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.

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.

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.

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.

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.

Hydrolysates of egg white proteins modulate T- and B-cell responses in mitogen-stimulated murine cells.

This work assessed the effects of hydrolysates of ovalbumin (OVA), lysozyme (LYS), ovomucoid (OM) and whole egg white (EW) on cytokine secretion, antibody production, oxidative stress and proliferation of murine spleen and mesenteric lymph node cells stimulated with T- (concanavalin A - ConA) or B-cell mitogens (lipopolysaccharide - LPS). The hydrolysates of OVA, LYS and EW with alcalase reduced ConA-stimulated lymphocyte proliferation and production of Th2-biased cytokines, such as IL-13 and IL-10, and decreased the secretion of the Th1 cytokine TNF-α. In addition, these hydrolysates considerably inhibited IgG1-class switching induced by LPS and counteracted the release of reactive oxygen species. EW peptides modulated the immune responses of murine cells to mitogen stimuli, revealing potential activities that could be used for different purposes as Th1- or Th2-skewing mediators.

Clinical efficacy and immunological changes subjacent to egg oral immunotherapy.

BACKGROUND: Evidence of the efficacy of food oral immunotherapy (OIT) is not robust enough to change clinical practice from current standard management. Furthermore, the immunologic changes underlying food desensitization are unknown. OBJECTIVE: To establish the immunologic basal status and differences between an egg-allergic group of children and a population of nonallergic children and to investigate the safety and efficacy of a specific egg OIT protocol to induce clinical desensitization and the associated immune responses. METHODS: Children with or without egg allergy were recruited. Allergic subjects underwent an OIT protocol based on weekly doses of egg protein and a maintenance phase. Immune profile and changes in all subjects were investigated by measuring T-helper cells types 1 and 2 (TH1 and TH2) and T-regulatory cytokines and transcription factors and egg-specific IgE and IgG4 levels. RESULTS: At baseline, a significantly lower production of ovalbumin-specific interleukin (IL)-10 and tumor necrosis factor-α and a trend toward higher IL-5 and IL-13 were found in allergic children. The egg OIT protocol enabled 60% of them to ingest 32 mL of egg white. Significant increases in egg-specific IgG4 levels and IL-10 production, with a trend toward lower IL-5 and IL-13 and higher tumor necrosis factor-α and interferon-γ levels, and significant decreases in egg-specific IgE concentration were observed. CONCLUSION: Egg-allergic individuals display a bias toward TH2 type cytokine production and decreased TH1 and IL-10 responses compared with nonallergic individuals. The OIT protocol was safe and effective in inducing egg desensitization, leading to a shift in the immune profile of allergic individuals toward a nonallergic phenotype.

In vitro digestibility of bovine ß-casein with simulated and human oral and gastrointestinal fluids. Identification and IgE-reactivity of the resultant peptides.

Stability during digestion is considered an important feature in determining the allergenicity of food proteins. This study aimed to provide an immunological characterisation of the digestion products of the major cow's milk allergen ß-casein (ß-CN) produced by in vitro orogastrointestinal hydrolysis with simulated and human digestive fluids. ß-CN was unaffected by oral digestion, but quickly broke down during the early stages of gastric digestion. The degradation with human fluids was faster than that with commercial enzymes. There were similarities in the peptide patterns of the hydrolysates produced in both models, showing 20 peptides in common after gastric digestion. After gastroduodenal digestion, the human fluids gave less numerous and shorter peptides. The IgE binding of most of the individual sera used to the hydrolysates produced with simulated and human fluids increased at the end of the gastric phase and decreased when the duodenal digestion was completed. Two IgE-binding synthetic peptides: ß-CN (57-68) and ß-CN (82-93), which matched fragments released by ß-CN following in vitro digestion with simulated and human fluids, consisted of the most immunoreactive areas of the protein. The similarities found between the in vitro simulated digestion system and that using human digestive fluids suggest that the former would provide a reasonably good estimation of the potential allergenicity of protein digests.

Identification of IgE-binding peptides in hen egg ovalbumin digested in vitro with human and simulated gastroduodenal fluids.

The digestibility of the major egg allergen ovalbumin (OVA, Gal d 2) with human and simulated digestive fluids was assessed. Degradation of OVA was faster when treated with human fluids, particularly following duodenal digestion, leading to gastrointestinal digests with lower IgE binding. Gastric digestion with both systems yielded 52 identical cleavage sites and a similar peptide pattern with 47 peptides in common. Subsequent duodenal digestion showed that the human fluid released fewer and shorter peptides. Several high-frequency IgE-binding epitopes were detected among the fragments of molecular mass lower than 3 kDa identified in the digests: OVA (141-154) and OVA (164-176) in the gastrointestinal digests produced with human fluids; and OVA (125-134), OVA (159-172), OVA (141-154), OVA (188-198), OVA (326-336), and OVA (370-385) in the gastrointestinal digests produced with simulated fluids. The high binding frequency of the fragment OVA (370-385), which reacted with 80% of the sera from allergic patients used, was noteworthy.

Identification of an IgE reactive peptide in hen egg riboflavin binding protein subjected to simulated gastrointestinal digestion.

Riboflavin binding protein (RfBP) is a minor protein in hen egg; its potential involvement in egg allergy has seldom been studied. The aim of this work was to investigate the IgE binding capacity of RfBP before and after simulated gastrointestinal digestion. It was shown that digestion of RfBP mainly occurred during the gastric phase. The protein fragments resulting from the subsequent duodenal phase remained linked through disulfide bonds. Both the intact protein and its digests were subjected to inhibition ELISA with sera obtained from patients allergic to egg. The results revealed significant IgE binding to intact RfBP, whereas the digests showed reduced but substantial IgE binding levels, with serum-to-serum variability. The RfBP digests were then subjected to immunoblot with allergic patients' sera, and the IgE-reactive peptides were further analyzed by MALDI-TOF/TOF mass spectrometry for sequence determination. The RfBP sequence 41-84 was identified as a novel IgE binding peptide in patients allergic to egg.

Vascular effects of egg white-derived peptides in resistance arteries from rats. Structure-activity relationships.

BACKGROUND: The vasodilator properties of several peptide sequences derived from egg white proteins were screened in mesenteric resistance arteries from Wistar-Kyoto rats. For this, third-order branches of the mesenteric arteries from 6-month-old male rats were used. The vasodilator responses, with or without endothelium, to several peptides (0.1 mmol L(-1)) were analysed in an isometric myograph. Moreover, the effect of nitric oxide (NO) synthase (L-NAME, 100 micromol L(-1)) and cyclooxygenase (indomethacin, 10 micromol L(-1)) inhibitors on the vasodilator response was tested. RESULTS: The peptides Arg-Ala-Asp-His-Pro-Phe-Leu, Arg-Ala-Asp-His-Pro-Phe, Arg-Ala-Asp-His-Pro, Tyr-Arg-Gly-Gly-Leu-Glu-Pro-Ile-Asn-Phe, Arg-Asp-Ile-Leu-Asn-Gln and Val-Pro-Pro showed a high endothelium-dependent vasorelaxation, whereas Phe-Arg-Ala-Asp-His-Pro-Phe-Leu was only partially endothelium-dependent. The relaxation induced by Arg-Ala-Asp-His-Pro-Phe-Leu, Arg-Ala-Asp-His-Pro-Phe, Arg-Ala-Asp-His-Pro, Arg-Asp-Ile-Leu-Asn-Gln and Val-Pro-Pro was mainly mediated by NO, since the response was inhibited only by L-NAME, while both L-NAME and indomethacin inhibited the vasodilator response induced by Phe-Arg-Ala-Asp-His-Pro-Phe-Leu and Tyr-Arg-Gly-Gly-Leu-Glu-Pro-Ile-Asn-Phe. The presence of Arg or Tyr at the N-terminal position could be related to the vasodilator activity of these compounds in this vascular bed. The well-known angiotensin-converting enzyme inhibitor captopril showed only a slight vasodilator effect. CONCLUSION: These peptides could reduce the vascular resistance and be used as functional ingredients in the prevention and/or treatment of hypertension and other associated disorders.

Vasodilator effects of peptides derived from egg white proteins.

The aim of this work was to investigate the effect of several peptides, identified before and after simulated gastrointestinal digestion of an egg white hydrolysate, on the vascular function, in rat aorta. The sequences IVF, RADHPFL and YAEERYPIL (0.1 mM) induced vasodilatation in intact aortic rings, with the maximum percentage of dilation corresponding to RADHPFL (40.5+/-7.0%). Two of the end products of the gastrointestinal digestion, RADHP and YPI, also showed vasodilator activity with degrees of relaxation higher than 50%. However, all these peptides failed to induce relaxation in endothelium-denuded aortic rings. The relaxation induced by RADHP was concentration-dependent and it was partially blocked by the nitric oxide synthase inhibitor L-NAME (100 microM) and by the B(1) bradykinin receptor antagonist Des-HOE 140 (30 nM), thus showing that it was mediated by NO production through the activation of B(1) bradykinin receptors. These findings suggest that these peptides could reduce the vascular resistance and could be used as functional food ingredients in the prevention and treatment of hypertension.