Development Of Oral Tolerance Research Articles

A34 DEVELOPING A NOVEL MOUSE MODEL TO ELUCIDATE MECHANISMS OF ORAL TOLERANCE IN EARLY LIFE

Abstract Background Oral tolerance is the active suppression of immune responses to antigens that are first encountered in the gastrointestinal tract. When this process fails, food allergies can arise. Food allergies affect up to 6% of Canadian children underlining the need for a thorough understanding of how oral tolerance develops in childhood. Despite this, tolerance has historically been studied using adult rodent models instead of focusing on more physiologically relevant early-life time points. Aims Our research aims to develop a robust mouse model to elucidate mechanisms of oral tolerance development in early life and we hypothesize that the mesenteric lymph nodes are a major site of food antigen-specific T cell development in early life. Methods Prior to weaning, actively suckling mouse pups were orally gavaged with ovalbumin (OVA), an egg white component and common food allergen or water as a control. Following a series of systemic challenges with OVA, circulating antibody levels were quantified by ELISA to measure the development of oral tolerance to OVA. Additionally, splenocytes isolated from tolerized and control mice were stimulated in vitro with exogenous OVA and cytokines were quantified in culture supernatants via a cytometric bead array assay to assess the suppression of T cell responses to OVA in tolerized mice. One week following oral OVA exposure flow cytometry was used to characterize the expansion of T cell populations in the mesenteric lymph nodes, spleen and thymus. Results Mice orally gavaged with OVA in early life show reduced systemic antibody production after subsequent challenges with OVA, compared to mice not orally exposed to OVA. We determined the minimum dosage of OVA required to confer tolerance in these mice and confirmed the validity of this model in both male and female C57BL/6 and BALB/c mice. Further, we detected an active suppression of IL-5 production, a cytokine indicative of a Type 2 immune response, in OVA-stimulated splenocyte cultures isolated from mice tolerized to OVA. We found a significant increase in the frequency of CD4 T cells in the mesenteric lymph nodes just 1-week following oral OVA exposure. Conclusions We have developed a robust mouse model of oral tolerance where mice are directly exposed to OVA in an early life period while they are still actively ingesting breastmilk, and their mucosal immune system and intestinal microbiota composition are under significant development. To date, our data suggests that the gut draining mesenteric lymph nodes may be a site of food antigen-specific T cell expansion in early life. Funding Agencies CIHRTRIANGLE

Early-life risk factors for food allergy: Dietary and environmental factors revisited.

There appears a steep increase in the prevalence of food allergy worldwide in the past few decades. It is believed that, rather than genetic factors, the recently altered dietary and environmental factors are the driving forces behind the rapid increase of this disease. Accumulating evidence has implied that external exposures that occurred in prenatal and postnatal periods could affect the development of oral tolerance in later life. Understanding the potential risk factors for food allergy would greatly benefit the progress of intervention and therapy. In this review, we present updated knowledge on the dietary and environmental risk factors in early life that have been shown to impact the development of food allergy. These predominantly include dietary habits, microbial exposures, allergen exposure routes, environmental pollutants, and so on. The key evidence, conflicts, and potential research topics of each theory are discussed, and associated interventional strategies to prevent the disease development and ameliorate treatment burden are included. Accumulating evidence has supported the causative role of certain dietary and environmental factors in the establishment of oral tolerance in early life, especially the time of introducing allergenic foods, skin barrier function, and microbial exposures. In addition to certain immunomodulatory factors, increasing interest is raised toward modern dietary patterns, where adequately powered studies are required to identify contributions of those modifiable risk factors. This review broadens our understanding of the connections between diet, environment, and early-life immunity, thus benefiting the progress of intervention and therapy of food allergy.

The Programming Effect of Plant-Based DHA, Along with Equivalent AA, on Immune System and Oral Tolerance Development in Six-Week Allergy Prone BALB/c Pups

BackgroundDocosahexaenoic acid (DHA) and arachidonic acid (AA) on oral tolerance (OT) development in allergy-prone infants is less known. ObjectivesWe aim to determine the effects of early life DHA supplementation (1% of total fat, from novel canola oil), along with AA, on OT toward ovalbumin (ova, egg protein) in allergy-prone BALB/c pups at 6-wk. MethodsBreastfeeding dams (n ≥ 10/diet) were fed DHA+AA (1% DHA, 1% AA wt/wt of total fat) or control (0% DHA, 0% AA) suckling period diet (SPD) during which pups consumed dam’s milk. At 3-wk, pups from each SPD group were assigned to either the control or DHA+AA weaning diet. For OT, pups from each diet group were either orally fed ova or placebo daily from 21–25 d. Systemic immunization to ova was induced through intraperitoneal injections before euthanizing 6-wk pups. Ova-specific immunoglobulin (ova-Ig) and splenocytes ex-vivo cytokine response to different stimuli were analyzed using a 3-factor analysis of variance. ResultsOT-induced suppression was seen in ova-stimulated splenocyte ex-vivo response, where ova-tolerized pups showed significantly lower total immunoglobulin (Ig)G, IgG1, interleukin (IL)-2 and IL-6 production than sucrose (placebo) pups. DHA+AA SPD was associated with 3 times lower plasma concentrations of ova-IgE (P = 0.03) than controls. DHA+AA weaning diet resulted in lower T helper type-2 cytokines (IL-4 and IL-6) with ova stimulation than controls, which may benefit OT. DHA+AA SPD resulted in significantly higher T cell cytokine response [IL-2, interferon-gamma, (IFNγ) and IL-1β] to anti-CD3/CD28 stimulation than controls. The splenocytes stimulated with lipopolysaccharide produced lower inflammatory cytokines (IFNγ, tumor necrosis factor-alpha, IL-6, and C-X-C motif ligand 1), which may be because of lower CD11b+CD68+ splenocytes proportion in pups from DHA+AA SPD than control (all P < 0.05). ConclusionsDHA and AA in early life may influence OT in allergy-prone BALB/c mouse offspring, as they effectively promote T helper type-1 immune responses.

Endogenous retroviruses act as a tonic signal for Treg induction and oral tolerance development

Abstract Endogenous retrovirus (ERVs) result from accumulated germline infections by ancient exogenous retroviruses and constitute a sizable proportion of the mammalian genome. Recent findings support the idea that these elements control tissue immune threshold of activation and inflammation. Within this context, our lab has recently described that, in the skin, the beneficial immunity to the microbiota depends on endogenous retrovirus expression. How the crosstalk between ERVs and the microbiota controls tissue immunity at other barrier sites and how dysregulation of this dialogue impairs tolerance responses remain unknown. Our results reveal that antiretroviral (anti-RT) treatment (blocking the conversion of ERV into cDNA) impairs gut regulatory T cell homeostasis and differentiation and compromises the induction of oral tolerance to food antigens. Consistently, our data demonstrate that antiretroviral treatment impairs type I IFN signaling in mucosal dendritic cells involved in the induction of regulatory T cells. Further, our results show that epithelial ERV expression is highly conserved across tissues and seems to be only partially affected by the microbiota in the small intestine. Altogether, our results propose that ERV control immunoregulation within the gastrointestinal tract by acting as a tonic signal required for Treg induction and differentiation thereby promoting oral tolerance and preventing allergic responses. This work was supported in part by intramural funds of NIAID, NIH. C.A.R is supported by Damon Runyon Fellowship program (DRG-2496-23).

Breast milk microRNAs: Potential players in oral tolerance development.

Breast milk is an essential source of nutrition and hydration for the infant. In addition, this highly complex biological fluid contains numerous immunologically active factors such as microorganisms, immunoglobulins, cytokines and microRNAs (miRNAs). Here, we set out to predict the function of the top 10 expressed miRNAs in human breast milk, focusing on their relevance in oral tolerance development and allergy prevention in the infant. The top expressed miRNAs in human breast milk were identified on basis of previous peer-reviewed studies gathered from a recent systematic review and an updated literature search. The miRNAs with the highest expression levels in each study were used to identify the 10 most common miRNAs or miRNA families across studies and these were selected for subsequent target prediction. The predictions were performed using TargetScan in combination with the Database for Annotation, Visualization and Integrated Discovery. The ten top expressed miRNAs were: let-7-5p family, miR-148a-3p, miR-30-5p family, miR-200a-3p + miR-141-3p, miR-22-3p, miR-181-5p family, miR-146b-5p, miR-378a-3p, miR-29-3p family, miR-200b/c-3p and miR-429-3p. The target prediction identified 3,588 potential target genes and 127 Kyoto Encyclopedia of Genes and Genomes pathways; several connected to the immune system, including TGF-b and T cell receptor signaling and T-helper cell differentiation. This review highlights the role of breast milk miRNAs and their potential contribution to infant immune maturation. Indeed, breast milk miRNAs seem to be involved in several pathways that influence oral tolerance development.

Identification of peptides sequence and conformation contributed to potential allergenicity of main allergens in yogurts.

Yogurts provide a good source of nutrition and may induce tolerance in people with cow's milk allergy (CMA). This study aimed to investigate the IgE-binding capacity of main allergens in the different yogurts which provide a reference for people with a high risk of CMA, and analyze the epitopes of major allergen peptides in yogurt. We assessed the degradation and the allergenic properties of major allergens in six commercial yogurts and fresh milk. The degradation of major allergens was analyzed by SDS-PAGE and RP-HPLC. Western blot and ELISA experiments detected allergenic characteristics by using specific sera. The results showed that β-lactoglobulin (Bos d 5) and α-lactalbumin (Bos d 4) were obviously degraded in yogurts but caseins were still present in abundance, which indicated that the proteases in yogurts were specific to whey proteins. IgE and IgG binding ability of major allergens were obviously reduced in yogurts, especially GuMi yogurt. In addition, 17 peptides of major allergens in GuMi yogurt were identified by LC-MS/MS and most of them were located in the interior of the spatial structure of proteins. Among them, 8 peptides had specific biological functions for health benefits, such as antibacterial, antioxidant, and ACE-inhibitory. We also found that 6 and 14 IgE epitopes of Bos d 5 and caseins were destroyed in GuMi yogurt, which could lead to the reduction of IgE-binding capacity. Meanwhile, peptides [Bos d 5 (AA15-40), Bos d 9 (AA120-151, AA125-151)] also preserved T cell epitopes, which might also induce the development of oral tolerance. Therefore, this study suggested that the sequence and conformation of peptides in yogurts contributed to hypoallergenicity.

Front Cover: Breast Milk‐Derived Limosilactobacillus reuteri Prevents Atopic Dermatitis in Mice via Activating Retinol Absorption and Metabolism in Peyer's Patches

Mol. Nutr. Food Res. 2023, 67, 202200444 DOI: 10.1002/mnfr.202200444 Limosilactobacillus reuteri Fn041 is a breast milk-derived probiotic from agricultural and pastoral areas. Eczema is prevented in mice pups when Fn041 is administrated to dams during later pregnancy and lactation and to pups after weaning. The mechanism is related to the promotion of retinol absorption and conversion to retinoic acid in Peyer's patches, which is critical for oral tolerance development. This is reported by Renqiang Yu, Jin Sun, and co-workers in article number 2200444.

Diet-induced obesity results in impaired oral tolerance induction.

Obesity increases the risk of several diseases, such as type 2 diabetes mellitus and cardiovascular disease. Obesity also affects the immune system. When dietary lipids are transported via the lymphatics, they pass the mesenteric lymph nodes (mLNs). In these secondary lymphoid organs, immune responses towards pathogens are generated, or tolerance against harmless antigens is induced. In this study, the effects of diet-induced obesity (DIO) on mLN induced oral tolerance induction were examined in C57BL/6NCrl mice. Therefore, mice were fed a high-fat or a low-fat diet for 14 weeks. After 10 weeks of feeding oral tolerance induction started, ending up in measuring the delayed-type hypersensitivity reaction, the cell subset composition and cytokine expression. We detected an impaired oral tolerance induction during DIO, but changes were reversible after switching the feed to standard chow. Thus, the altered immunological function of mLNs depends on the intake of dietary lipids. Additionally, our results show an influence of the microenvironment on the development of oral tolerance during DIO as oral tolerance was induced in transplanted peripheral lymph nodes. This indicates a functional influence of dietary lipids on stromal cells involved in immune system induction in the mLNs.

The Role of the Gut Microbiome in Cow's Milk Allergy: A Clinical Approach.

Cow's milk allergy (CMA) is the most prevalent food allergy (FA) in infancy and early childhood and can be present with various clinical phenotypes. The significant increase in FA rates recorded in recent decades has been associated with environmental and lifestyle changes that limit microbial exposure in early life and induce changes in gut microbiome composition. Gut microbiome is a diverse community of microbes that colonize the gastrointestinal tract (GIT) and perform beneficial functions for the host. This complex ecosystem interacts with the immune system and has a pivotal role in the development of oral tolerance to food antigens. Emerging evidence indicates that alterations of the gut microbiome (dysbiosis) in early life cause immune dysregulation and render the host susceptible to immune-mediated diseases later in life. Therefore, the colonization of the gut by "healthy" microbes that occurs in the first years of life determines the lifelong health of the host. Here, we present current data on the possible role of the gut microbiome in the development of CMA. Furthermore, we discuss how gut microbiome modification might be a potential strategy for CMA prevention and treatment.

The Role of HnRNPA1 in T Cell-Mediated Gut Tolerance

Abstract Our previous research showed that Akt phosphorylation of the RNA-binding protein, (RBP) heterogeneous nuclear ribonucleoprotein (hnRNP) A1, is dependent on TCR signal strength, and occurs under conditions of induced T regulatory (Treg) cell differentiation. We have shown hnRNPA1 is required for optimal Treg differentiation by performing knockdown experiments, and our present research is focused on identifying a role for Akt phosphorylation in hnRNPA1 function. HnRNPA1 is phosphorylated by Akt at S199 and our lab has generated a new mutant mouse model, hnRNPA1-S199A (mA1), in which hnRNPA1 will no longer be Akt-phosphhorylated. Our initial characterization of mA1 mouse model failed to reveal major abnormalities in immune cells populations at steady state except for changes in the Treg proportion in the mesenteric lymph nodes. To investigate the effect of the mA1 on the development of oral tolerance we used an adoptive transfer model in which OT-II mA1 or OT-II WT naïve T cells were transferred into congenic strains and then the mice were given OVA food. Results indicate that mA1 OT-II T cells have a proliferation defect in vivo and we also observed a significant reduction in the induction of OVA-specific Treg in the mA1 T cells. Similar results were obtained when adoptively transferred OT-II mA1 T cells were challenged with limiting doses of specific peptide, given iv. RNASeq analysis of the transferred mA1 and WT T cells is being performed. These results suggest that Akt phosphorylation of hnRNPA1 is necessary for T cell response to low dose antigen and Treg induction in vivo. Our studies identify a new mechanism by which RBP influence T cell proliferation and Treg differentiation which has implications for tolerance to food antigens in the gut. Supported by NIH (F31-AI152320-02) NIH (R01-AI125513-03)

Ефективність пробіотичної терапії гастроінтестинальної форми харчової алергії в дітей

Актуальність. Останніми роками багато досліджень зосереджені на розумінні механізмів виникнення алергічної реакції. Кишкова мікробіота бере участь у розвитку оральної толерантності, тому виникає зацікавленість у використанні пробіотиків, які сприятимуть колонізації кишечника та становленню кишкового гомеостазу, та існує потреба в дослідженнях ефективності застосування пробіотиків з метою профілактики та лікування алергічних захворювань у дітей. Мета дослідження: оцінити ефективність застосування біопрепарату, створеного на основі спороутворюючого мікроорганізму Bacillus subtilis, у комплексній терапії дітей із гастроінтестинальними проявами харчової алергії. Матеріали та методи. Обстежені 34 дитини віком від 3,5 до 12 років із харчовою алергією зі скаргами з боку шлунково-кишкового тракту. Основна група — 18 дітей, група порівняння — 16 дітей. Діти основної групи додатково отримували споровий пробіотик на основі Bacillus subtilis по 1 дозі 2 рази на день протягом 20 днів. Ефективність терапії оцінювалась за швидкістю й повнотою згасання гастроінтестинальних симптомів та проявів коморбідної алергічної патології. Результати. У пацієнтів основної групи згасання симптоматики з боку шлунково-кишкового тракту та симптомів супутньої алергічної патології відзначалося швидше, ніж у пацієнтів групи порівняння. Через 10 днів використання спорового пробіотика на основі Bacillus subtilis відмічалося поступове зниження рівня специфічних IgE як до харчових, так і до інгаляційних алергенів на 25–30 % у дітей, які отримували комплексну терапію, а через 21 добу — на 35–40 %. При бактеріологічному дослідженні випорожнень у всіх дітей були виявлені зміни за рахунок дефіциту біфідо- і лактобактерій, збільшення кількості умовно-патогенної флори і змін її видового співвідношення. При повторному дослідженні калу на дисбактеріоз у дітей основної групи нормалізація мікробіоценозу кишечника відзначалася в 78 % випадків. Споровий пробіотик на основі Bacillus subtilis добре переносився всіма пацієнтами без будь-яких ускладнень. У подальші 3 місяці абдомінального болю та порушень із боку випорожнень у 83,33 % пацієнтів основної групи не спостерігалося, а в дітей групи порівняння продовжували відзначатися епізоди абдомінального болю та порушень випорожнень. Протягом 3-тижневого курсу застосування спорового пробіотика на основі Bacillus subtilis відзначалося помірне покращання клінічної симптоматики коморбідної патології — алергічного риніту та атопічного дерматиту, а загострень бронхіальної астми не було зовсім на противагу групі порівняння, у якій не відзначалося суттєвої позитивної клінічної динаміки коморбідних захворювань. Висновки. Застосування спорового пробіотика на основі Bacillus subtilis у комплексній терапії дітей з гастроінтестинальною формою харчової алергії сприяє швидкому регресу клінічних проявів з боку шлунково-кишкового тракту та покращанню загального стану хворих.

Retinoic Acid Induces Functionally Suppressive Foxp3+RORγt+ T Cells In Vitro.

IntroductionCD4+ T cells with regulatory function co-expressing Foxp3 and RORγt are linked to the development of oral tolerance towards innocuous food antigens in mice. This study aimed to discern the role played by IL-6 and retinoic acid (RA) in the in vitro generation of Foxp3+RORγt+ T cells and to investigate whether such cells have suppressive properties.MethodsCD4+CD25- T cells isolated from the spleen of BALB/c mice, were stimulated in the presence of IL-2 alone or together with TFG-β and different concentrations of IL-6 and/or RA. Percentage of Foxp3+, RORγt+, IL-17+, Foxp3+RORγt-, Foxp3+RORγt+, and Foxp3-RORγt+ T cells within the total CD4+ T cell population, production of cytokines (IL-10 and IL-17A) and gene expression (Foxp3, Rorc, Tgfb1, Il6, Il10, and Il17) were assessed at different time points. The phenotype and ability of cells generated from CD4+CD44-CD62L+ cells in the presence of RA to suppress effector T cell proliferation was assessed.ResultsTGF-β plus IL-6 induced the generation of Foxp3+ and double positive Foxp3+RORγt+ T cells to a higher extent than TGF-β alone at the beginning of the incubation period, although expression of Foxp3 subsequently declined. RA, added to TGF-β, increased Foxp3 and Rorc expression and Foxp3 and RORγt transcription and promoted the differentiation of Foxp3+RORγt- and Foxp3+RORγt+ cells that expressed and secreted IL-17. Foxp3+ T cells generated in vitro in presence of RA were functionally suppressive.ConclusionsUnder the influence of IL-2 and TGF-β, suppressive Foxp3+RORγt+ T cells that express and secrete IL-17 can be produced in vitro and RA further contributes to stabilize this phenotype.

Strategies and Future Opportunities for the Prevention, Diagnosis, and Management of Cow Milk Allergy.

The prevalence of food allergy has increased over the last 20-30 years, including cow milk allergy (CMA) which is one of the most common causes of infant food allergy. International allergy experts met in 2019 to discuss broad topics in allergy prevention and management of CMA including current challenges and future opportunities. The highlights of the meeting combined with recently published developments are presented here. Primary prevention of CMA should start from pre-pregnancy with a focus on a healthy lifestyle and food diversity to ensure adequate transfer of inhibitory IgG- allergen immune complexes across the placenta especially in mothers with a history of allergic diseases and planned c-section delivery. For non-breastfed infants, there is controversy about the preventive role of partially hydrolyzed formulae (pHF) despite some evidence of health economic benefits among those with a family history of allergy. Clinical management of CMA consists of secondary prevention with a focus on the development of early oral tolerance. The use of extensive Hydrolysate Formulae (eHF) is the nutrition of choice for the majority of non-breastfed infants with CMA; potentially with pre-, probiotics and LCPUFA to support early oral tolerance induction. Future opportunities are, among others, pre- and probiotics supplementation for mothers and high-risk infants for the primary prevention of CMA. A controlled prospective study implementing a step-down milk formulae ladder with various degrees of hydrolysate is proposed for food challenges and early development of oral tolerance. This provides a more precise gradation of milk protein exposure than those currently recommended.

Influences of non-IgE-mediated cow's milk protein allergy-associated gut microbial dysbiosis on regulatory T cell-mediated intestinal immune tolerance and homeostasis

Gut microbial dysbiosis is closely associated with cow's milk protein allergy (CMPA) during infancy. Recent research has highlighted the crucial role of the commensal microbiota-induced intestinal regulatory T (Treg) cell response in the development of oral tolerance and protection against IgE-mediated food allergies. However, the influences of CMPA (particularly non-IgE-mediated CMPA)-associated microbial dysbiosis on Treg cell-mediated intestinal immune tolerance and homeostasis remain poorly characterized. To investigate this issue, fecal microbiota from infant donors with food protein-induced allergic proctocolitis (FPIAP) associated with cow's milk, which is the most frequent clinical type of non-IgE-mediated gastrointestinal CMPA, and from age-matched healthy controls were transplanted into germ-free mice in this study. Two weeks post fecal microbiota transplantation, the gut microbiome of the recipient mice was analyzed by 16S rRNA gene sequencing, and the intestinal immunological alterations associated with the Treg cell compartment and intestinal immune homeostasis were detected. The specific gut microbial phylotypes that were potentially responsible for the disruption of intestinal immune homeostasis were also analyzed. We observed that the main characteristics of the gut microbiome in infant donors could be stably maintained in recipient mice. We also found that mice colonized with the gut microbiome from infants with cow's milk-induced FPIAP showed significant deficiencies in the accumulation and function of intestinal Treg cells. Furthermore, these mice showed disrupted intestinal immune homeostasis, which was characterized by an overactivated Th2 biased immune response. We further identified two potentially pathogenic genera that contribute to this disruption. Overall, our results highlight a destructive effect of non-IgE-mediated CMPA-associated microbial dysbiosis on intestinal immune tolerance and homeostasis. We believe these findings will help improve our understanding of the gut microbiota-mediated pathogenesis of non-IgE-mediated CMPA in the future.

The role of immunoglobulin A in oral tolerance and food allergy

Mucosal surfaces, which are frequently exposed to external antigens, are a major part of the immune system. The development of oral tolerance is a result of a complex interplay between a variety of innate and adaptive immune functions; deficiencies in these mechanisms may contribute to the development of allergic disorders. Although regulatory T cells have a clear role in enforcing oral tolerance, the role of the dominant immunoglobulin in the gut, immunoglobulin A (IgA), is less clear. This perspective will focus on IgA and its role in tolerance to oral antigens and the development of food allergies.

Recombinant Lactococcus lactis Carrying IL-4 and IL-10 Coding Vectors Protects against Type 1 Diabetes in NOD Mice and Attenuates Insulitis in the STZ-Induced Model.

Type 1 diabetes (T1D) is an autoimmune disease that culminates in beta cell destruction in the pancreas and, subsequently, deficiency in insulin production. Cytokines play a crucial role in the development of diabetes, orchestrating the recruitment and action of immune cells, to not only destroy insulin-producing cells but also preserve them. Therefore, the aim of this study was to investigate the effect of orally administered Lactococcus lactis MG1363 FnBPA+ strains carrying plasmids encoding IL-4 and IL-10 in the streptozotocin- (STZ-) induced diabetes model and in nonobese diabetic (NOD) mice. The STZ-induced mice that were treated with combined bacterial strains carrying plasmids encoding IL-4 and IL-10 showed lower incidence of diabetes and more preserved pancreatic islets than the mice that received the individual bacterial strains. Combined administration of L. lactis MG1363 FnBPA+ (pValac::dts::IL-4) and L. lactis MG1363 FnBPA+ (pValac::IL-10) resulted in protection against diabetes in NOD mice. It was shown that the combined treatment with recombinant bacterial by oral route prevented hyperglycemia and reduced the pancreatic islets-destruction in NOD mice. In addition, increased levels of IL-4 and IL-10 in serum and pancreatic tissue revealed a systemic effect of the treatment and also favored an anti-inflammatory microenvironment. Reduced concentrations of IL-12 in pancreas were essential to the regulation of inflammation, resulting in no incidence of diabetes in treated NOD mice. Normal levels of intestinal sIgA after long-term treatment with the L. lactis strains carrying plasmids encoding IL-4 and IL-10 indicate the development of oral tolerance and corroborate the use of this potent tool of mucosal delivery. For the first time, L. lactis MG1363 FnBPA+ strains carrying eukaryotic expression vectors encoding IL-4 and IL-10 are tested in STZ-induced and NOD mouse models. Therefore, our study demonstrates this innovative strategy provides immunomodulatory potential for further investigations in T1D and other autoimmune diseases.

Health and growth indices in children with cow’s milk protein allergy depending on oral tolerance development

Relevance. Dairy-free diet is the basis of cow’s milk allergy (CMA) patient’s management. Duration of the diet is individual, but some children don’t develop tolerance.The aim of the study was to investigate health and growth indices in children with CMA depending on tolerance development by the age of five. Materials and methods. 153 children from 1 to 18 months with diagnosed CMA were included in the prospective study (76.5% with IgE-mediated form). The tolerance was determined after 6–12 months of a milk-free diet and at the age of 5 using an open challenge test. Anthropometry indices (Anthro Plus), levels of specific IgE (Immuno CAP 250) and IgG4 (ELISA) to dietary proteins and the presence of allergic diseases in children also were assessed.Results. 50.3% of children developed tolerance to cow’s milk protein after 6–12 months of a milk-free diet. By the age of 5 63.3% of patients had complete tolerance of dairy products. Partially formed tolerance was observed in 22.9%. 13.8% of patients still had clinical reactions to cow’s milk proteins. Patients with persistent CMA typically had IgE-mediated form, lower growth indices and multiple allergic pathology.Conclusion. The consideration of tolerance development predictors can allow to personalize the management of CMA. Additional researches are needed to clarify the causes of growth indices decline in children with persistent CMA.

Effects of Early Nutrition and Sanitary Conditions on Oral Tolerance and Antibody Responses in Broiler Chickens

Greater antigenic exposure might accelerate activation and maturation of the humoral immune system. After hatch, commercial broiler chickens can have early (EN) or delayed (DN) access to nutrition, up to 72 h after hatch. The immune system of EN versus DN broilers is likely more exposed to antigens after hatch. This might contribute to activation and maturation of the immune system, but might also influence the development of oral tolerance, thereby altering later life antibody responses. We studied antibody (IgM, IgY, IgA) responses between 21 and 42 d of age in fast-growing EN and DN broilers, kept under low (LSC) or high sanitary conditions (HSC). In a first experiment (n = 51 broilers), we tested whether early oral exposure to bovine serum albumin (BSA) affected later life antibody responses towards BSA and a novel antigen—rabbit γ-globulin (RGG), under HSC. In a second experiment, a total of 480 EN and DN broilers were housed under either LSC or HSC, and we studied antibody responses against both BSA and RGG (n = 48 broilers per treatment) and growth performance. Broilers kept under LSC versus HSC, had higher antibody levels and their growth performance was severely depressed. Interactions between feeding strategy (EN versus DN) and sanitary conditions, or main effects of feeding strategy, on natural and specific antibody levels, and growth performance were not observed. Levels of IgA were elevated in EN versus DN broilers, in experiment I and in batch 2 of experiment II, but not in the other batches of experiment II. We concluded that EN versus DN contributes minimally to the regulation of antibody responses, irrespective of antigenic pressure in the rearing environment.

Exploring the Molecular Mechanisms Underlying the Protective Effects of Microbial SCFAs on Intestinal Tolerance and Food Allergy.

A body of evidence suggests that food allergy (FA) has increased in prevalence over the past few decades. Novel findings support the hypothesis that some commensal bacteria and particularly microbial metabolites might contribute to development of oral tolerance and prevention from FA. Recently, beneficial effects of short-chain fatty acids (SCFAs), the main class of gut microbiota-derived metabolites, on FA have been proposed. The intestinal SCFAs are major end products during bacterial fermentation of complex and non-digestible carbohydrates such as dietary fiber. The multifaceted mechanisms underlying beneficial effects of SCFAs on the mucosal immune system comprise the regulation of diverse cellular pathways in epithelial, dendritic, and T cells, as well as the impact on the immunometabolism and epigenetic status of regulatory lymphocytes. Of note, SCFAs are effective inhibitors of histone deacetylases (HDACs). As a consequence, SCFAs appear to be implicated in attenuation of intestinal inflammation and autoimmune diseases. In this review, we will discuss the recent development in this research area by highlighting the role of the individual SCFAs acetate, propionate, butyrate, and pentanoate in promoting the differentiation of regulatory T and B cells and their potential beneficial effects on the prevention of FA. In this context, targeted alterations in the gut microbiota in favor of SCFA producers or supplementation of medicinal food enriched in SCFAs could be a novel therapeutic concept for FA.

Toll-like receptor 2 impacts the development of oral tolerance in mouse pups via a milk-dependent mechanism

The role of breast-feeding in the development of oral tolerance and allergic diseases is controversial, which could be related to variability in milk components. Toll-like receptor 2 (TLR2) is an innate immune receptor implicated in regulating allergic disease development. We examined whether deficiency of maternal TLR2 affects the normal development of oral tolerance and related immune parameters during lactation in a mouse model. Heterozygous TLR2+/- pups from wild-type (WT) or TLR2-/- dams were fed either by their biologic dam or a dam of the alternate genotype. Development of oral tolerance to ovalbumin, levels of tolerogenic CD103+ dendritic cells, and regulatory T (Treg) cells, as well as intestinal permeability, were evaluated in these pups. The levels of key immune mediators in milk from TLR2-/- and WT mothers were also examined. Heterozygous TLR2+/- pups that were born to and nursed by TLR2-/- dams exhibited impaired oral tolerance. This was prevented by cross-fostering onto WT (TLR2+/+) dams. Impairments included selective elevation in anti-ovalbumin IgE in plasma following immunization, reduced numbers of tolerogenic dendritic cells and Treg cells in the intestinal tract, and increased intestinal permeability. TLR2 deficiency also affected milk content of insulin-like growth factor-1, IFN-γ, IL-6, and IL-13. Our results underline a critical role for TLR2 in regulating milk components that are essential for development of oral tolerance in early life and demonstrate the importance of considering the immune status of nursing mothers in studies of immune development and responses.