Memory CD25 cells promote mild gastritis and persistent H. pylori colonization

Our data demonstrate that adoptive transfer of purified CD25 cells from H. pylori-infected donors into immunodeficient mice promotes inflammation that is hostile to H. pylori upon challenge (Figs. 3 and 4). This is in contrast to the hyporesponsiveness we observed in vitro. We next determined whether transfer of bulk CD25 cells from infected mice, as performed above, might include a memory and naïve population of CD25 cells capable of generating distinct responses. The CD25 population from H. pylori-infected mice was fractionated into CD45RBhi (naïve) and CD45RBlo (memory) populations and then separately transferred into rag1−/− recipients followed by challenge with H. pylori. As demonstrated in Fig. 5, H. pylori challenge of rag1−/− mice reconstituted with either bulk CD25 cells or purified CD4+CD25CD45RBhi naïve cells from H. pylori-infected mice resulted in significantly decreased levels of bacterial colonization relative to infected WT controls (p<0.05). In contrast, challenge of rag1−/− mice reconstituted with purified CD4+CD25CD45RBlo memory cells from H. pylori-infected mice failed to reduce bacterial colonization and had bacterial loads that were significantly greater than that from transfer of unfractionated CD25 cells or purified CD4+CD25CD45RBhi naïve cells from infected mice (p<0.05). The increased levels of bacteria in these mice were comparable to H. pylori-infected WT control mice. These data demonstrate that after in vivo challenge with H. pylori, the H. pylori-specific memory CD25 cells remain unresponsive despite the absence of CD25+ Treg cells resulting in persistent colonization.

thumbnail image

Figure 5. Memory CD4+CD25 cells from H. pylori-infected mice remain hyporesponsive after in vivo challenge. Bulk CD25 cells, CD25 CD45RBhi (naïve) cells, or CD25 CD45RBlo (memory) cells were prepared from the spleens of H. pylori-infected mice and transferred into immunodeficient recipient mice on day 0. Mice were challenged with live H. pylori on days 1 and 2 and sacrificed 28 days post-challenge. Gray bars represent the median for each group. N=6 mice per group.

Download figure to PowerPoint

Histologic examination of the gastric mucosa in these mice revealed an inverse relationship between the bacterial load and the degree of gastritis (Fig. 6). Although protected rag1−/− mice that had been reconstituted with CD25 bulk cells or CD25 naïve cells from infected donor mice displayed severe gastritis compared with infected WT control mice (p<0.003), mice reconstituted with H. pylori-specific CD25 memory cells had a heavy bacterial load and only minimal inflammation. The gastric inflammation was equivalent to infected WT controls and statistically reduced relative to mice reconstituted with bulk or naïve CD25 cells (p<0.003). These results are consistent with previous reports, suggesting that increased gastritis correlates with decreased bacterial colonization 24, 30, 31. These data suggest that after anergy is induced in H. pylori-specific CD25 cells, the CD25+ Treg cells are no longer necessary to maintain persistent infection.

thumbnail image

Figure 6. Memory CD4+CD25 cells induce mild gastritis in response to H. pylori infection. Bulk CD25 cells, CD25 CD45RBhi (naïve) cells, or CD25 CD45RBlo (memory) cells were prepared from the spleens of H. pylori-infected mice and transferred into immunodeficient recipient mice on day 0. Mice were challenged with live H. pylori on days 1 and 2 and sacrificed 28 days post-challenge. Gastric sections were removed and the degree of inflammation based on amount and degree of cellular infiltrate and changes in tissue architecture was measured. Transfer of splenocytes from infected WT mice served as a control. N=6–7 mice per group.

Download figure to PowerPoint

Treg cells suppress previously activated H. pylori-specific CD25 cells in vivo

The data presented above indicate that the presence of Treg cells during activation of H. pylori-specific CD25 cells is sufficient to induce a permanent state of anergy in some H. pylori-specific cells. Because H. pylori-infected mice can be protected by therapeutic immunization 32, 33 we next tested whether previously activated CD25 T cells are capable of promoting inflammation that reduces the bacterial load when introduced to WT mice either before or after H. pylori infection, a system in which the recipient mice have a full complement of CD25+ T cells. First, rag1−/− mice reconstituted with CD25 cells from naïve WT mice were challenged with H. pylori. Challenge of these mice resulted in a population of CD25 T cells associated with a significant reduction in bacterial load relative to our infected WT control group (Fig. 7, p<0.05). These CD25 cells were then isolated from the spleens of the immunodeficient mice and transferred again into either naïve WT mice or H. pylori-infected WT recipients that contain a normal repertoire of CD25+ Treg cells. The naïve recipients were challenged 1 wk after transfer. Mice were harvested at 4 wk post-transfer and gastric sections were removed to determine the level of bacterial colonization and gastric inflammation as described in Materials and methods.

thumbnail image

Figure 7. Previously activated CD25 cells are not capable of reducing bacterial load in WT recipients. CD25 splenocytes isolated from naïve WT donor mice were transferred into rag1−/−recipient mice on day 0 followed by challenge with live H. pylori on days 1 and 2. Bulk splenocytes isolated from these recipient mice were isolated on day 29 and transferred into either naïve or H. pylori-infected WT mice. Naïve mice were challenged with live H. pylori on days 35 and 36. Mice were sacrificed on day 64 and the level of bacterial colonization was compared with infected WT control mice. Gray bars represent the median for each group. N=5–7 mice per group.

Download figure to PowerPoint

Transfer of the CD25 cells into naïve WT mice that were challenged after adoptive transfer resulted in high levels of bacterial colonization, statistically greater than transfer into rag1−/− mice (p<0.05) and equivalent to that of infected control mice (Fig. 7). Similar results were observed when these CD25 cells were transferred into previously infected WT mice. Bacterial colonization levels remained high, not significantly distinct from infected WT controls. These data suggest that in addition to naïve CD25 cells, previously activated CD25 cells succumb to the regulatory control of CD25+ T cells in vivo.

Gastric sections were isolated from these mice to assess the level of inflammation. As shown in Fig. 8, infected WT control mice developed mild gastritis as we have observed previously. The control group of immunodeficient mice that had received CD25 cells associated with reduced bacterial loads resulted in significantly increased levels of inflammation relative to infected controls (p<0.03). Transfer of these CD25 cells into either infected WT or naïve WT mice followed by challenge resulted in statistically equivalent levels of gastritis (1.4±1.0, 1.3±1.2, respectively). Furthermore, both groups of WT recipients of the CD25 cells displayed mild gastritis statistically similar to infected controls. These data are in accordance with other models of H. pylori immunity in which high gastritis correlates with decreased colonization 24, 30, 31.

thumbnail image

Figure 8. Transfer of previously activated CD25 cells into WT recipients results in mild gastric inflammation. CD25 splenocytes isolated from naïve WT donor mice were transferred into rag1−/− recipients on day 0 followed by challenge with live H. pylori on days 0 and 2. Bulk splenocytes isolated from these recipients were isolated on day 29 and transferred into either naïve or H. pylori-infected WT mice. Naïve mice were challenged with live H. pylori on days 35 and 36. Mice were sacrificed on day 64 and gastric inflammation was measured as described in Materials and methods and compared with infected WT control mice. N=8 mice per group.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

This study was designed to investigate the extent of CD25+ T-cell regulatory activity during H. pylori infection. T cells from H. pylori-infected mice remained hyporesponsive to H. pylori antigens in vitro even in the absence of CD25+ Treg cells. The ability of high-dose IL-2 together with H. pylori antigen to activate these CD25 T cells is indicative of anergy. We confirmed these observations in vivo as transfer of H. pylori-specific CD25 memory cells fractionated from the T-cell population of infected mice failed to promote gastritis or reduce the bacterial load when transferred into rag1−/− mice that were subsequently challenged with H. pylori. Therefore, using both in vitro and in vivo techniques, we demonstrate here that CD25+ Treg cells induce a CD25 cell hyporesponse that helps give rise to the mild inflammation and persistent colonization observed during chronic H. pylori infection in mice.

Several reports have documented the involvement of CD4+CD25+ Treg cells in the host immune response of human subjects to H. pylori17, 26–28, 34, 35. Foxp3 expression, a surrogate marker for CD25+ Treg cells, has been documented in the gastric mucosa of infected humans 17, 26 and the number of CD25+ Treg cells is elevated in children compared with adults, which is consistent with reduced levels of gastritis in the pediatric population 35.

In vivo studies by Raghavan et al. 27 in the mouse model have shown that transfer of lymph node CD25 cells into immunodeficient nu/nu mouse recipients followed by H. pylori challenge results in severe gastritis and significantly decreased bacterial colonization relative to control nu/nu mice reconstituted with unfractionated lymph node cells. More recently, Rad et al. 26 depleted the CD25+ cell population in vivo using CD25-specific antibodies to achieve significantly increased gastritis in H. pylori-infected mice and a reduction in bacterial load. The results obtained by these two laboratories are similar to other models of pathogenic microbial infections in which CD25 cells activated in the absence of Treg cells mount a protective immune response 14, 15. These studies and others by Eaton et al. discussed below are significant in that they provide evidence that Treg cells are part of the natural host response to H. pylori infection and that they contribute to the persistence of H. pylori at the gastric mucosa. However, while these studies have investigated the potential of CD25 T cells to respond to H. pylori infection in the absence of Treg cells, they have not addressed the potential activity of CD25 T cells activated in the presence of Treg cells.

As discussed above, Raghavan et al. 27 transferred CD25 cells from naïve mice into immunodeficient recipients prior to challenge. Rad et al. 26 employed a different strategy in that CD25+ cells were depleted in vivo by application of CD25-specific antibodies. This depletion, however, was accomplished before infection was established and therefore the H. pylori-specific CD25 cells were activated in the absence of Treg cells 26. Eaton et al. have developed a useful model for studying host immunity against H. pylori infection using adoptive transfer of WT T cells into SCID mouse recipients that subsequently get challenged with H. pylori36–38. These mice develop severe gastritis and over time significantly reduce and in some cases eliminate the H. pylori load from the gastric mucosa 37. When fractionated populations were transferred into SCID mice, naïve T cells (CD45RBhigh) were capable of promoting severe gastritis that depleted the Helicobacter population while memory T cells (CD45RBlow) did not 36. Similar to the study by Raghavan et al., the donor cells used by Eaton et al. were obtained from naïve mice. The present report is distinct from these prior studies in that the donor populations employed to investigate the potential activity of CD25 T cells were obtained from H. pylori-infected mice and therefore the H. pylori-specific memory cells were originally activated in the presence of Treg cells.

In vitro analysis of CD25+ T-cell regulatory activity often is performed by depleting CD25+ T cells from the lymphocyte population to assess the ability of the CD25 cell to respond to antigen in their absence 7. Evidence that the CD25+ T cells are required for ongoing suppression is obtained when the CD25 T cells proliferate or produce cytokine in recall assays. We recently demonstrated that H. pylori-specific CD25 cells from infected mice remain hyporesponsive even in the absence of CD25+ T cells, an observation consistent with the presence of anergic cells 24. We relied upon the adoptive transfer model in immunodeficient mice developed by Eaton et al. to test these observations in vivo36–38. Initially, adoptive transfer of CD25 cells from H. pylori-infected WT donor mice resulted in significant gastritis and a reduction in bacterial load. This observation was contrary to expectations given the anergic response of these cells noted in vitro. Our subsequent transfer in which CD25 memory cells were compared with the CD25 naïve cells from H. pylori-infected mice demonstrated that the naïve CD25 cells were associated with bacterial clearance, whereas memory CD25 cells from the same donor mice remained hyporesponsive upon infection. Although Eaton et al. have described the role of CD45RBhigh cells in promoting inflammation capable of killing H. pylori36, this is the first study to demonstrate that the H. pylori-specific CD25 cells induced during infection of WT mice are in fact hyporesponsive and do not require ongoing suppression to remain down-regulated.

The present study also demonstrates that CD25 T cells activated in the absence of Treg cells and which reduce the bacterial load in the SCID adoptive transfer model become ineffective when transferred into WT mice. Transfer into WT recipients reintroduces dominant Treg cells into the response and results in mild inflammation and persistent infection. Using this population of CD25 cells, we were able to determine that Treg cells are not only capable of influencing naïve CD25 cells but also capable of rendering previously activated CD25 cells ineffective. These findings are in accordance with previous studies investigating the extent of CD25+ regulatory control in a murine model of colitis 39. Transfer of CD4+CD25+ Treg cells 4 wk after established CD4+CD45RBhi-induced colitis resulted in resolution of disease as early as 2 wk after transfer of Treg cells. Similarly, we demonstrate here that CD25+ Treg cells maintain the ability to influence previously activated responsive CD25 cells during H. pylori infection.

The ability of resident Treg cells to suppress previously activated donor CD25 T cells is in contrast with studies demonstrating that animals harboring an existing Helicobacter infection can be protected by therapeutic immunization 32, 33, 40. Therapeutic immunization studies indicate that activation of T cells under certain circumstances results in a population of T cells that is not influenced by the Treg cells that are part of the host response to infection. Therefore, either the nature or the frequency of proinflammatory T cells produced here in the absence of Treg must be different from the T-cell response induced by immunization. Further analysis will be required to characterize these two types of responses.

Several recent reports have begun to elucidate the development of the host T-cell response to H. pylori infection. This response consists, in part, of immunoregulatory CD25+ Treg cells that actively suppress other H. pylori-specific cells from promoting heightened inflammation 26, 27, 41. In the mouse model, we have demonstrated that the presence of CTLA-4 on Treg is necessary to suppress H. pylori-associated protective gastritis 24. The presentation of antigen by the gastric epithelium and the involvement of the co-receptor B7H1 may also be promoting the induction of suppressive CD4+CD25+ Treg cells that have been demonstrated in vitro to decrease the proliferative activity of activated T cells 42. Mechanistically, it appears that IL-10 production by Treg cells may play a role in the suppressive activity of these cells 36, 43, 44 although IL-10-deficient Treg cells were also capable of significantly reducing the proinflammatory effects of H. pylori-specific CD25 cells in an adoptive transfer model 41.

CD25+ Treg cells are typically associated with active suppression. The present study, however, demonstrates an alternate mechanism of immune down-regulation by this cell type. The observation that H. pylori-specific CD25 T cells remain unresponsive even in the absence of CD25+ T cells both in vitro and in vivo provides compelling evidence for the induction of anergy. These observations should enhance our understanding of H. pylori immunopathogenesis and may be relevant to our understanding of the immunopathogenesis of other microbial infections and the maintenance of immunologic homeostasis in the gastrointestinal tract.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Reagents

Complete cell media consisted of RPMI 1640 supplemented with 10% FBS (Gibco Life Sciences, Carlsbad, CA). MACS CD4+ cell purification reagents and columns were purchased from Miltenyi Biotech (Auburn, CA) and used according to the manufacturer's instructions. Low-tox M rabbit complement and Lymphocyte-M were purchased from Cedarlane Laboratories (Hornby, Ontario). Anti-CD28 antibody and IFN-γ ELISA reagents were purchased from ebioscience (San Diego, Ca) and IL-2 ELISA reagents purchased from R&D (Minneapolis, MN) were used according to the manufacturer's instructions. Anti-CD25, anti-CD45RB, and PE-conjugated anti-CD25 and anti-FCγRIII antibodies were purchased from BD Biosciences (Franklin Lakes, NJ). H. pylori lysates were prepared by probe sonication of H. pylori suspensions in PBS. Sonicate was sterile filtered using 0.2 μm acrodisc filters (Pall, Ann Arbor, MI). The Foxp3 assay on demand (Mm00475156_m1) was purchased from ABI (Foster City, CA).

Mice

Six-to-ten-week-old C57BL/6 female mice and lymphocyte-deficient rag1−/− male mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed under specific pathogen-free conditions in microisolator units. All studies involving the use of mice were reviewed and approved by the Case Western Reserve University Institutional Animal Care and Use Committee.

Bacteria

H. pylori Sydney Strain 1 (SS1) 45 was grown on Columbia agar (Difco Laboratories, Detroit, MI) supplemented with horse blood and antibiotics at 37°C for 96 h under microaerobic conditions (5% O2, 10% CO2). For inoculation of mice, bacteria were transferred to 10 mL Brucella broth (Difco Laboratories) supplemented with 10% FBS (Invitrogen, Carlsbad, CA) and amphotericin B (2.5 μg/mL). Liquid cultures were established in T25 flasks and maintained at 37°C with 5% CO2.

Infection/immunization

Infections were performed with H. pylori SS1 by gavage using flexible tubing on the end of an 18 G needle. Each mouse received 500 μL of an actively growing bacterial culture of at least 0.3 OD450 nm on two consecutive days. Immunization was performed by intranasal administration of 100 μg of H. pylori lysate plus 5 μg of cholera toxin adjuvant in 20 μL of PBS on days 0, 7, 14, and 28 as described previously 25.

Complement-mediated lysis of CD25+ cells

Single cell suspensions were prepared from spleens and red blood cells were removed by lysis in a hypotonic solution. Cells were resuspended in 2% FBS/PBS and incubated for 30 min at 4°C with anti-mouse CD25+ antibody at 0.65 μg per 1×107 cells. Cells were then washed and resuspended in PBS with Low-tox M rabbit complement (20:1 v/v) and incubated at 37°C for 45 min. Viable lymphocytes were concentrated using a Lymphocyte-M gradient followed by several washes in PBS. Deletion of CD25+ cells was confirmed by staining with PE-conjugated anti-CD25 antibody and assessment by flow cytometry.

Foxp3 quantitative PCR

The ABI Foxp3 assay was set up in accordance with the manufacturer's instructions and run against all the samples in the study using GAPDH as an endogenous control on a 384-well plate. RNA was accurately quantified using a nanodrop-1000 spectrophotometer (Nanodrop Industries). Archive cDNA was made for all samples by means of an RT reaction using the ABI high-Capacity cDNA archive kit and using similar amounts of total RNA as the starting material in a 100 μL reaction in an ABI 9700 PCR unit. Results were generated using ABI SDS software and are presented as relative fold changes versus a designated calibrator sample. Results include 95% confidence limits.

Isolation of CD25+ and CD25 cell populations by flow cytometry

CD4+ splenocytes were positively selected according to the manufacturer's instructions using the MACS CD4+ cell purification reagents and medi-MACS columns on a magnetic support. Purified CD4+ cells were incubated with FCγRIII for 15 min followed by incubation with PE-conjugated anti-CD25 antibody. Sorting of CD25+ and CD25 cells was performed using a BD FACSAria (Franklin Lakes, NJ) at the Flow Cytometry Core Facility of the Comprehensive Cancer Center of Case Western Reserve University.

In vitro recall assay

Bulk spleen cells or CD25 spleen cells from immunized or infected mice were prepared as described above and plated in 96-well plates in 200 μL complete media and stimulated with 10 μg/mL H. pylori lysate. Supernatants were removed at 36 h to determine the amount of IFN-γsecretion. Designated groups were also treated with high-dose (1000 U/mL) IL-2 for 1 h prior to stimulation with antigen. Similar assays were also performed with CD4+ spleen cells prepared by positive selection using the MACS CD4+ cell purification reagents and columns purchased from Miltenyi Biotech, and in some cases with CD4+ cells further fractionated into CD25+ and CD25 population by affinity isolation using a CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotech). Stimulation of CD4+ cells fractionated by CD25 status was accomplished with bone-marrow-derived macrophages from C57BL/6 mice. Briefly, bone marrow cells isolated from hind leg femurs and tibias were grown in DMEM supplemented with 10% FBS and supplemented with 20% GM-CSF-conditioned media from Ladmac cell culture. Each well was seeded with 1×104 macrophages and pulsed with 10 μg/mL H. pylori lysate antigen for 6 h. The macrophages were washed three times and then the fractionated cells were added to the wells for stimulation as described above.

Evaluation of inflammation and CFU determination

For all adoptive transfer studies, recipient mice were sacrificed 28 days post-infection by CO2 asphyxiation and gastric biopsies from the greater curvature of the stomach were collected to assess the degree of gastritis and bacterial load. To determine gastric inflammation, a biopsy strip was fixed in 10% buffered formalin. H&E staining was performed at the Willard Alan Bernaum Cystic Fibrosis Research Center core facility at the CWRU School of Medicine (Cleveland, OH). As previously described 46, 47 the area of the tissue section displaying the most severe inflammation was evaluated blindly and assigned a global score from 0–5 based on the following parameters: 0, no significant lesions; 1, mild infiltrate of inflammatory cells, typically along the base of the glands; 2, larger focus of inflammation extending between glands and/or in submucosa; 3, patch(es) of inflammation extending between glands toward the lumen and in the underlying submucosa. Moderate mucous cell metaplasia and mild-to-moderate epithelial hyperplasia may be present. 4, intense transmucosal inflammatory infiltrate extending across the field, distorting glandular architecture, marked epithelial hyperplasia and extensive mucous cell metaplasia often present; 5, extensive mucosal and submucosal inflammation with disruption of glandular architecture and ulceration. To determine bacterial colonization, a biopsy strip was placed in a pre-weighed tube of 200 μL Columbia broth, the wet weight was determined, and the tissue was homogenized using a disposable pellet pestle (Kontas Glass Company, Vineland, NJ). Serial dilutions were prepared and 10 L aliquots were plated for growth as described above. Bacterial load was determined as CFU/gram of stomach tissue.

Adoptive transfer studies

Transfer of CD25 cells to rag1−/− mice (Figs. 3 and 4): Bulk CD4+ or CD4+CD25 cells were resuspended in PBS and 2×106 cells were injected i.p. into each rag1−/− mouse on day 0. Mice were challenged with H. pylori SS1 on days 1 and 2.

Transfer of naïve or memory CD25 cells into rag1−/− mice (Figs. 5 and 6): CD4+CD25 bulk, CD4+CD25CD45RBhi (brightest 20%) or CD4+CD25CD45RBlo (dullest 10%) cells were resuspended in PBS and 1.5×105 cells were injected i.p. into each rag1−/− mouse on day 0. On days 1 and 2, mice were infected with H. pylori SS1.

Transfer of CD25 cells from reconstituted rag1−/− mice into WT mice (Figs. 7 and 8): On day 0, 4.5×106 CD25 spleen cells were injected i.p. into rag1−/− recipient mice. Mice were challenged with H. pylori SS1 on days 1 and 2. On day 29, mice were sacrificed and splenocytes were removed and prepared for a second round of adoptive transfer by i.p. injection of 1×107 bulk splenocytes (16% CD4+ measured by flow staining) into naïve or infected WT mice. Naïve recipients were then challenged on days 35 and 36.

Statistics

Differences between experimental groups in each experiment were evaluated by Student's t-test. Differences were considered statistically significant if p values were less than 0.05.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

This research was supported by NIH grants AI055710 (T.G.B.) and DK046461 (S.J.C.) and by the Flow Cytometry Core Facility of the Comprehensive Cancer Center of Case Western Reserve University and University Hospitals of Cleveland grant P30 CA43703.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References