Dietary
Polyphenols and Food Allergies:
Effect on
Bioaccessibility
and Bioavailability of Milk Allergens
Rodolfo Dinis Pinto Simões
Mestrado em Bioquímica da Universidade do Porto
FCUP/ICBAS 2022
Supervisor
Maria Rosa Perez Gregorio, LAQV – REQUIMTE, Faculdade de Ciências da Universidade do Porto
Co-supervisor
Victor Armando Pereira Freitas, LAQV – REQUIMTE, Faculdade de Ciências da Universidade do Porto
Susana Isabel Pinto Teixeira Pereira Soares - LAQV – REQUIMTE, Faculdade de
Ciências da Universidade do Porto
I, Rodolfo Dinis Pinto Simões, enrolled in the Master Degree in Biochemistry at the Faculty of Sciences of the University of Porto hereby declare, in accordance with the provisions of paragraph a) of Article 14 of the Code of Ethical Conduct of the University of Porto, that the content of this dissertation reflects perspectives, research work and my own interpretations at the time of its submission.
By submitting this dissertation, I also declare that it contains the results of my own research work and contributions that have not been previously submitted to this or any other institution.
I further declare that all references to other authors fully comply with the rules of attribution and are referenced in the text by citation and identified in the bibliographic references section. This dissertation/ internship report/ project [choose accordingly] does not include any content whose reproduction is protected by copyright laws.
I am aware that the practice of plagiarism and self-plagiarism constitute a form of academic offense.
Rodolfo Dinis Pinto Simões 26/09/2022
Agradecimentos
Este trabalho só foi possível com o apoio de várias pessoas. Em particular, gostaria de agradecer:
À Doutora Rosa Pérez e à Doutora Susana Soares, por me ajudarem a crescer não só como cientista, mas também como pessoa. Por serem excelentes mentoras e por nunca se cansarem de me ajudar quando algo nas experiências inevitavelmente corria mal.
À Rita, à Catarina Bessa e ao Carlos por todo o apoio ao longo deste trabalho e por estarem sempre disponíveis para responder às minhas perguntas.
Aos Professores Victor de Freitas e Nuno Mateus, pela oportunidade de poder desenvolver este trabalho no Food Polyphenol Lab.
À Sofia, à Mônica, à Catarina, à Alexandra, à Leonor, ao Francisco e ao José, não só por toda a ajuda ao longo da realização deste trabalho, mas também por me
receberem de braços abertos no laboratório. Por todas as pausas para tomar café onde se falava de tudo e não se aprendia nada.
A todas as pessoas do Food Polyphenol Lab pela simpatia e por toda a ajuda.
Gostaria também de agradecer a outras pessoas que não estiveram diretamente envolvidas na realização deste trabalho, mas mesmo assim foram indispensáveis:
Aos meus Pais, Generosa e Dinis, e à Beatriz, por estarem sempre presentes ao longo desta viagem.
À Beatriz Costa, por seres a minha companheira ao longo destes dois anos de mestrado. Por todos os cafés à porta dos auditórios do ICBAS e pelas conversas à entrada da FCUP.
À Beatriz Silva, ao João, à Rafaela e à Diana, por estarem sempre presentes ao longo de todos os altos e baixos destes últimos 5 anos.
Por fim, à Mariana, pelo teu apoio incondicional não só ao longo desta tese, mas todos os dias.
Resumo
O aumento da prevalência de alergias alimentares, entre elas a alergia alimentar ao leite de vaca, tornou necessário o desenvolvimento de novas estratégias terapêuticas para prevenir esta patologia e reduzir os seus sintomas. O uso de compostos fenólicos para modular a digestão de alergénios, para regular o sistema imune e para modular as populações comensais de bactérias presentes no trato digestivo constitui uma nova abordagem para manutenção da tolerância oral a alergénios.
Os compostos fenólicos são uma classe de químicos presentes em tecidos vegetais que apresentam um elevado número de atividades biológicas, como por exemplo atividade anti-inflamatória e antioxidante. Estes compostos podem modular a digestão de diferentes alergénios e alterar a sua biodisponibilidade. Para além disso, os compostos fenólicos modular diretamente a resposta imune, atuando nas cascatas de sinalização que controlam a resposta alérgica a alergénios alimentares. Por fim, os compostos fenólicos podem modular as populações de microbiota comensal presentes ao longo do trato digestivo. Estes microrganismos têm um papel preponderante não só no processo digestivo, mas também na imunomodulação dos tecidos intestinais.
Deste, esta tese foca-se no estudo das diferentes formas como os compostos fenólicos podem modular a resposta imune a alergénios derivados do leite de vaca. A digestão e biodisponibilidade de leite magro e de extratos proteicos derivados do leite de vaca serão monitorizados, assim como o efeito de compostos fenólicos nestes processos. Um modelo in vitro para a desgranulação de basófilos humanizados foi também utilizado para estudar o efeito de compostos fenólicos na ativação destas células. Por fim, microbiota oral humana foi recolhida e cultivada em placas de agar com o objetivo de estudar o seu papel na manutenção da tolerância oral a alergénios.
Este trabalho demonstrou que tanto a matriz alimentar como os compostos fenólicos derivados chá verde e de mirtilos têm a capacidade não só modular a biodisponibilidade e bioacessibilidade de alguns dos principais péptidos imunogénicos do leite, mas também de modular a desgranulação de basófilos de rato humanizados in vitro.
Palavras-chave: alergias alimentares, compostos fenólicos, digestão in vitro, bioacessibilidade, biodisponibilidade, ativação de basófilos, microbiota.
Abstract
The rapid increase in the prevalence of food allergies, among them cow’s milk allergy, as prompted the need for the development of new therapeutic strategies to prevent this pathology and reduce its symptoms. The use of dietary phenolic compounds to modulate the digestion of allergens, the host immune system and the hot’s microbial populations constitutes an exciting new approach for the maintenance of oral tolerance to allergens.
Phenolic compounds are a class of plant-derived chemicals with a myriad of described biological activities, like antioxidant and anti-inflammatory activity. These compounds can modulate the digestion of different allergens and alter their bioavailability. Besides that, phenolic compounds can modulate the host’s immune response directly, acting on the signalling cascades involved in the allergic response to dietary allergens. Finally, phenolic compounds can modulate the hot’s microbial populations. It is well known that the intestinal microbiota pays a crucial role not only on the host’s digestive process, but also on the immunoregulation of the intestinal tissues.
The aim of this thesis was thus to study the different ways in which food phenolic compounds can be used to modulate the immune response to cow’s milk derived allergens. The digestion and bioavailability of skimmed milk and extracted cow’s milk proteins were monitored, as well as the effect of phenolic compounds extracts on these phenomena. An in vitro model to study the degranulation of humanized basophils was also used to study the effect of dietary phenolic compounds on the activation of these cells. Finally, human oral microbiota was sampled, stored and cultivated to study its role on the maintenance of oral tolerance to allergens.
This work demonstrated that the food matrix of milk and that polyphenols extracted from green tea and blueberries are not only capable of altering the bioaccessibility and bioavailability of some immunogenic cow’s milk peptides, but can also modulate the degranulation of humanized rat basophils in vitro.
Keywords: food allergies, phenolic compounds, in vitro digestion, bioaccessibility, bioavailability, basophil activation, microbiota.
Table of Contents
List of Tables
………viiiList of Figures
...ixList of Abbreviations
………xiI. Introduction
………...……….11. Food allergy……….2
1.1 Pathophysiology of IgE-mediated food allergy………..3
1.2 Mast Cell and basophil degranulation as a major component of the immune response do dietary allergens………..7
1.3. Factors that modulate oral tolerance………..9
1.4. Cow’s milk allergy……….10
1.5. Analytical techniques for the analysis of milk proteins………...12
1.6. In vitro degranulation studies and their role in the study of IgE-mediated food allergies………..14
2. Importance of microbiota in food allergy………...15
3. Phenolic Compounds: from chemistry to their role in food allergy………18
3.1 Nature and chemistry of phenolic compounds………..………18
3.2 Phenolic compounds human metabolization………....20
3.3. Phenolic compounds and food allergy: main role and mechanisms………...23
II. Study design
………...29III. Reagents and Methods
………311. Reagents and samples..……… 31
2. Extraction, characterization and quantification of milk proteins………32
2.1. Optimization of the extraction of milk proteins……….32
2.2. Protein quantification of milk proteins in milk protein extract………33
2.3. Optimization of the characterization of milk proteins……….….34
2.4. Scale-up of the protein extraction process……….………..35
3. Extraction and characterization of dietary phenolic compounds………..……….36
3.1 Extraction of green tea phenolic compounds………..36
3.2. Extraction of blueberry phenolic compounds………..36
3.3. Characterization of phenolic compounds……….37
4. In Vitro digestion of milk proteins………..37
4.1. Preparation of digestion fluids, enzymatic solutions and bile salts solutions….37 4.2. In vitro Digestion………..38
4.3. Sample preparation by solid-phase extraction (SPE)………40
4.4. Characterization of digestion of milk proteins………..41
4.5. Analysis of immunogenic peptides by targeted LC-MS/MS………...41
5. Bioavailability of immunogenic peptides………43
5.1. Digestion of milk proteins………43
5.2. Culture of Caco-2 cells………43
5.3. Bioavailability assays ………44
6. Degranulation of humanized basophils ………45
6.1. Culture of RBL cells ………...45
6.2 Optimization of the basophil degranulation assays………..46
7. Oral Microbiota……….47
7.1. Sampling and cryopreservation of oral microbiota………...47
7.2. Optimization of growth conditions for oral microbiota………48
IV. Results
……….501. Extraction, characterization and quantification of milk proteins……….50
1.1. Quantification of milk proteins……….50
1.2. Characterization of milk proteins………52
2. Extraction and Characterization of Dietary Polyphenols………55
3. In Vitro digestion of milk proteins………59
4. Bioavailability of immunogenic peptides………62
5. Degranulation of humanized basophils……….65
6. Oral Microbiota………..66
V. Discussion
………70VI. Conclusion and Future Perspectives
………73References
………..……….76List of Tables
Table 1 – Bioactivity of dietary polyphenols ... 24 Table 2 – Volumes and concentrations of used in the preparation of casein and BSA calibration curves ... 34 Table 3 – Final concentration of salts used in in vitro digestion fluids. ... 38 Table 4 – Ions monitored in SIM mode. Cas - casein; LA - α-lactalbumin; LG – β- lactoglobulin. ... 42 Table 5 – Main PCs identified in GTE ... 56 Table 6 – Main PCs identified in BE ... 57
List of Figures
Figure 1 – Comparative overview of the tolerogenic versus allergic response to food
antigen in the gut. ... 7
Figure 2 – Schematic representation of the activation and degranulation of mast cells and basophils in the presence of allergens ... 9
Figure 3 – Schematic representation of the mechanism involved in the activation of the reporter RBL cell lines ... 15
Figure 4 – Basic structure of flavonoids ... 18
Figure 5 – Basic structure of the six subgroups of flavonoids ... 19
Figure 6 – Basic structure of hydroxycinnamic and hydroxybenzoic ... 20
Figure 7 – Schematic representation of the metabolic pathway of phenolic compounds in humans. ... 21
Figure 8 – Schematic representation of through which PCs can modulate the hosts immune response ... 27
Figure 9 – Schematic representation of the workflow ... 29
Figure 10 – Schematic representation of the milk protein extraction process ... 33
Figure 11 – Schematic representation of the in vitro digestion experiments ... 40
Figure 12 – Schematic representation of the SPE process ... 41
Figure 13 – Schematic representation of the trans-wells used in the bioavailability experiments ... 44
Figure 14 – Schematic representation of the 96-well plate used in the basophil degranulation experiment ... 46
Figure 15 – Schematic representation of the collection and storage of oral microbiota..47
Figure 16 – Schematic representation of the plating and culture of oral microbiota ... 48
Figure 17 – Calibrations curves resulting from BSA and Casein standards ... 50
Figure 18 – Quantification of proteins extracts. M stands for mean value... 51
Figure 19 – SDS-gel resulting from the skimmed milk and powdered milk extracts ... 52
Figure 20 – SDS-gel resulting from the skimmed milk and powdered milk extracts ... 53
Figure 21 – Chromatogram of GTE ... 55
Figure 22 – Chromatogram of BE ... 56
Figure 23 – SDS-gel resulting from the skimmed milk extracts ... 59
Figure 24 – Peptides monitored during the bioaccessibility experiments……….60
Figure 25 – Percentage of peptides in the apical chamber, compared with the timepoint zero of the bioavailability experiments……….63-64 Figure 26 – Degranulation of humanized basophils. A) Non lysed cells; B) lysed cells ... 65
Figure 27 – Oral microbiota pool plated in TSA plates supplemented with 5% horse blood ... 67
Figure 28 – Close up of the colonies of oral microbiota ... 67
List of Abbreviations
APC BSA BTK CMA CTLA-4 DAG DC DTT EGF EGFR ESI FA FAB FBS GALT GAP GM GPCR GT HDAC HMO HPLC IgA IgE IKK IL ILC2 iNOS IP3 ITAM KDAC
Antigen presenting cell Bovine serum albumin Bruton’s tyrosine kinase Cow’s milk allergy
Cytotoxic t-lymphocyte antigen 4 Diacylglycerol
Dendritic cell DL-Dithiolthretiol
Epidermal growth factor
Epidermal growth factor receptor Electrospray ionization
Food allergy
Fast atom bombardment Fetal bovine serum
Gut associated lymphoid tissue Goblet cell associated passage Gut microbiota
G protein-coupled receptors Gastrointestinal tract
Histone deacetylase
Human milk olygosacharides
High-performance liquid chromatography Immunoglobulin A
Immunoglobulin E IKB kinase
Interleukin
Type 2 innate lymphoid cells Inducible nitric oxide synthase Inositol-1,4,5-trisphosphate
Immunoreceptor tyrosine-based activation motif Lysine deacetylase
LAT MALDI MAPK MHC OX40L PC PCR PI(3)K PIP3 PKC PLCγ1 PP RBL SCFA SDS TCR TGF TH2 TNFSF4 TREG TSA TSB TSLP
Linker for activation of t cells
Matrix assisted laser desorption ionization Mitogen-activated protein kinase
Major histocompatibility complex OX40 ligand
Phenolic Compound Polymerase chain reaction Phosphoinositide 3-kinases
Phosphatidylinositol (3,4,5)-trisphosphate Protein kinase C
Phospholipase Cγ1 Peyer’s patch
Rat Basophilic Leukaemia cells Short-chain fatty acid
Sodium docedyl sulfate T-cell receptor
Transforming growth factor T-helper 2 cells
Tumor necrosis factor ligand superfamily member 4 T regulatory cell
Tryptic soy agar Tryptic soy broth
Thymic stromal lymphopoietin
I. Introduction
In the past years, allergic diseases have been considered as a global problem, with a rapidly increasing prevalence. Among them food allergy, and more specifically, allergy to cows’ milk has been estimated between 0.25% and 4.9%, being higher in children than adults (Flom and Sicherer, 2019). Their rapid raise into prominence, coupled with the high burden of such diseases on healthcare systems, has placed a high emphasis on the development of therapeutic strategies that focus not only on ameliorating symptoms, but also on addressing the underlying causes of the dysregulation of the normal immune system function.
Phenolic compounds (PCs) are a class of plant-derived compounds with many biological activities descripted, which confer them a high therapeutically value. PCs can modulate oral tolerance do dietary allergens directly, regulating various components of the host immune system to limit immune responses. They can also carry out that modulation indirectly, interacting with the dietary allergens, cell receptors and/digestive enzymes, thus modulating their bioaccessibility and bioavailability or structurally altering their epitopes. They can also alter the composition of the hosts Gut Microbiota (GM), which can itself modulate the hosts’ immune system. The host immune system/GM/dietary PCs axis is, therefore, a prime therapeutic target, capable not only of addressing the symptoms of food allergy (FA), but also of reinstating oral tolerance to dietary allergens.
1. Food allergy
Food allergy (FA) can be defined as an adverse health effect arising from an immune response that occurs reproducibly on exposure to a given food. This immune response can be IgE-mediated or non-IgE mediated. Over the past years, the prevalence of allergic disorders has been increasing worldwide. It is estimated that FA affects up to 8% of children and 3% of adults in industrialized countries(Messina and Venter, 2020). This, combined with the severity of the clinical manifestations associated with FA and the fact that this disorder can persists past childhood, confers great importance to the development of new therapeutic approaches to treat and prevent this disorder (Boyce, 2010; M. Christopher, 2016; Sicherer and Sampson, 2018a; Goldberg et al., 2020).
Many factors can modulate the development of FA. Typically, this disorder begins to manifest in early childhood, being resolved by the time patients reach adulthood.
However, in a small percentage of patients, FA will persist into adolescence and adulthood. It is also noticeable that this disorder is more prevalent in developed countries, compared with developing countries. This can be due to the reduced exposure to food allergens during early childhood, which according to the hygiene hypothesis, modulates the development of the immunity system (Elizur et al., 2012; Lack, 2012; Hoi et al., 2017; Goldberg et al., 2020; Kreft, Hoffmann and Ohnmacht, 2020). Some of the most common allergenic food items include milk, eggs, fish, shellfish, tree nuts, peanuts, wheat and soy beans (Thompson, Kane and Hager, 2006; Wang et al., 2021). Factors such as timing of resolution, age at diagnosis, nature of immune responses and associated comorbid allergic diseases dictate the severity of FAs (Savage, Sicherer and Wood, 2016).
The immune responses resulting from oral exposure to food allergens can be divided into three categories: cell-mediated reactions, immunoglobulin E (IgE) mediated reactions and reactions mediated not only by IgE, but also by other classes of immunoglobulins (Sicherer and Sampson, 2009). Numerous diagnostic procedures exist; with some of the most common being serum food specific IgE tests and skin prick tests. The choice of an adequate diagnostic method should be made in accordance with the nature and severity of FA, with the medical history of the patient also being an important factor. The current approach to the management substantially relies heavily on allergen avoidance via dietary elimination combined with a preparation to promptly treat allergic reactions with injectable epinephrine. Quick management is crucial to prevent complications related to anaphylactic reactions. This presents many problems, as it fails to address the underlying dysregulation of immune response associated with
FA, thus not being a definite therapeutic approach. Besides, both require a great level of patient education.
1.1 Pathophysiology of IgE-mediated food allergy
Mechanistically, FA is a direct result of oral tolerance failure relative to dietary allergens. The establishment and maintenance of oral tolerance depends on the interplay between several biological systems, with the most significant being the immune system, the gut epithelium and the GM (Smith et al., 2013) (Maslowski et al., 2009). IgE-mediated food allergies result from a loss of integrity in the key immune components. These components maintain a state of tolerance and prevent benign food antigens from being recognized as pathogens (Anvari et al., 2019).
The intestinal uptake of allergens is one of the crucial steps not only on the sensitization process, but also on the loss of oral tolerance to dietary proteins. As such, the integrity of the intestinal mucosa is crucial for the maintenance of a normal immune function (Xiong et al., 2022). Structurally, the intestinal mucosa can be divided into three distinct layers: the outer layer is the mucus layer, the central layer, and the lamina propria. Goblet cells and mucinous cells present in the mucus layer are responsible for the secretion of mucins, which protect the protect the intestinal epithelial cells from digestive enzymes. The mucus layer is also constantly exposed to the microorganism that populate the gastrointestinal tract (GT), preventing the excessive proliferation of potentially photogenic microorganisms (Bansil and Turner, 2018) (Johansson, Sjövall and Hansson, 2013) (Schroeder, 2019). The central layer is made up of intestinal epithelial cells, which can be divided into a variety of subsets: absorptive enterocytes, goblet cells, Paneth cells, enteroendocrine cells, microfold cells (M cells), among others (Salim and Söderholm, 2011). These intestinal epithelial cells are linked by a complex set of intercellular junctions. The three main type of junctions present in central layer are tight junctions, adherent junctions and desmosomes (Turner, 2009). This complex junction system is crucial for the normal function of the intestinal barrier. Finally, the inner most layer of the intestinal barrier is the lamina propria. This layer contains cells from both the innate and adaptative immune system, such as macrophages, dendritic cells (DCs), B cells and T cells. These cells are involved in the not in the hots defense against potentially hazardous substances, but also play an important role in the immune regulation of the intestinal microenvironment (Vancamelbeke and Vermeire, 2017).
Foreign substances can cross the intestinal barriers through a great variety of pathways. Small molecules present in the intestinal tract can cross the intestinal barrier through the paracellular pathway, mainly through the tight junctions present in the intestinal barrier (Lee, Moon and Kim, 2018) (Zuo, Kuo and Turner, 2020). Under normal circumstances, this pathway of intestinal uptake is highly regulated, with the transport of substances being highly dependent on their size and charge (Lee, Moon and Kim, 2018).
However, when the intestinal barrier is damaged, the selectivity of this pathway decreases drastically (Grozdanovic et al., 2016). This in turn increases the permeability of the intestinal barrier and can result in a disruption of the oral tolerance to dietary antigens and in the inflammation of the intestinal tissues. For example, the reduced expression of proteins necessary for the correct function of tight junctions, like claudins, is a hallmark of inflammatory bowel disease (Fries, Belvedere and Vetrano, 2013).
Foreign substances can also cross the intestinal barrier via the transcellular pathways. Small molecules can cross the intestinal barrier through transporters present in the membrane of epithelial cells, such as sodium dependent transports of glucose, alanine, and glutamine. Larger molecules can also be transported across the intestinal barrier through vesicles (Allaire et al., 2018). A variety of intestinal epithelial cells, like M cells and goblet cells, are also involved in the sampling of luminal antigens into the gut associated lymphoid tissues (GALTs). These antigens are then presented to intestinal dendritic cells (DCs) which subsequently present them to other cells of the immune system, thus establishing an oral tolerance to various dietary allergens (Bockman and Cooper, 1973) (Jang et al., 2004).
M cells are specialized epithelial cells located in the follicle-associated epithelium overlying Peyer’s patches (PP) (Bockman and Cooper, 1973). Due to their location, M cells can easily translocate a wide range of compounds to underlying DCs. Besides the delivery of intact antigens into the underlying lymphoid tissue of the GALT (Bockman and Cooper, 1973) (Gebert, Rothkötter and Pabst, 1996), M cells also participate in antigen processing and presentation, as it has been reported that these cells express MHC class II molecules and acidic endosomal-lysosomal compartments (Wolf and Bye, 1984).
However, additional studies have also reported that these cells are not detrimental in the establishment of oral tolerance, as the disruption of these cells alone was not enough to sensitize mouse model to dietary allergens (Spahn et al., 2001) (Spahn et al., 2002).
The secretory products of goblet cells, including mucins, trefoil factors, and other proteins, are crucial for the integrity and normal function of the intestinal barrier, thus preventing harmful substances crossing the intestinal barrier (McCauley and Guasch,
2015). Goblet cells are also responsible for the formation of a goblet cell associated antigen passage (GAP) to transport luminal allergens to antigen-presenting cells (APC) (McDole et al., 2012). First, goblet cells capture food antigens from the lumen, enclose them in internal sack-like vesicles, and transport them across the cell. Then the APCs in the lamina propria sample luminal antigens and present them to other cells of the immune system (Kulkarni et al., 2020). Recent studies have also revealed that acetylcholine can trigger both the sampling of antigens and the production of mucus (Gustafsson et al., 2021). This regulation by acetylcholine allows goblet cells to dynamically modulate the mucosal environment of the GT. Goblet cells can also deliver luminal substances, including food antigens and microbial antigens, and induce intestinal tolerance through the GAP process (Kulkarni et al., 2020). Finally, antigens can be directly sampled from the GT by CX3CR1 macrophages, that then transport antigens to DCs in the gut lamina propria (Pabst and Mowat, 2012).
After sampling dietary allergens, DCs transport them to the draining lymph nodes. In the presence of non-inflammatory mediators, such as retinoic acid and transforming growth factor-beta (TFG-β), DCs present antigens to the T cell receptors (TCR) on naive T cells by way of the major histocompatibility complex (MHC) (Worbs et al., 2006).
Antigens presented via this mechanism promote the differentiation of naive T cells into food antigen-specific T regulatory cells (Tregs) (Evans and Reeves, 2013). Food specific Tregs are then transported to the lamina propria via integrin α4β7. There, they maintain tolerance to antigens present in food via CTLA-4 expression and the release of cytokines TGF-β and IL-10. CTLA-4 inhibits Th2 T cells, while TGF-β and IL-10 suppress mast cells. Moreover, TGF-β and IL-10 also facilitate the maintenance of IgA in the lumen (Hadis et al., 2011).
In FA, these cellular mechanisms are altered and an initial exposure to food allergens results in a sensitization process, with subsequent exposures to the same antigen resulting triggering an immune response (Pulendran and Artis, 2012). The secretion of pro-inflammatory cytokines such as IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), promotes the expansion of type 2 innate lymphoid cells (ILCs) and activation of DCs (Paul and Zhu, 2010). Activated dendritic cells express surface OX40L, also known as tumor necrosis factor ligand superfamily member 4 (TNFSF4). These activated DCs also transport antigens to draining lymph nodes. However, when they present antigen to naive T cells OX40L binds to OX40 on naive T cells, thus promoting the differentiation off these cells into T-helper 2 cells (Th2), thus promoting the allergic state (Blázquez and Berin, 2008). ILCs and Th2 cells secrete proinflammatory cytokines such IL-5 and IL-13,
which promote eosinophil and basophil recruitment in the gut lamina propria. This leads to downstream target effects that promote allergic sensitization. Th2 cells also secrete IL-4, which allows for B cell class switching to promote food specific IgE production (Saenz et al., 2013) (Halim et al., 2014). After this, re-exposure to the same allergen results in FAs, as IgE recognize the allergen epitopes and bind to them, leading to mast cells and basophils degranulation, releasing mediators, such as histamine, serotonin, proteases, and proteoglycans (Galli and Tsai, 2012). In addition to Th2 cell activation, naive T cells have also been shown to differentiate to T helper 9 cells. These cells secret IL-9, therefore promoting the accumulation of tissue residing mast cells, further contributing to the development of the allergic (Sehra et al., 2015). These mechanisms are schematized in Figure 1.
Figure 1 - Comparative overview of the tolerogenic versus allergic response to food antigen in the gut. Adapted from (Anvari et al., 2019).
1.2 Mast cell and basophil degranulation as a major component of the immune response do dietary allergens
The degranulation of mast cells and basophils is a major biological event associated with IgE-mediated food allergies (Oettgen and Burton, 2015) (Kanagaratham et al., 2020). When sensitized immune system cells are exposed to dietary allergens, IgEs specific to that allergen are secreted. These IgE bind to allergens and to high-affinity FcεRI present on the membrane of basophils and mast cells (Miyake et al., 2021). The
activation of the cells is initiated when allergens cross-link two adjacent IgEs on sensitized mast cells or basophils. FceRIα chains are aggregated by crosslinking of bound IgE by multivalent antigen (Letourneur et al., 1995). This aggregation leads to the activation of Lyn by CD45 phosphatese and Src kinase, although the exact mechanism of activation is not fully understood yet. Activated Lyn phosphorylates the β- and γ-chain Immunoreceptor tyrosine-based activation motifs (ITAMs) of FcεRI, with the latter being then able to recruit the kinase Syk. This kinase is in turn activated by Lyn (Honda et al., 1997) (Moarefi et al., 1997).
After their recruitment to the β and γ chains of FceRI, Lyn and Syk target many different proteins and enzymes involved in the activation of mast cells and basophils.
Syk phosphorylates PI(3)K (Phosphoinositide 3-kinases), thus activating it and enabling it to produce PtdIns(3,4,5)P3 (PIP3) (Vanhaesebroeck et al., 1997). Syk is also responsible for the phosphorylation of the membrane-localized adapter protein linker for activation of T cells (LAT) and for the activation of phospholipase Cγ1 (PLCγ1) that binds to phosphorylated LAT (Zhang et al., 1998). Lyn, on the other hand, is responsible for the phosphorylation of Bruton’s tyrosine kinase (BTK). This kinase is bound to PIP3, and when phosphorylated by Lyn, it also phosphorylates PLCγ1 (Salim et al., 1996) (Rameh et al., 1997).
PLCγ1 catalyzes the breakdown of membrane phospholipids to generate two second messengers: inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (Rosenstein et al., 2014) (Falcone, Haas and Gibbs, 2000). These signaling molecules are responsible for calcium release from intracellular stores and activation of various protein kinase C (PKC) isoforms, respectively. The rise of intracellular calcium then results in the Snare- dependent release of vesicles, thus resulting in mast cell and basophil degranulation and the release of various immune mediators (Neher, 1988) (Dyatlova, 2004). This processes is schematically represented in Figure 2.
Figure 2 – Schematic representation of the activation and degranulation of mast cells and basophils in the presence of allergens.
1.3. Factors that modulate oral tolerance
Many factors can regulate the expression of Treg cells in the intestinal epithelium, thus regulating the oral tolerance to food allergens, either positively or negatively. As stated previously, tolerance to food allergens is usually established in early life. As such, there has been an growing interest in the early life events that can contribute to the establishment of this tolerance to allergens (Yokanovich, Newberry and Knoop, 2021).
Early life exposure to antigens can determine the immunological response later in life.
Studies revealed a lower incidence of peanut allergy by the age of seven was significantly reduced if the mothers consumed peanuts while breastfeeding and if the infants were exposed to this food in before 12 months of age (Yokanovich, Newberry and Knoop, 2021). On the other hand, sensitization to peanut by age sevens was increased if the children were not exposed to the allergens before 12 months of age.
This suggests the importance of early-life exposure to allergens in the establishment of oral tolerance in later life.
Early exposure to microbes, in particular the ones that normally compose the GM, is also a key player in the induction of oral tolerance. In early life, the pioneering microbiota in the gut eventually develops into the normal microbiota found in later stages.
Decreased exposure to microbes during early life can lead to an increase in allergenic
responses to food allergens, with germ free mice presenting a preferential differentiation of Th2 cells, instead of tolerance promoting Treg cells. However, not all microbes contribute to the establishment of oral tolerance. The dysbiosis of the normal GM, either from lack of exposure in early life or due to various events in later life, can alter the immunologic response to various foods allergens. Cohort studies of patients with CMA revealed a significant intestinal microbial dysbiosis, compared to healthy controls. The CMA patients had decreased levels of Clostridia species (Feehley et al., 2019).
Another key player in the development of oral tolerance is maternal milk.
Breastfeeding regulates microbial colonization and development, providing nutrients such as human milk oligosaccharides (HMOs), maternal IgAs and lactoferrins, all of which shape microbial populations in the gut. Besides that, maternal milk also contains cytokines with tolerogenic properties, like TGF-β. Higher levels of TGF-β in maternal milk have been reported to contribute to a reduced allergy prevalence (Soto-Ramírez et al., 2012) (Joseph et al., 2014). Finally, breast milk contains high levels of epidermal growth fact receptor (EGFR) ligands, like HB-EGF, EGF and TFG-α. All those ligands promote intestinal barrier development and maintenance of IgA levels (Tang et al., 2016) (Lee et al., 2006).
The establishment of oral tolerance to food allergens is a time sensitive process, meaning that exposure to allergens and must be done inside a “window of tolerance” in order to establish a normal later immunological response to allergens present in food.
Exposure to antigens during this window of tolerance promotes the development of Treg cells populations that promote oral tolerance. A failure to establish these populations of cells can result a in a deregulation of immunological responses later in life, with the action of pro-inflammatory Th2 cells populations being favoured (Stern, Wold and Östman, 2013) (Pezoldt et al., 2018).
1.4. Cow’s milk allergy
Cow’s milk allergy (CMA) is one of the most common food allergies, with an prevalence ranging from 0,25% to 4.9% in the general population affecting approximately 1% of children up to 1 year old, in developed countries (Savage and Johns, 2015). As previously referred, food allergies are defined as an immune-mediated response to and specific orally ingested antigen, and can be categorized as IgE-dependent, non-IgE dependent or mixed (Lifschitz and Szajewska, 2015) (Boyce et al., 2011). The IgE dependent variation of CMA is characterized by a type 1 hypersensitivity, with symptoms manifesting within minutes to 1-2 hours after ingestion. If oral tolerance is established,
IgE antibodies to proteins to proteins in cow’s milk bind to mast cells. This leads to degranulation and to the release of inflammatory mediators, like histidine and proteoglycans (Flom and Sicherer, 2019).
Immediate symptoms of CMA include angioedema, urticaria, throat tightness, respiratory symptoms like difficulty breathing, coughing and wheezing; gastrointestinal symptoms like vomiting, diarrhea and abdominal pain; and cardiovascular symptoms which include hypotension and dizziness (Sampson et al., 2014) (Sicherer and Sampson, 2018b) (Høst, 2002). A delayed onset can also occur, being characterized by eosinophilic gastroenteropathy, allergic proctocolitis, food protein-induced enterocolitis syndrome and enteropathy (du Toit et al., 2010). Although estimates vary greatly between studies, it is estimated that 60% of CMAs are of the IgE-mediated variant (Sampson, 1999).
As previously referred, cow’s milk contains various allergens that can initiate an immune response in CMA patients. Caseins and whey proteins constitute the bulk of milk proteins, representing about 80% and 20% of total proteins, respectively (Wal, 2004).
Caseins can be further dived into αs1, αs2, β and κ-caseins (Bos d 8), with αs1 and β being the most abundant. These proteins do not have a clear three-dimensional structure. This in turn suggests that casein epitopes are preferentially linear, with six major and 3 minor IgE binding epitopes identified (Lisson, Novak and Erhardt, 2014). In milk, casein aggregates to form casein micelles, characterized by a central hydrophobic part and a peripheral hydrophilic layer which contains major phosphorylation sites (Wal, 2004).
In the case of whey allergens, α-lactalbumin (ALA, Bos d 4) and β-lactoglobulin (BLG, Bos d 5) represent 5% and 10% of total milk protein, respectively (Docena et al., 1996) (Natale et al., 2004).
α-lactalbumin is a monomeric globular calcium binding protein, with a high affinity site for calcium. This protein interacts with β-1,4-galactosyltransferase to form the lactose synthase complex. α-lactalbumin modifies the substrate specificity of β-1,4- galactosyltransferase allowing the formation of lactose from glucose and UDP-galactose (Brew, 2013). β-lactoglobulin is the most abundant allergen in the whey fraction, accounting for 50% of the total protein of this fraction. Being a part of the lipocalin superfamily, β-lactoglobulin is capable of binding to a wide range of molecules like β- carotene, retinol, aliphatic hydrocarbons and saturated and unsaturated fatty acids.
Many IgE binding epitopes have been described for this proteins (Kontopidis, Holt and Sawyer, 2004).
Other allergens are also present, albeit in smaller quantities. These include lactoferrin and immunoglobulins (Bos d 7) and bovine serum albumin (BSA, Bos d 6) (Zeiger et al., 1999).
It has been reported that cooking can diminish the allergenicity of whey proteins, particularly that of β-lactoglobulin. This presumably occurs by denaturation of heat-labile proteins, which in turn results in loos of conformational epitopes, therefore preventing recognition by IgE in the GI tract (Cocco et al., 2007).
Enzyme-linked immunosorbent assays (ELISA) can be utilized to evaluate the immunogenicity of cow’s milk allergens through the binding of specific IgE to CM proteins, such as casein, α-lactalbumin and β-lactoglobulin. Through such assays, it is possible to estimate the concentration of specific IgE in patient serum. Studies have demonstrated that only a small percentage of CMA patients are monosensitized, with most of them being sensitized to several proteins. Casein, α-lactalbumin and β- lactoglobulin appear to be the major allergens of cow’s milk. However, many patients were also sensitized to proteins present in smaller concentrations, such as lactoferrin.
1.5. Analytical techniques for the analysis of milk proteins
There are many biochemical analytical techniques available for the analysis of milk proteins. Immunoassays are sensitive and specific systems use antibodies to detect allergens in samples. Most immunoassays use the ELISA format for the detection of milk antigens (Msagati, 2017).
Two of the most common ELISA assays are the Sandwich ELISA (S-ELISA) assay and the Competitive ELISA (C-ELISA) assay. In the S-ELISA assay, proteins are captured by an antibody bound to a solid phase support, and later detected by a second protein-specific enzyme-labelled antibody. After the binding of the second antibody, a substrate of the enzyme bound to the second antibody is added. Finally, the product of the catalysed reaction can be measured with colorimetric or electrochemical methods, therefore allowing for the detection and quantification of allergens (Schmidt et al., 2012).
In the C-ELISA essay, samples are incubated with unlabelled antibodies before being
added to wells containing antibodies immobilized in a solid phase. These wells are then washed to remove antigens that are not bound to the immobilized antibodies in the solid phase. If the antigens present in the sample bind with the specific antibodies in the first incubation, they will be unable to bind to the immobilized antibodies in the solid phase, thus being removed from the well in the washing step. After the washing step, a second enzyme labelled antibody is added (similarly to the sandwich assay), followed by a specific substrate. However, in the C-ELISA assay, the binding of antigens to the immobilized phase indicates the inability of said antigens to bind to the specific antibodies in the first incubation. Thus, signal detected in inversely proportional to the concentration of a specific antigen in the sample (Castillo and Cassola, 2017).
Polymerase chain reactions (PCRs) may also be used to detect allergens in samples via the amplification of a specific DNA sequence. This kind of assays are widespread and used for the detection of a vast range of food allergens, including CM allergens.
These assays, however, do not detect the presence of antigens directly in samples, detecting instead the presence of a specific DNA sequence. As such, a positive result in a PCR assay cannot prove the presence of a allergen in a sample. This can be disadvantageous in the detection of allergens in cooked and processed foods, seeing as the allergens are degraded by those processes, while DNA sequences are not. This in turn leads to false-positives (Besler, Steinhart and Paschke, 2001).
The coupling of protein separation techniques like SDS-PAGE, Capillary Electrophoresis (EC), Liquid Chromatography (LC) and High-Performance Liquid Chromatography (HPLC) with a Mass Spectrometry (MS) analysis allows not only for the identification of proteins in samples but also for the study of the post-translational modifications, conformation and folding/unfolding process of said protein. Moreover, it also allows for the study of protein-protein interactions (Alomirah, Alli and Konishi, 2000) (Nuwaysir and Stults, 1993). Soft ionization techniques such as fast atom bombardment (FAB) electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) allow for the analysis of proteins with MS without a significant chemical decomposition during the generation of ions (Medhe, 2018). The use of tandem mass spectrometry (MS/MS), in which ions formed during the ionization process are further fragmented into ion fragments. Analyses of protein containing samples via MS/MS spectrometry results in the formation of a peptide sequence tag that can be used to identify peptides via comparation of results with a protein data bank, thus allowing for the analysis of complex biological sample (Roepstorff and Fohlman, 1984).
Fragmentation of precursor ions can be achieved using collision-induced dissociation methods, electron capture and transfer methods or photodissociation (Domon, 2006).
1.6. In vitro degranulation studies and their role in the study of IgE-mediated food allergies
Cellular models are useful tools for studying a myriad of pathologies, as well as developing new therapeutic approaches. In line with this premise, mast cell basophiles models have been developed for studying the molecular basis of allergen mediated food allergies. Rat Basophilic Leukaemia (RBL) cells were generated by injecting rats with the chemical carcinogen β-chloroethylamine (Eccleston et al., 1973). Tumours that subsequently formed in inject rats were then cultured (Kulczycki, Isersky and Metzger, 1974). The RBL- 2H3 subline resulted from subsequent cloning of the original tumoral cells. This subline was capable of histamine release, either by chemical stimulation of by IgE binding (Barsumian et al., 1981). This cell can be used to study the exocytosis of pro-inflammatory compounds associated with both basophils and mast cells (Woska and Gillespie, 2012).
The expression of the high affinity IgE receptor FcεRI on RBL cells also allows the use of this cell line in the allergen binding assa. However, while human IgE receptors are capable of binding both rodent and human IgEs (Conrad, Wingard and Ishizaka, 1983), the rodent receptor does not bind human IgE (Hakimi et al., 1990). Thus, to use human IgE in degranulation studies, RBL cells had to be transfected with genes that allow for the expression human FcεRI receptor (Wan et al., 2020).
A further improvement of humanized RBL cells consisted in introducing a reporter gene (firefly luciferase), allowing easy and highly sensitive measurement of RBL activation and degranulation (Nakamura et al., 2010). Nuclear Factor of Activated T- cells (NFAT) proteins are a transcription factors whose subcellular localization is ultimately determined by intracellular calcium concentrations. In a resting state, the Nuclear Localization Signal (NLS) located in the N- terminal of this protein is masked due to the action of serine/threonine kinases, that phosphorylate this portion of the protein (Macian, 2005). Activation of RBL cells results in sustained calcium influx, leading to the activation of Calcineurin. This enzyme dephosphorylates NFAT, unmasking the NLS and allow for nuclear translocation (Hogan et al., 2003). In the nucleus, NFAT binds to specific
promoter sites and initiates gene transcription of the luciferase reporter gene. As such, this reporter cell line allows for the quantification of basophil/RBL activation by lysing the cells some hours after applying the stimuli and measuring luciferase activity with appropriate substrates and a luminescence detector (Nakamura et al., 2010). Other proteins can be used as fluorescent report systems, as is the case of the DsRed protein.
The expression of this protein in the NFAT-DsRed RBL cell line is also dependent of the nuclear translocation of NFAT due to a calcium influx in cells (Xiaowei Wang et al., 2014) (Wan et al., 2014). Finally, reporter cells lines that measure the degranulation event directly instead of measuring prior events (Ca2+ influx in cells) were also developed using subcellular compartment targeting properties of neuropeptide Y (NPY). When synthetized, this protein is located in the electron-dense secretory granules of RBL cells (Tatemoto, Carlquist and Mutt, 1982). As such, the use of a NPY-GFP recombinant protein allows for the measurement of RBL degranulation through fluorescence (Barwary, Wan and Falcone, 2020). The activation of RBL cells is schematically represented in Figure 3.
Figure 3 – Schematic representation of the mechanism involved in the activation of the reporter RBL cell lines.
2. Importance of microbiota in food allergy
More than 1000 different species of microorganism colonize the human intestinal tract. These commensal bacteria have an important role in many of the hosts physiological processes, namely in food digestion, differentiation of epithelial cells and
in the development and maintenance of the host’s immune system (Shu et al., 2019) (Abelius et al., 2014).
Many techniques can be used to study to characterize the hosts microbiota, with the analysis of the 16S rRNA gene using a clone library being one of the most prominent.
GM is composed primarily by four major microbial phyla: Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria. The taxonomic composition of the microbiota is not static, with different compositions being characteristic of different age groups. In the early stages of life, the GM is primarily composed by Proteobacteria. Then, the composition of GM is composed mainly of Actinobacteria, before finally maturing into the adult-like microbiota, dominated by Firmicutes and Bacteroidotes (Rautava et al., 2012) (Jiménez et al., 2008).
The metabolism of these commensal bacteria leads to the formation of many molecules with the ability to modulate the oral tolerance to food allergens. One of these secondary metabolites are short-chain fatty acids (SCAFS), which are the end-product of the fermentation of dietary fibers in the GT. Some of the main SCFAs produced during GM metabolism include acetate, propionate, butyrate and pentanoate. SCFAs have a pivotal role in the modulation of the human immune system, with many of them possessing beneficial effects on autoimmune and inflammatory disorders (Luu, Monning and Visekruna, 2020) (Luu and Visekruna, 2019).
Under normal conditions, ingested allergens are vastly degraded by gastric acid and by luminal digestive enzymes. As discussed previously, a large variety of cells of the immune system work in tandem to capture the allergens in the intestinal tract, to transport across the intestinal epithelial cells and present them to dendritic cells and further acting towards T Cells. The presence of different cytokines can alter the resulting immune response or allow the maintenance of the oral tolerance to specific allergens (Nowak- Wegrzyn, Szajewska and Lack, 2017a). SCFAs can act as diffusible signaling molecules, acting on cells expressing G protein-coupled receptors (GPRs), such as the intestinal epithelial cells and cells of the immune system, namely DCs and Tregs. The binding of SCFAs to GPCRs like GRP43, GRP41 and GRP109a results in the activation of various signaling cascades. This in turn promotes the proliferation of Treg cells, thus contributing to the maintenance of oral tolerance to dietary food allergens (Sivaprakasam, Prasad and Singh, 2016). SCFAs are also strong histone deacetylase (HDAC) and lysine deacetylase (KDAC) inhibitors. As such, they modulate the expression of various genes, with some of them being involved in cell proliferation and differentiation of immune system cells. An increase in histone acetylation via the inhibition of HDACs and KDACs
also results in as decreased production of inflammatory cytokines like TNF-α; IL-6 and IL-8; and an increase of intestinal Treg levels and of IgA production in the lumen of the intestine (Goverse et al., 2017), (Park et al., 2015).
Due to the crucial role of these microorganisms in the development of an adequate immune response, changes in the composition of the populations present in the GT can have severe effects in that development. As such, dysbiosis of the host’s microbiota is one of the primary hallmarks of food allergy (Nowak-Wegrzyn, Szajewska and Lack, 2017b). Studies with germ-free mice confirmed the key role of microbiota in the development of adequate immune responses, as these animals do not present a normal immune response when exposed to food allergens. However, restauration of different microbial populations leads to a normalization of immune response, with the establishment of Treg cells populations (Pannaraj et al., 2017), (Fernández et al., 2013).
Reestablishment of normal GM population constitutes a new therapeutic approach to FA.
Elimination of the dysbiosis characteristic of FA allows increase in SCFAs production, thus allowing a restauration of oral tolerance to dietary allergens.
Many factors can modulate the composition of the GM, with some of the more prominent being the mode of birth, breastfeeding, the chronic use of antibiotics and, in particular, the type of diet. As stated previously, SCFAs result from the fermentation of insoluble fibers in the GT. Dietary fibers have a prebiotic effect, potentiating the growth of specific bacteria and having a deep effect in the fermentative metabolism of the commensal bacteria. Furthermore, PCs have been recently described as prebiotic agents given by their ability to modulate GM ecology and function (Alves-Santos et al., 2020). On the other hand, a high-fat diet potentiates dysbiosis, being associated with a dysregulation of immune system function and potential loss of oral tolerance to food allergens (Bibbò et al., 2016), (McKenzie et al., 2017).
The use of probiotics also continues new therapeutic approach to reset dysbiosis in the microbial population of the GT. Probiotics are defined as live microorganisms that confer a health benefit on the host when administered in adequate amounts. Probiotics modulate GM by competing with pathogenic bacteria and, conversely, increasing the levels of beneficial SCAFs producing microorganisms (Eslami et al., 2020).
3. Phenolic compounds: from chemistry to their role in food allergy
3.1. Nature and chemistry of phenolic compounds
There are more than 8000 varieties of PCs produce by plants as secondary metabolites. These compounds present a great structural variety, but all of them contain at least one phenol ring with one or more hydroxyl groups in their structure. The vast diversity in the structure of these compounds allows for a great variety of biological functions (Carrillo-López and Yahia, 2019) (Muller et al., 2019) (Williamson, Kay and Crozier, 2018).
PCs are divided into two main categories: flavonoids and non-flavonoids, with flavonoids accounting for about two thirds of dietary flavonoids. The basic structure of this subclass of PCs consists in an arrangement of two phenolic rings (A and B) connect to a heterocyclic pyran ring (C) (Figure 4).
Figure 4 – Basic structure of flavonoids (Carrillo-López and Yahia, 2019).
Flavonoids can be further divided into six groups, according to the structure of the heterocyclic ring. These six subclasses are: flavanols, anthocyanins, flavanones, flavones, flavonols and isoflavones (Figure 5) (Carrillo-López and Yahia, 2019) (Vauzour et al., 2010).
Figure 5 – Basic structure of the six subgroups of flavonoids (Carrillo- López and Yahia, 2019).
PCs that do not possess the basic structure of a flavonoid are classified as non- flavonoids. The chemical structure of these compounds varies greatly, with most of them being generally smaller and simpler than flavonoids. However, some are these compounds possess complex structures and high molecular weights. Non-flavonoids are divided into many subgroups, with the most common being the phenolic acids, which account for almost one third of dietary PCs. Phenolic acids can be divided into two subgroups: phenolic acids derived from hydroxybenzoic acid, and phenolic acids derived from hydroxycinnamic acids (Figure 6) (Heleno et al., 2015a) (Carrillo-López and Yahia, 2019) (Heleno et al., 2015b) .
Figure 6 – Basic structure of hydroxycinnamic and hydroxybenzoic (Heleno et al., 2015a).
3.2. Phenolic compounds human metabolization
Most of the PCs in plant tissues occur in the form of glycosides and esters and are extensively metabolized after ingestion (Tarko, Duda-Chodak and Zajac, 2013) (Xu et al., 2016). The metabolization process can be divided into two distinct phases. The first phase involves processes of reduction, oxidation and hydrolyzation, and the second phase involves processes of glucuronidation methylation. Those reactions can start to take place as soon as the PCs are orally ingested and continues even after absorption in the GI track. The metabolization of PCs varies greatly, with some compounds being extensively metabolized, not only in the GI track, but in the various tissues of the body, while other compounds, especially those with lower molecular weights and simpler
structures, are observed in the large intestine with minimal changes in their chemical structure (Figure 7). The GM also plays an important role in the metabolization of PCs.
Compounds that are not absorbed throughout the small intestine are metabolized by the microflora present in the colon (Williamson, Kay and Crozier, 2018) (Bilal Hussain et al., 2019) (D’Archivio et al., 2010) (Domínguez-Avila et al., 2017).
Figure 7 – Schematic representation of the metabolic pathway of phenolic compounds in humans. Adapted from (Bilal Hussain et al., 2019).
The first stage of metabolism involves the biotransformation of PCs through reactions of oxidation, reduction and hydrolysis. The changes on the chemical structure of PCs resulting from these reactions increases of the polarity these compounds, thus increasing their solubility and facilitating their excretion. Most of these reactions occur in the GI tract, with the enzymes that catalyse said reactions being either constitutively expressed by the various tissues that compose the GI tract (like the CPY450 superfamily of enzymes) or secreted by the GM (Bilal Hussain et al., 2019) (Domínguez-Avila et al., 2017).
Hydrolysis of functional groups in catalysed mainly by a variety of enzymes, with carboxylesterases, aldehyde dehydrogenase, carbonic anhydrase, proteases and lipases being some of the most important in this process. Oxidation of PCs is primarily facilitated through enzyme-based oxidation processes, controlled by the CPY450 superfamily of enzymes. Lastly, the reduction of PCs takes place mainly in the large intestine, with native enzymes and enzymes secreted by the GM having an equally import role(Curtis D. Klaassen, 2015) (Scalbert and Williamson, 2000) (Misaka et al., 2013) (Selma, Espín and Tomás-Barberán, 2009).
The second phase of metabolism begins in the small intestine and involves the incorporation of different chemical radicals into the chemical structure of the PCs. The most common conjugation reactions are glucuronidations and methylations. The second phase of metabolization continuous after the absorption of these compounds into the blood stream, with most of the PCs being metabolized in the liver. Glucuronidation is the major conjugation-based reaction in humans and involves the binding o glucuronic acid to form more water-soluble compounds, which are more readily excreted via urine. The enzyme responsible for catalysing this process is the UDP-glucuronide transferase.
Methylation of PCs is not as common, because they normally decrease the hydro solubility of those compounds. Furthermore, the changes in the chemical structure resulting from these reactions also prevent further conjugation reactions. However, many PCs are methylated, with some of the most notable examples being catechins and epicatechins. These reactions are catalysed by O-methyltransferases (Steffen et al., 2008) (Das and Rosazza, 2006) (Walle, 2009).
PCs are extensively metabolized, either in tissues or in the lower portions of the GI track. Due to this, virtually all the PCs in circulation are glucoronidated and/or sulphated.
Also, very few free aglycones are found in plasma (Alkhalidy, Wang and Liu, 2018).
Notable exceptions to this include flavonoids like phloretin, quercetin and caffeic acid.
These compounds are present both a conjugated and a non-conjugated form in plasma (Crespy et al., 2001) (Gee et al., 1998) (Hollman et al., 1995). Free flavonoid aglycones can however be detected when pharmacological doses are administered. This indicates a possible saturation of the conjugation pathways for these compounds (Das, 1971).
Although the conjugation of PCs has been recognized and vastly studied, most of the biological studies involving these compounds have been carried out using only the free aglycone form. As such, knowledge regarding the biological properties of conjugated derivatives is poor (Scalbert et al., 2002).
Even though sulphate esters and glucuronides were shown to retain part of their bioactivity, such as their antioxidant properties (Manach et al., 1998), recent studies also showed that the glucuronidation of flavonoids greatly reduced their bioactivity. Therefore, this metabolized PCs had an reduced potential has antioxidants in the protection of neurons and fibroblasts against oxidative stress (Zhang et al., 1999). Other conjugation reactions, such as methylations, can also affect the bioactivity and bioavailability of PCs (Morand et al., 2000).
3.3. Phenolic compounds and food allergy: main role and mechanisms
The in vitro and in vivo bioactivity of PCs has been vastly described in the past few years, namely their antioxidant and anti-inflammatory properties (Luca et al., 2020). Due to these properties, many studies have been proposed to study the use of dietary polyphenols as modulator of immune reactions (Table 1).
Despite this, the health benefits of dietary PCs are relatively unexplored, as these compounds are vastly metabolized in phase I and II metabolism and are poorly absorbed in the GT. More studies are necessary to fully comprehend not only the effect metabolization on the bioactivity and bioavailability of phenolic compounds, but also the possible applications of metabolites resulting from the metabolization of phenolic compounds, as these may conserve a substantial portion of their bioactivity (Luca et al., 2020) (Skrovankova et al., 2015).
Table 1 – Bioactivity of dietary polyphenols
Polyphenol Experimental study
Biological
action Food Allergy References
Epigallocatechin gallate
Protein- polyphenols complexation
Conformational changes
Milk allergy (albumin)
(Xiaoya Wang et al.,
2014) Epigallocatechin
gallate
Protein- polyphenol complexation
Conformational changes
Shrimp allergy (tropomyosin)
(Lv et al., 2021)
Resveratrol Mouse model
Inhibition of Th2 differentiation
and APCs
Ovalbumin (Okada et al., 2012)
Red wine and coffee polyphenols
In-vivo gut microbiota
Increase Bacteroides
Inflammation biomarkers of rhinitis allergic
(Singh et al., 2017)
Apple polyphenol
extract
Mouse model
Reduction of allergy symptoms in a dose dependent
manner
Ovalbumin (Lv et al., 2021)
Apple polyphenol
extract
In vitro mast cell degranulation
Reduced histamine release
Universal allergy model
(TOKURA et al., 2005)
Phenolic acids
Protein- polyphenol complexation
Binding to peanut allergy
specific IgE
Peanut allergy
(Chung and Champagne,
2009)
PCs can directly modulate the hosts immune system and contribute to the maintenance of oral tolerance. Different studies have shown that these compounds can modulate the immunological response to food allergens by inhibiting different enzymes involved in this process. For example, the PC resveratrol has been shown to inhibit the cyclooxygenase family of enzymes in mice, preventing the formation of prostaglandins, while curcumin inhibits IκB kinase (IKK) and Mitogen-Activated Protein Kinase (MAPK), downregulating the NFκ B Signalling Pathway and the MAPK Signalling Pathway
respectably (Leiherer, Mündlein and Drexel, 2013) (Basnet and Skalko-Basnet, 2011).
PCs can also downregulate the expression of certain enzymes involved in the IgE mediated allergic response to food allergens. The administration of Epigallocatechin gallate in human epithelial cells downregulates the expression of inducible nitric oxide synthase (iNOS) in macrophages, resulting in a decreased production of key inflammation mediators (Kanwar, 2012) (Singh, Shankar and Srivastava, 2011).
Dietary PCs can also affect the number of specific immune system cells and their differentiation process. Male C3h/HeN mice treated with PCs extracted from fruit of the date palm tree had an increased count of Th1 and DCs in the intestinal lumen, while mice threated with the PCs baicalin and apigenin presented a reduced count of Th2 cells, being that an increased count of this type of cells is one of the hallmarks of loss of oral tolerance to dietary antigens (Karasawa et al., 2011) (Choi et al., 2009) (Magrone and Jirillo, 2012).
Lastly, PCs can also regulate the equilibrium between the production of pro- inflammatory cytokines like (IL-1β, IL-2, TNFα, Il-6 and IL-8); and anti-inflammatory cytokines like (IL-10; IL-4 and TGFβ). Changes in this equilibrium will affect immune response homeostasis. Several PCs have been shown to inhibit the expression of various pro-inflammatory cytokines in different cell types, namely lipopolysaccharide- activated mouse primary macrophages and activated human mast cell lines (Comalada et al., 2006) (Min et al., 2007). Likewise, quercetin has been shown to enhance the production of the anti-inflammatory cytokines, like IL-10 (Drummond et al., 2013).
The interactions between food allergens and PCs can also modulate the hosts immune response. PCs have the ability to form soluble and insoluble complexes with proteins, thus rendering them hypoallergenic, either by changes in the epitope structure or by diminishing their bioavailability (Singh, Holvoet and Mercenier, 2011). Dietary proteins, like the ones present in milk, can form complexes via reversible non-covalent interactions, like hydrogen bonding, hydrophobic interactions and ionic bonds; or via irreversible covalent interactions. Both types of interactions can alter the proteins secondary and tertiary structure. (Zhang et al., 2020a) Both caffeic and chlorogenic acid have been shown to form complexes with whey proteins, thus reducing their ability to bind to food specific IgE (Pessato et al., 2018) (Wu et al., 2018)
These changes in structure can alter the digestion of the complexed protein. The complexation of chlorogenic acid with whey proteins and casein resulted in an enhanced digestion. This resulted in a decrease of the bioavailability of these proteins, therefore
