Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp Complexation of whey protein with caffeic acid or (-)-epigallocatechin-3- gallate as a strategy to induce oral tolerance to whey allergenic proteins Tássia B. Pessato a , Natália C. de Carvalho a , Daniella de Figueiredo b , Talita C. Colomeu b , Luís Gustavo R. Fernandes b , Flavia M. Netto a , Ricardo de L. Zollner b, ⁎ a Department of Food and Nutrition, School of Food Engineering, University of Campinas, São Paulo, Brazil b Laboratory of Translational Immunology, School of Medical Sciences, University of Campinas, São Paulo, Brazil ARTICLE INFO Keywords: Protein-phenolic interactions Cow's milk allergy Hypoallergenic products Oral immunotherapy Anti-allergic capacity ABSTRACT Proteins and phenolic compounds can interact and form soluble and insoluble complexes. In this study, the complexation of whey protein isolate (WPI) with caffeic acid (CA) or (-)‑epigallocatechin‑3‑gallate (EGCG) is investigated as a strategy to attenuate oral sensitization in C3H/HeJ mice against WPI. Treatment with WPI-CA reduced the levels of IgE, IgG1, IgG2a and mMCP-1 in serum of mice measured by ELISA. This might be related to CD4 + LAP + Foxp3 + T and IL-17A + CD4 + T (Th17) cell activation, evidenced by flow cytometry of spleno- cytes. Treatment with WPI-EGCG, in turn, decreased the levels of IgG2a and mMCP-1 in serum of mice, possibly by the modulation of Th1/Th2 response and the increase of CD4 + Foxp3 + LAP - T and IL-17A + CD4 + T (Th17) cell populations. In conclusion, WPI-CA and WPI-EGCG attenuated oral sensitization in C3H/HeJ mice through different mechanisms. We consider that the complexation of whey proteins with CA and EGCG could be a promising strategy to induce oral tolerance. 1. Introduction Cow's milk (CM) is the most common food allergen to infants, since it is usually the first food protein offered when breastfeeding is not possible, which could lead to an earlier allergic sensitization [1]. Cow's milk allergy (CMA) is an immunologically mediated adverse reaction to one or more CM proteins and mainly manifests against caseins and the two main whey proteins, α‑lactoalbumin (α‑la) and β‑lactoglobulin (β‑Lg). It is considered that food allergy is caused by a failure or loss of oral tolerance induction, however, the specific moment when that failure occurs is not yet completely clarified [2]. Tolerance induction is a normal immune phenomenon that occurs in the intestinal mucosa after exposure to allergens. The mechanism through which the oral tolerance is induced is partially related to the ingested antigen dose [3]. In the presence of high doses of antigen, tolerance is mainly caused by the clonal deletion, due to apoptosis or anergy of antigen-specific T cell clones. When the food antigen is at lower concentrations, tolerance is induced by regulatory T cells (Tregs), which is considered to be a central mechanism of tolerance induction by food antigens [4]. Nowadays, it is known that oral tolerance instead of an un- responsive state is an active phenomenon which involves the suppres- sion of specific immune responses to antigens first encountered in the gastrointestinal tract [5,6]. For instance, Castro-Junior and collabora- tors [6] have shown that tolerant and immunized mice presented the same number of regulatory and activated T cells in the spleen after immunization to ovalbumin. The authors suggested that, in addition to the immune response suppression mechanisms performed by Tregs, the earlier expression of regulatory cytokines, such as TGF-β and IL-10 and a transitory expression of effector cytokines (IL-2 and IFN-γ), play a role in the induction of tolerance. Lymphocytes with active regulatory properties in the mucosa are crucial to induce tolerance since they could permeate the body and modulate the immune response in a systemic manner [6,7]. CD4 + T cells which express TGF-β in the membranes linked to latency-associated peptide (LAP), known as LAP + Treg cells, may be one of the markers of Tregs induced by oral tolerance [7]. Furthermore, a reliable intracellular marker of naturally-occurring Treg cells is the transcription factor forkhead box p3 (Foxp3), since the frequency of CD4 + Foxp3 + regulatory T cells tend to be lower in al- lergic individuals [8,9]. The Th17 response also tends to be impaired in food-allergic individuals [9]. Since it has recently been suggested that Th17 cells could transdifferentiate into regulatory T cells depending on the environmental context, IL-17 might be a potential biomarker for tolerance to food allergens as well [10–12]. Due to the lack of clear understanding of the molecular mechanisms https://doi.org/10.1016/j.intimp.2018.12.047 Received 22 July 2018; Received in revised form 19 December 2018; Accepted 20 December 2018 ⁎ Corresponding author. E-mail address: zollner@unicamp.br (R. de L. Zollner). International Immunopharmacology 68 (2019) 115–123 1567-5769/ © 2018 Elsevier B.V. All rights reserved. T