Intestinal factors promoting the development of RORγt+ cells and oral tolerance.

The gastrointestinal tract has to harmonize the two seemingly opposite functions of fulfilling nutritional needs and avoiding the entry of pathogens, toxins and agents that can cause physical damage. This balance requires a constant adjustment of absorptive and defending functions by sensing environmental changes or noxious substances and initiating adaptive or protective mechanisms against them through a complex network of receptors integrated with the central nervous system that communicate with cells of the innate and adaptive immune system. Effective homeostatic processes at barrier sites take the responsibility for oral tolerance, which protects from adverse reactions to food that cause allergic diseases. During a very specific time interval in early life, the establishment of a stable microbiota in the large intestine is sufficient to prevent pathological events in adulthood towards a much larger bacterial community and provide tolerance towards diverse food antigens encountered later in life. The beneficial effects of the microbiome are mainly exerted by innate and adaptive cells that express the transcription factor RORγt, in whose generation, mediated by different bacterial metabolites, retinoic acid signalling plays a predominant role. In addition, recent investigations indicate that food antigens also contribute, analogously to microbial-derived signals, to educating innate immune cells and instructing the development and function of RORγt+ cells in the small intestine, complementing and expanding the tolerogenic effect of the microbiome in the colon. This review addresses the mechanisms through which microbiota-produced metabolites and dietary antigens maintain intestinal homeostasis, highlighting the complementarity and redundancy between their functions.

Egg yolk lipids induce sensitization to egg white proteins in a mouse model without adjuvant and exacerbate Th2 responses to egg white in cells from allergic patients.

This study evaluates the influence of egg lipid fractions in the induction of allergic sensitization to egg white (EW) proteins, using a mouse model of orally adjuvant-free induced allergy. Egg triglycerides (TG) and phospholipids (PL), and to a higher extent the whole egg lipid fraction (EL), induced allergy to EW proteins characterized by increased EW-specific IgG1. EL also increased EW-specific IgE. The administration to mice of a mixture of EW and EL increased the intestinal expression of Il33, Il25, and Tslp, the secretion of IL-33 and IL-6, the expansion of group 2 innate lymphoid cells, the regulation of Gata3, Il4 and Il13, dendritic cell (DC) activation and expression of DC molecules that drive Th2 differentiation. TG promoted the absorption of proteins through the intestinal epithelium, enhancing local Th2 responses, while PL favoured the delivery of antigens to the Peyer's Patches. This differential modulation of the site of absorption of egg proteins determined the different behaviour of TG and PL. Egg yolk lipids also induced activation of Th2-inducing innate responses on intestinal human cells in vitro and enhanced adaptive Th2 functions through the activation of DCs in egg-allergic subjects.

IgE-Binding and Immunostimulating Properties of Enzymatic Crosslinked Milk Proteins as Influenced by Food Matrix and Digestibility.

Dairy foods are essential in the diet, although in some susceptible individuals they may cause allergy to cow's milk proteins. Therefore, alternative methods are sought to reduce their allergenicity. Transglutaminase (TG) is widely used in dairy products mainly to improve texture. Although it has been claimed that TG can be used to modify the digestibility and allergenicity of foods, its impact within a real matrix has been rarely studied. The aim of this work was to assess the allergenic potential of crosslinked skim milk (SM), milk casein fraction (CN), and whey protein (WP). To this purpose, inhibition ELISA with sera from milk allergic patients, in vitro activation tests of mouse mast cells and splenocytes, and simulated gastrointestinal digestion assays were performed. The results showed that cross-linking increased the binding of IgE to WP, but decreased IgE-binding to SM and CN. However, no differences were observed in the ability of cross-linked proteins to induce mast cell degranulation compared to native proteins. The cross-linking of SM and CN reduced Th2 cytokine release from the splenocytes of sensitized mice. All TG-treated samples exhibited more resistance to in vitro digestion than the untreated proteins and the human IgE binding capacity after digestion was higher. In conclusion, TG treatment of milk proteins does not reduce the risk of eliciting allergic symptoms in cow's milk allergic patients.

Triacylglycerides and Phospholipids from Egg Yolk Differently Influence the Immunostimulating Properties of Egg White Proteins.

As part of a whole egg, egg white proteins are embedded in a lipid matrix that could modify their presentation to the immune system and their allergenic properties. The present study examines the impact of the main egg lipid components, triacylglycerides and phospholipids, in the early events of sensitization to egg. To this end, BALB/c mice were exposed intragastrically to egg lipids and egg lipid fractions, alone and in mixtures with egg white proteins, and Th2-promoting and proinflammatory effects were investigated. Our results highlight that the egg lipid fraction is responsible for Th2 adjuvant effects and point at a different influence of triacylglycerides and phospholipids on the bioavailability and immunomodulating properties of egg white proteins. While triacylglycerides promote type 2 responses at the small intestine level, phospholipids reduce the solubility of EW proteins and induce Th2 skewing in lymphoid intestinal tissues, which may have a direct impact on the development of egg allergy.

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

Introduction: CD4+ 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. Methods: CD4+CD25- T cells isolated from the spleen of BALB/c mice, were stimulated in the presence of IL-2 alone or together with TFG-ß 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. Results: TGF-ß plus IL-6 induced the generation of Foxp3+ and double positive Foxp3+RORγt+ T cells to a higher extent than TGF-ß alone at the beginning of the incubation period, although expression of Foxp3 subsequently declined. RA, added to TGF-ß, 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. Conclusions: Under the influence of IL-2 and TGF-ß, suppressive Foxp3+RORγt+ T cells that express and secrete IL-17 can be produced in vitro and RA further contributes to stabilize this phenotype.

Oral Exposure to House Dust Mite Activates Intestinal Innate Immunity.

SCOPE: House dust mite (HDM) induces Th2 responses in lungs and skin, but its effects in the intestine are poorly known. We aimed to study the involvement of HDM in the initial events that would promote sensitization through the oral route and eventually lead to allergy development. METHODS AND RESULTS: BALB/c mice were exposed intragastrically to proteolytically active and inactive HDM, as such, or in combination with egg white (EW), and inflammatory and type 2 responses were evaluated. Oral administration of HDM, by virtue of its proteolytic activity, promoted the expression, in the small intestine, of genes encoding tight junction proteins, proinflammatory and Th2-biasing cytokines, and it caused expansion of group 2 innate immune cells, upregulation of Th2 cytokines, and dendritic cell migration and activation. In lymphoid tissues, its proteolytically inactivated counterpart also exerted an influence on the expression of surface DC molecules involved in interactions with T cells and in Th2 cell differentiation, which was confirmed in in vitro experiments. However, in our experimental setting we did not find evidence for the promotion of sensitization to coadministered EW. CONCLUSION: Orally administered HDM upregulates tissue damage factors and also acts as an activator of innate immune cells behaving similarly to potent oral Th2 adjuvants.

A Mouse Model of Oral Sensitization to Hen's Egg White.

Egg allergy is one of the most common food allergies in children, being the most important allergenic proteins found in the egg white (EW). Allergy to EW shows a complex phenotype that involves a multifaceted reaction that can only be assessed in vivo. Although other routes of sensitization have been described, oral exposure to food antigens is one of the most suitable in humans. In mice, oral administration of allergenic proteins results in the development of tolerance, and the use of adjuvants, such as cholera toxin (CT), is required to promote Th2-biased immune responses over tolerogenic responses. In this regard, among the mouse strains that readily display Th2 responses, Balb/c has been widely used. Here, we describe a frequently used protocol of oral EW sensitization by using CT as an adjuvant and we explain in detail the methods that we have developed to analyze the sensitizing and eliciting capacity of EW proteins including evaluation of signs, measurement of serum levels of specific immunoglobulins, mast cell degranulation, cytokine secretion profile of allergen-reactive T cells, phenotyping of mesenteric lymph node- and spleen-derived dendritic and T cells by flow cytometry, and quantification of intestinal gene expression.

Ovalbumin-Derived Peptides Activate Retinoic Acid Signalling Pathways and Induce Regulatory Responses Through Toll-Like Receptor Interactions.

This study investigates the potential of a hydrolysate of ovalbumin with pepsin (OP) to preclude Th2-type immunity by the enhancement of tolerogenic dendritic cells (DCs) and regulatory T (Treg) cells. Through Toll-like receptor (TLR) stimulation, OP enhances the retinoic acid pathway on DCs by means of the induction of aldehyde dehydrogenase enzymes and transforming growth factor beta (TGF-ß), and it confers upon DC the ability to upregulate interleukin 10 (IL-10) as well as other tolerance-promoting mediators downstream of TRL signalling, such as IL-27, IL-33, Notch ligands, OX40L, and the transcription factors IRF4 and IRF8. OP-conditioned DCs induce the expansion of Foxp3+ and Tr1 cells in co-culture with CD4+ T cells. Furthermore, OP directly conditions CD4+ T cells from naïve mice, without the mediation of DCs, to express aldehyde dehydrogenase (ALDH) enzymes and, in the presence of the Th2 cytokine IL-4 and exogenous TGF-ß, it enhances Foxp3 expression. It is noteworthy that, on CD4+ T cells isolated from egg-allergic mice, OP significantly enriches the levels of Foxp3+ and Foxp3+ RORγt+ CD4+ T cells. In conclusion, we show that food peptides may work, analogously to microbial-driven signals, through TLRs, to promote a tolerogenic phenotype on cells of the innate and adaptive immune system, a property that is further enhanced in the context of a Th2 cytokine-rich environment.

Egg yolk augments type 2 immunity by activating innate cells.

PURPOSE: Egg yolk (EY) may play a role during the sensitizing phase of egg allergy by exerting intestinal type 2-biasing effects. We aimed to identify the mechanism and role of EY in the induction of allergy to egg white (EW). METHODS: BALB/c mice were exposed intragastrically to EW, EY, or the mixture of EW:EY. In addition in vitro experiments were conducted with intestinal epithelial cells (IECs), dendritic cells (DCs), and T cells from naïve mice. Inflammatory and type 2 responses were evaluated. RESULTS: Administration of EW upregulated duodenal expression of factors that influence epithelial barrier integrity and function, such as Muc2 and Cldn2, type 2-promoting epithelial cytokines Il33 and Il25, DC genes Irf4 and Tnfsf4, and Th2-cytokines Il14 and Il13. EW:EY further increased the expression of Il25 and Tslp in the duodenum, Il33 and Tslp in the jejunum, and the proportion of lamina propria group 2 innate immune cells (ILC2s) over EW alone. Moreover, it distinctively enhanced the expression of Irf4 and Cd1d1 in the Peyer's patches (PPs), and of Il6, Il33, Gata3, and Il13, both in PPs and mesenteric lymph nodes. In co-cultures of DCs and T cells, EW:EY induced a higher expression of Gata3, Il4, and Il13, secretion of IL-13 and expansion of CD4+ T cells expressing ST2, the IL-33 receptor, than EW or EY added individually. CONCLUSION: Co-administration of EY may promote sensitization to EW through activation of innate immune cells, such as IECs, DCs and ILC2s, that are central to the progress of allergies.

Role of dietary lipids in food allergy.

The prevalence of food allergy is raising in industrialized countries, but the mechanisms behind this increased incidence are not fully understood. Environmental factors are believed to play a role in allergic diseases, including lifestyle influences, such as diet. There is a close relationship between allergens and lipids, with many allergenic proteins having the ability to bind lipids. Dietary lipids exert pro-inflammatory or anti-inflammatory functions on cells of the innate immunity and influence antigen presentation to cells of the adaptive immunity. In addition to modifying the immunostimulating properties of proteins, lipids also alter their digestibility and intestinal absorption, changing allergen bioavailability. This study provides an overview of the role of dietary lipids in food allergy, taking into account epidemiological information, as well as results of mechanistic investigations using in vivo, ex vivo and in vitro models. The emerging link among high-fat diets, obesity, and allergy is also discussed.

Oral Immunotherapy with Egg Peptides Induces Innate and Adaptive Tolerogenic Responses.

SCOPE: The mechanism through which peptide-based immunotherapy provides effective desensitization toward food allergy is investigated. METHODS AND RESULTS: Ex vivo experiments are conducted with intestinal epithelial cells (IECs), dendritic cells (DCs), and T cells from mice sensitized to egg white (EW) and either left untreated or tolerized by the oral administration of a hydrolysate of ovalbumin with pepsin (OP). IECs from EW-sensitized mice upregulate Il33 and Tslp to a higher extent than those from tolerized mice and induce bone marrow (BM)-DCs to express Tnfsf4 and produce pro-inflammatory cytokines. On the other hand, incubation with OP upregulates Aldh1a1 in IEC cultures and BM-DCs conditioned with supernatants of OP-pulsed IECs also overexpress Aldh1a2 and Tgfb1. DCs from tolerized mice, in co-culture with CD4+ T cells from sensitized mice, reduce the secretion of IL-5, IFN-γ, and IL-17, following stimulation with EW, to a level similar than DCs from sham-sensitized mice. Furthermore, incubation with OP of DCs and CD4+ T cells, regardless of the mouse sentitization status, promotes the secretion of TGF-ß and the generation of Foxp3+ RORγt+ cells. CONCLUSION: OP induces the expression of aldehyde dehydrogenase enzymes in cells of the innate immune system and the development of Foxp3+ RORγt+ T cells.

Egg Yolk Provides Th2 Adjuvant Stimuli and Promotes Sensitization to Egg White Allergens in BALB/c Mice.

SCOPE: Egg is the second most frequent source of allergic reactions in children. Egg yolk (EY) amounts to one-third in weight of a fresh whole egg, but its contribution to egg allergy has not been investigated in depth. This study assesses whether EY influences the capacity of egg white (EW) to sensitize and trigger allergic responses. METHODS AND RESULTS: BALB/c mice were exposed to EW, EY, and their mixture, using models of orally (with and without adjuvant) and adjuvant-free intraperitoneally induced allergy. In vitro assays were also conducted to examine epithelial and dendritic cell (DC) functions. Results showed that EY played a role during the sensitizing phase of allergy. EY exerted local Th2-biasing effects through the upregulation of intestinal IL-33 expression and it also favored Th2 polarization directly during DC presentation of allergens to T cells. CONCLUSION: The results obtained reveal that EY provides Th2-adjuvant stimuli to the immune system that may increase the susceptibility to develop egg allergy. The joint administration of EW and EY may be a trigger for initiation or maintenance of egg allergy with implications in prevention strategies regarding egg introduction in the diet of susceptible children.

Pepsin Egg White Hydrolysate Improves Glucose Metabolism Complications Related to Metabolic Syndrome in Zucker Fatty Rats.

The purpose of this study was to evaluate the effect of the administration of two egg white hydrolysates on glucose metabolism complications related to Metabolic Syndrome (MS) in Zucker fatty rats (ZFR). ZFR were given 750 mg/kg/day of egg white hydrolyzed with pepsin (HEW1) or with aminopeptidase (HEW2) for 12 weeks in their drinking water or just water. Zucker lean rats (ZLR), which received water, were used as a control. The presence of tactile allodynia, which is a sign of peripheral neuropathy, was assessed. Blood samples and pancreas were collected to determine the effect of the hydrolysates on glucose metabolism. The intake of HEW1 significantly lowered plasma insulin levels and improved the quantitative indexes of insulin resistance, insulin sensitivity, and pancreatic ß-cell functionality (HOMA-IR, HOMA-ß, and QUICKI, respectively), but non-significant changes were observed in group treated with HEW2. Compared to ZLR, ZFR showed tactile allodynia, but the consumption of both hydrolysates significantly increased mechanical sensitivity in ZFR. In conclusion, HEW1 pepsin could improve the glucose metabolism abnormalities associated with MS in obese Zucker rats.

Assessment of the Allergenic Potential of the Main Egg White Proteins in BALB/c Mice.

This work aimed to assess the contribution of the major egg white proteins, ovalbumin, ovomucoid, and lysozyme, to the induction and elicitation of allergenic responses. For this purpose, BALB/c mice were orally administered either the individual egg allergens or a mixture of the three proteins in the same proportion, to evaluate their relative allergenicity avoiding their different abundance in egg white. Cholera toxin was used as a T helper 2 (Th2)-polarizing adjuvant. Ovomucoid and lysozyme triggered the most severe anaphylaxis reactions upon oral challenge. In comparison to ovalbumin and ovomucoid, lysozyme was a more active promotor of early immunoglobulin E and immunoglobulin G1 production and stimulated stronger Th2-biased responses from both mesenteric lymph node and spleen cells. These results indicate that lysozyme is highly immunogenic and should be considered as a major allergen, whose clinical usefulness in the diagnosis, prognosis, and therapeutic approaches of egg allergy deserves further consideration.

Immunomodulating peptides for food allergy prevention and treatment.

Among the most promising strategies currently assayed against IgE-mediated allergic diseases stands the possibility of using immunomodulating peptides to induce oral tolerance toward offending food allergens or even to prevent allergic sensitization. This review focuses on the beneficial effects of food derived immunomodulating peptides on food allergy, which can be directly exerted in the intestinal tract or once being absorbed through the intestinal epithelial barrier to interact with immune cells. Food peptides influence intestinal homeostasis by maintaining and reinforcing barrier function or affecting intestinal cell-signalling to nearby immune cells and mucus secretion. In addition, they can stimulate cells of the innate and adaptive immune system while supressing inflammatory responses. Peptides represent an attractive alternative to whole allergens to enhance the safety and efficacy of immunotherapy treatments. The conclusions drawn from curative and preventive experiments in murine models are promising, although there is a need for more pre-clinical studies to further explore the immunomodulating strategy and its mechanisms and for a deeper knowledge of the peptide sequence and structural requirements that determine the immunoregulatory function.

Sensitizing and Eliciting Capacity of Egg White Proteins in BALB/c Mice As Affected by Processing.

This study assesses to what extent technological processes that lead to different degrees of denaturation of egg white proteins affect their allergenicity. We focused on heat (80 °C, 10 min) and high-pressure (400 MPa and 37 °C, 10 min) treatments and used a BALB/c mouse model of food allergy. Oral sensitization to egg white using cholera toxin as adjuvant induced the production of IgE and IgG1 isotypes and led to severe clinical signs following challenge with the allergen. Extensive protein denaturation caused by heat treatment increased its ability to induce Th1 responses and reduced both its sensitizing and eliciting capacity. Heated egg white stimulated the production of IgE over IgG1 antibodies directed, at least in part, toward new epitopes exposed as a result of heat treatment. Conversely, partial denaturation caused by high-pressure treatment increased the ability of egg white to stimulate Th2 responses and its allergenic potential.

Assessment of IgE Reactivity of ß-Casein by Western Blotting After Digestion with Simulated Gastric Fluid.

Cow's milk allergy is defined as an immunologically mediated adverse reaction to cow's milk proteins and it is usually, along with hen's egg allergy, the first food allergy identified in childhood.One of the main aspects to consider when evaluating the allergenic potential of food proteins is the effect of gastric digestion. It is known that allergens are usually able to survive the harsh acidic environment of the stomach, tolerate the presence of surfactants, and resist digestion by pepsin. They might also be digested into high molecular weight peptide fragments, which retain the same, or sometimes increased, IgE-binding. In this respect, western blotting is a highly sensitive and efficient technique that we have used to detect IgE-binding to the digests of milk and egg proteins. Given the importance of the resistance of food proteins to gastric digestion in their capacity to modulate the immune response, we describe in this chapter the assessment of IgE reactivity of a relevant cow's milk allergen, ß-casein, by western blotting after simulated digestion under relevant physiological conditions.

Pepsin egg white hydrolysate modulates gut microbiota in Zucker obese rats.

There is limited information that relates the intake of food-derived bioactive peptides and the gut microbiota. We have previously described a pepsin hydrolysate of egg white (EWH) that ameliorates fat accumulation and dyslipidemia, while reducing oxidative stress and inflammation markers in obese Zucker rats. The aim of this study was to associate the beneficial effects of EWH with gut microbiota changes in these animals. Obese Zucker rats received daily 750 mg kg-1 EWH in drinking water for 12 weeks and faeces were analysed for microbial composition and metabolic compounds in comparison with Zucker lean rats and obese controls. EWH supplementation modulated the microbiological characteristics of the obese rats to values similar to those of the lean rats. Specifically, counts of total bacteria, Lactobacillus/Enterococcus and Clostridium leptum in EWH fed obese Zucker rats were more similar to the lean rats than to the obese controls. Besides, feeding the obese Zucker rats with EWH reduced (P < 0.05) the faecal concentration of lactic acid. The physiological benefits of EWH in the improvement of obesity associated complications of Zucker rats could be associated with a more lean-like gut microbiota and a tendency to diminish total short-chain fatty acids (SCFA) production and associated obesity complications. The results warrant the use of pepsin egg white hydrolysate as a bioactive food ingredient.

Oral Food Desensitization in Children With IgE-Mediated Cow's Milk Allergy: Immunological Changes Underlying Desensitization.

PURPOSE: This study aimed to evaluate the safety and efficacy to induce clinical desensitization to cow's milk (CM) of an oral immunotherapy (OIT) protocol in a pediatric population with cow's milk allergy (CMA). In addition, the immune responses against ß-casein, of peripheral blood mononuclear cells (PBMCs) from CMA patients, before and after the protocol were evaluated and compared to a nonallergic population. METHODS: A group of 20 children with IgE-mediated CMA and 15 nonallergic children were recruited. Allergic subjects underwent an OIT protocol based on weekly doses of commercial semi-skimmed ultra-high temperature treated (UHT) CM, followed by a maintenance phase. Immune profiles and changes in all subjects were investigated by measuring Th1, Th2, and Treg cytokines, transcription factors, and specific IgE and IgG4 levels. RESULTS: The CM-OIT protocol enabled to desensitize 70% of the allergic patients. Successful OIT was accompanied by significant increases in casein-specific IgG4 levels, together with a reduction in the concentration of antigen-specific IgE and in IL-5, IL-13, and IL-10 production by ß-casein-stimulated PBMCs. Baseline significant differences observed between allergic and nonallergic children in IL-13 and IL-5 levels were no longer found once the protocol had finished. CONCLUSIONS: The OIT protocol was safe and effective in inducing milk desensitization in 70% of the children with CMA, leading to alterations in their immune profiles toward a nonallergic phenotype.

Regulation of Exacerbated Immune Responses in Human Peripheral Blood Cells by Hydrolysed Egg White Proteins.

The anti-allergic potential of egg white protein hydrolysates (from ovalbumin, lysozyme and ovomucoid) was evaluated as their ability to hinder cytokine and IgE production by Th2-skewed human peripheral blood mononuclear cells (PBMCs), as well as the release of pro-inflammatory factors and generation of reactive oxygen species from Th1-stimulated peripheral blood leukocytes (PBLs). The binding to IgE of egg allergic patients was determined and the peptides present in the hydrolysates were identified. The hydrolysates with alcalase down-regulated the production of Th2-biased cytokines and the secretion of IgE to the culture media of Th2-skewed PBMCs, and they significantly neutralized oxidative stress in PBLs. The hydrolysates of ovalbumin and ovomucoid with pepsin helped to re-establish the Th1/Th2 balance in Th2-biased PBMCs, while they also inhibited the release of pro-inflammatory mediators and reduced oxidative stress in PBLs treated with inflammatory stimuli. The hydrolysates with alcalase, in addition to equilibrating Th2 differentiation, exhibited a low IgE-binding. Therefore, they would elicit mild allergic reactions while retaining T cell-stimulating abilities, which might correlate with an anti-allergic benefit.