Mechanisms Of Oral Tolerance Research Articles

Oral Tolerance in Birds and Mammals: Digestive Tract Development Determines the Strategy

Primary Audience: Researchers, Immunologists, Veterinarians SUMMARY Development of oral (or airway) vaccines to be used for the vaccination of poultry is clearly a necessity because the process of individual bird vaccination is time-consuming and costly. One of the key issues to resolve in this context is whether the vaccine is protected from oral tolerance. Oral tolerance is a form of immune tolerance that is induced following the feeding of protein antigens dissolved in water or aqueous buffers. The issue is particularly relevant in cases in which nonpathogenic subunit protein vaccines are considered as vaccines. These short peptides/proteins contain the immunogenic moiety of the parent pathogen, but they lack the capacity to induce inflammation, and an antigen in the absence of inflammation might induce tolerance. The following review summarizes our main observations on mechanisms of oral tolerance in the chicken, compares these findings to what is known in the mammal, and provides some insight for developing strategies for oral vaccination of poultry.

Mechanisms of oral tolerance revisited

Oral tolerance induction is thought to depend on special antigen presenting cells in the gut. A new report in the previous issue of Arthritis Research & Therapy supports this idea by demonstrating that indoleamine 2,3-dioxygenase-expressing dendritic cells in Peyer's patches from orally tolerized mice suppress T-cell responses via the generation of CD4+CD25+ regulatory T cells. This finding provides novel input into the mechanisms of oral tolerance that could further facilitate its use for the treatment of autoimmunity and chronic inflammatory reactions.

Oral Tolerance Increased the Proportion of CD8+ T Cells in Mouse Intestinal Lamina Propria

Oral tolerance is defined by the inhibition of immune responsiveness to a protein previously exposed via the oral route. Protein antigens exposed via the oral route can be absorbed through the mucosal surfaces of the gastrointestinal tract and can make physical contact with immune cells residing in the intestinal lamina propria (LP). However, the mechanisms of oral tolerance and immune regulation in the intestines currently remain to be clearly elucidated. Methods: In order to determine the effect of oral protein antigen intake (ovalbumin, OVA) on the intestinal LP, we assessed the expression profile of the T cell receptor and the co-receptors on the cells from the intestines of the tolerant and immune mouse groups. Results: We determined that the proportion of OVA-specific B cells and γδ T cells had decreased, but the CD8αβ and D8αα T cells were increased in the LP from the tolerant group. The proportion of CD8＋ T cells in the spleen did not evidence any significant differences between treatment groups. Conclusion: These results indicate that CD8＋ T cells in the intestinal LP may perform a regulatory role following antigen challenge via the oral route.

ZP3 peptides administered orally suppress murine experimental autoimmune ovarian disease

Experimental autoimmune ovarian disease (AOD) is a T cell-mediated chronic inflammatory disease that may lead to premature ovarian failure. Autoimmune disease can be suppressed by oral administration of autoantigens leading to tolerance. One of the major mechanisms of oral tolerance is induction of regulatory CD4 + T cells that can mediate active suppression by producing immunomodulatory cytokines. However, the role of oral tolerance as a treatment for experimental AOD has received little attention. Therefore, the purpose of this study was to examine the conditions necessary to produce oral tolerance in experimental AOD in B6AF1 female mice. In this study, mice received different doses of peptides of the mouse zona pellucida 3 (pZP3) via gastric intubation for 7 times. After 4 times of oral administration, AOD was induced by immunization with pZP3. The optimal tolerating regimen for oral administration of pZP3 in mice was 10 μg, which decreased morbidity of oophoritis compared to the control group. In this moderate-dose therapeutic group (MD), alterations in the estrous cycle were normalized and CD4 + T cells that were CD25 + increased while those that were CD25 decreased. The severity of autoimmune oophoritis and the titer of ZP autoantibodies were also significantly reduced. These findings suggest that oral administration of pZP3 may be successfully used as an oral tolerance strategy for suppression of AOD.

Update on Coeliac Disease and Type 1 Diabetes Mellitus in Childhood

The increased prevalence of coeliac disease (CD) among children with type 1 diabetes mellitus (DM1) implies that there is more than a simple association. A link between the gut immune system and DM1 has been suggested both in animal models and in humans. We review the literature on the epidemiology and genetic and clinical aspects shared by these two diseases and speculate on the role of gluten on the possible relationship between CD and DM1, on the basis of recent animal and human studies. The data suggest a failure in oral tolerance mechanisms in DM1 other than that in CD. It remains to be understood why only a small proportion of patients with DM1 proceed to the production of coeliac-associated antibodies and to overt enteropathy.

Oral administration of type-II collagen peptide 250–270 suppresses specific cellular and humoral immune response in collagen-induced arthritis

Oral antigen is an attractive approach for the treatment of autoimmune and inflammatory diseases. Establishment of immune markers and methods in evaluating the effects of antigen-specific cellular and humoral immune responses will help the application of oral tolerance in the treatment of human diseases. The present article observed the effects of chicken collagen II (CII), the recombinant polymerized human collagen II 250–270 (rhCII 250–270) peptide and synthesized human CII 250–270 (syCII 250–270) peptide on the induction of antigen-specific autoimmune response in rheumatoid arthritis (RA) peripheral blood mononuclear cells (PBMC) and on the specific cellular and humoral immune response in collagen-induced arthritis (CIA) and mice fed with CII (250–270) prior to immunization with CII. In the study, proliferation, activation and intracellular cytokine production of antigen-specific T lymphocytes were simultaneously analyzed by bromodeoxyuridine (BrdU) incorporation and flow cytometry at the single-cell level. The antigen-specific antibody and antibody-forming cells were detected by ELISA and ELISPOT, respectively. CII (250–270) was found to have stimulated the response of specific lymphocytes in PBMC from RA patients, including the increase expression of surface activation antigen marker CD69 and CD25, and DNA synthesis. Mice, fed with CII (250–270) before CII immunization, had significantly lower arthritic scores than the mice immunized with CII alone, and the body weight of the former increased during the study period. Furthermore, the specific T cell activity, proliferation and secretion of interferon (IFN)-γ in spleen cells were actively suppressed in CII (250–270)-fed mice, and the serum anti-CII, anti-CII (250–270) antibody activities and the frequency of specific antibody-forming spleen cells were significantly lower in CII (250–270)-fed mice than in mice immunized with CII alone. These observations suggest that oral administration of CII (250–270) can suppress the cellular and humoral immune response in collagen-induced arthritis, and the simultaneous analysis of antigen-specific cellular and humoral immune responses at single-cell level will help the understanding of the oral tolerance mechanisms in CIA and the development of innovative therapeutic intervention for RA.

Is It Dietary Insulin?

In humans the primary trigger of insulin-specific immunity is a modified self-antigen, that is, dietary bovine insulin, which breaks neonatal tolerance to self-insulin. The immune response induced by bovine insulin spreads to react with human insulin. This primary immune response induced in the gut immune system is regulated by the mechanisms of oral tolerance. Genetic factors and environmental factors, such as the gut microflora, breast milk-derived factors, and enteral infections, control the development of oral tolerance. The age of host modifies the immune response to oral antigens because the permeability of the gut decreases with age and mucosal immune response, such as IgA response, develops with age. The factors that control the function of the gut immune system may either be protective from autoimmunity by supporting tolerance, or they may induce autoimmunity by abating tolerance to dietary insulin. There is accumulating evidence that the intestinal immune system is aberrant in children with type 1 diabetes (T1D). Intestinal immune activation and increased gut permeability are associated with T1D. These aberrancies may be responsible for the impaired control of tolerance to dietary insulin. Later in life, factors that activate insulin-specific immune cells derived from the gut may switch the response toward cytotoxic immunity. Viruses, which infect beta cells, may release autoantigens and potentiate their presentation by an infection-associated "danger signal." This kind of secondary immunization may cause functional changes in the dietary insulin primed immune cells, and lead to the infiltration of insulin-reactive T cells to the pancreatic islets.

Oral immunogenicity of the plant proteinase bromelain

Bromelain is a natural mixture of proteolytic enzymes derived from pineapple stem that has been shown to have anti-inflammatory activity when administered orally. Although most proteins given orally without adjuvant (e.g., food) result in tolerance, we previously reported that long-term oral exposure to bromelain stimulated the development of high serum anti-bromelain antibody titers. The purpose of these studies was to further investigate the mechanisms responsible for the immunogenicity of oral bromelain. Results showed that repeated exposure was required for development of anti-bromelain antibodies, with strong antibody responses in all mice that received at least 12 doses of bromelain either orally or intragastrically over 3–6 weeks. Proteolytic activity was required for strong oral immunogenicity in the absence of conventional adjuvant, with strong serum antibody responses generated against proteolytically active bromelain and trypsin, but not against ovalbumin, lysozyme, or inactivated bromelain. Significantly higher anti-bromelain antibody titers were seen in IL-10-deficient versus wild-type mice, suggesting that simultaneous treatments that decrease IL-10 activity may further enhance systemic antibody responses following oral exposure. The antibodies generated did not affect the proteolytic activity of bromelain. The data demonstrate that proteolytically active antigens such as bromelain can stimulate both systemic and mucosal immune responses following repeated oral exposure. Further studies of the mechanisms involved in generation of immune responses following oral exposure to proteolytically active antigens can lead to a better understanding of mechanisms of oral tolerance and to the development of novel adjuvants for oral vaccines.

Oral CD3-specific antibody suppresses autoimmune encephalomyelitis by inducing CD4+CD25−LAP+ T cells

A major goal of immunotherapy for autoimmune diseases and transplantation is induction of regulatory T cells that mediate immunologic tolerance. The mucosal immune system is unique, as tolerance is preferentially induced after exposure to antigen, and induction of regulatory T cells is a primary mechanism of oral tolerance. Parenteral administration of CD3-specific monoclonal antibody is an approved therapy for transplantation in humans and is effective in autoimmune diabetes. We found that orally administered CD3-specific antibody is biologically active in the gut and suppresses autoimmune encephalomyelitis both before induction of disease and at the height of disease. Orally administered CD3-specific antibody induces CD4+ CD25- LAP+ regulatory T cells that contain latency-associated peptide (LAP) on their surface and that function in vitro and in vivo through a TGF-beta-dependent mechanism. These findings identify a new immunologic approach that is widely applicable for the treatment of human autoimmune conditions.

Defect of oral tolerance in NC/Nga mice

The NC/Nga mouse is a model animal for human atopic dermatitis. In this study, we investigated oral tolerance induction in NC/Nga mice. In BALB/c mice, oral administration of ovalbumin (OVA) resulted in suppression of both OVA-specific T and B cell responses induced by OVA immunization. In NC/Nga mice, OVA-induced antigen (Ag) -specific T and B cell responses were significantly less than those in BALB/c mice. Furthermore, oral administration of OVA did not suppress OVA-specific immunoresponses in NC/Nga mice. We further examined antibody (Ab) response against food Ag by feeding mice an experimental diet that contained OVA or casein as a protein source. The level of serum OVA or casein-specific IgG was significantly higher in NC/Nga mice than in BALB/c mice. These results indicate that NC/Nga mice have a defect in the induction of oral tolerance. NC/Nga mice can therefore be used as a model for investigating the mechanism of oral tolerance.

Oral immune tolerance mediated by suppressor T cells may be responsible for the poorer prognosis of foregut cancers

The poor prognosis of foregut cancers might, in part, be due to the immune tolerising effect of tumour antigens which are shed into the gastrointestinal tract and processed by the gut immune system. This would create a tumour specific tolerance without compromise of global immune functions. Experimental data shows that orally fed cancer tissue induces a non cross reactive attenuation of the cellular anti tumour host responses and confers a growth advantage specific to individual cancers. Although the cellular basis of such pro-tumourogenic responses has yet to be established, it is likely, based on studies of oral tolerance mechanisms, that recruitment of immune suppressive T cells (T(regs)) may be responsible. Abrogation of oral immune tolerance to the tumour by immune based therapy could represent a significant advance in the management of upper gastrointestinal cancers.

Oral tolerance in the absence of naturally occurring Tregs

Mucosal tolerance prevents pathological reactions against environmental and food antigens, and its failure results in exacerbated inflammation typical of allergies and asthma. One of the proposed mechanisms of oral tolerance is the induction of Tregs. Using a mouse model of hyper-IgE and asthma, we found that oral tolerance could be effectively induced in the absence of naturally occurring thymus-derived Tregs. Oral antigen administration prior to i.p. immunization prevented effector/memory Th2 cell development, germinal center formation, class switching to IgE, and lung inflammation. Oral exposure to antigen induced development of antigen-specific CD4CD25Foxp3CD45RB cells that were anergic and displayed suppressive activity in vivo and in vitro. Oral tolerance to the Th2 allergic response was in large part dependent on TGF-beta and independent of IL-10. Interestingly, Tregs were also induced by single i.p. immunization with antigen and adjuvant. However, unlike oral administration of antigen, which induced Tregs but not effector T cells, i.p. immunization led to the simultaneous induction of Tregs and effector Th2 cells displaying the same antigen specificity.

Mechanisms of Oral Tolerance: Dendritic Cells—Summary of Part II

Mechanisms of Oral Tolerance: Dendritic Cells—Summary of Part II

Mechanisms of Oral Tolerance: Regulatory T Cells—Summary of Part III

Mechanisms of Oral Tolerance: Regulatory T Cells—Summary of Part III

Insights into the Mechanism of Oral Tolerance Derived from the Study of Models of Mucosal Inflammation

Murine models of mucosal inflammation are frequently due to the inability of the mouse to mount a regulatory T cell response. To the extent that such responses arise from oral tolerance mechanisms, these models provide a unique way of studying oral tolerance. In this paper we focus on the regulatory cells generated in two of the most well-studied of such models, the cell-transfer model and the TNBS-colitis model. Our analysis leads to the view that regulatory cells generated by the oral tolerance seen in mucosal inflammation are, at least in part, cells that recognize self-antigens or antigens in the mucosal microflora whose effector function relies on the expression of TGF-beta.

Oral tolerance in a murine model of relapsing experimental autoimmune uveoretinitis (EAU): induction of protective tolerance in primed animals.

Oral administration of uveitogenic retinal antigens suppresses the expression of EAU induced by a subsequent immunization with these antigens. Effectiveness and mechanisms of oral tolerance in EAU have mainly been studied in the acute, monophasic model in Lewis rats by feeding antigen prior to induction of disease. In this study we investigated the effect of oral tolerance induction in the acute as well as the chronic-relapsing models in the B10.A mouse. In acute murine EAU we could effectively suppress disease by induction of oral tolerance prior to immunization. In the chronic-relapsing EAU, antigen feeding was started only after the animals had recovered from their first attack of uveitis. Under these experimental conditions the subsequent relapse was largely prevented. These experiments demonstrate that oral tolerance may have practical clinical implications in uveitis, which is predominantly a chronic-relapsing condition in humans.

Reduced transforming growth factor-beta1-producing T cells in the duodenal mucosa of children with food allergy.

Infant food allergies are increasing, and many breast-fed infants now sensitize to maternally-ingested antigens. As low-dose oral tolerance requires generation of suppressor lymphocytes producing TGF-beta1 (Th3 cells), we studied these cells in duodenal biopsies after diagnostic endoscopy. Spontaneous production of Th1, Th2 and Th3 cytokines by duodenal lymphocytes was studied using flow cytometry in 20 children with no eventual clinico-pathological diagnosis (controls), 30 children with multiple food allergy, nine with celiac disease and six with inflammatory enteropathies. Immunohistochemistry and in situ hybridization were used to localize TGF-beta1 protein and mRNA in matched biopsies. We found no significant Th1/Th2 skewing amongst mucosal lymphocytes in allergic children compared to controls, although celiac and inflammatory enteropathy patients showed increased Th1 responses. By contrast, the allergic children showed reduction of TGF-beta1(+) lymphocytes in both epithelial and lamina propria compartments. Reduction of TGF-beta1 expression was also seen in mononuclear cells and epithelium in food allergy by immunohistochemistry and in situ hybridization. The dominant mucosal abnormality in food allergic children was, thus, not Th2 deviation but impaired generation of Th3 cells. As generation of these cells requires innate immune response to enteric bacteria, we suggest that changing infectious exposures may inhibit primary establishment of basic oral tolerance mechanisms.

Evaluation of immunologic profile in patients with nickel sensitivity due to use of fixed orthodontic appliances

The aim of this study was to develop a new approach to testing the impact of nickel antigen on in vitro cell-proliferation assay, to identify adverse reactions to casting alloys among orthodontic patients. Cell-proliferation assay in vitro was used as the basic methodology to assess the influence of such variables as source of nickel antigen, type of serum used to supplement the culture medium, and number of cells in the culture. We selected 35 orthodontic patients who were classified as nickel sensitive and non–nickel sensitive, based on their clinical records. Our results showed that hexahydrated nickel sulfate at 10 μg/mL, 10% of autologous sera, and 2 × 10 5 cells was the best condition for inducing the most marked nickel proliferation response in vitro. This optimized method was able to distinguish nickel-sensitive from non–nickel-sensitive dental patients and also to discriminate those with positive skin tests. Our data suggest that continuous exposure to nickel casting alloys might lead to oral tolerance mechanisms that modulate nickel sensitivity, as evidenced by the lower cell proliferation index in patients undergoing orthodontic treatment over 24 months. Finally, our findings demonstrated a known nickel-induced type 2 immune response and a marked lack of type 1 immunity (interferon γ) as the hallmarks of nickel-sensitive patients. Further studies are needed to clarify the major cell phenotype associated with this type 2 immune response and the lack of type 1 immunity observed in nickel-sensitive people.

Chronic T cell-mediated enteropathy in rural west African children: relationship with nutritional status and small bowel function.

Previous studies from The Gambia have shown that poor childhood growth is resistant to all but the most intense nutritional intervention and highly dependent on small bowel permeability related to enteropathy. We thus aimed to characterize the mucosal inflammatory response in rural Gambian children in relation to intestinal permeability and nutritional status. Small bowel biopsies were taken from 38 rural Gambian children (age, 0.5-3 y) with a range of nutritional and clinical states (median weight z score, -4.6; range, 0.5 to -6.4), 75% of whom had diarrhea. Morphometry was performed with immunohistochemical analysis for a range of lineage and activation markers, including proinflammatory and regulatory cytokines, and related to current clinical status and gut permeability. Comparison was made with 19 age-matched U.K. controls. All Gambian children, regardless of nutritional status, had evidence of chronic cell-mediated enteropathy with crypt hyperplasia, villous stunting, and high numbers of intraepithelial lymphocytes. CD25+ cells were 20-fold higher than in U.K. controls. Although small bowel architecture was independent of nutritional status, T cell numbers rose and B cell numbers fell with worsening nutrition, and mucosal cytokine production became biased toward a proinflammatory response, with progressive decrease of transforming growth factor-beta expression. Tropical enteropathy predates the onset of marasmus and is characterized by a cell-mediated TH1 response. Protein-energy malnutrition is associated with reduction of regulatory immune responses in the mucosal microenvironment, potentially impairing the mechanisms of oral tolerance.

Oxazolone and diclofenac-induced popliteal lymph node assay reactions are attenuated in mice orally pretreated with the respective compound: potential role for the induction of regulatory mechanisms following enteric administration☆

The murine popliteal lymph node assay (PLNA) was examined as a preclinical assay with the potential to identify low-molecular-weight compounds (LMWCs) that are likely to be associated with immune-mediated drug hypersensitivity reactions (IDHRs) in humans. We hypothesized that the contact sensitizer oxazolone (OX) would cause a strong PLN reaction in naive mice and that the PLN reaction would be attenuated in mice orally pretreated with OX due to the induction of oral tolerance. In naive mice, OX induced a strong PLN reaction and caused dose-dependent increases in PLN size, weight, cellularity, percentage of CD4 + PLN T cells, and percentage of PLN B cells, with a concomitant decrease in the percentage of CD8 + PLN T cells. Next, the PLNA was conducted in mice gavaged three times with either OX or vehicle alone (olive oil). Mice pretreated with OX had suppressed PLN reactions following the footpad injection of OX (decrease in PLN size, weight, and cellularity), which was associated with an increase in the percentage of PLN CD8 +T cells. In contrast, oral pretreatment with OX had no observable effect on the PLN reaction induced following footpad injection of the irrelevant hapten dinitrochlorobenzene (DNCB). Adoptive transfer studies were conducted to examine the mechanism of PLN hyporesponsiveness. It was found that either (1) unfractionated splenocytes or (2) purified CD8 + splenocytes, but not (3) purified CD4 + splenocytes isolated from mice gavaged with OX adoptively transferred PLN suppression to naive BALB/c mice. Because OX is not a pharmaceutical, we also examined the NSAID diclofenac (DF) (Voltaren). Like OX, DF caused dose-dependent increases in PLN size, weight, and cellularity in naive mice. Furthermore, like OX, the diclofenac-induced PLN reaction was attenuated in mice that had been orally pretreated three times with DF. However, splenocytes from mice orally treated with DF were not able to adoptively transfer PLN hyporesponsiveness. Collectively, these observations demonstrate that both OX and DF are potent immunostimulators in the PLNA. As importantly, these results demonstrate that the immunostimulating potential of OX and DF in the PLNA is significantly decreased in mice orally exposed to the respective drug, possibly due to the presence of a cellular mechanism of oral tolerance. For OX, the mechanism appears to involve, in part, CD8 + T cells, whereas the mechanism(s) associated with PLN hyporesponsiveness using DF remain to be defined.