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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.

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.

This study investigates the mechanism by which food peptides are more effective than intact allergens in providing desensitization against food allergy. BALB/c mice sensitized to egg white (EW) were subjected to oral immunotherapy (OIT) with intact and pepsin-hydrolysed EW (EP). Treatment with EP was superior to that with EW in terms of reduction of anaphylaxis and levels of specific antibodies. OIT with EP, but not with EW, modulated the microbiota by restoring the levels of some members of the order Clostridiales (clusters IV and XIVa) that were affected by sensitization. Mice treated with EP exhibited upregulated intestinal expression of Il22 and Muc2, which encode for factors that contribute to reinforce the epithelial barrier function, as well as Aldh1a1, Aldh1a2, Csf2 and Tfgb1, that take part in the conversion of vitamin A into retinoic acid. Tolerance induced by EP paralleled the development of Foxp3+ cells that simultaneously expressed RORγt.

Oral food challenge (OFC) remains the gold standard for the diagnosis of food allergies. However, this test is not without risks, given that severe allergic reactions can be triggered while it is conducted. The purpose of this study is to identify potential demographic variables, clinical characteristics of the patients and biomarkers that may be associated with severe reactions during the hazelnut oral challenge test. The sample included 22 children allergic to hazelnut who underwent a tree nut skin prick test (SPT), specific IgE (sIgE) to hazelnut, component-resolved diagnosis (CRD) with different hazelnut allergens (Cor a 1, Cor a 8, Cor a 9, Cor a 11, Cor a 14), and a single-blind placebo-controlled challenge with hazelnut. A statistically significant relationship was found between the severity of the reaction and the highest values of sIgE to hazelnut, Cor a 11 and Cor a 14, cumulative symptom-triggering dose and sunflower seed sensitization. The use of the CRD is a useful tool to identify patients at higher risk of developing a severe reaction. In this pediatric population sample from Spain, storage proteins were confirmed to be most involved in hazelnut allergy and the development of severe reactions.

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.

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.

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-β), 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-β, 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.