Transient activation of mucosal effector immune responses by resident intestinal bacteria in normal hosts is regulated by interleukin‐10 signalling

Interleukin‐10 (IL‐10) is a key regulator of mucosal homeostasis. In the current study we investigated the early events after monoassociating germ‐free (GF) wild‐type (WT) mice with an Escherichia coli strain that we isolated previously from the caecal contents of a normal mouse housed under specific pathogen‐free conditions. Our results show that interferon‐γ (IFN‐γ) secreted by mesenteric lymph node (MLN) cells from both IL‐10 deficient mice and WT mice, stimulated ex vivo with E. coli lysate, was dramatically higher at day 4 after monoassociation compared with IFN‐γ secreted by cells from GF mice without E. coli colonization. Production of IFN‐γ rapidly and progressively declined after colonization of WT but not IL‐10‐deficient mice. The E. coli lysate‐stimulated WT MLN cells also produced IL‐10 that peaked at day 4 and subsequently declined, but not as precipitously as IFN‐γ. WT cells that express CD4, CD8 and NKp46 produced IFN‐γ; WT CD4‐positive cells and B cells produced IL‐10. Recombinant IL‐10 added to E. coli‐stimulated MLN cell cultures inhibited IFN‐γ secretion in a dose‐dependent fashion. MLN cells from WT mice treated in vivo with neutralizing anti‐IL‐10 receptor antibody produced more IFN‐γ compared with MLN cells from isotype control antibody‐treated mice. These findings show that a resident E. coli that induces chronic colitis in monoassociated IL‐10‐deficient mice rapidly but transiently activates the effector immune system in normal hosts, in parallel with induction of protective IL‐10 produced by B cells and CD4⁺ cells that subsequently suppresses this response to mediate mucosal homeostasis.     

CD47-deficient mice have decreased production of intestinal IgA following oral immunization but a maintained capacity to induce oral tolerance

Signal regulatory protein α (SIRPα/CD172a), expressed by myeloid cells including CD11b(+) dendritic cells, interacts with ubiquitously expressed CD47 to mediate cell-cell signalling and therefore, may be pivotal in the development of tolerance or immunity. We show that in mice deficient in CD47 (CD47(-/-) ) the cellularity in gut-associated lymphoid tissues is reduced by 50%. In addition, the frequency of CD11b(+) CD172a(+) dendritic cells is significantly reduced in the gut and mesenteric lymph nodes, but not in Peyer's patches. Activation of ovalbumin (OVA)-specific CD4(+) T cells in the mesenteric lymph nodes after feeding OVA is reduced in CD47(-/-) mice compared with wild-type however, induction of oral tolerance is maintained. The addition of cholera toxin generated normal serum anti-OVA IgG and IgA titres but resulted in reduced intestinal anti-OVA IgA in CD47(-/-) mice. Replacing the haematopoietic compartment in CD47(-/-) mice with wild-type cells restored neither the cellularity in gut-associated lymphoid tissues nor the capacity to produce intestinal anti-OVA IgA following immunization. This study demonstrates that CD47 signalling is dispensable for oral tolerance induction, whereas the expression of CD47 by non-haematopoietic cells is required for intestinal IgA B-cell responses. This suggests that differential CD4 T cell functions control tolerance and enterotoxin-induced IgA immunity in the gut.     

Alcohol-induced gastritis prevents oral tolerance induction in mice

Despite several reports on the immunological relationship between inflammatory bowel diseases and immunoregulatory mechanisms in the gut, systematic studies addressing the impact of inflammatory processes in the gastric mucosa on events, such as oral tolerance, are still limited. Herein, we report the establishment of a novel murine model of gastritis induced by short-term administration of ethanol. The major immumological features of this clinical entity are characterized, as well as its impact on the induction of oral tolerance. Our data demonstrate that ethanol ingestion during 4 consecutive days triggered an acute inflammatory reaction in the stomach referred as ethanol-induced gastritis and characterized by hyperaemia, oedema and mixed mononuclear/polymorphonuclear cell infiltrate. Besides local immunological changes, such as high levels of gastric interleukin (IL)-4 and interferon (IFN)-gamma, systemic alterations are also observed, including increased IL-4 synthesis, enhanced levels of serum IgE and absence of IL-10 production by spleen cells. Moreover, ethanol-induced gastritis prevents oral tolerance induction to ovalbumin (OVA) as demonstrated by unaltered anti-OVA humoral and cellular immune responses in treated animals. Tissue eosinophilia after footpad immunization with OVA suggests that oral treatment with ethanol induced an allergic-type reaction. Taken together, our findings indicate that short-term ethanol ingestion is associated with gastric inflammatory events able to break immunoregulatory mechanisms that maintain mucosal homeostasis and oral tolerance.     

Amorphous nanosilica particles block induction of oral tolerance in mice

The mucosal immune system is exposed to non-self antigens in food and the gut microbiota. Therefore, the recognition of orally ingested non-self antigens is suppressed in healthy individuals to avoid excessive immune responses in a process called “oral tolerance”. The breakdown of oral tolerance has been cited as a possible cause of food allergy, and amorphous silica nanoparticles (nSP) have been implicated in this breakdown. As nSP are widely used in foodstuffs and other products, exposure to them is increasing; thus, investigations of any effects of nSP on oral tolerance are urgent. This study evaluated the effects of nSP30 (particle diameter =?39?nm) on immunological unresponsiveness induced in mice with oral ovalbumin (OVA). Specifically, production of OVA-specific antibodies, splenocyte proliferation in response to OVA, and effects on T-helper (T_H)-1, T_H2, and T_H17 responses (in terms of cytokine and IgG/IgE subclass expression) were evaluated. nSP30 increased the levels of OVA-specific IgG in OVA-tolerized mice and induced the proliferation of OVA-immunized splenocytes in response to OVA in a dose-related manner. nSP30 also increased the expression of OVA-specific IgG₁, IgE, and IgG_2a, indicating stimulation of the T_H1 and T_H2 responses. The expression of interferon (IFN)-γ (T_H1), interleukin (IL)-4 and IL-5 (T_H2), and IL-17 (T_H17) was also stimulated in a dose-related manner by nSP30 in splenocytes stimulated ex vivo with OVA. The induction of tolerance by OVA, the production of anti-OVA IgG antibodies, and proliferation of splenocytes in response to OVA was inhibited by nSP30 in conjunction with OVA and was dose-related. The nSP30 enhanced T_H1 and T_H2 responses that might prevent the induction of oral tolerance. Overall, this study showed that the abrogation of OVA-induced oral tolerance in mice by exposure to nSP30 was dose-related and that nSP30 stimulated T_H1, T_H2, and T_H17 responses.     

Tolerosome-induced oral tolerance is MHC dependent

Oral administration of a protein antigen generates a serum factor that induces tolerance when transferred into naïve recipients. This serum factor has been described in rats as consisting of exosome‐like structures or tolerosomes, which express major histocompatibility complex class II molecules (MHCII) and mediate antigen‐specific tolerance. In this study, we investigated the functions of serum‐derived tolerosomes both in vivo and in vitro. Tolerosomes were purified from the 100 000 g pellet fraction of serum from ovalbumin (OVA)‐fed mice. When transferred into naïve recipient mice, the tolerosomes mediated OVA‐specific tolerance. We also found that tolerosomes from OVA‐fed mice induced the activation of OVA‐specific T cells both in vivo and in vitro. The inoculation of severe combined immunodeficiency (SCID) mice with an interferon‐γ‐producing cell line normalized the expression of MHCII in the intestinal epithelial cells and restored their ability to generate tolerosomes. Syngeneic but not allogeneic transfer of tolerosomes from OVA‐fed donors induced tolerance in the recipients. Our results show that tolerosomes can be isolated from mouse serum, that tolerosome‐induced oral tolerance requires MHCII expression in intestinal epithelial cells, and that tolerosomes are functional only in syngeneic recipients.     

Long-term tolerance to skin commensals is established neonatally through a specialized dendritic cell subgroup

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Peripheral tolerance in T cell receptor-transgenic mice: Evidence for T cell anergy

T cell tolerance can be induced in adult mice by injection of soluble antigenic peptide. The underlying mechanism has been difficult to establish in normal mice due to the low precursor frequency of T cells specific for any given antigen. Therefore, we examined peripheral tolerance in mice transgenic for a T cell receptor specific for a cytochrome c peptide bound to I-E^k. Antigen-specific hyporesponsiveness could be induced in the transgenic mice. We followed the transgene-bearing T cells with a clonotypic monoclonal antibody and found similar numbers of clonotypic T cells in tolerized and control mice. To prevent de novo differentiation of T cells we analyzed thymectomized mice in which antigen-specific hyporesponsiveness was induced. Our analysis of thymectomized transgenic mice showed that antigen-specific T cell hyporesponsiveness following injection of peptide intravenously is not caused by gross elimination of T cells. These data provide evidence for the role of anergy in peripheral tolerance.     

Innate profiles of cytokines implicated on oral tolerance correlate with low- or high-suppression of humoral response

Oral tolerance (OT) is being studied with great interest because of its therapeutic potential in allergy and autoimmunity. In the present study, two mouse strains with extreme phenotypes of OT susceptibility (TS) or resistance (TR) to ovalbumin (OVA) were used to demonstrate whether the tr and ts genes, cumulated during 18 generations of bi‐directional genetic selection, influence expression of immunobiological traits in naive or antigen‐gavaged TR/TS mice. The difference in anti‐OVA titres was 2048‐fold between OVA‐gavaged TS and TR mice. Tolerance susceptibility to OVA gavage in individuals from a (TS × TR)F₂ population was 24% high‐susceptibility, 62% low‐susceptibility and 14% non‐tolerant. Different antigens, unrelated to OVA, were tested by gavage and TS mice were generally susceptible while TR mice were resistant. The stability of TS and TR phenotypes was not affected by the use of strict protocols of intraperitoneal immunization or feeding over 30 consecutive days. The levels of interleukin‐2 (IL‐2), IL‐4, interferon‐γ and IL‐10 cytokines evaluated in concanavalin A‐stimulated spleen cells from naive mice and in OVA‐stimulated spleen cells from OVA‐gavaged mice were higher in TS mice. Interleukin‐10 was up‐regulated in OVA‐gavaged TS mice and down‐regulated in TR mice. In naive mice, the percentage of CD4⁺ CD25⁺ and CD4⁺ Foxp3⁺ spleen cells and IL‐10 expression by CD4⁺ cells was significantly higher in TS mice. These results indicate that regulation of IL‐10 expression could be an important factor contributing to the mechanisms controlling OT susceptibility, and that the OT responses of TR and TS individuals strongly correlate with their innate potential to secrete this cytokine.     

Accumulation of immunoglobulin-containing cells in the gut mucosa and presence of faecal immunoglobulin in severe combined immunodeficient (scid) mice with T cell-induced inflammatory bowel disease (IBD)

Scid mice transplanted either with a gut wall graft or with low numbers of purified CD4⁺ T cells from immunocompetent syngeneic donor mice show clinical signs of IBD 3–4 months post-transplantation. The disease is mediated by mucosa-infiltrating CD4⁺ TCRαβ⁺ T cells. The pathology of 52 individual colon segments obtained from 20 gut wall- or CD4⁺ T cell-transplanted diseased scid mice was evaluated by histology and the numbers of infiltrating immunoglobulin-containing cells were determined. In particular, cells positive for IgM, IgA and non-inflammatory immunoglobulin isotypes such as IgG1 and IgG2b were found to accumulate in colon segments displaying the most severe histopathology, including inflammatory cellular infiltration, epithelial hyperplasia and ulcerative lesions. Compared with colon segments of normal C.B-17 mice, the lesional scid colon shows increased levels of cells positive for the IgG classes. Faecal extracts of the CD4⁺ T cell-transplanted scid mice revealed the presence of all six murine immunoglobulin isotypes. Disease progression was accompanied by an increased level of excreted IgM and IgG3 and decreased levels of IgA. It is concluded that locally secreted immunoglobulins may play an immunomodulating role in the pathological changes observed in the present model of T cell-induced inflammatory bowel disease.     

Gut T cell receptor‐γδ+ intraepithelial lymphocytes are activated selectively by cholera toxin to break oral tolerance in mice

The gut immune system is usually tolerant to harmless foreign antigens such as food proteins. However, tolerance breakdown may occur and lead to food allergy. To study mechanisms underlying food allergy, animal models have been developed in mice by using cholera toxin (CT) to break tolerance. In this study, we identify T cell receptor (TCR)‐γδ⁺ intraepithelial lymphocytes (IELs) as major targets of CT to break tolerance to food allergens. TCR‐γδ⁺ IEL‐enriched cell populations isolated from mice fed with CT and transferred to naive mice hamper tolerization to the food allergen β‐lactoglobulin (BLG) in recipient mice which produce anti‐BLG immunoglobulin (Ig)G1 antibodies. Furthermore, adoptive transfer of TCR‐γδ⁺ cells from CT‐fed mice triggers the production of anti‐CT IgG1 antibodies in recipient mice that were never exposed to CT, suggesting antigen‐presenting cell (APC)‐like functions of TCR‐γδ⁺ IELs. In contrast to TCR‐αβ⁺ cells, TCR‐γδ⁺ IELs bind and internalize CT both in vitro and in vivo. CT‐activated TCR‐γδ⁺ IELs express major histocompatibility complex (MHC) class II molecules, CD80 and CD86 demonstrating an APC phenotype. CT‐activated TCR‐γδ⁺ IELs migrate to the lamina propria, where they produce interleukin (IL)‐10 and IL‐17. These results provide in‐vivo evidence for a major role of TCR‐γδ⁺ IELs in the modulation of oral tolerance in the pathogenesis of food allergy.     

Estren-mediated inhibition of T lymphopoiesis is estrogen receptor-independent whereas its suppression of T cell-mediated inflammation is estrogen receptor-dependent

Estrogen has extensive effects on the immune system. The aim of the present experiments was to compare the effects of 17beta-estradiol (E2) and 4-estren-3alpha,17beta-diol (estren) on T lymphopoiesis and T cell-dependent inflammation. In order to investigate the role of estrogen receptors (ER) in the effects of E2 and estren on the immune system, ER knock-out mice lacking both ERalpha and ERbeta (DERKO) were used. T lymphopoiesis and T cell-dependent inflammation were studied by investigating thymus cellularity, the delayed-type hypersensitivity (DTH) reaction, CD4(+) T cells in spleen and serum levels of interleukin (IL)-6. As expected, the presence of ERs was mandatory for all the effects of E2. In contrast, treatment with estren reduced thymus cellularity in ER knock-out mice, indicating an effect through ER-independent pathways. Interestingly, estren suppressed only DTH, the frequency of CD4(+) T cells in spleen and serum levels of IL-6 in wild-type (WT) mice, but not in mice lacking ERs. Thus, our study is the first to show that estren inhibits T lymphopoiesis via ER-independent pathways, whereas its suppressive effects on inflammation are ER-dependent.     

Effect of in vivo administration of anti-CTLA-4 monoclonal antibody and IL-12 on the induction of low-dose oral tolerance

Oral tolerance has been characterized as an immunological hyporesponsiveness to fed antigen. Previous studies have suggested that high-dose oral tolerance involves the preferential interaction of B7 with CTLA-4 on the T cell. To determine whether similar mechanisms are involved in the induction of low-dose oral tolerance, mice were treated with anti-CTLA-4 monoclonal antibody (MoAb), with or without IL-12, at the time of feeding. Results showed that anti-CTLA-4 MoAb alone failed to restore cellular proliferation, antibody titres and IFN-gamma levels; however, IL-4 cytokine levels in OVA-fed mice were partially restored. In contrast, administration of IL-12 along with anti-CTLA-4 MoAb to mice during feeding completely prevented the suppression of Th1 immune responses, as shown by increased serum IgG2a titres, IFN-gamma production and cell proliferation. These results suggest that blocking B7-CTLA-4 interactions in the presence of IL-12 prevents the induction of low-dose oral tolerance at the Th1 cell level.     

Specific oral tolerance induction in food allergy

Specific oral tolerance in food allergy can be induced by oral administration of the offending food, starting with very low dosages, gradually increasing the daily dosage up to an amount equivalent to a usually relevant dose for daily intake, followed up by a daily maintenance dose. Unfortunately, the body of scientific evidence concerning specific oral tolerance induction (SOTI) is still rather poor. Following a couple of case reports, only a few studies on a limited number of patients including different allergens are available. So far, no placebo-controlled, long-term study has been published. Concerning the underlying immunological mechanism, a limited number of studies have reported on changes in antibody production, and more recently on the role of different T-cell populations. The individual pattern of clinical reaction during SOTI seems to vary considerably between patients and from allergen to allergen. Arguments in favour of SOTI are the safety for an inadvertent intake of the offending food and the increased quality of life. Arguments against SOTI are the necessity for a regular intake and possible long-term compliance problems. Indications to consider SOTI in the future might be (i) importance of the incriminated food for the individual nutritional regimen, (ii) avoidance of the corresponding food cannot be assured and (iii) persistent severe food allergy. However, before SOTI can be recommended for the daily praxis, more studies are warranted to clarify whether certain patients may profit from SOTI and to understand the underlying mechanism.