Oral Tolerization Research Articles

Migratory CD103+CD11b+ cDC2s in Peyer’s patches are critical for gut IgA responses following oral immunization

Induction and regulation of specific intestinal IgA responses critically depend on dendritic cell subsets and the T cells they activate in the Peyer’s patches (PP). We found that oral immunization with cholera toxin (CT) as an adjuvant resulted in migration-dependent changes in the composition and localization of PP DC subsets with increased numbers of CD103- cDC2s and LysoDCs in the subepithelial dome and of CD103+ cDC2s that expressed CD101 in the T cell zones (TZ), while oral OVA tolerization was instead associated with larger quantities of TZ cDC1s and pTregs. Decreased IgA responses were observed after CT adjuvanted immunization in huCD207DTA mice lacking CD103+ cDC2s, while oral OVA tolerization was inefficient in cDC1-deficient Batf3-/- mice. Using Ovalbumin (OVA) TCR transgenic CD4 T cell adoptive transfer models, we found that co-transferred endogenous WT CD4 T cells can hinder the induction of OVA-specific IgA responses through secretion of IL-10. CT could overcome this blocking effect, apparently through a modulating effect on peripherally induced Tregs (pTreg) while promoting an expansion of follicular helper T cells (Tfh). The data support a model where cDC1-induced pTreg normally supress PP responses for any given antigen and where CT’s oral adjuvanticity effect is dependent on promoting Tfh responses through induction of CD103+ cDC2s.

Oral administration of human carbonic anhydrase I suppresses colitis in a murine inflammatory bowel disease model

The incidence of inflammatory bowel disease (IBD) is increasing; hence, effective treatments are warranted. The therapeutic effect of human carbonic anhydrase I (hCA I) in IBD remains unknown. Therefore, we investigated whether oral tolerization to hCA I would induce antigen-specific protection from intestinal inflammation in vivo. Severe combined immunodeficient mice received hCA I, keyhole limpet hemocyanin (KLH), or phosphate-buffered saline (PBS) orally for 7 days. Colons and mesenteric lymph nodes (MLNs) were collected 4 weeks after cell transfer. Additionally, the mechanisms underlying the therapeutic effects were investigated. The comparison between the effects of well-established drugs and hCA I oral administration was investigated. Oral administration of hCA I ameliorated colitis remarkably. hCA I reached the cecum and ameliorated colitis more effectively than mesalazine and similarly to prednisolone. Compared with PBS treatment, hCA I treatment reduced interleukin (IL)-17a, IL-6, and retinoic acid-related orphan receptor gamma t (RORγt) expression in the colon or MLNs; moreover, hCA I markedly reduced IL-6, IL-17, and interferon-gamma (IFN-γ) levels in the MLN. Oral administration of hCA I induced immune tolerance and suppressed colitis in vivo. Thus, hCA I administration could be proposed as a new treatment option for IBD.

Role of undenatured collagen type II and Aflapin combination in the management of osteoarthritis: a review

<p class="abstract">Osteoarthritis (OA) is the most common joint disease affecting millions worldwide. Osteoarthritis typically affects the knees, hands, hips, and feet. It is characterized by complex pathologic changes in cartilage which haven’t been fully elucidated yet. However, recent research has shown the involvement of two contributing pathways namely the mechanical and the immune pathways which interlink to cause cartilage destruction. Patients with OA on current treatment options still inevitably progress to a more severe stage becoming candidates for total joint replacement. The cornerstones of OA management in the early stage include exercises, weight loss, education—complemented by topical or oral nonsteroidal anti-inflammatory drugs (NSAIDs) and nutraceuticals like Undenatured type II collagen and Aflapin. Both Undenatured type II collagen and Aflapin offer great promise in OA management by targeting both the immune and mechanical pathways respectively. Undenatured type II collagen works by oral tolerization turning off the immune response in the inflammatory damage (T cell response) against endogenous Type II collagen in the cartilage thus reducing joint inflammation and degradation and stimulates anti-inflammatory cytokine release. Aflapin inhibits 5-LOX and exerts anti-inflammatory action thus providing symptomatic relief of pain and inflammation. This review focusses on the role of mechanical and immune pathways in the pathogenesis of OA and the impact of the combination of Undenatured type-II collagen and Aflapin in targeting these pathways thus improving the clinical outcomes.</p>

Small intestinal immune-environmental changes induced by oral tolerance inhibit experimental atopic dermatitis

Atopic dermatitis is a chronic skin inflammatory disease mediated by Th2-type immune responses. Although intestinal immune responses have been shown to play a critical role in the development or prevention of atopic dermatitis, the precise influence of intestinal immunity on atopic dermatitis is incompletely understood. We show here that orally tolerized mice are protected from experimental atopic dermatitis induced by sensitization and epicutaneous (EC) challenge to ovalbumin. Although the expression of Th2-type cytokines in the small intestine of orally tolerized and EC-challenged mice did not change significantly, these mice showed decreased inflammatory responses in the small intestine with restoration of microbial change elicited by the EC challenge. Interestingly, an increase in small intestinal eosinophils was observed with the EC challenge, which was also inhibited by oral tolerance. The role of small intestinal eosinophils and microbiota in the pathogenesis of experimental atopic dermatitis was further substantiated by decreased inflammatory mediators in the small intestine and attenuated Th2-type inflammation in the skin of eosinophil-deficient and microbiota-ablated mice with EC challenges. Based on these data, we propose that the bidirectional interaction between the skin and the intestine has a role in the pathogenesis of atopic dermatitis and that modulation of the intestinal microenvironments could be a therapeutic approach to atopic dermatitis.

Comprehensive analysis of DNA methylation and gene expression in orally tolerized T cells

T cell anergy is known to be a crucial mechanism for various types of immune tolerance, including oral tolerance. The expression of several anergy-specific genes was reportedly up-regulated in anergic T cells, and played important roles in the cells. However, how the genes were up-regulated has not been understood. In this study, we comprehensively analyzed the altered gene expression and DNA methylation status in T cells tolerized by oral antigen in vivo. Our results showed that many genes were significantly up-regulated in the orally tolerized T cells, and most of the genes found in this study have not been reported previously as anergy related genes; for example, ribosomal protein L41 (FC = 3.54E06, p = 3.70E-09: Fisher's exact test; the same applies hereinafter) and CD52 (FC = 2.18E05, p = 3.44E-06). Furthermore, we showed that the DNA methylation statuses of many genes; for example, enoyl-coenzyme A delta isomerase 3 (FC = 3.62E-01, p = 3.01E-02) and leucine zipper protein 1 (FC = 4.80E-01, p = 3.25E-02), including the ones distinctly expressed in tolerized T cells; for example, latexin (FC = 3.85E03, p = 4.06E-02 for expression; FC = 7.75E-01, p = 4.13E-01 for DNA methylation) and small nuclear ribonucleoprotein polypeptide F (FC = 3.12E04, p = 4.46E-04 for expression; FC = 8.56E-01, p = 5.15E-01 for DNA methylation), changed during tolerization, suggesting that the distinct expression of some genes was epigenetically regulated in the tolerized T cells. This study would contribute to providing a novel clue to the fine understanding of the mechanism for T cell anergy and oral tolerance.

Oral tolerance modulates the skin transcriptome in mice with induced atopic dermatitis.

Defective gut immune reactions have been implicated in the development of atopic dermatitis (AD), whereas oral tolerance (OT), that is, the immune unresponsiveness induced by oral antigen administration, protects mice against AD. To investigate this protective role of OT, the transcriptomic profiles of skin were obtained by RNA sequencing from mice that were epicutaneously sensitized, orally tolerized prior to epicutaneous sensitization, or neither (control). Oral tolerance inhibited the upregulation of keratin- and allergic inflammation-associated genes that occurred in the epicutaneously sensitized group. Compared to the controls, mice that were orally tolerized and epicutaneously sensitized showed an upregulation of genes that regulate inflammation or keratinocyte differentiation. Knocking down two of those genes, SCGB1A1 and TSC22D3, upregulated Th2 inflammatory mediators and downregulated a cornified cell envelope-related gene. Based on our findings, OT may protect skin against allergic inflammation by promoting the expression of genes that regulate Th2 inflammatory responses and skin barrier function.

Oral Tolerization with Mycobacterial Heat Shock Protein 65 Reduces Chronic Experimental Atherosclerosis in Aged Mice

Background: Atherosclerosis is a chronic inflammatory disease of the artery wall where both innate and adaptive immunity play important roles. Modulation of the immune response against the stress protein antigen, heat shock protein (HSP) 60, by administration of mycobacterial HSP65 (mbHSP65) orally and/or nasally shows promising therapeutic results in young animals in the sense of less severe experimental atherosclerosis; however, the case of aged animals with already established atherosclerosis has so far never been investigated. Objective: To investigate if mbHSP65 immunization would further accelerate atherosclerotic progression in aged ApoE^-/- mice (18 months old) with already long-established atherosclerosis and if these mice could be orally tolerized against mbHSP65. Methods: Aged wild-type (WT) and ApoE^-/- mice (65 weeks) were immunized and/or orally treated with mbHSP65 and then either kept on normal chow or changed to high-cholesterol diet (HCD). Atherosclerosis was assessed by en face analysis and the number of CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) was assessed by flow cytometry in lymph node and spleen cells. Total cholesterol and triglyceride levels were determined. Soluble mammalian HSP60 and anti-mouse HSP60 (mHSP60) and anti-mbHSP65 antibodies were detected by enzyme-linked immunosorbent assay. Results: As expected, aged WT mice had only minor lesions in the aorta, which did not change under HCD for 14 weeks. Aged ApoE^-/- mice already had large complicated plaques, which increased in size under HCD. mbHSP65 immunization led to a significant aggravation of atherosclerosis in both WT and ApoE^-/- mice irrespective of the nature of their diet. This increase was accompanied by increased titers of both anti-mHSP60 and anti-mbHSP65 antibodies in the circulation. The increased plaque formation could be significantly diminished with oral mbHSP65 tolerization. An increased number of Tregs and lower or unchanged levels of cholesterol and triglycerides were associated with the reduced size of aortal lesions. Conclusion: Oral tolerization against mbHSP65 could be used both to prevent and to treat chronic atherosclerosis in aged individuals.

Mycobacterial heat shock protein 65 (mbHSP65)-induced atherosclerosis - Preventive oral tolerization and definition of atheroprotective and atherogenic mbHSP65-peptides

Mycobacterial heat shock protein 65 (mbHSP65)-induced atherosclerosis - Preventive oral tolerization and definition of atheroprotective and atherogenic mbHSP65-peptides

Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce

Anti-drug antibodies in hemophilia patients substantially complicate treatment. Their elimination through immune tolerance induction (ITI) protocols poses enormous costs, and ITI is often ineffective for factor IX (FIX) inhibitors. Moreover, there is no prophylactic ITI protocol to prevent anti-drug antibody (ADA) formation. Using general immune suppression is problematic. To address this urgent unmet medical need, we delivered antigen bioencapsulated in plant cells to hemophilia B dogs. Commercial-scale production of CTB-FIX fusion expressed in lettuce chloroplasts was done in a hydroponic facility. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds, and pentamer assembly after 30-month storage at ambient temperature. Robust suppression of immunoglobulin G (IgG)/inhibitor and IgE formation against intravenous FIX was observed in three of four hemophilia B dogs fed with lyophilized lettuce cells expressing CTB-FIX. No side effects were detected after feeding CTB-FIX-lyophilized plant cells for >300 days. Coagulation times were markedly shortened by intravenous FIX in orally tolerized treated dogs, in contrast to control dogs that formed high-titer antibodies to FIX. Commercial-scale production, stability, prolonged storage of lyophilized cells, and efficacy in tolerance induction in a large, non-rodent model of human disease offer a novel concept for oral tolerance and low-cost production and delivery of biopharmaceuticals.

Restoring immune tolerance in neuromyelitis optica: Part II.

Neuromyelitis optica spectrum disorder (NMO/SD) and its clinical variants have at their core the loss of immune tolerance to aquaporin-4 and perhaps other autoantigens. The characteristic phenotype is disruption of astrocyte function and demyelination of spinal cord, optic nerves, and particular brain regions. In this second of a 2-part article, we present further perspectives regarding the pathogenesis of NMO/SD and how this disease might be amenable to emerging technologies aimed at restoring immune tolerance to disease-implicated self-antigens. NMO/SD appears to be particularly well-suited for these strategies since aquaporin-4 has already been identified as the dominant autoantigen. The recent technical advances in reintroducing immune tolerance in experimental models of disease as well as in humans should encourage quantum leaps in this area that may prove productive for novel therapy. In this part of the article series, the potential for regulatory T and B cells is brought into focus, as are new approaches to oral tolerization. Finally, a roadmap is provided to help identify potential issues in clinical development and guide applications in tolerization therapy to solving NMO/SD through the use of emerging technologies. Each of these perspectives is intended to shine new light on potential cures for NMO/SD and other autoimmune diseases, while sparing normal host defense mechanisms.

Feeding a Maternal Diet Enriched in Docosahexaenoic Acid to Lactating Dams Improves the Ex Vivo Tolerance Response to Egg Protein in Suckled Pups

An important T cell mediated process that occurs early in life is the development of oral tolerance (OT). Food allergies are believed to be the result of a failure to develop OT to a dietary antigen. The objective of this study was to determine the effect of feeding a maternal diet supplemented with docosahexaenoic acid (DHA) while containing adequate amounts of arachidonic acid (AA) during the suckling period on the development of the immune system and OT in offspring.Sprague‐Dawley dams were randomized to one of the two nutritionally adequate experimental diets 24–48h prior to parturition: control diet (N=12, 0.4% AA and 0% DHA of total fatty acids) or high DHA diet (N=8, 0.4% AA and 0.9% DHA of total fatty acids). Diets were fed to dams ad libitum throughout the suckling period (21 days) and matched for macronutrient, micronutrient and fatty acid content differing only in the composition of omega‐3. At 14‐days pups from each dam were randomly assigned to a mucosal OT challenge for 5 days: placebo (sucrose) or ovalbumin (OVA). At 3 weeks, pups were euthanized and immune cell phenotypes and cytokine production by mitogen‐ (Concanavalin A (ConA), lipopolysaccharide (LPS)) or OVA‐stimulated splenocytes were measured by direct immunofluorescence assay and ELISA, respectively.Feeding dams a high DHA diet increased the proportion of DHA and AA found in breast milk vs. the control diet (both P<0.01). Regardless of the OT challenge, feeding a high DHA diet led to a higher production of interferon‐γ by splenocytes stimulated with ConA and a higher production of interleukin (IL)‐6, IL‐10 and tumor necrosis factor‐α after LPS stimulation (all P<0.05). OVA‐stimulated splenocytes from suckled pups fed the high DHA diet produced significantly more IL‐10 and less transforming growth factor‐β compared to the control diet (both P<0.02), suggestive of better OT. The OT challenge with OVA resulted in a lower production of IL‐6 by LPS‐stimulated splenocytes and led to a higher proportion of cytotoxic T cells expressing CD28 and total cells expressing CD27 (all P<0.02) compared to non‐tolerized pups.Our results suggest that feeding a maternal diet enriched in DHA during the suckling period improves the ability of splenocytes to respond ex vivo to mitogens and the tolerance response to a dietary antigen. Irrespective of the suckling diet, pups who had been orally tolerized to OVA had a lower response to a bacterial antigen despite the presence of more activated T cells and memory immune cells.Support or Funding InformationSupported from the Natural Sciences and Engineering Research Council of Canada. CR holds a scholarship from Canadian Institutes for Health Research and Fonds de recherche en santé du Québec.

Mycobacterial heat shock protein 65 (mbHSP65)-induced atherosclerosis: Preventive oral tolerization and definition of atheroprotective and atherogenic mbHSP65 peptides.

The aim of this study was to identify atherogenic and atheroprotective peptides of bacterial HSP60 [taking mycobacterial HSP65 (mbHSP65) as a potent paradigmatic representative] that could be used as candidates for an orally applied tolerizing vaccine against atherosclerosis. ApoE(-/-) mice were immunized with mbHSP65 protein or peptides, given mbHSP65 orally and then kept either on chow or high cholesterol diet. Atherosclerosis was assessed by en face and immunohistological analysis. Anti-HSP autoantibodies were detected by ELISA. The number and in vitro suppressive function of splenic and lymph node regulatory T cells (Tregs) were analyzed by flow cytometry. Specific T cell reactivity against mbHSP65 protein or peptides was assessed by proliferation assay. Decreased lesion size was accompanied by (a) increased splenic Treg numbers; (b) increased interleukin (IL)-10 mRNA levels in the aorta; (c) increased levels of anti-mbHSP65 and anti-mouse HSP60 antibodies pointing to pro-eukaryotic HSP60 humoral crossreaction, not curtailed by oral tolerization; (d) most importantly, we identified and functionally characterized novel atherogenic and atheroprotective mbHSP65 epitopes. Atheroprotective mbHSP65 peptides may be considered as potential candidates for the development of a tolerizing vaccine to prevent and treat atherosclerosis, while keeping protective immunity to non-atherogenic domains of mbHSP65 intact.

The effect of immune status, age and genetic background on induction of oral tolerance to Actinomyces viscosus in mice

The aim of the present study was to determine the effect of immune status, age and genetic background on the induction of oral tolerance to Actinomyces viscosus. Suppression of delayed type hypersensitivity (DTH) response and antigen-specific serum antibody levels could be induced in DBA/2 mice intragastrically and systemically immunized with A. viscocus, suggesting the induction of oral tolerance. In contrast, this immune suppression could be abrogated if the animals had been systemically immunized prior to the induction of oral tolerance with the same bacterium. Long-term systemic immunization prior to intragastric immunization with A. viscocus suppressed DTH response only. Cell transfer of this group of animals also suppressed DTH response in the donors, indicating the action of suppressor cells for inhibition of DTH response. Furthermore, oral tolerance to A. viscocus failed to occur in mice aged at 3days and 1, 2, 4, 6 and 36weeks old. Mice bearing H-2d haplotype were the most susceptible to oral tolerization, followed by H-2b and H-2k. Therefore, the results of the presence study suggest that the induction of oral tolerance to A. viscosus in mice may be dependence on the immune status and genetic background but not age.

Spontaneously relapsing–remitting experimental autoimmune uveitis in rats allows successful therapeutic oral tolerance induction in ongoing disease

Antigen-specific tolerance induction is a desired therapy for uveitis patients. Our relapsing–remitting rat model of experimental autoimmune uveitis (EAU) induced with IRBP peptide R14 enables us to test the effect of oral tolerance on the prevention of relapsing uveitis. We investigated several peptides overlapping the sequence of R14 for prevention and different doses of R14 for therapy to determine the tolerogenic epitope and the most effective therapeutic regimen for uveitis. Lewis rats were immunized with R14-CFA to induce EAU. Oral tolerance was induced prior to immunization (prevention) or after onset of EAU to prevent relapses (therapy). Therapeutic feeding was performed with high and/or low doses of oral antigen for clonal deletion of effector and induction of regulatory T cells. Uveitis was determined clinically and histologically; mesenteric lymph node (mLN) cells of tolerized rats were tested for surface markers, cytokines and Foxp3 expression. Preventive feeding of R14 and its major epitope R16, but none of the overlapping peptides significantly suppressed EAU and also prevented relapses, irrespective of their pathogenicity. Therapeutic feeding with R14 dramatically reduced relapses, while only the consecutive feeding of high and low-dose R14 had an ameliorating effect on the first course of disease. IL-10-producing T cells from mLN decreased after oral tolerization, and with R14-stimulation in vitro the TCRαβ+/Foxp3+ population increased in the low-dose fed group. No mLN population could be clearly assigned to successful oral tolerance induction during active autoimmune uveitis.

Oral administration of carbonic anhydrase I ameliorates murine experimental colitis induced by Foxp3−CD4+CD25− T cells

IBDs are thought to involve uncontrolled innate and adaptive immunity against intestinal self-antigens and bacterial antigens. Mouse CA I is a major cecal bacterial antigen in fecal extracts and is implicated in the pathogenesis of IBD. We show here that oral tolerization to CA I induced antigen-specific protection from intestinal inflammation in a murine model. Oral administration of CA I but not irrelevant antigen (KLH) ameliorated CD4(+)CD25(-) T cell transfer murine colitis and DSS-induced murine colitis. Next, we investigated the mechanisms involved in the therapeutic effects of oral administration, such as induction of ALDH1a2, transcription factors, cytokines, CD103(+)CD11c(+) DCs, and generation of Tregs. Oral administration of CA I induced ALDH1a2 mRNA expression in the MLN and colon. When compared with PBS-treated mice, CA I-treated mice had higher Foxp3(+)CD4(+)CD25(+) Treg and CD103(+)CD11c(+) DC numbers in the MLN and colon; had higher TGF-β production in the MLN and colon; had lower RORγt mRNA expression in the MLN and colon; and had lower IL-17 mRNA expression and production in the MLN. These results demonstrate that oral administration of CA I induced antigen-specific immune tolerance by generating Foxp3(+)CD4(+)CD25(+) Tregs and inhibiting Th17 cells in a murine colitis model, thus suggesting that oral tolerization with CA I is an effective therapeutic strategy for IBD regulation.

Modulation of the Effects of Lung Immune Response on Bone Marrow by Oral Antigen Exposure

Allergic airway inflammation is attenuated by oral tolerization (oral exposure to allergen, followed by conventional sensitization and challenge with homologous antigen), which decreases airway allergen challenge-induced eosinophilic infiltration of the lungs and bone marrow eosinophilia. We examined its effects on bone marrow eosinophil and neutrophil production. Mice of wild type (BP-2, BALB/c, and C57BL/6) and mutant strains (lacking iNOS or CD95L) were given ovalbumin (OVA) or water (vehicle) orally and subsequently sensitized and challenged with OVA (OVA/OVA/OVA and H2O/OVA/OVA groups, resp.). Anti-OVA IgG and IgE, bone marrow eosinophil and neutrophil numbers, and eosinophil and neutrophil production ex vivo were evaluated. T lymphocytes from OVA/OVA/OVA or control H2O/OVA/OVA donors were transferred into naïve syngeneic recipients, which were subsequently sensitized/challenged with OVA. Alternatively, T lymphocytes were cocultured with bone marrow eosinophil precursors from histocompatible sensitized/challenged mice. OVA/OVA/OVA mice of the BP-2 and BALB/c strains showed, relative to H2O/OVA/OVA controls, significantly decreased bone marrow eosinophil counts and ex vivo eosinopoiesis/neutropoiesis. Full effectiveness in vivo required sequential oral/subcutaneous/intranasal exposures to the same allergen. Transfer of splenic T lymphocytes from OVA/OVA/OVA donors to naive recipients prevented bone marrow eosinophilia and eosinopoiesis in response to recipient sensitization/challenge and supressed eosinopoiesis upon coculture with syngeneic bone marrow precursors from sensitized/challenged donors.

Antigen Up-take by Intestinal Epithelial Cells in IBD

Table 1. Percentage of the cells stained with CD45 (leukocytes) and EpCAM (epithelial cells) antibodies for flow cytometry analysis of the mucosal fractions obtained from surgical specimens from UC inflamed. UC non-inflamed. CD and NL patients. Statistic comparisons performed between UC I, CD and NL (***p<0.001. **p<0.01 and *p<0.05). Previous work of our group showed that inflammatory bowel disease (IBD) patients could not be orally tolerized against keyhole limpet hemocyanin (KLH) [1]. Our aim is to test in vitro the antigen up-take ability of the same antigen in isolated IECs from IBD patients and non-inflammatory controls. 1. Kraus TA et al. Failure to induce oral tolerance to a soluble protein in patients with inflammatory bowel disease. Gastroenterology (2004) 126: 1771-1778. Surgical specimens were obtained from intestinal resections performed in Mount Sinai Medical Center. Five ulcerative colitis (with inflamed tissue (UC I) and four out of them also with non-inflamed tissue (UC NI)), and five Crohn's disease (CD) patients and 8 normal mucosal samples (NL) were included. Briefly, the protocol of IECs isolation consisted in intense cleaning of surgical pieces with PBS; the mucosa was stripped from the submucosa in small pieces (3-5 mm). The pieces were incubated in DTT (1 mM in RPMI medium) for 15 min followed by 2 incubations of 30 min each in 25 mL of Dispase II (1.5 mg/mL in RPMI) at 37 °C. After washing and centrifugation, IECs were recovered from the supernatant and were counted and adjusted to a final concentration of 106 cells/mL in RPMI (plus antibiotics and Fungizone). 105 cells/well were plated in 96 wells plates in a total volume of 200 μl/well of RPMI with or without 100 μg of KLH. At different time points (0.5, 1, 2, 4 and 18 h) IECs were surface stained for flow cytometry analysis with CD45 (as a marker of leukocytes) and Ep-CAM (as a marker of IECs) antibodies, fixed and permeabilized and intra-cellular stained with an anti-KLH antibody. In 4 UC inflamed, 4 CD and 7 NL samples from same patients MHCII expression was analyzed by flow cytometry. In other set of experiments, after overnight (ON) incubation with or without KLH, MHCII expression was analyzed by flow cytometry in Ep-CAM + cells from 3 NL and 3 UC inflamed samples. Comparisons between groups were statistically analyzed by Mann-Whitney test for unpaired samples. As showed in Table 1, the proportion of leukocytes and IECs in mucosa reflected the inflammatory state of the tissue with increased proportion of leukocytes and decreased levels of IECs in IBD samples. IECs from UC inflamed showed an increased earlier up-take of KLH compared to CD, however CD IECs had larger overnight uptake of KLH than UC inflamed (Fig. 1). The percentage of IECs that are MHCII + is significantly higher in inflamed UC patients compared to NL, although KLH incubation does not have any effect on% of IECs expressing MHCII+ (Fig. 2). The different kinetics of KLH up-take by IECs from CD and UC patients can affect the way through which KLH can be presented to immune cells. Although KLH presence does not seem to be important to IECs expression of MHCII molecules, differences in MHCII expression suggests a difference in the ability of IECs from both types of IBD to stimulate different immune cells subsets.

A distinct tolerogenic subset of splenic IDO+CD11b+ dendritic cells from orally tolerized mice is responsible for induction of systemic immune tolerance and suppression of collagen-induced arthritis

In oral tolerance, locally instigated tolerance in the gut propagate to systemic tolerance. In order to investigate the mechanism, we analyzed indoleamine 2,3-dioxygenase (IDO) expression in splenic dendritic cell (DC) subsets and tested whether DCs suppress collagen-induced arthritis (CIA) by inducing regulatory T cells (Tregs). The proportion of IDO-expressing cells was higher in the CD11b+ subset of splenic DCs from orally tolerized CIA mice. These DCs suppressed type II collagen-specific T cell proliferation and promoted Treg induction from CD4+CD25− T cells using transforming growth factor-β. These DCs also increased the expression of cytotoxic T lymphocyte antigen-4 and programmed death-1 on Tregs. When adoptively transferred, spenic IDO-expressing CD11b+ DCs from tolerized animals suppressed the development of arthritis, increased the Treg/Th17 cell ratio, and decreased the production of inflammatory cytokines in the spleen. Taken together, a distinct subset of splenic IDO+CD11b+DCs is responsible for the systemic immune regulation in oral tolerance.

Gut-Tropic T Cells That Express Integrin α4β7 and CCR9 Are Required for Induction of Oral Immune Tolerance in Mice

Induction of oral immune tolerance (OT) blocks proinflammatory responses to orally administered antigens and might be used to treat autoimmune conditions. We investigated whether gut-tropic T cells that express the integrin α4β7 and the chemokine receptor CCR9 are required for OT. Skin delayed-type hypersensitivity and experimental autoimmune encephalomyelitis were used to monitor OT in mice. To assess the role of receptors that mediate localization of lymphocytes to the gut (gut-homing receptors) in induction of OT, we studied CCR9(-/-) and β7(-/-) mice and also blocked the α4β7 ligand MAdCAM-1 in wild-type mice. We used DEREG and Scurfy mice to assess the role of Foxp3(+) regulatory T cells (Treg) and IL-10(-/-) and IL-10Rβ(-/-) mice to examine the role of interleukin (IL)-10 in induction of OT. OT could not be induced in CCR9(-/-) or β7(-/-) mice, or when MAdCAM-1 was blocked in wild-type mice, indicating that gut-homing receptors are required for oral tolerization. Consistent with the role of all-trans retinoic acid in inducing gut-homing T cells, OT could not be induced in mice depleted of vitamin A. OT was rescued in CCR9(-/-) mice following adoptive transfer of wild-type T cells, but not CCR9(-/-) or β7(-/-) T cells. Gut-homing T cells are therefore necessary and sufficient to induce OT. Wild-type Treg and IL-10 were required to restore OT to CCR9(-/-) mice, indicating that homing and functional differentiation of IL-10-producing Treg in the gut is required for OT. Conversely, transfer of CCR9(-/-) or β7(-/-) T cells to wild-type mice partially inhibited OT. Expression of CCR9 and α4β7 on T cells and their subsequent localization to the gut is required for induction of OT in mice. Therapies designed to block gut-homing receptors might, under some conditions, interfere with normal tolerogenic mechanisms in the intestinal mucosa.

Oral tolerance inhibits pulmonary eosinophilia in a cockroach allergen induced model of asthma: a randomized laboratory study

BackgroundAntigen desensitization through oral tolerance is becoming an increasingly attractive treatment option for allergic diseases. However, the mechanism(s) by which tolerization is achieved remain poorly defined. In this study we endeavored to induce oral tolerance to cockroach allergen (CRA: a complex mixture of insect components) in order to ameliorate asthma-like, allergic pulmonary inflammation.MethodsWe compared the pulmonary inflammation of mice which had received four CRA feedings prior to intratracheal allergen sensitization and challenge to mice fed PBS on the same time course. Respiratory parameters were assessed by whole body unrestrained plethysmography and mechanical ventilation with forced oscillation. Bronchoalveolar lavage fluid (BAL) and lung homogenate (LH) were assessed for cytokines and chemokines by ELISA. BAL inflammatory cells were also collected and examined by light microscopy.ResultsCRA feeding prior to allergen sensitization and challenge led to a significant improvement in respiratory health. Airways hyperreactivity measured indirectly via enhanced pause (Penh) was meaningfully reduced in the CRA-fed mice compared to the PBS fed mice (2.3 ± 0.4 vs 3.9 ± 0.6; p = 0.03). Directly measured airways resistance confirmed this trend when comparing the CRA-fed to the PBS-fed animals (2.97 ± 0.98 vs 4.95 ± 1.41). This effect was not due to reduced traditional inflammatory cell chemotactic factors, Th2 or other cytokines and chemokines. The mechanism of improved respiratory health in the tolerized mice was due to significantly reduced eosinophil numbers in the bronchoalveolar lavage fluid (43300 ± 11445 vs 158786 ± 38908; p = 0.007) and eosinophil specific peroxidase activity in the lung homogenate (0.59 ± 0.13 vs 1.19 ± 0.19; p = 0.017). The decreased eosinophilia was likely the result of increased IL-10 in the lung homogenate of the tolerized mice (6320 ± 354 ng/mL vs 5190 ± 404 ng/mL, p = 0.02).ConclusionOur results show that oral tolerization to CRA can improve the respiratory health of experimental mice in a CRA-induced model of asthma-like pulmonary inflammation by reducing pulmonary eosinophilia.