Oral Antigen Research Articles

Antibiotic Administration Early in Life Impairs Specific Humoral Responses to an Oral Antigen and Increases Intestinal Mast Cell Numbers and Mediator Concentrations

In this study, we assessed the effect of administering the antibiotic amoxicillin to rat pups on the immune response to orally fed ovalbumin (OVA). We first established that amoxicillin administration durably altered the gut microbiota of these animals. In parallel, we observed that the induction of the specific humoral response to ovalbumin was impaired when it occurred during antibiotic administration to the rat pups. We also examined the consequences of those observations on further allergic reactions. Amoxicillin administration had no significant impact on subsequent sensitization to OVA, as nonexacerbated systemic allergic responses were induced in antibiotic-treated animals. However, increased rat mast cell protease II levels and higher mast cell numbers were detected in their small intestines, independently of the antigen administration. Globally, our data suggest that antibiotic administration early in life negatively affects the specific immune response to a luminal antigen when it is first introduced during antibiotic administration. The increased mast cell numbers and mediator concentrations in the intestinal mucosae of the antibiotic-treated animals may testify to the early stages of an altered immune system homeostasis.

Production of monoclonal antibodies against Streptococcus mutans antigens

Several studies have been conducted in the last decades aiming to obtain an anti-caries vaccine, however some studies have demonstrated cross reactivity between Streptococcus mutans surface antigens and the human cardiac tissue. In this work, the reactivity of five anti-Streptococcus mutans monoclonal antibodies (MoAb) (24A, 56G, C8, E8 and F6) was tested against oral streptococci, cardiac antigens and skeletal and cardiac myosins, aiming to evaluate the specificity of these MoAb. The hybrid producers of immunoglobulins of the IgG2b class were cloned by limit dilution and expanded in vivo. MoAb were tested by ELISA. The hybrid 24A reacted with S. mutans CCT 1910, S. salivarius CCT 0365 and S. pyogenes T23. No reactivity difference was observed among the tested species. Cross reactivity with heart and cardiac myosin was not confirmed and only reaction with myosin of skeletal muscle was observed (p = 0.0381). The hybrid 56G reacted with all the tested microorganisms and there was statistically significant difference between S. mutans and S. pyogenes T23 (p < 0.001). This hybrid also reacted with myosin of skeletal muscle (p = 0.0095). C8, E8 and F6 presented low reactivity against oral streptococci strains and no reactivity against cardiac antigens. The data of this study showed that the 24A and 56G anti-S. mutans MoAb presented reactivity with S. pyogenes and S. salivarius, reinforcing the occurrence of common antigens between these species. The tested MoAb presented low cross-reactivity with myosin of skeletal muscle, but anti-heart activity could not be confirmed.

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.

Breakdown of mucosal immunity in gut by 2,3,7,8-tetraclorodibenzo-p-dioxin (TCDD)

Mucosal immunity plays a pivotal role for body defense against infection and allergy. The aim of this study was to clarify the effects of 2,3,7,8-tetraclorodibenzo-p-dioxin (TCDD) on mucosal immunity in the gut. Fecal IgA level and oral tolerance induction were examined in TCDD-treated mice. Flow cytometric and histological analyses were also performed. Single oral administration of low dose 2,3,7,8-TCDD resulted in a marked decrease in IgA secretion in the gut without any effects on the cellular components of gut-associated lymphoid tissues (GALT) including Peyer's patches (PPs) and mesenteric lymph nodes (LNs). Decressed IgA secretion by TCDD was not observed in aryl hydrocarbon receptor (AhR)-deficient mice. Flow cytometric analysis revealed that IgA B cells in PPs and the mesenteric LNs remained unchanged in the TCDD-treated mice. An immunofluorescence study also demonstrated that a significant number of cytoplasmic IgA cells were present in the lamina propria of the gut in the TCDD-treated mice. Furthermore, oral tolerance induction by ovalbumin (OVA) was impaired in the TCDD-treated mice and OVA-specific T cell proliferation occurred in the peripheral lymphoid tissues including the spleen and LNs. These results suggest that a relatively low dose of TCDD impairs mucosal immunity in the gut and induces systemic sensitization by oral antigens.

Regulation of T cell activation and tolerance by PDL2

T cell activation and tolerance are regulated by costimulatory molecules. Although PD-1 serves as a crucial negative regulator of T cells, the function of its ligands, PDL1 and PDL2, is still controversial. In this study, we created a PDL2-deficient mouse to characterize its function in T cell activation and tolerance. Antigen-presenting cells from PDL2-/- mice were found to be more potent in activation of T cells in vitro over the wild-type controls, which depended on PD-1. Upon immunization with chicken ovalbumin, PDL2-/- mice exhibited increased activation of CD4(+) and CD8(+) T cells in vivo when compared with WT animals. In addition, T cell tolerance to an oral antigen was abrogated by the lack of PDL2. Our results thus demonstrate that PDL2 negatively regulates T cells in immune responses and plays an essential role in immune tolerance.

CCR6 and CCL20

The CCR6 chemokine receptor is expressed by most B cells and subsets of T cells and dendritic cells (DCs) found in the gut mucosal immune system. CCL20, the single chemokine ligand for CCR6, is selectively made by the follicle-associated epithelium (FAE) overlying Peyer's patches (PPs) and isolated lymphoid follicles (ILFs). CCL20 contributes to the recruitment of CCR6-expressing B cells to these structures. CCL20 expression by the intestinal epithelium is also highly inducible in response to inflammatory stimuli. Thus, CCL20 functions as both an inflammatory and homeostatic chemokine. Interactions between CCR6 and CCL20 play a role at several stages in the development of intestinal lymphoid structures. A subset of the c-kit(+) lymphoid precursors found in cryptopatches (CPs) expresses CCR6. Recruitment of B cells to CPs and the subsequent expansion and organization of these B cells allows differentiation of some of these structures into ILFs. In CCR6 knockout mice, PPs are smaller with fewer follicles and the development of ILFs is compromised. These defects in the development of mucosal inductive sites in CCR6-deficient mice are responsible for decreased IgA production to oral antigens. CCR6 can be included with CXCR5 and CCR7 in a list of chemokine receptors that participate in shaping the organized lymphoid structures that are part of the intestinal immune system.

This Month in Gastroenterology

This Month in Gastroenterology

Dendritic cells: the commanders-in-chief of mucosal immune defenses

Intestinal dendritic cells have emerged as key regulators of immunity to pathogens, oral tolerance and intestinal inflammation. Studies have begun to elucidate the regulatory mechanisms responsible for defining region- and compartment-specific phenotypes and functions of dendritic cells in mucosal tissues. Specific subsets of dendritic cells appear to be associated with the various routes for antigen acquisition in the intestine. The constant sampling of intestinal antigenic content ensures establishment of tolerance to commensal bacteria and food antigens. Tolerance development to oral antigens is restricted to the mucosal immune system. Other advances have provided insight into the molecular basis of microbial recognition and innate immune responses by intestinal dendritic cells. Differences in the involvement of dendritic cells have begun to emerge in Crohn's disease and ulcerative colitis and link gene regulation in dendritic cells to therapeutic responses. A major focus of mucosal immunology will be to understand how diverse dendritic cell subsets cooperate in regulating homeostasis and host defense in the different intestinal immune compartments. This will be pivotal to understanding how the mucosal immune system makes the distinction between commensal microbiota, pathogens and self antigens.

New advances in coeliac disease: serum and intestinal expression of HLA-G

Coeliac disease (CD) is a common autoimmune disorder characterized by an immune response to ingested gluten and has a strong HLA association with HLA-DQ2 and HLA-DQ8, but as human HLA-DQ risk factors do not explain the entire genetic susceptibility to gluten intolerance. Our aim was to investigate whether HLA-G, a gene located in the MHC class I region, and with important role in the induction of immunotolerance, may contribute to CD susceptibility. We demonstrated the expression of soluble HLA-G (sHLA-G) forms in intestinal biopsy and in serum of patients with CD. Indeed, all patients tested showed a positive expression of HLA-G in intestinal mucosa with different grade of immunoreaction. The serum levels of sHLA-G found in coeliac patients depend on the association with other diseases of autoimmune nature or genetics, and also depend on the transgressions in the diet with gluten ingested. The enhancer expression of sHLA-G in CD could be due as part of a mechanism to try restore the tolerance process towards oral antigens in a disease caused by loss of tolerance to dietary antigens and counteract the inflammation. In summary, in this paper, we demonstrate the association of CD with sHLA-G expression.

T cell-mediated oral tolerance is intact in germ-free mice

Commensal enteric bacteria stimulate innate immune cells and increase numbers of lamina propria and mesenteric lymph node (MLN) T and B lymphocytes. However, the influence of luminal bacteria on acquired immune function is not understood fully. We investigated the effects of intestinal bacterial colonization on T cell tolerogenic responses to oral antigen compared to systemic immunization. Lymphocytes specific for ovalbumin-T cell receptor (OVA-TCR Tg(+)) were transplanted into germ-free (GF) or specific pathogen-free (SPF) BALB/c mice. Recipient mice were fed OVA or immunized subcutaneously with OVA peptide (323-339) in complete Freund's adjuvant (CFA). Although the efficiency of transfer was less in GF recipients, similar proportions of cells from draining peripheral lymph node (LN) or MLN were proliferating 3-4 days later in vivo in GF and SPF mice. In separate experiments, mice were fed tolerogenic doses of OVA and then challenged with an immunogenic dose of OVA 4 days later. Ten days after immunization, lymphocytes were restimulated with OVA in vitro to assess antigen-specific proliferative responses. At both high and low doses of OVA, cells from both SPF and GF mice fed OVA prior to immunization had decreased proliferation compared to cells from control SPF or GF mice. In addition, secretion of interferon (IFN)-gamma and interleukin (IL)-10 by OVA-TCR Tg(+) lymphocytes was reduced in both SPF and GF mice fed OVA compared to control SPF or GF mice. Unlike previous reports indicating defective humoral responses to oral antigen in GF mice, our results indicate that commensal enteric bacteria do not enhance the induction of acquired, antigen-specific T cell tolerance to oral OVA.

Induction of oral tolerization in CD86 deficient mice: A role for CD86 and B cells in the up-regulation of TGF-β

Feeding myelin oligodendrocyte glycoprotein (MOG) followed by immunization results in induction of oral tolerance evidenced by the amelioration of experimental autoimmune encephalomyelitis (EAE). Oral tolerization is characterized by the suppression of Th1 responses and up-regulation of Th2 responses and TGF-β. To identify the costimulatory molecules and cell types involved in cytokine-mediated suppression we examined wild type mice and mice deficient for either CD86 (CD86 −/−) or B cells (μMT). Oral tolerance was found in CD86 −/− mice evidenced by amelioration of disease severity, decreased proliferative responses and IFN-γ production and increased IL-4. TGF-β was not up-regulated in CD86 −/− or μMT mice but was increased in wild type mice. Analysis of the gut associated lymphoid tissue (GALT) of different mouse strains (C57BL/6 and PLJ × SJL F1) fed distinct myelin antigens (MOG and myelin basic protein, MBP) showed that TGF-β was increased in wild type mice of both strains by 3 days post-immunization and further increased with time. In contrast, no up-regulation of TGF-β was found in the GALT of CD86 −/− or μMT mice. These results demonstrate that CD86 is not required for oral tolerization and that both CD86 and B cells are important for the up-regulation of TGF-β following oral antigen.

Splenic Dendritic Cells Induced by Oral Antigen Administration Are Important for the Transfer of Oral Tolerance in an Experimental Model of Asthma

Peripheral tolerance can be induced after the feeding of Ag, which is referred to as oral tolerance. We demonstrated in this study that the oral administration of OVA induced tolerance in an experimental model of asthma in mice, and investigated which cells function as the regulatory cells in the transfer of this oral tolerance. In OVA-fed mice, the percentage of eosinophils in bronchoalveolar lavage fluid, serum IgE levels, airway hyperresponsiveness, and mRNA levels of IL-13 and eotaxin were significantly lower than found in nonfed mice. Histological examination of lung tissue showed a suppression of the accumulation of inflammatory cells in the peribronchial area of OVA-fed mice. Feeding after the first immunization or between the first and the second immunization suppressed these findings, whereas feeding just before the airway Ag challenge did not. The suppression of disease in OVA-fed mice was successfully transferred by injection of whole spleen cells of OVA-fed mice. When CD11c+ dendritic cells (DCs) were removed from splenocytes, this transfer of suppression was completely abolished. The injection of splenic DCs purified from OVA-fed mice alone transferred the suppression, whereas the injection of splenic DCs from naive mice that were cocultured with OVA in vitro did not. These data suggest that not only CD4+ T cells, but also CD11c+ DCs induced by Ag feeding are important for the active transfer of oral tolerance in this murine experimental model of asthma.

The N-terminal half part of the oral streptococcal antigen I/IIf contains two distinct binding domains.

In order to investigate the binding properties of the antigen I/IIf from Streptococcus mutans, we analyzed the binding activity of five I/IIf derivatives expressed by I/IIf gene derivatives obtained by insertion of a kanamycin resistance marker. ELISA-derived binding assays showed that the derivatives containing both the N-terminal alanine-rich domain (A-region) and an A-region distal domain extending to amino-acid 766 were the most effective in binding biotinylated (Biot-) human salivary components (SAC) and Biot-epithelial cell membrane components. Sodium metaperiodate treatment of SAC inhibited these interactions, suggesting a binding specificity of the A-region distal domain for carbohydrate residues. All the I/IIf derivatives were found to bind Biot-type I collagen, Biot-laminin, Biot-keratin, and Biot-fibronectin, the derivatives containing the A-region but lacking the A-region distal domain exhibiting the highest binding levels. Sodium metaperiodate treatment of laminin had no effect on its binding to the derivatives, suggesting that carbohydrate residues of the ligand were not involved.

Involvement of the Liver in the Induction of CD8 T Cell Tolerance towards Oral Antigen

The liver is an organ with unique immune regulatory potential. This review highlights the experimental evidence for the involvement of hepatic cell populations in the induction of oral tolerance. Although immune tolerance towards oral antigens is mainly induced in the gastrointestinal tract within gut associated lymphatic tissue via generation of regulatory CD4 T cells, there is a further need for tolerance induction outside the gastrointestinal tract, because oral antigens rapidly distribute within minutes systemically through the blood stream. Besides hepatic dendritic cells, liver sinusoidal endothelial cells are active in the uptake and cross-presentation of oral antigens from portal venous blood and engage in the induction of CD8 T cell tolerance towards these antigens. These reports strengthen the notion that the liver participates in the induction of oral tolerance.

Commensal oral bacteria antigens prime human dendritic cells to induce Th1, Th2 or Treg differentiation

In various immunopathologic conditions, bacterial flora induce an immune response which results in inflammatory manifestations, e.g. periapical granuloma. Dendritic cells provide the main orchestration of specific immune responses. The aim of our study was to test the capacity of distinct oral bacterial antigens (prepared from Streptococcus mitis, Propionibacterium acnes, and Bacteroides spp.) to prime human dendritic cells for stimulation of the T-lymphocyte response. To assess the T-lymphocyte response, the expression of CD25, CD69, intracellular interferon gamma (cIFN-gamma), and intracellular interleukin 4 (cIL-4) was determined. Dendritic cells were prepared from leukocyte buffy coat from healthy blood donors. Monocytes were stimulated with IL-4 and GM-CSF and dendritic cells activated with bacterial lysates. Cell suspensions contained up to 90% dendritic cells, which represented 2-12% of the initial number of mononuclear cells. Lymphocyte subsets that developed in lymphocyte cultures after 1 week of stimulation were analyzed by flow cytometry. Dendritic cells, primed with antigens of Bacteroides fragilis have shown significantly higher activation and expression of intercellular IFN-gamma by T lymphocytes compared to negative controls. The dendritic cells primed with antigens of P. acnes had no effect on T-lymphocyte activation or cytokine production; instead they induced differentiation of T lymphocytes into CD25bright cells (regulatory T cells) with a potentially inhibitory effect on immune response. Dendritic cells primed with antigens of S. mitis induced increased expression of cIL-4. We conclude that commensal oral bacteria antigens prepared from B. fragilis, S. mitis, and P. acnes prime human dendritic cells to induce Th1, Th2, and T(reg) differentiation, respectively. This may advance our understanding of immunopathologic manifestations in the oral cavity and offer new possibilities for redirecting immune responses in mucosal vaccination.

Chronic Psychological Stress in Rats Induces Intestinal Sensitization to Luminal Antigens

There is increasing evidence that stress plays a role in the pathophysiology of chronic intestinal disorders, but the mechanisms remain unclear. Previous studies in rats have revealed that stress decreases gut barrier function and allows excessive uptake of luminal material. Here, we investigated whether chronic psychological stress acts to induce sensitization of intestinal tissues to oral antigens. Rats were subjected to 1 hour per day of water avoidance stress or sham stress daily for 10 days, and horseradish peroxidase (HRP) was delivered by gavage on day 5. Studies to determine sensitization were conducted on day 20. All stressed rats developed HRP-specific IgE antibodies, antigen-induced intestinal secretion, and increased numbers of inflammatory cells in gut mucosa. Luminal HRP was absorbed more readily by enterocytes of stressed animals. In addition, stressed rats had increased expression of interleukin-4 and decreased expression of interferon-gamma in gut mucosa, a cytokine profile that is typical of allergic conditions. Treatment of stressed rats with an antagonist to corticotropin-releasing hormone (previously shown to inhibit stress-enhanced gut permeability) eliminated the manifestations of intestinal hypersensitivity. Our results indicate that the presence of oral antigen during chronic psychological stress alters the immune response (to sensitization rather than oral tolerance) and causes subsequent antigen-induced gut pathophysiology.

Development of polysaccharide-capped niosomes for oral immunization of tetanus toxoid

Niosomes appear to be a promising and potential carrier system for oral antigen delivery. In the present study, polysaccharide-capped-niosomes were prepared, characterized and evaluated for immunostimulating potential. Tetanus toxoid (TT) antigen was encapsulated in the niosomes using the reverse phase evaporation method and coated with a pullulan derivative i.e. O-palmitoyl pullulan (OPP). Synthesized OPP was characterized by IR and proton NMR spectroscopy. The prepared niosomes were evaluated for size, shape, encapsulating efficiency and stability in simulated gastric fluid (pH 1.5). Serum IgG antibody level was measured to assess the immune stimulating effect of these carriers following oral administration. OPP-capped TT loaded niosomes could produce better humoral response as compared to their plain counterpart. Further, OPP appended niosomes produce immune response almost equivalent to intramuscularly given alum adsorbed TT. Mucosal IgA titers suggest polysaccharide-capped niosomal systems could be a potential oral vaccine delivery system.

Expression of cholera toxin B subunit and the B chain of human insulin as a fusion protein in transgenic tobacco plants

A DNA construct containing the cholera toxin B subunit (CTB) gene genetically fused to a nucleotide sequence encoding three copies of tandemly repeated diabetes-associated autoantigen, the B chain of human insulin, was produced and transferred into low-nicotine tobaccos by Agrobacterium. Integration of the fusion gene into the plant genome was confirmed by polymerase chain reaction (PCR). The results of immunoblot analysis verified the synthesis and assembly of the fusion protein into pentamers in transgenic tobacco. GM1-ELISA showed that the plant-derived fusion protein retained GM1-ganglioside receptor binding specificity. The fusion protein accounted for 0.11% of the total leaf protein. The production of transgenic plants expressing CTB-InsB3 offers a new opportunity to test plant-based oral antigen therapy against autoimmune diabetes by inducing oral tolerance.

Functional specialization of gut CD103+ dendritic cells in the regulation of tissue-selective T cell homing

Gut-associated lymphoid tissue (GALT) dendritic cells (DCs) display a unique ability to generate CCR9+ α 4 β 7 + gut-tropic CD8+ effector T cells. We demonstrate efficient induction of CCR9 and α 4 β 7 on CD8+ T cells in mesenteric lymph nodes (MLNs) after oral but not intraperitoneal (i.p.) antigen administration indicating differential targeting of DCs via the oral route. In vitro, lamina propria (LP)–derived DCs were more potent than MLN or Peyer's patch DCs in their ability to generate CCR9+ α 4 β 7 + CD8+ T cells. The integrin α chain CD103 (α E) was expressed on almost all LP DCs, a subset of MLN DCs, but on few splenic DCs. CD103+ MLN DCs were reduced in number in CCR7−/− mice and, although CD8+ T cells proliferated in the MLNs of CCR7−/− mice after i.p. but not oral antigen administration, they failed to express CCR9 and had reduced levels of α 4 β 7. Strikingly, although CD103+ and CD103− MLN DCs were equally potent at inducing CD8+ T cell proliferation and IFN-γ production, only CD103+ DCs were capable of generating gut-tropic CD8+ effector T cells in vitro. Collectively, these results demonstrate a unique function for LP-derived CD103+ MLN DCs in the generation of gut-tropic effector T cells.