# Application of the adverse outcome pathway (AOP) concept to structure the available in vivo and in vitro mechanistic data for allergic sensitization to food proteins

Jolanda van Bilsen, Edyta Sienkiewicz Szlapka, Daniel Lozano Ojalvo, Linette E. M. Willemsen, Celia M Antunes, Elena Molina, Joost J. Smit, Barbara Wróblewska, Harry J. Wichers J, Edward F. Knol, et al.

► To cite this version:

Jolanda van Bilsen, Edyta Sienkiewicz Szlapka, Daniel Lozano Ojalvo, Linette E. M. Willemsen, Celia M Antunes, et al. Application of the adverse outcome pathway (AOP) concept to structure the available in vivo and in vitro mechanistic data for allergic sensitization to food proteins. Clinical and Translational Allergy, 2017, pp.1-18. 10.1186/s13601-017-0152-0 . hal-01604242

HAL Id: hal-01604242

https://hal.science/hal-01604242

Submitted on 26 May 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
REVIEW

Open Access

<figure><img src="image_2.png" /></figure>

CrossMark

# Application of the adverse outcome pathway (AOP) concept to structure the available in vivo and in vitro mechanistic data for allergic sensitization to food proteins

Jolanda H. M. van Bilsen<fnref n="1" />*, Edyta Sienkiewicz-Szłapka², Daniel Lozano-Ojalvo³, Linette E. M. Willemsen⁴, Celia M. Antunes⁵, Elena Molina³, Joost J. Smit⁴, Barbara Wróblewska⁶, Harry J. Wichers⁷, Edward F. Knol⁸, Gregory S. Ladics⁹, Raymond H. H. Pieters⁴, Sandra Denery-Papini¹⁰, Yvonne M. Vissers¹¹, Simona L. Bavaro¹², Colette Larré¹⁰, Kitty C. M. Verhoeckx<fnref n="1" /> and Erwin L. Roggen¹³

**Abstract**

**Background:** The introduction of whole new foods in a population may lead to sensitization and food allergy. This constitutes a potential public health problem and a challenge to risk assessors and managers as the existing understanding of the pathophysiological processes and the currently available biological tools for prediction of the risk for food allergy development and the severity of the reaction are not sufficient. There is a substantial body of in vivo and in vitro data describing molecular and cellular events potentially involved in food sensitization. However, these events have not been organized in a sequence of related events that is plausible to result in sensitization, and useful to challenge current hypotheses. The aim of this manuscript was to collect and structure the current mechanistic understanding of sensitization induction to food proteins by applying the concept of adverse outcome pathway (AOP).

**Main body:** The proposed AOP for food sensitization is based on information on molecular and cellular mechanisms and pathways evidenced to be involved in sensitization by food and food proteins and uses the AOPs for chemical skin sensitization and respiratory sensitization induction as templates. Available mechanistic data on protein respiratory sensitization were included to fill out gaps in the understanding of how proteins may affect cells, cell-cell interactions and tissue homeostasis. Analysis revealed several key events (KE) and biomarkers that may have potential use in testing and assessment of proteins for their sensitizing potential.

**Conclusion:** The application of the AOP concept to structure mechanistic in vivo and in vitro knowledge has made it possible to identify a number of methods, each addressing a specific KE, that provide information about the food allergenic potential of new proteins. When applied in the context of an integrated strategy these methods may reduce, if not replace, current animal testing approaches. The proposed AOP will be shared at the www.aopwiki.org platform to expand the mechanistic data, improve the confidence in each of the proposed KE and key event relations (KERs), and allow for the identification of new, or refinement of established KE and KERs.

**Keywords:** Adverse outcome pathway, Key events, Key event relations, Food allergy, Sensitization, Mechanistic understanding, Food proteins, Molecular initiating event

<footnote>*Correspondence: j.vanbilsen@tno.nl</footnote>

<footnote n="1">¹TNO, PO Box 360, 3700 AJ Zeist, The Netherlands</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
**Background**

Consumers are exposed to increasing numbers of novel proteins or protein-containing products (e.g. insect burgers or proteins derived from bacteria grown on waste streams). These sustainable protein-rich food products are to solve the food insecurity problem but require a comprehensive risk assessment complying with the European 'Novel Food' law. Additional knowledge and biological tools are needed to support the prediction of the risk for food allergy development and the potential severity of the reaction [1, 2]. This constitutes a major public health problem and a challenge to risk assessors and managers [3, 4].

Like other allergies, food allergy has a non-symptomatic sensitization phase and a symptomatic elicitation phase. Food-associated adverse reactions can be immunoglobulin E (IgE) mediated, non-IgE mediated or both [5]. This paper focusses on the current understanding of the cellular and molecular mechanisms driving sensitization induction resulting in IgE-mediated allergy.

The mode of action (MOA) of sensitization and IgE mediated allergy to food proteins in predisposed individuals is poorly understood [6]. It is recognized that food processing, oral uptake and digestion affect the characteristics of food and food proteins [7]. Thus, acquiring a good understanding of the MOA requires well-characterized food and food protein samples for in vivo challenges in animals or preferentially humans. Such samples are now made available by the INFOGEST Cost Action [8].

There is a substantial body of in vivo and in vitro data describing molecular and cellular events potentially involved in food sensitization. However, these events have not been organized in a sequence of related events that is plausible to result in sensitization, and useful to challenge current hypotheses [9, 10]. The aim of this paper is to collect and structure the current mechanistic understanding of sensitization induction to food proteins by applying the concept of adverse outcome pathway (AOP).

**Main text**

**AOPs for food sensitization: a proposal**

The AOP concept is a framework for collecting and organizing information relevant to an adverse outcome at different levels of biological organization. It is believed that AOPs based on available information on substance-response and response-response relationships allow the development of relevant predictive animal-free test methods and approaches, as well as the contextualization of the results across a diverse range of biological mechanisms and toxicity endpoints. The Organisation for Economic Cooperation and Development (OECD) provides an international hub for constructing, reviewing,

and using AOPs with the help of a suite of tools comprising the AOP knowledge base, including the AOP Wiki (www.aopwiki.org). This AOP Wiki provides a collaborative platform for constructing AOPs and can be used by groups that have a proposal for an AOP [11].

This manuscript proposes an AOP for food sensitization (Fig. 1). The information on mechanisms and pathways evidenced to be involved in sensitization by food and food proteins is structured using the AOP for chemical skin sensitization [12] and respiratory sensitization [13] induction as templates. Available mechanistic data on protein respiratory sensitization [14] were considered to fill out gaps in the understanding of how proteins may affect cells, cell-cell interactions and tissue homeostasis. Figure 1 depicts a proposal for the AOP of food sensitization including all key events and cellular players with substantial (solid lines) or circumstantial (dashed lines) evidence for a role in the sensitization induction to food proteins. In this review, the successive sections describe events following the common structure of an AOP consisting of a molecular initiating events (MIE) and a series of key events (KEs) that eventually lead to the occurrence of clinical symptoms upon repeated exposure in the elicitation phase.

**Molecular processes in the AOP for food sensitization**

**Bioavailability: protein and protein fragments acquire access to the relevant immune cells**

*Acquiring access to the underlying immune system via the gastrointestinal tract* The multiple facets of intestinal permeability and epithelial handling of dietary antigens were previously reviewed [15]. The following sections intend to capture the current understanding of the role of transport in sensitization induction as schematically outlined in Fig. 2.

Impairment of tight junctions may be implicated in sensitization induction. Paracellular transport is mainly determined by pore size in tight junction and only concerns molecules of MW < 600 Da [16, 17]. Proteins that are transported via this paracellular route are not exposed to lysosomes in the enterocyte and therefore are not degraded [18, 19]. Passage of intact protein may allow sensitization of immune cells in the subepithelial compartment (Fig. 2a).

Several in vitro studies suggest that gluten (gliadin), kiwi (Act d 1) and peanut (Ara h 2) allergens facilitate absorption by modulating tight junctions [20–23]. Act d 1, in analogy with house dust mite (HDM) Der p 1 [24], may cause a protease-dependent disruption of the tight junctions of the epithelial cell (EC) layer (Fig. 2a) For Ara h 2 the effect on the tight junctions and barrier integrity appears to involve endocytosis and downregulation of the zink finger protein A20, a crucial gatekeeper preserving
<figure><img src="image_3.png" /></figure>

tissue homeostasis with ubiquitin-regulatory activities [21].

Among various intestinal innate immune cells, mast cells (MCs) play critical roles in maintaining gut immune homeostasis. Studies on sensitized individuals revealed that the integrity of the tight junctions is decreased by mast cell-derived mediators like tryptase and IL-4 [25–30]. In murine models, the importance of MCs for the development of food allergies has also been clearly shown. Mice presensitized with alum and OVA develop allergic diarrhoea after oral OVA inoculation [31]. Inhibitors of serotonin and histamine as well as depletion of MCs by an anti-c-kit monoclonal antibody reduce the occurrence of allergic diarrhoea. Therefore, MCs as well as serotonin and histamine derived from them are involved in the allergic diarrhoea in this model [31]. These murine and human data clearly show the importance of MCs in the gut homeostasis and the effect of MC activity by allergens resulting in inflammatory responses in mucosal compartments [32]. However, in the context

of food allergy, the importance of MCs has only been described in already sensitized animals/individuals. To date there is no data available supporting the role of mast cells in sensitization induction to food proteins as depicted in Fig. 2b.

Transcellular transport mechanisms provide intact protein with free passage to the sub-epithelial compartment. Transcellular transport of proteins and peptides can occur via different mechanisms as illustrated in Fig. 2a, b. Most dietary proteins or peptides are absorbed actively by ECs in non-sensitised persons by endocytosis at the apical membrane and undergo transcytosis toward the lamina propria (LP). An estimate of 70–90% of the transported protein is degraded by intracellular enzymes into amino acids and peptides [33, 34].

In vitro studies showed that intact soybean allergen P34 and bovine β-lactoglobulin cross the epithelial barrier by transcytosis, while tryptic fragments of β-lactoglobulin followed para- and transcellular routes [35, 36]. Thus, protein resisting proteolysis in the gastrointestinal tract
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Molecular initiating events (MIE) that initiate food sensitization. **a** Food allergen interaction with mucosal surfaces of the gut intestine may result in disruption of tight junctions, receptor mediated or unspecific transcellular transport of food allergens across the gut epithelium. These events can be the initiating events that results in the activation of epithelial and innate cells such as dendritic cells. **b** Mechanisms and cells involved in intestinal protein transport in non-sensitized individuals. **Solid boxes** represent events with substantial evidence of being involved in food protein transport. **Dashed boxes** represent events that are possibly involved in food protein transport. **Crosses** indicate events and cell types of which there is no indication that they play a role in the transport of food proteins across the gut barrier in non-sensitized individuals</figcaption></figure>

is transported intact through the intestinal epithelial barrier, and may enhance the risk for sensitisation induction.

In sensitized persons, T<sub>h</sub>2 or innate lymphoid cells (ILC)-2-derived IL-13 with other inflammatory mediators (e.g. IFN-γ, TNF-α and IL1-β) stimulate transport via the CD23 receptor, which is overexpressed by enterocytes in sensitised persons. Antigen-IgE complexes are transcellularly transported by the CD23 receptor [18]. A role for CD23-mediated transport in sensitization induction must still be documented.

In other studies, receptor-mediated transcellular transport via the CD23 receptor appears to protect proteins from lysosomal degradation [18, 19], however as explained above, CD23-mediated transport has only been described in already sensitized individuals.

M cells transport protein directly to the underlying immune cells. M cells are specialized ECs covering the Peyer's, caecal and colonic patches. These cells deliver intact proteins and particles directly to the gut associated lymphoid tissues (GALT), and therefore may play an
important role in the regulation of immunological reactions to dietary antigens [37, 38].

The current perception is that particulate or aggregated antigens are taken up by M cells, resulting in antigen-specific local or systemic immune responses [39, 40]. This perception is supported by a study in mice showing that pasteurization shifted the transport of soluble β-lactoglobulin and α-lactalbumin from transcytosis through enterocytes to transport of protein aggregates by M cells to the Peyer's patches, and higher IgE and cytokine (IL-5, IL-13, IFN-γ, IL-10) levels [41]. However, another study demonstrating the presence of soluble protein as well as protein bodies of digested peanut in the cytoplasm of M cells within intestinal Peyer's patches in BALB/c mice [42] suggests that additional factors may contribute to the induction of adverse responses.

Monocytes and DCs sample protein directly from in the gut lumen. Both cells can sample the gut lumen using their pseudopodia. Tight junctions are opened and after sampling new tight junctional complexes between adjacent ECs are formed. Luminal sampling is upregulated in inflammatory conditions [43, 44]. However, information is lacking whether protein transport occurs via these cells.

The role of goblet cells in protein up-take and sensitization induction seems limited. In healthy human ileum and jejunum goblet cells can transport small peptides (10 kDa) and particles across the intestinal wall [45]. A role for these cells in uptake of food proteins has not yet been documented, nor their role in sensitization induction to food proteins.

*Acquiring access to the underlying immune system after cutaneous and respiratory exposure* There is growing evidence ascribing food proteins the capacity to induce sensitization when contacting the skin, and potentially the respiratory tract. How these proteins acquire access to the cutaneous immune cells that drive sensitization of the gastrointestinal tract is not sufficiently understood yet. In contrast, the mechanistic understanding of protein-induced sensitization of the respiratory tract is growing [14].

Evidence for gastrointestinal tract sensitization following cutaneous exposure exists. Mice exposed via different routes (intragastrically, cutaneously, intranasally or sublingually) to α-lactalbumin in the presence of cholera toxin were all sensitized, however sensitization via the skin resulted in the highest IgE levels [46]. Supporting evidence emerged from studies using hazelnut protein [47] and ovalbumin [48] as model proteins. In each case, oral as well as intragastric challenge led to anaphylactic symptoms after skin sensitization.

Even though human skin appears to be less penetrable than murine skin, epidemiological studies in human populations seem to confirm the skin as a relevant route for food sensitization induction and allergy in man.

High cutaneous exposure to peanut increases the risk of peanut allergy in human [49]. The use of peanut-protein containing skin creams was found to correlate with peanut allergy in children. These creams were mostly used to relieve eczema, i.e. damaged skin with increased permeability for e.g. proteins [50]. The importance of the quality of the skin barrier was demonstrated by the impact of filaggrin mutation/expression on human skin integrity and permeability, and its correlation with increased incidence of sensitization [51]. In Japan, at least 1800 individuals were sensitized following application of a facial soap containing hydrolysed wheat proteins (HWP). An epidemiological relationship was documented between wheat allergy and contact exposure to HWP in Japanese women [52]. Cutaneous sensitization to latex correlates with allergic symptoms upon ingestion of cross-reacting foods e.g. avocado, banana and kiwi. However, it cannot be excluded that oral exposure to these foods may lead to allergic reactions to cutaneous latex exposure [53].

Against the evidence in favor of the skin being a route for sensitization to food and food proteins stand data from studies in mice and humans showing that sensitization can be achieved to food and aero-allergens by applying antigen cutaneously [46].

Evidence for gastrointestinal tract sensitization following respiratory exposure is more ambiguous. The contribution of the respiratory tract to sensitization induction of the gastrointestinal tract is less clear, despite a growing understanding of how proteins may acquire access to immune cells and trigger inflammation [14].

Dunkin et al. [46] presented data suggesting that mice are sensitized by exposure of the respiratory tract to α-lactalbumin in the presence of cholera toxin. However, respiratory sensitization was less effective than cutaneous sensitization as judged from the IgE levels induced by subsequent oral challenges.

In humans, the second most significant risk factor associated with life-threatening asthma is a history of an asthma attack precipitated by food. A clear link between the pathophysiology of food and respiratory allergy is not yet established, but two hypotheses are currently pursued: (i) effector cells resulting from gastric sensitization populate the respiratory tract; (ii) chronic inhalation of food particles results in direct sensitization of the respiratory tract [54]. These hypotheses are not mutually exclusive. The oral allergy syndrome is currently believed to result from respiratory sensitization to pollen (e.g. Bet v 1 protein) in conjunction with oral exposure to apple
or carrot, containing cross-reactive Bet v 1 homologues. However, it cannot be excluded that oral ingestion of apple or carrot, results in sensitization and subsequent cross-reactivity to inhaled pollen [55]. There is also evidence suggesting that respiratory sensitization to food proteins not necessarily develops into food allergy. Indeed, to it has been reported that bakers with occupational asthma following exposure to wheat flower can safely consume wheat products [56].

**_The molecular initiation event (MIE): lessons learned from respiratory sensitization_**

The innate immune system is an evolutionary conserved system equipped with a range of pattern recognition receptors (PRRs) with specificity for pathogen-associated molecular patterns (PAMPs) on microorganisms, parasites and fungi, and danger-associated molecular patterns (DAMPs). These interactions trigger an inflammatory response driving an adaptive immune response [57].

The molecular basis for the propensity of specific proteins to induce sensitization and subsequent allergic responses is still poorly defined. Studies addressing sensitization of the respiratory tract revealed that the ability of proteins to trigger sensitization and allergy is a function of their ability to interact with pathways of innate immune recognition and activation at mucosal surfaces. Potential MIEs relate to proteolytic activity, engagement of PRRs, molecular mimicry of Toll-like receptor (TLR) signalling complex molecules, lipid binding activity, and oxidant potential [14].

The relevance of these pathways for food sensitization induction in general remains to be fully established. The current understanding of the mechanisms applied by food and food proteins (e.g. gluten, kiwi, peanut allergens) suggest that interaction with tight junctions may represent a MIE (MIE-1). Receptor-mediated transcellular transport via e.g. the CD23 receptor may be an alternative route (MIE-2) although its relevance for sensitization induction is not fully established. Endocytosis by either ECs or M cells may be a less well defined MIE (MIE-3) resulting in regulation of gene expression and disturbance of tissue homeostasis or induction of inflammation by direct contact of the potential allergen (e.g. peanut allergens) with antigen presenting cells (APC)s.

Animal experimentation has clearly presented evidence that matrix components are relevant in the sensitisation process. For instance, Van Wijk et al. [58] showed that, in the popliteal lymph node assay in C3H/HeOuJ-mice, purified peanut allergens were unable to induce immune activation, opposed to immunisation with whole peanut extract. In line with this, Wavrin et al. [59] demonstrated a much higher Th2-like response to whole milk in BALB/cJ mice, than to purified β-lactoglobulin. Matrix-derived (poly-)sarcharides

are involved, via heat-induced glycation, in sensitisation for peanut allergen in BALB/c-mice [60]. Heat-induced glycation of ovalbumin led to the induction of a Th2-skewing milieu in a human DC/T cell co-culture model, in which a role for the mannose receptor is suggested [61].

Similarly, c-type lectins receptors (CLRs) have been highlighted as important factors in initiating and modulation allergic responses [62]. For example, Dectin-1 recognizes β-glucans, a carbohydrate present in cells walls of many, if not all fungal species, and thereby promotes lung immunopathology during fungal allergy [63]. Dectin-1 has also been shown to limit gut mucosal inflammation induced by fungi [64]. In HDM allergy, Dectin-2 can recognize HDM and induce the release of cysteinyl leukotriene, which is, as well as IL-33, essential for the initiation of airway inflammation and promotion of subsequent Th2 immunity in response to HDM [65, 66]. In murine models, Dectin-2 is involved in the development of HDM-allergy during both the sensitization and challenge stages [66, 67]. Likewise, the mannose receptor has been shown to act on human DCs as receptor for HDM (Der p 1 and Der p 2), cockroach (Bla g 2), dog (Can f 1) and peanut (Ara h 1) and shown in mice to play a crucial role in Th2 cell polarization (reviewed by [62]). Several DC-SIGN-binding glycoproteins were identified in common allergenic foods at the difference of food that less frequently induce allergy [68]. Also lipids are claimed to be involved in the sensitization process [69]. Recently, an adjuvant role for the peanut allergen Ara h 1 in sensitization for Ara h 6 was suggested [70].

Details on the molecular mechanisms of the adjuvant effects that can be exerted by matrix components are mostly still lacking, but this does not preclude their relevance and importance. Additional knowledge into the identification of such matrix components is required and will help to develop tools to incorporate such adjuvancy into model systems. Specific PRRs, such as the mannose receptor, appear to be involved and (co-)activation of e.g. other CLRs, TLRs or G-protein coupled receptors may turn out to be involved.

**_Key events (KE): cellular 'innate' events at epithelial (KE 1) and DC (KE 2 and 3) level—setting the scene for inflammation_**

The intestinal epithelium is a cell monolayer (with diverse cells: enterocytes, Goblet cells, Paneth cells, endocrine cells, M cells) between the lumen and the immune compartment with inductive Peyer's and colonic patches, draining mesenteric lymph nodes (MLN) and the scattered immune cells in the LP [71]. The role of intestinal epithelium in allergic sensitization induction is recognized but the mechanistic understanding of the processes involved is limited when compared to skin and respiratory sensitization.
**KE 1: Sensitizer-related inflammatory responses at epithelial level** It is widely accepted that sensitization involves factors from gut epithelium which are activated during cellular stress. Such factors include radical oxygen species (ROS), Th2 driving mediators (e.g. IL-1, IL-18, IL-25, IL-33, TSLP) and mucus. There is limited availability of mechanistic data on pathways driving the allergic sensitization via the intestinal mucosa, however, much can be learned from pathways involved in skin and lung sensitization.

The role of ROS in allergen-induced skin sensitization was reviewed [72]. Evidence for a role of ROS in respiratory sensitization induction is provided by allergens with proteolytic activity (e.g. HDM allergen Der p 1). There is evidence that cysteine proteases may induce stress and elevated ROS levels e.g. through cleavage and activation of the Protease-Activated Receptor (PAR) 2 [24], and inactivation of lung surfactant proteins (e.g. SP-A, SP-D). Consequently, tight junction proteins (e.g. ZO-1 and occluding) become accessible for protease activity, leading to increased transepithelial access of allergens to immune cells [73, 74]. If and how this information can be translated to gastrointestinal tract is not yet clear, but it is clear that its epithelial lining is used to deal with protease activity involved in digestion.

ROS activation is also implicated in the sensitization to pollen allergens exerting [NAD(P)H] oxidase activity, an enzyme activity also found in mitochondria and driving intracellular ROS production [75]. Endocytosis of this pollen-derived enzyme increases ROS production and results in degradation of endogenous hyaluronic acid (HA), and TLR2 and TLR4 activation. This may be of relevance as a variety of studies implicate MyD88-mediated TLR2 and TLR4 signalling in the induction of T<sub>h</sub>2 immune responses leading to allergic respiratory inflammation and promotion of T<sub>h</sub>17 responses [14, 76].

ROS production may be induced directly via TLR activation. The HDM allergen Der p 2 reveals sequential homology to the MD-2-related lipid-recognition (ML) domain family, which is a well-characterized member of the TLR4 signalling complex. There is compelling evidence demonstrating that Der p 2 exhibits the same function as ML. Thus, the intrinsic adjuvant activity of MD-2 homologous allergens and their lipid cargo is likely to have wide generality as a mechanism underlying sensitization induction [77].

Activation of TLR requires phosphorylation by c-Src signals and activation of phosphoinositide 3-kinase and phospholipase C γ for activating NF-κB and chemokine expression leading to lymphocyte recruitment to the lung and increase in mucus production. Subsequently, Ca²⁺-dependent proteases cleave the transmembrane proteins occludin and e-cadherin on ECs promoting

transmigration of leukocytes [78]. NFκB activation is causally related to increased release by ECs of IL-33, IL-25 and TSLP, endogenous danger factors (e.g. high-mobility group box-1 (HMGB-1), uric acid and ATP, DC activation and migration, and the induction of ovalbumin and Der p 2 sensitization [79]. The release of ATP and uric acid drives the activation of the NLRP3 inflammasome complex resulting in cleavage of pro-IL-1β to mature IL-1β through caspase 1. IL-1β creates a pro-inflammatory micro-environment with the production of IL-6 and chemokines that mobilize neutrophils and enhance T<sub>h</sub>17 cell differentiation [80]. Uric acid may play an important role in T<sub>h</sub>2 skewing [81].

The limited mechanistic data suggest that similar TLR-dependent and -independent pathways may drive allergic sensitization via the intestinal mucosa. Epithelium-derived factors (IL-33, TSLP, IL-25) identified in studies focusing on HDM-induced sensitization were observed during intestinal peanut sensitization in mice, of which it was shown that IL-33 is important for OX40L expression on DC and the development of peanut allergy (see KE 2) [82]. Furthermore, α-amylase inhibitor from cereals activates TLR4 mediated processes [83]. Kong et al. demonstrated that uric acid is a critical signal for the induction of peanut allergy [84].

**KE 2: Sensitizer-related inflammatory responses at DC level** PRR, TLR and ROS signalling pathways are also active in endothelial cells, macrophages, fibroblasts as well as DCs. These cell types may therefore contribute to the induction of inflammation and sensitization by employing the same mechanisms as described for ECs.

In vitro data revealed that the allergen Pru p 3, but not the non-allergenic LPT 1 variant, crosses the epithelial barrier and induces production of the T<sub>h</sub>2 skewing cytokines TSLP, IL-25, and IL-33. In addition, Pru p 3, but not LPT 1, triggered the expression of inflammatory cytokines such as IL1β, IL6, and IL10 in a co-culture of Caco-2 cells and human peripheral blood mononuclear cells. The highest induction was observed for IL1β, which is related to a T<sub>h</sub>2 response and antibody production [85, 86].

The importance of EC-derived IL-33 for in vivo DC activation was demonstrated using a murine model for intragastric induced peanut allergy. It was observed that peanut administered in the presence of cholera toxin increased the expression of OX40L, a co-signaling molecule required for proper T cell activation [82]. In IL-33 receptor knock out mice this increase was not observed. TSLP and IL-25 protein levels were increased in duodenal tissue of peanut allergic mice, but TSLP or IL-25 receptor (IL-17-RB) knock out (KO) mice revealed no driving role for IL-25 or TSLP in peanut sensitization.
These data show that IL-33 is sufficient for the observed increase in OX40L expression, expansion of ILCs and the development of peanut allergy [82].

DCs, among other cells, can sample food protein directly from the gut lumen using pseudopodia. Alternatively, they can come in direct contact with intact protein after paracellular or transcellular transport through the ECs, or by M cell mediated transport. Driven by microenvironment changes and contact with the protein, DCs upregulate the expression of MHC II, costimulatory molecules such as CD54, CD80, and CD86, and receptors that are essential for migration (e.g. CCR7) [87].

While proteins are recognized to drive the activation of DCs in an inflammatory context, the observed suppression of LPS-induced IL-12 production suggest that HDM and peanut allergens may be capable of suppressing $T_h1$ driving DC responses [82].

Eosinophils constitute about 20% of the leukocytes in the LP and appear to be required for the induction of CCR7 and CD86 expression on CD103⁺DC. Hence eosinophils may contribute to activation and instructed migration of DCs from the LP to the MLN [88, 89]. Upon eosinophil depletion $T_h2$ priming and induction of peanut allergy was abolished in an IL-4 independent fashion [89]. While supporting data were presented, these data need to be further substantiated to strengthen the case for eosinophils playing a key role in the induction of food sensitization.

### KE 3: Dendritic cell and macrophage migration: translating innate responses into specific T and B cell responses

DCs and macrophages are the major APCs in the intestine, and play both a role in the induction of immune responses. However, only DCs determine the balance between tolerance and sensitization guided by local intestinal co-players e.g. ECs and ILCs. These co-players stimulate the expression of chemokine receptors facilitating sampling of antigens from the lumen (e.g. CX3CR1) and migration (e.g. CCR7, CXCR4) of CD103⁺DC from the epithelial lining or in Peyer's patches to draining lymph nodes [71].

Intestinal DCs and macrophages, as well as of individual intestinal DC subsets are poorly defined. In the murine intestine, CD103, CD11b, and CX3CR1 expression identifies three major populations of CD11c⁺MHCII⁺ DCs. CD103⁺CD11b⁺CX3CR1⁻ and CD103⁺CD11b⁻CX3CR1⁻ DCs arise from a non-monocytic origin and are considered classical DCs. They express CCR7 and migrate to MLN under steady-state and inflammatory conditions where they affect T cell homeostasis. CD103⁻CD11b⁺CX3CR1⁺/int DCs arise from monocytes and resemble macrophages despite lacking F4/80 or CD64 [90, 91]. They do not express CCR7

in the steady state and their ability to migrate to MLN remains controversial. Mice treated with cytokine Fms-related tyrosine kinase 3 ligand (Flt3L), a hematopoietic growth factor, increase the numbers of these DC subsets and a plasmacytoid CD11c⁺B220⁺mPDCA⁺ subset (pDC). The increase correlates with a decrease in IgE response to peanut extract and is reversed by depletion of pDC using e.g. monoclonal antibody 120G8. Thus, pDCs may control food allergic responses and possibly tolerance development [92].

Under basal conditions, CD103⁺CCR7⁺ DCs are known to instruct oral tolerance via the induction of $T_{reg}$ in the MLN. However, CCR7 KO mice lacking CD103⁺MHCII⁺CD86⁺ migratory DCs in the MLN do not develop sensitization and allergy after intragastric exposure to peanut, suggesting this subset may instruct immunity [84, 93–98].

Macrophages may also play a role in food allergic reactions [99], either by exhibiting regulatory or pro-inflammatory processes [100, 101]. To date comprehensive investigations deciphering the precise role of macrophages in food allergy is lacking.

### KE 4 and 5: Organ responses: initiation and amplification of specific responses

**KE 4: T cell priming, proliferation and polarization** Molecular profiling of human T cell pathways indicate that the signal supplied by the T cell receptor (TCR) (Fig. 3, event 1) only participates in the development of the $T_h1$ cell phenotype, whereas formation of the $T_h2$ phenotype depends on the CD28 signalling, even in the absence of specific TCR activation [102] (Fig. 3, events 2). Stimulation of CD28 activates NF-κB signalling and the expression of GATA3, a signal favouring $T_h2$ differentiation [103] (Fig. 3, event 3).

In the process of $T_h1/T_h2$ polarization also CTLA-4 (CD152) appears important. CTLA-4 mediated dephosphorylation of 'linker for activation of T cells' (LAT) results in TCR signal inhibition, decreased GATA3 expression, and inhibition of CD28 mediated $T_h2$ differentiation [104, 105] (Fig. 3). During sensitization, both CD28 and CTLA-4 molecules may interact with either CD80 or CD86 on the surface of APCs [106, 107] (Fig. 3, events 2). Following exposure to an allergen CD86 expression increases faster than for CD80, suggesting that CD86 is required for initiating the immune response, whereas CD80 has a more regulatory function [106]. Kuchroo et al. [108] showed that CD28-CD86 interaction relates to $T_h2$ cell responses, whereas CD28-CD80 interaction favours $T_h1$ cells responses. Thus, compounds (e.g. allergens) affecting the kinetics of CD86 and CD80 expression on APCs and CD28 and CTLA-4 expression on T cells, may favour a $T_h2$ response.
<figure><img src="image_5.png" /></figure>

The abundance of GATA3 in T<sub>h</sub>2 cells (Fig. 3, event 3) triggered the hypothesized that naïve CD4<sup>+</sup>T cells are “naturally” programmed for switching into T<sub>h</sub>2 cells and that T<sub>h</sub>2 cell differentiation is repressed by T-bet during T<sub>h</sub>1 cell differentiation in healthy individuals [109] (Fig. 3, event 4). The importance of GATA3 for the T<sub>h</sub>2 cell phenotype is well established [110]. It promotes the IL-4/IL-5/IL-13 gene translation (Fig. 3, event 5) by regulating methylation of histones which grants access for transcription factors (e.g. c-Maf) that activate IL-4 gene expression. GATA3 also binds specific DNA sites controlling activation (e.g. IL-5 and IL-13) or inhibition (e.g. T-bet, IFN-γ and IL-12Rβ2) of cytokine expression [110, 111].

IL-2 from activated CD4⁺T cell plays a crucial role in
the T<sub>h</sub>2 cell polarization [112] (Fig. 3, event 6). Activat-
ion of the IL-2 signalling via STAT5 results in an early
and IL-4-independent induction of the IL-4Rα subu-
nit with formation of a functional IL-4 type I receptor,
the signalling pathway for IL-4 [113]. IL-2/STAT5 may
also participate in the early initiation of the IL-4 gene
expression [114]. The mechanisms driving the estab-
lishment of IL-4 signalling by activated CD4⁺T cells
resembles OX40L/OX40 signalling [115]. OX40 (CD134)

expression is induced on the surface of naïve T cells
hours after exposure to antigen [116]. Allergen-induced
T<sub>h</sub>2 polarization requires only endogenous IL-4 and
is controlled by the OX40L/OX40 signalling pathway
[115]. OX40 signalling sustains TCR, CD28 and IL-2R
expression [116].

The contribution of TSLP, IL-25 and IL-33 to T<sub>h</sub>2
polarization is recognized in a murine asthma model.
Influence of TSLP on the T<sub>h</sub>2 response development is
indirect and occurs by inhibiting IL-12 secretion and by
inducing OX40L co-signalling in DCs. The presence of
IL-25R and IL-33R on CD4<sup>+</sup>T cells suggests that IL-25
and IL-33 exert a direct effect on these cells. In vitro
IL-25 promotes T<sub>h</sub>2 differentiation and IL-4 expression
through NFATc1 and IL-4/STAT6-dependent mecha-
nisms [117]. This observation was not confirmed by a
murine model for HDM allergy [82]. Epithelial IL-33
affects OX40L expression by DCs and activates signalling
pathways that are relevant for the T<sub>h</sub>2 polarization (e.g.
ERK, MAPKs and NF-κB) via IL-33R. Abolishing IL-33
signalling in allergic mice decreased the production of
specific IgE with 50% [82, 118]. However, whether IL-33
is inducing or maintaining a T<sub>h</sub>2 phenotype cytokine
remains to be clarified.
Activation and differentiation of naïve CD4+T cells may be under hormonal and metabolic control. A key event for both immune and metabolic signalling is mTOR protein kinase (PK) activity. A HDM/OVA mouse model for asthma showed that mTORC1 activity relates to T<sub>h</sub>1 and T<sub>h</sub>17, while mTORC2 relates to T<sub>h</sub>2 differentiation [119]. A role of mTOR PK, if any, in sensitization seems plausible. Inhibition of mTOR PK prevents activation of STATs and T-bet expression, known to be involved in sensitization, and drives iT<sub>reg</sub> cell differentiation by blocking polarization signals even in the presence of the T<sub>h</sub>1 and T<sub>h</sub>2 polarization cytokines. A leptin-dependent increase of the mTOR signalling is one of the causes of iT<sub>reg</sub> cell developmental disturbances resulting in food allergies [119, 120].

**KE 5: B cell activation and class switching** Synthesis of specific IgE requires two different levels of qualitative changes in the DNA of immunoglobulin (Ig) genes.

Somatic hypermutation (SHM) in the variable regions of heavy and light chain leads to changes in the affinity to antigen. Direct or sequential Ig class-switch recombination (CSR) constitutes strictly controlled intrachromosomal DNA fragment deletion (class segments) in the constant region of heavy chain (IGH) locus [121]. In atopic dermatitis patients the CSR for IgE antibodies may occur in a direct way (IgM > IgE) or an indirect one (IgM > IgG > IgE) [122–124].

The surface B cell antigen-recognizing receptor (BCR) identifies the antigen in its native form. BCR stimulation triggers a complex cascade of signalling events leading to B cell activation [125] (Fig. 4). The CD19/CD81/CD21 complex on B-cells is important for B cell activation with CD81 playing a crucial role in B-cell/T-cell communication through the MHCII-TCR (Fig. 4). Activated B cells establish contact with T<sub>h</sub>2 cells by interaction of the CD80/86–CD28 and CD40–CD40L molecules (Fig. 4).

<figure><img src="image_6.png" /><figcaption>**Fig. 4** IgE isotype switching in B cells. The surface immunoglobulin that serves as the B-cell antigen receptor (BCR) has two roles in B-cell activation. First, in combination with the BCR co-receptors, it transmits signals directly to the cell's interior when it binds antigen (event 1). Second, the B-cell antigen receptor delivers the antigen to intracellular sites where it is degraded and returned to the B-cell surface as peptides bound to MHC class II molecules (event 2). Subsequent isotype switching of B cells to IgE production is induced by two separate signals, both of which can be provided by polarized Th2 cells. The first signal is provided by the cytokine IL-4 interacting with the IL4 receptor on the B cells (event 3), which leads to activation of STAT-6 (event 4). The second signal for IgE switching is a co-stimulatory CD40-CD40L interaction (event 5). Both signals are important for the initiation of the antibody class switching (event 6), resulting in IgE production (event 7). It is also possible to induce a CD40-independent induction of isotype switching which involves the interaction of BAFF-BAFFR and APRIL-TACI interactions between DC and B cells (event 8)</figcaption></figure>
Effective induction of the activation-induced cyti-
dine deaminase (AID) expression requires a cooperation
between the CD40/TRAFs/NFκB signalling pathway, IL-4
and IL-13 as well as CD40 and IL-4 activation of HoxC4
in the B cell nucleus (Fig. 4). CD40 signalling in combina-
tion with IL-4 and IL-13 activates the synthesis of GLTε.
Activation of the Iε promoter by transcription factors
Pax5, PU.1, C/EBP, AP1, and E-box for E2A enhances
GLTε transcription. GLTε synthesis is regulated by
IL-4 only, since IL-4 depletion is sufficient to block Ig
CSR to IgE [126–128]. Ig CSR to IgE may occur also in
the absence of CD40 signalling [122–124]. BAFF and
APRIL molecules on DCs activate surface BAFFR and
TACI molecules on B cells, and stimulate NFκB through
a pathway involving NIK (NFκB-inducing kinase) and
p52 activation (Fig. 4). IL-4-secreting mast cells, eosino-
phils, basophils, and γδ T cells, are alternative inducers of
CD40 independent IgE synthesis [104, 128].

That IgE synthesis is tightly regulated is suggested by
the minute amounts of serum IgE as compared to IgG,
even in allergy or parasitic diseases. One regulatory
mechanism involves rapid capturing and subsequent
endocytosis by cells expressing a high affinity receptor
FceRI (e.g. on mast cells, basophils or eosinophils), and
to a lesser extent by the cells expressing a low affinity
receptor FceRII/CD23 (e.g. intraepithelial cells (IECs)
or B cells). Due to technical challenges the mechanistic
understanding of IgE synthesis regulation and Ig class
switch at cellular level is limited, especially in humans
[122, 129]. IgE switching may result in suboptimal poly-
adenylation of 3'-untranslated region of membrane IgE
resulting in less stable mRNA for membrane IgE (mIgE)
and lower expression of mIgE BCRs [129]. A plasma
membrane molecule specifically involved in the nega-
tive feedback regulation of IgE-CSR is CD23 (FcεRII),
which plays a role in in vivo sensitization induction
by cysteine proteases (e.g. HDM Der p 1) [130]. In the
presence of high levels of IgE or IgE-antigen complexes
membrane-bound CD23 (mCD23) suppresses the pro-
duction of IgE. Endogenous or exogenous (e.g. by Der
p 1) proteolytic cleavage of mCD23 renders CD23 solu-
ble (sCD23) which reinforces the production of IgE by
inhibiting binding of IgE to mCD23. In combination
with CD21 mediated signalling, sCD23 enhances B cell
proliferation and transformation into IgE + plasma
cells. Three cytokines may also be involved in down-
regulation of IgE synthesis. IL-21 represses IL-4 stimu-
lated GLTε synthesis in a STAT6-independent manner.
IFN-γ increases IgG1-CSR over IgE-CSR, whereas
TGF-β induces transcriptional factor ID2 (inhibitor of
DNA binding-2) that suppresses the E2A binding with
Iε promoter [131, 132].

Defining sensitization induction mechanisms by getting a
grip on tolerance

The current perception is that soluble antigens are pre-
dominantly absorbed by EC and suppress the immune
response, while particulate or aggregated antigens
are taken up by M cells and activate local or systemic
immune responses [39, 40]. Nonetheless, divergent
results exist on the contribution of these two main routes
of intestinal transport for food proteins and on influ-
ence of the protein physico-chemical properties [37, 41].
Linking entry route of food proteins and elicited immune
response deserves thus further investigation [133].

Oral tolerance to food proteins is the result of anergy
and/or deletion of antigen-responsive T<sub>h</sub>2 cells (high-
dose tolerance), and differentiation of regulatory T
(T<sub>reg</sub>) cells (low-dose tolerance) [134]. The best charac-
terized T<sub>reg</sub> cells in terms of induction of oral tolerance
are FOXP3<sup>+</sup> T<sub>reg</sub>. Among human FOXP3<sup>+</sup> cells, thymus-
derived CD25<sup>+</sup>FOXP3<sup>+</sup> natural T<sub>reg</sub> (nT<sub>reg</sub>) and periph-
eral induced CD25<sub>bright</sub>FOXP3<sup>+</sup>CD127<sup>-</sup> T<sub>reg</sub> (iT<sub>reg</sub>) exist.
Both employ several mechanisms to inhibit the activity of
allergen-specific T<sub>h</sub>2 cells and promote the synthesis of
allergen-specific IgG4 by B cells [135, 136].

Oral tolerance depends on the ability of CD103<sup>+</sup> DCs to induce FoxP3<sup>+</sup>α4β7<sup>hi</sup> CCR9<sup>hi</sup> iT<sub>reg</sub> in the MLN. The molecular conditions for tolerance to develop include conversion of vitamin A into retinoic acid (RA), upregulation of TGF-β, enzyme indoleamine 2,3-dioxygenase (IDO), intestinal Muc2, GM-CSF and co-signalling molecule 4-IBB [137–139]. This micro-environment facilitates gut-homing receptors CCR9 and α4β7 mediated migration of primed T<sub>reg</sub> cells from the MLN to the LP of the small intestine [140]. In the LP T<sub>reg</sub> cells are expanded and maintained by IL-10 producing CD103<sup>-</sup>CX3CR1<sup>+</sup> macrophages resulting in oral tolerance development [141, 142]. IL-10 and IL-27 producing CD11b<sup>+</sup>DCs enhance the secretion of IL-10 by T<sub>reg</sub> cells [143].

Induction of iT<sub>reg</sub> cells depends on a low density of
high-affinity ligands on DCs, sub-optimally activation
of TCR signalling, induction of CD28, and lack of the
CTLA-4 signalling. These cells synthesize IL-2 and their
transient CD25 expression coincides with CD25 expres-
sion by T<sub>h</sub>2 cells [144, 145]. Both in vitro and in vivo
iT<sub>reg</sub> cells require the presence of the GATA3 for main-
taining FOXP3 expression and accumulation in inflam-
matory sites. In contrast to T<sub>h</sub>2 cells, GATA3 expression
on iT<sub>reg</sub> cells regulation is IL-4/STAT6-independent but
controlled by IL-2/STAT5 [146]. Extracellular adeno-
sine reinforces CD25, CTLA-4 and FOXP3 expression
by increasing the cAMP concentration in naïve CD4<sup>+</sup>T
cells and, at the same time, preventing ZAP70 phospho-
rylation, and the AKT and ERK1/2 activation, which
inhibits stimulation, differentiation and proliferation of
the remaining T cell subsets [147].

Regulatory B cells ($B_{reg}$) with tolerogenic functions exist in human and mice. These $B_{reg}$ also express FoxP3 and produce IL-10, TGF-$\beta$ and IL-35 [148]. In humans with cow's milk allergy the frequency of specific IL-10-producing B cells is higher than in healthy individuals [149, 150]. Studies using mouse models of food allergy revealed that allergen-specific CX3CR1$^+$ $B_{reg}$ cells producing TGF-$\beta$ and IL-10 are expanded in response to high-dose allergen exposure [151].

## The currently most substantiated building blocks of the AOP for food sensitization

This proposed AOP used the one outlined for skin sensitization [12] and the AOP suggested for sensitization of the respiratory tract [13] to capture data that are available for proteins in general, and supplemented it with data on sensitizing food and food proteins in particular. Available mechanistic data on protein respiratory sensitization [14] were included to fill out gaps in the understanding of how proteins may affect cells, cell-cell interactions and tissue homeostasis. Therefore, caution should be considered since the skin and the respiratory tract may differ in view of food intake, tolerance, microbiome, presence of digestive enzymes, hormones etc.

Efforts to discern the mechanisms involved in food sensitization are motivated by the need for test methods and strategies to identify novel food with sensitizing potential. An AOP approach allows to transparently gather all available and relevant information following recognized criteria [11]. This assessment subsequently informs potential regulatory applications, which may include support for grouping and read-across of food and food proteins, identification of relevant and biologically plausible test methods, support for the development of integrated approaches to testing and assessment (IATA), identification or characterization of hazard, or quantitative risk assessment.

The potential regulatory applicability of any AOP is informed by the degree of confidence in the biological plausibility of each of the key event relations (KERs), and the identified KEs, and the empirical support for each of the KERs and the overall AOP [11].

### KER 1: MIE triggers innate responses and inflammation at epithelial level (KE 1)

The MIEs for food sensitization induction by food and food proteins are poorly understood. The knowledge and understanding acquired for proteins sensitizing the respiratory tract provides insight into MIEs potentially triggered by proteins: modification of tight junctions (MIE-1), receptor-mediated effects (MIE-2) (e.g. PAR and

TLR signalling) and endocytosis facilitating modification of intracellular processes (MIE-3) [14].

The scarce data on food proteins suggest that, in analogy with respiratory sensitizing proteins, cellular danger signals, induction of oxidative stress and pro-inflammatory cytokines and chemokines are involved in sensitization induction. The proposed MIEs for food proteins include modification of tight junctions (MIE-1) by proteolytic (e.g. kiwi Act d 1) but also non-proteolytic (e.g. gluten gliadin and peanut Ara h 2) allergens, receptor mediated induction of inflammation (MIE-2) (e.g. CD23 mediated) and unspecific endocytosis with impact on intracellular events (MIE-3). The proposed MIE-2 for food proteins involves CD23-mediated uptake by the exposed cells. Whether this event occurs also during sensitization induction by food proteins and not only during elicitation remains to be substantiated. Alternative MIE-2 mechanisms for proteolytic food allergens (e.g. kiwi Act d 1) may exist. CD23 was reported to play a role in sensitization induction in the respiratory tract, but not as a carrier for endocytosis. HDM Der p 1 and Der f 1, both cysteine proteases, release CD23 from the membrane and increase the concentration of sCD23, resulting in a disturbance of the negative control of IgE production [130]. A role of PAR-2 in receptor mediated induction of sensitization by proteolytic allergens is not established yet. However, an analogy with Der p 1 and Der f 1 cannot be excluded [24].

The causal relation between these MIEs and the induction of ROS, $T_h2$-driving cytokines (e.g. IL-33, TSLP and IL-25) and eventually inflammation is well established. Especially epithelial IL-33 is a major player both in the induction of sensitization, and the activation of e.g. DCs, basophils, mast cells and eosinophils [152]. IL-33 activates signalling pathways (e.g. ERK, MAPKs, NF-$\kappa$B) with relevance for inflammation and $T_h2$ polarization. TSLP inhibits IL-12 secretion while stimulating OX40L co-signalling on DCs. The presence of IL-25R on $CD4^+$ T cells suggests that IL-25 exerts a direct effect on these cells [117].

### KER 2: MIE 2–3 trigger innate responses and inflammation at DC level (KE 2)

Except MIE-1 (tight-junction modification), the identified MIEs may also have relevance for DCs since these cells share the innate mechanisms (e.g. PRRs, TLRs) reacting with e.g. PAMPs and DAMPs.

Differences between MIE-2 (receptor-mediated) effects on ECs (expressing PAR-1, 2, 3 and 4) and DCs (expressing primarily PAR-1) may exist. In vitro studies using human primary ECs and cell lines revealed that the cysteine protease Der p 1 activates PAR-2 but inactivates PAR-1 [153]. Since PAR-1 is the dominant PAR on
the surface of DCs, it may be anticipated that DC activa-
tion is reduced by allergens that engage PAR-1. While it
remains to be substantiated mechanistically, the available
data suggest that e.g. HDM may directly suppress T<sub>h</sub>1
driving DC responses [82].

In vitro and in vivo data underscore the importance of
IL-33 for proper activation of DCs as determined by e.g.
increased OX40L expression. Allergenic proteins in the
presence of IL-33 drive activated DCs to release IL-1β,
IL-6 and IL-10, with IL-1β known to be involved in T<sub>h</sub>2
cell stimulation and antibody production [82, 86].

KER 3: KE 1–2 drive DC migration (KE 3)

The ECs (IL-33, TSLP, IL-25), OX40L⁺ DCs (IL-1β, IL-6 and IL-10) and eosinophils (EPO) derived signals constitute an allergen-induced inflammatory microenvironment that triggers DCs maturation and migration.

Among the currently identified DC subsets,
CD103⁺CD11b⁻CX3CR1⁻ and CD103⁺CD11b⁺CX3CR1⁻
DCs appear to be the most relevant for sensitization induc-
tion. Informed by changes in the microenvironment at
epithelial level, CD103⁺ DCs express chemokine recep-
tors facilitating sampling of protein from the lumen (e.g.
CX3CR1), MHC II, co-stimulatory molecules (e.g. CD54,
CD80, CD86 and OX40L)) as well as receptors that are
required for migration to the LP and MNL (e.g. CCR7 and
CXCR4).

Especially CD103⁺MHCII⁺CD86⁺ DCs expressing
CCR7 seem to be important for sensitization induction.

KER 4: KE 3 leads to T and B cell activation in the lymphoid
organs (KE 4–5)

It is well documented that epithelial TSLP, IL-25 and
IL-33 play an important role also in T<sub>h</sub>2 polarization, by
inhibiting IL-12 secretion while stimulating OX40L co-
signalling on DCs (TSLP) and IL-33R mediated activa-
tion of DCs and CD4<sup>+</sup> T cells (IL-33). The mechanism
by which IL-25 contributes to T<sub>h</sub>2 polarization is not
clear, but the presence of IL-25R on CD4<sup>+</sup> T cells sug-
gests that IL-25 exerts a direct effect on these cells [117]
[82, 118].

Polarization of the T cell response towards T<sub>h</sub>2 requires GATA3 which promotes the expression of IL-4, IL-5 and IL-13 while inhibiting T-bet, IFN-γ and IL-12Rβ2 [110, 111]. Furthermore, activation of autocrine IL-2 signalling drives the establishment of the IL-4 signalling pathway by an IL-4 independent induction of the IL-4R subunit and formation of a functional IL-4 type I receptor [113].

Proper T<sub>h</sub>2 activation requires the expression by T cells
of CD28, OX40 (CD134) and CTLA-4 (CD152) and bind-
ing of these marker proteins to CD80, CD86 and OX40L
on the surface of the activated DCs [104, 105]. In this
context, CD86 seems to be most relevant for induction

of sensitization [106]. Indeed, CD28–CD86 interaction
relates to T<sub>h</sub>2 cell responses, whereas CD28–CD80 inter-
action favours T<sub>h</sub>1 cells responses [108]. It is suggested
that the allergenic potential of proteins is defined by their
capacity to affect the kinetics of the CD86/CD80 expres-
sion on DCs and CD28/CTLA-4 expression on T cells, in
the context of MHCII-peptide-TCR interaction.

The ultimate marker for sensitization to protein is the
production of specific IgE. The CD19/CD81/CD21 com-
plex on B-cells is important CD81 playing a crucial role
in B-cell/T-cell communication through the MHCII-
TCR. Interaction between T<sub>h</sub>2 cells and activated B cells
requires functional CD86–CD28 and CD40–CD40L
interactions [125].

IL-4 appears to be sufficient for Ig CSR to IgE to occur.
This cytokine may originate from activated T<sub>h</sub>2 cells
(CD40 dependent) or mast cells, eosinophils, basophils
and γδ T cells (CD40 independent). CD40 signalling
in combination with IL-4 and IL-13 activates the syn-
thesis of GLTε [126–128]. CD40 independent mecha-
nisms involve the interaction between BAFF and APRIL
on DCs, and BAFFR and TACI on the surface of B cells
[122–124]. This interaction stimulates NFκB signal-
ling through a pathway involving NIK (NFκB-inducing
kinase) and p52 activation [104, 128].

Tolerance

Developing AOPs for sensitization induction by foods
and food proteins requires sufficient understanding of
the mechanisms behind tolerance development.

Overall, tolerance induction seems to be the result of CD103⁺ DCs mediated stimulation of FoxP3⁺α4β7⁺hi CCR9⁺hi iTₜreg in the context of complex RA-rich environment in the MLN. Establishment of iTₜreg in the LP requires IL-10 from CD103⁻CX3CR1⁺ macrophages.

Despite iT<sub>reg</sub> cells believed to be involved in tolerance
development, food antigen-specific iT<sub>reg</sub> cells have been
detected in both allergic patients and healthy people. It is
hypothesized that the equilibrium between allergen-spe-
cific T<sub>h</sub>2 cells and iT<sub>reg</sub> cells, which recognize the same
epitope, predisposes to an allergic response (T<sub>h</sub>2) or a
healthy tolerogenic response [154].

In contrast to iT<sub>reg</sub>, nT<sub>reg</sub> cells constitutively express CTLA-4 and are believed to limit the access to and activation of naïve CD4<sup>+</sup> T cells by aggregation with DCs, by IL-10 and TGF-β mediated inhibition of MHC-II expression on DCs, and effector functions and migration of activated T and B cells, while promoting iT<sub>reg</sub> cell differentiation [154].

Although the mechanisms underlying these effects
remain unclear, the identification of new B<sub>reg</sub> subsets and
functions encourages intensifying the research into their
share in tolerization versus sensitization to food proteins.
Conclusion

This manuscript has collected, structured and evaluated
molecular and cellular information on protein sensitiza-
tion in general, and food sensitization in particular with
the aim to build AOPs for food sensitization. Analysis
revealed several KEs and biomarkers (Fig. 1) that may
have potential use in testing and assessment of proteins
for their sensitizing potential. In the future, this may
help to identify a number of methods, each addressing
a specific KE, that provide information about the food
allergenic potential of new proteins. When applied in
the context of an integrated strategy these methods may
reduce, if not replace, current animal testing approaches.

The proposed AOPs will be shared at the www.aopwiki.org platform to expand the mechanistic data, improve the confidence in each of the proposed KEs and KERs, and allow for the identification of new, or refinement of established, KEs and KERs.

Abbreviations

Ag: antigen; AOP: adverse outcome pathway; B<sub>reg</sub>: regulatory B cells; CLRs: c-type lectin receptors; CSR: class-switch recombination; DAMPs: danger-associated molecular patterns; DC: dendritic cell; EC: epithelial cell; GALT: gut-associated lymphoid tissue; GI: gastro-intestinal; HA: hyaluronic acid; HDM: house dust mite; HWP: hydrolysed wheat proteins; IEL: intraepithelial lymphocytes; IgE: immunoglobulin E; IL: interleukin; ILC: innate lymphoid cells; KE: key event; KER: key event relation; KO: knock out; LP: lamina propria; MC: mast cells; Mφ: macrophages; MIE: molecular initiating event; MLN: mesenteric lymph node; MOA: mode of action; MW: molecular weight; NKT: natural killer cells; OECD: Organisation for Economic Cooperation and Development; OVA: ovalbumin; PAMPS: pathogen-associated molecular patterns; pDC: plasmacytoid dendritic cell; PRR: pattern recognition receptor; ROS: radical oxygen species; TCR: T cell receptor; TLR: toll-like receptor; T<sub>reg</sub>: regulatory T cells.

Authors' contributions

ELR and KV wrote the background section; ELR wrote the general introduc-
tion on AOP for food sensitization; KV, YV, CL and SD wrote the bioavailability
section; ELR and HW wrote the MIE section; EM, JS, LW and RP wrote the
cellular innate event section (KE 1–3); ESS, WB, JB and LW wrote the initiation
and amplification of specific responses section (KE 5–6); DLO and EM wrote
the tolerance section; LW, ESS, JB prepared the figures; EK and GL reviewed the
manuscript; JB and ELR wrote the discussion and conclusions and coordi-
nated the drafting of the manuscript. All authors read and approved the final
manuscript.

Author details

¹TNO, PO Box 360, 3700 AJ Zeist, The Netherlands. ²University of Warmia and Mazury, Olsztyn, Poland. ³Instituto de Investigación en Ciencias de la Alimentación, Madrid, Spain. ⁴Utrecht University, Utrecht, The Netherlands.
⁵University of Evora, Evora, Portugal. ⁶Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland. ⁷Wageningen University and Research, Wageningen, The Netherlands. ⁸University Medical Center Utrecht, Utrecht, The Netherlands. ⁹DuPont Company, Newark, NJ, USA. ¹⁰INRA, Nantes, France. ¹¹Nestlé Ltd., Nestlé Research Center, Lausanne, Switzerland. ¹²Institute of Sciences of Food Production, National Research Council, Bari, Italy. ¹³3Rs Managing and Consulting ApS, Lyngby, Denmark.

Acknowledgements

The authors are all part of the COST Action FA1402 entitled: Improving Allergy Risk Assessment Strategy for New Food Proteins (ImpARAS).

Competing interests

The authors declare that they have no competing interests.

Availability of data and material

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Funding

The research described in this manuscript was not funded.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Received: 20 January 2017 Accepted: 3 May 2017

Published online: 12 May 2017

References

1. Nwaru BI, Hickstein L, Panesar SS, Muraro A, Werfel T, Cardona V, Dubois AE, Halken S, Hoffmann-Sommergruber K, Poulsen LK, Roberts G, Van Ree R, Vlieg-Boerstra BJ, Sheikh A. EAACI food allergy and anaphylaxis guidelines group: the epidemiology of food allergy in Europe: a systematic review and meta-analysis. Allergy. 2014;69(1):62–75.

2. Vetander M, Ly DH, Hakansson N, Lilja G, Nilsson C, Ostblom E, Wickman M, Bergstrom A. Recurrent reactions to food among children at paediatric emergency departments: epidemiology of allergic disease. Clin Exp Allergy. 2014;44(1):113–20.

3. Jansson SA, Heibert-Arnlind M, Middelveld RJ, Bengtsson UJ, Sundqvist AC, Kallstrom-Bengtsson I, Marklund B, Rentzos G, Akerstrom J, Ostblom E, Dahlen SE, Ahlstedt S. Health-related quality of life, assessed with a disease-specific questionnaire, in Swedish adults suffering from well-diagnosed food allergy to staple foods. Clin Transl Allergy 2013;3:21-7022-3-21 (eCollection 2013).

4. Mills ENC, Mackie AR, Burney P, Beyer K, Frewer L, Madsen C, Botjes E, Crevel RWR, Van Ree R. The prevalence, cost and basis of food allergy across Europe. Allergy Eur J Allergy Clin Immunol. 2007;62(7):717–22.

5. Sampson HA. Update on food allergy. J Allergy Clin Immunol. 2004;113(5):805–19.

6. Untersmayr E, Jensen-Jarolim E. Mechanisms of type I food allergy. Pharmacol Ther. 2006;112(3):787–98.

7. Masilamani M, Commins S, Shreffler W. Determinants of food allergy. Immunol Allergy Clin North Am. 2012;32(1):11–33.

8. Minekus M, Alminger M, Alvito P, Ballance S, Bohn T, Bourlieu C, Carriere F, Boutrou R, Corredig M, Dupont D, Dufour C, Egger L, Golding M, Karakaya S, Kirkhus B, Le Feunteun S, Lesmes U, Macierzanka A, Mackie A, Marze S, McClements DJ, Menard O, Recio I, Santos CN, Singh RP, Vegarud GE, Wickham MS, Weitschies W, Brodkorb A. A standardised static in vitro digestion method suitable for food—an international consensus. Food Funct. 2014;5(6):1113–24.

9. Bischoff S, Crowe SE. Gastrointestinal food allergy: new insights into pathophysiology and clinical perspectives. Gastroenterology. 2005;128(4):1089–113.

10. Akdis CA, Akdis M. Mechanisms of allergen-specific immunotherapy and immune tolerance to allergens. World Allergy Organ J 2015;8(1):17-015-0063-2 (eCollection 2015).

11. OECD: Users' Handbook supplement to the Guidance Document for developing and assessing Adverse Outcome Pathways. OECD Series on Adverse Outcome Pathways 2016.

12. OECD: The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins: Organisation for Economic Co-operation and Development; 2014.

13. https://aopwiki.org/wiki/index.php/Aop:39.

14. Wills-Karp M, Nathan A, Page K, Karp CL. New insights into innate immune mechanisms underlying allergenicity. Mucosal Immunol. 2010;3(2):104–10.

15. Menard S, Cerf-Bensussan N, Heyman M. Multiple facets of intestinal permeability and epithelial handling of dietary antigens. Mucosal Immunol. 2010;3(3):247–59.

16. Heyman M. Gut barrier dysfunction in food allergy. Eur J Gastroenterol Hepatol. 2005;17(12):1279–85.
17. Kondoh M, Takahashi A, Yagi K. Spiral progression in the development of absorption enhancers based on the biology of tight junctions. Adv Drug Deliv Rev. 2012;64(6):515-22.

18. Bevilacqua C, Montagnac G, Benmerah A, Candalh C, Brousse N, Cerf-Bensussan N, Perdue MH, Heyman M. Food allergens are protected from degradation during CD23-mediated transepithelial transport. Int Arch Allergy Immunol. 2004;135(2):108-16.

19. Shimizu M. Food-derived peptides and intestinal functions. BioFactors. 2004;21(1-4):43-7.

20. Sander GR, Cummins AG, Henshall T, Powell BC. Rapid disruption of intestinal barrier function by gliadin involves altered expression of apical junctional proteins. FEBS Lett. 2005;579(21):4851-5.

21. Song CH, Liu ZQ, Huang S, Zheng PY, Yang PC. Probiotics promote endocytic allergen degradation in gut epithelial cells. Biochem Biophys Res Commun. 2012;426(1):135-40.

22. Grozdanovic MM, Cavic M, Nesic A, Andjelkovic U, Akbari P, Smit JJ, Gavrovic-Jankulovic M. Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions. Biochim Biophys Acta. 2016;1860(3):516-26.

23. Price DB, Ackland ML, Burks W, Knight MI, Suphioglu C. Peanut allergens alter intestinal barrier permeability and tight junction localisation in Caco-2 cell cultures. Cell Physiol Biochem. 2014;33(6):1758-77.

24. Hong JH, Lee SI, Kim KE, Yong TS, Seo JT, Sohn MH, Shin DM. German cockroach extract activates protease-activated receptor 2 in human airway epithelial cells. J Allergy Clin Immunol. 2004;113(2):315-9.

25. Jacob C, Yang PC, Darmoul D, Amadesi S, Saito T, Cottrell GS, Coelho AM, Singh P, Grady EF, Perdue M, Bunnett NW. Mast cell tryptase controls paracellular permeability of the intestine. Role of protease-activated receptor 2 and beta-arrestins. J Biol Chem. 2005;280(36):31936-48.

26. Berin MC, Yang PC, Ciok L, Waserman S, Perdue MH. Role for IL-4 in macromolecular transport across human intestinal epithelium. Am J Physiol Cell Physiol. 1999;276(5):C1046-52.

27. Heller F, Florian P, Bojarski C, Richter J, Christ M, Hillenbrand B, Mankertz J, Gitter AH, Burgel N, Fromm M, Zeitz M, Fuss I, Strober W, Schulzke JD. Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions, apoptosis, and cell restitution. Gastroenterology. 2005;129(2):550-64.

28. Ferrier L, Mazelin L, Cenac N, Desreumaux P, Janin A, Emilie D, Colombel JF, Garcia-Villar R, Fioramonti J, Bueno L. Stress-induced disruption of colonic epithelial barrier: role of interferon-gamma and myosin light chain kinase in mice. Gastroenterology. 2003;125(3):795-804.

29. Willemsen LE, Hoetjes JP, van Deventer SJ, van Tol EA. Abrogation of IFN-gamma mediated epithelial barrier disruption by serine protease inhibition. Clin Exp Immunol. 2005;142(2):275-84.

30. Dhawan S, Hiemstra IH, Verseijden C, Hilbers FW, Te Velde AA, Willemsen LE, Stap J, den Haan JM, de Jonge WJ. Cholinergic receptor activation on epithelia protects against cytokine-induced barrier dysfunction. Acta Physiol (Oxf). 2015;213(4):846-59.

31. Brandt EB, Strait RT, Hershko D, Wang Q, Munteel EE, Scribner TA, Zimmermann N, Finkelman FD, Rothenberg ME. Mast cells are required for experimental oral allergen-induced diarrhea. J Clin Invest. 2003;112(11):1666-77.

32. Kurashima Y, Kiyono H. New era for mucosal mast cells: their roles in inflammation, allergic immune responses and adjuvant development. Exp Mol Med. 2014;46:e83.

33. Heyman M, Desjeux JF. Significance of intestinal food protein transport. J Pediatr Gastroenterol Nutr. 1992;15(1):48-57.

34. Caillard I, Tome D. Transport of beta-lactoglobulin and alpha-lactalbumin in enterocyte-like Caco-2 cells. Reprod Nutr Dev. 1995;35(2):179-88.

35. Bernasconi E, Fritsche R, Corthesy B. Specific effects of denaturation, hydrolysis and exposure to *Lactococcus lactis* on bovine beta-lactoglobulin transepithelial transport, antigenicity and allergenicity. Clin Exp Allergy. 2006;36(6):803-14.

36. Sewekow E, Bimczok D, Kahne T, Faber-Zuschratter H, Kessler LC, Seidel-Morgenstern A, Rothkotter HJ. The major soyabean allergen P34 resists proteolysis in vitro and is transported through intestinal epithelial cells by a caveolae-mediated mechanism. Br J Nutr. 2012;108(9):1603-11.

37. Rytkonen J, Valkonen KH, Virtanen V, Foxwell RA, Kyd JM, Cripps AW, Karttunen TJ. Enterocyte and M-cell transport of native and heat-denatured bovine beta-lactoglobulin: significance of heat denaturation. J Agric Food Chem. 2006;54(4):1500-7.

38. Zlotkowska D, Maddaloni M, Riccardi C, Walters N, Holderness K, Callis G, Rynda-Apple A, Pascual DW. Loss of sialic acid binding domain restricts protein sigma1 to enhance M cell-directed vaccination. PLoS ONE. 2012;7(4):e36182.

39. Brandtzaeg PE. Current understanding of gastrointestinal immunoregulation and its relation to food allergy. Ann NY Acad Sci. 2002;964:13-45.

40. Sampson HA. Food allergy. Part 1: immunopathogenesis and clinical disorders. J Allergy Clin Immunol. 1999;103(5 Pt 1):717-28.

41. Roth-Walter F, Berin MC, Arnaboldi P, Escalante CR, Dahan S, Rauch J, Jensen-Jarolim E, Mayer L. Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer's patches. Allergy. 2008;63(7):882-90.

42. Chambers SJ, Wickham MS, Regoli M, Bertelli E, Gunning PA, Nicoletti C. Rapid in vivo transport of proteins from digested allergen across pre-sensitized gut. Biochem Biophys Res Commun. 2004;325(4):1258-63.

43. Escrigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio R, Granucci F, Kraehenbuhl JP, Ricciardi-Castagnoli P. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol. 2001;2(4):361-7.

44. Rubas W, Grass GM. Gastrointestinal lymphatic absorption of peptides and proteins. Adv Drug Deliv Rev. 1991;7(1):15-69.

45. McDole JR, Wheeler LW, McDonald KG, Wang B, Konjufca V, Knoop KA, Newberry RD, Miller MJ. Goblet cells deliver luminal antigen to CD103⁺ dendritic cells in the small intestine. Nature. 2012;483(7389):345-9.

46. Dunkin D, Berin MC, Mayer L. Allergic sensitization can be induced via multiple physiologic routes in an adjuvant-dependent manner. J Allergy Clin Immunol 2011;128(6):1251-58.e2.

47. Birmingham NP, Parvataneni S, Hassan HM, Harkema J, Samineni S, Navuluri L, Kelly CJ, Gangur V. An adjuvant-free mouse model of tree nut allergy using hazelnut as a model tree nut. Int Arch Allergy Immunol. 2007;144(3):203-10.

48. Hsieh KY, Tsai CC, Wu CH, Lin RH. Epicutaneous exposure to protein antigen and food allergy. Clin Exp Allergy. 2003;33(8):1067-75.

49. Fox AT, Sasieni P, du Toit G, Syed H, Lack G. Household peanut consumption as a risk factor for the development of peanut allergy. J Allergy Clin Immunol. 2009;123(2):417-23.

50. Lack G, Fox D, Northstone K, Golding J. Avon longitudinal study of parents and children study team: factors associated with the development of peanut allergy in childhood. N Engl J Med. 2003;348(11):977-85.

51. Agrawal R, Woodfolk JA. Skin barrier defects in atopic dermatitis. Curr Allergy Asthma Rep 2014;15(4):433-014-0433-9.

52. Fukutomi Y, Taniguchi M, Nakamura H, Akiyama K. Epidemiological link between wheat allergy and exposure to hydrolyzed wheat protein in facial soap. Allergy. 2014;69(10):1405-11.

53. Beezhold DH, Sussman GL, Liss GM, Chang NS. Latex allergy can induce clinical reactions to specific foods. Clin Exp Allergy. 1996;26(4):416-22.

54. Bird JA, Burks AW. Food allergy and asthma. Prim Care Respir J. 2009;18(4):258-65.

55. Bischoff SC, Mayer JH, Manns MP. Allergy and the gut. Int Arch Allergy Immunol. 2000;121(4):270-83.

56. Armentia A, Diaz-Perales A, Castrodeza J, Duenas-Laita A, Palacin A, Fernandez S. Why can patients with baker's asthma tolerate wheat flour ingestion? Is wheat pollen allergy relevant? Allergol Immunopathol (Madr). 2009;37(4):203-4.

57. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell. 2010;140(6):805-20.

58. van Wijk F, Nierkens S, Hassing I, Feijen M, Koppelman SJ, de Jong GA, Pieters R, Knippels LM. The effect of the food matrix on in vivo immune responses to purified peanut allergens. Toxicol Sci. 2005;86(2):333-41.

59. Wavrin S, Bernard H, Wal JM, Adel-Patient K. Influence of the route of exposure and the matrix on the sensitisation potency of a major cows' milk allergen. Clin Transl Allergy 2015;5(1):3-015-0047-x (eCollection 2015).
60. Moghaddam AE, Hillson WR, Noti M, Gartlan KH, Johnson S, Thomas B, Artis D, Sattentau QJ. Dry roasting enhances peanut-induced allergic sensitization across mucosal and cutaneous routes in mice. *J Allergy Clin Immunol.* 2014;134(6):1453–6.

61. Hilmenyuk T, Bellinghausen I, Heydenreich B, Ilchmann A, Toda M, Grabbe S, Saloga J. Effects of glycation of the model food allergen ovalbumin on antigen uptake and presentation by human dendritic cells. *Immunology* 2010;129(3):437–45.

62. Salazar F, Sewell HF, Shakib F, Ghaemmaghami AM. The role of lectins in allergic sensitization and allergic disease. *J Allergy Clin Immunol.* 2013;132(1):27–36.

63. Lilly LM, Gessner MA, Dunaway CW, Metz AE, Schwiebert L, Weaver CT, Brown GD, Steele C. The beta-glucan receptor dectin-1 promotes lung immunopathology during fungal allergy via IL-22. *J Immunol.* 2012;189(7):3653–60.

64. Iliev ID, Funari VA, Taylor KD, Nguyen Q, Reyes CN, Strom SP, Brown J, Becker CA, Fleshner PR, Dubinsky M, Rotter JI, Wang HL, McGovern DP, Brown GD, Underhill DM. Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. *Science.* 2012;336(6086):1314–7.

65. Barrett NA, Rahman OM, Fernandez JM, Parsons MW, Xing W, Austen KF, Kanaoka Y. Dectin-2 mediates Th2 immunity through the generation of cysteinyl leukotrienes. *J Exp Med.* 2011;208(3):593–604.

66. Clarke DL, Davis NH, Campion CL, Foster ML, Heasman SC, Lewis AR, Anderson IK, Corkill DJ, Sleeman MA, May RD, Robinson MJ. Dectin-2 sensing of house dust mite is critical for the initiation of airway inflammation. *Mucosal Immunol.* 2014;7(3):558–67.

67. Parsons MW, Li L, Wallace AM, Lee MJ, Katz HR, Fernandez JM, Saijo S, Iwakura Y, Austen KF, Kanaoka Y, Barrett NA. Dectin-2 regulates the effector phase of house dust mite-elicited pulmonary inflammation independently from its role in sensitization. *J Immunol.* 2014;192(4):1361–71.

68. Kamalakannan M, Chang LM, Grishina G, Sampson HA, Masilamani M. Identification and characterization of DC-SIGN-binding glycoproteins in allergenic foods. *Allergy.* 2016;71(8):1145–55.

69. Publin M, Eiwegger T, Breiteneder H. Do lipids influence the allergic sensitization process? *J Allergy Clin Immunol.* 2014;134(3):521–9.

70. Guillon B, Bernard H, Drumare MF, Hazebrouck S, Adel-Patient K. Heat processing of peanut seed enhances the sensitization potential of the major peanut allergen Ara h 6. *Mol Nutr Food Res.* 2016;60(12):2722–35.

71. Mowat AM, Agace WW. Regional specialization within the intestinal immune system. *Nat Rev Immunol.* 2014;14(10):667–85.

72. Corsini E, Galbiati V, Nikitovic D, Tsatsakis AM. Role of oxidative stress in chemical allergens induced skin cells activation. *Food Chem Toxicol.* 2013;61:74–81.

73. Runswick S, Mitchell T, Davies P, Robinson C, Garrod DR. Pollen proteolytic enzymes degrade tight junctions. *Respirology.* 2007;12(6):834–42.

74. Brandt EB, Mingler MK, Stevenson MD, Wang N, Khurana Hershey GK, Whitsett JA, Rothenberg ME. Surfactant protein D alters allergic lung responses in mice and human subjects. *J Allergy Clin Immunol* 2008;121(5):1140–7.e2.

75. Boldogh I, Bacsi A, Choudhury BK, Dharajiya N, Alam R, Hazra TK, Mitra S, Goldblum RM, Sur S. ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation. *J Clin Invest.* 2005;115(8):2169–79.

76. Janssens S, Beyaert R. A universal role for MyD88 in TLR/IL-1R-mediated signaling. *Trends Biochem Sci.* 2002;27(9):474–82.

77. Roggen EL. In vitro approaches for detection of chemical sensitization. *Basic Clin Pharmacol Toxicol.* 2014;115(1):32–40.

78. Gregory LG, Lloyd CM. Orchestrating house dust mite-associated allergy in the lung. *Trends Immunol.* 2011;32(9):402–11.

79. Ather JL, Hodgkins SR, Janssen-Heininger YM, Poynter ME. Airway epithelial NF-kappaB activation promotes allergic sensitization to an innocuous inhaled antigen. *Am J Respir Cell Mol Biol.* 2011;44(5):631–8.

80. Hartwig C, Tschernig T, Mazzega M, Braun A, Neumann D. Endogenous IL-18 in experimentally induced asthma affects cytokine serum levels but is irrelevant for clinical symptoms. *Cytokine* 2008;42(3):298–305.

81. Kool M, Willart MA, van Nimwegen M, Bergen I, Pouliot P, Virchow JC, Rogers N, Osorio F, Reis e Sousa C, Hammad H, Lambrecht BN. An unexpected role for uric acid as an inducer of T helper 2 cell immunity to inhaled antigens and inflammatory mediator of allergic asthma. *Immunity* 2011;34(4):527–40.

82. Chu DK, Llop-Guevara A, Walker TD, Flader K, Goncharova S, Boudreau JE, Moore CL, Seunghyun In T, Waserman S, Coyle AJ, Kolbeck R, Humbles AA, Jordana M. IL-33, but not thymic stromal lymphopoietin or IL-25, is central to mite and peanut allergic sensitization. *J Allergy Clin Immunol* 2013;131(1):187-200.e1-8.

83. Junker Y, Zeissig S, Kim SJ, Barisani D, Wieser H, Leffler DA, Zevallos V, Libermann TA, Dillon S, Freitag TL, Kelly CP, Schuppan D. Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4. *J Exp Med.* 2012;209(13):2395–408.

84. Kong J, Chalcraft K, Mandur TS, Jimenez-Saiz R, Walker TD, Goncharova S, Gordon ME, Naji L, Flader K, Larche M, Chu DK, Waserman S, McCarry B, Jordana M. Comprehensive metabolomics identifies the alarmin uric acid as a critical signal for the induction of peanut allergy. *Allergy.* 2015;70(5):495–505.

85. Tordesillas L, Cuesta-Herranz J, Gonzalez-Munoz M, Pacios LF, Compes E, Garcia-Carrasco B, Sanchez-Monge R, Salcedo G, Diaz-Perales A. T-cell epitopes of the major peach allergen, Pru p 3: identification and differential T-cell response of peach-allergic and non-allergic subjects. *Mol Immunol.* 2009;46(4):722–8.

86. Tordesillas L, Gomez-Casado C, Garrido-Arandia M, Murua-Garcia A, Palacin A, Varela J, Konieczna P, Cuesta-Herranz J, Akdis CA, O'Mahony L, Diaz-Perales A. Transport of Pru p 3 across gastrointestinal epithelium—an essential step towards the induction of food allergy? *Clin Exp Allergy.* 2013;43(12):1374–83.

87. Tan JK, O'Neill HC. Maturation requirements for dendritic cells in T cell stimulation leading to tolerance versus immunity. *J Leukoc Biol.* 2005;78(2):319–24.

88. Cherry WB, Yoon J, Bartemes KR, Iijima K, Kita H. A novel IL-1 family cytokine, IL-33, potently activates human eosinophils. *J Allergy Clin Immunol.* 2008;121(6):1484–90.

89. Chu DK, Jimenez-Saiz R, Verschoor CP, Walker TD, Goncharova S, Llop-Guevara A, Shen P, Gordon ME, Barra NG, Bassett JD, Kong J, Fattouh R, McCoy KD, Bowdish DM, Erjefalt JS, Pabst O, Humbles AA, Kolbeck R, Waserman S, Jordana M. Indigenous enteric eosinophils control DCs to initiate a primary Th2 immune response in vivo. *J Exp Med.* 2014;211(8):1657–72.

90. Varol C, Vallon-Eberhard A, Elinav E, Aychek T, Shapira Y, Luche H, Fehling HJ, Hardt WD, Shakhar G, Jung S. Intestinal lamina propria dendritic cell subsets have different origin and functions. *Immunity.* 2009;31(3):502–12.

91. Scott CL, Bain CC, Wright PB, Sichien D, Kotarsky K, Persson EK, Luda K, Guilliams M, Lambrecht BN, Agace WW, Milling SW, Mowat AM. CCR2(+)-CD103(−) intestinal dendritic cells develop from DC-committed precursors and induce interleukin-17 production by T cells. *Mucosal Immunol.* 2015;8(2):327–39.

92. Smit JJ, Bol-Schoenmakers M, Hassing I, Fiechter D, Boon L, Bleumink R, Pieters RH. The role of intestinal dendritic cells subsets in the establishment of food allergy. *Clin Exp Allergy.* 2011;41(6):890–8.

93. de Kivit S, van Hoffen E, Korthagen N, Garssen J, Willemsen LE. Apical TLR ligation of intestinal epithelial cells drives a Th1-polarized regulatory or inflammatory type effector response in vitro. *Immunobiology.* 2011;216(4):518–27.

94. Bashir ME, Louie S, Shi HN, Nagler-Anderson C. Toll-like receptor 4 signaling by intestinal microbes influences susceptibility to food allergy. *J Immunol.* 2004;172(11):6978–87.

95. Bol-Schoenmakers M, Braber S, Akbari P, de Graaff P, van Roest M, Kruijssen L, Smit JJ, van Esch BC, Jeurink PV, Garssen J, Fink-Gremmels J, Pieters RH. The mycotoxin deoxynivalenol facilitates allergic sensitization to whey in mice. *Mucosal Immunol.* 2016;9(6):1477–86.

96. Pabst O, Mowat AM. Oral tolerance to food protein. *Mucosal Immunol.* 2012;5(3):232–9.

97. Worbs T, Bode U, Yan S, Hoffmann MW, Hintzen G, Bernhardt G, Forster R, Pabst O. Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells. *J Exp Med.* 2006;203(3):519–27.

98. Laffont S, Siddiqui KR, Powrie F. Intestinal inflammation abrogates the tolerogenic properties of MLN CD103⁺ dendritic cells. *Eur J Immunol.* 2010;40(7):1877–83.

99. Kumar S, Dwivedi PD, Das M, Tripathi A. Macrophages in food allergy: an enigma. *Mol Immunol.* 2013;56(4):612–8.
100. Bain CC, Mowat AM. Macrophages in intestinal homeostasis and inflam-
mation. Immunol Rev. 2014;260(1):102-17.

101. Cerovic V, Bain CC, Mowat AM, Milling SW. Intestinal macrophages and dendritic cells: what's the difference? Trends Immunol. 2014;35(6):270-7.

102. Smeets RL, Fleuren WW, He X, Vink PM, Wijnands F, Gorecka M, Klop H, Bauerschmidt S, Garitsen A, Koenen HJ, Joosten I, Boots AM, Alkema W. Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling. BMC Immunol 2012;13:12-2172-13-12.

103. Acuto O, Michel F. CD28-mediated co-stimulation: a quantitative support for TCR signalling. Nat Rev Immunol. 2003;3(12):939-51.

104. Kumar S, Verma AK, Das M, Dwivedi PD. A molecular insight of CTLA-4 in food allergy. Immunol Lett. 2013;149(1-2):101-9.

105. Nasta F, Corinti S, Bonura A, Colombo P, Di Felice G, Pioli C. CTLA-4 regulates allergen response by modulating GATA-3 protein level per cell. Immunology. 2007;121(1):62-70.

106. van Wijk F, Nierkens S, de Jong W, Wehrens EJ, Boon L, van Kooten P, Knippels LM, Pieters R. The CD28/CTLA-4-B7 signaling pathway is involved in both allergic sensitization and tolerance induction to orally administered peanut proteins. J Immunol. 2007;178(11):6894-900.

107. Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol. 2012;12(3):180-90.

108. Kuchroo VK, Das MP, Brown JA, Ranger AM, Zamvil SS, Sobel RA, Weiner HL, Nabavi N, Glimcher LH. B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell. 1995;80(5):707-18.

109. Zhu J, Jankovic D, Oler AJ, Wei G, Sharma S, Hu G, Guo L, Yagi R, Yamane H, Punkosdy G, Feigenbaum L, Zhao K, Paul WE. The transcription factor T-bet is induced by multiple pathways and prevents an endogenous Th2 cell program during Th1 cell responses. Immunity. 2012;37(4):660-73.

110. Wei G, Abraham BJ, Yagi R, Jothi R, Cui K, Sharma S, Narlikar L, Northrup DL, Tang Q, Paul WE, Zhu J, Zhao K. Genome-wide analyses of transcription factor GATA3-mediated gene regulation in distinct T cell types. Immunity. 2011;35(2):299-311.

111. Ho IC, Tai TS, Pai SY. GATA3 and the T-cell lineage: essential functions before and after T-helper-2-cell differentiation. Nat Rev Immunol. 2009;9(2):125-35.

112. Zhu J, Paul WE. Peripheral CD4⁺T-cell differentiation regulated by networks of cytokines and transcription factors. Immunol Rev. 2010;238(1):247-62.

113. Liao W, Lin JX, Leonard WJ. IL-2 family cytokines: new insights into the complex roles of IL-2 as a broad regulator of T helper cell differentiation. Curr Opin Immunol. 2011;23(5):598-604.

114. Cote-Sierra J, Foucas G, Guo L, Chiodetti L, Young HA, Hu-Li J, Zhu J, Paul WE. Interleukin 2 plays a central role in Th2 differentiation. Proc Natl Acad Sci USA. 2004;101(11):3880-5.

115. Chu DK, Mohammed-Ali Z, Jimenez-Saiz R, Walker TD, Goncharova S, Llop-Guevara A, Kong J, Gordon ME, Barra NG, Gillgrass AE, Van Seggelen H, Khan WI, Ashkar AA, Bramson JL, Humbles AA, Kolbeck R, Waserman S, Jordana M. T helper cell IL-4 drives intestinal Th2 priming to oral peanut antigen, under the control of OX40L and independent of innate-like lymphocytes. Mucosal Immunol. 2014;7(6):1395-404.

116. Croft M, So T, Duan W, Soroosh P. The significance of OX40 and OX40L to T-cell biology and immune disease. Immunol Rev. 2009;229(1):173-91.

117. Angkasekwinai P, Park H, Wang YH, Wang YH, Chang SH, Corry DB, Liu YJ, Zhu Z, Dong C. Interleukin 25 promotes the initiation of proallergic type 2 responses. J Exp Med. 2007;204(7):1509-17.

118. Zhao Q, Chen G. Role of IL-33 and its receptor in T cell-mediated autoimmune diseases. Biomed Res Int. 2014;2014:587376.

119. Maciolek JA, Pasternak JA, Wilson HL. Metabolism of activated T lym-
phocytes. Curr Opin Immunol. 2014;27:60-74.

120. Melnik BC. The potential mechanistic link between allergy and obesity development and infant formula feeding. Allergy Asthma Clin Immunol 2014;10(1):37-1492-10-37 (eCollection 2014).

121. Xu Z, Zan H, Pone EJ, Mai T, Casali P. Immunoglobulin class-switch DNA recombination: induction, targeting and beyond. Nat Rev Immunol. 2012;12(7):517-31.

122. Berkowska MA, Heeringa JJ, Hajdarbegovic E, van der Burg M, Thio HB, van Hagen PM, Boon L, Orfao A, van Dongen JJ, van Zelm MC. Human IgE(+). B cells are derived from T cell-dependent and T cell-independent pathways. J Allergy Clin Immunol. 2014;134(3):688-97.e6.

123. Xiong H, Dolpady J, Wabl M, Curotto de Lafaille MA, Lafaille JJ. Sequential class switching is required for the generation of high affinity IgE antibodies. J Exp Med. 2012;209(2):353-64.

124. Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol. 2014;14(3):141-53.

125. Vascotto F, Lankar D, Faure-Andre G, Vargas P, Diaz J, Le Roux D, Yuseff MI, Sibarita JB, Boes M, Raposo G, Mougneau E, Glaichenhaus N, Bonnerot C, Manoury B, Lennon-Dumenil AM. The actin-based motor protein myosin II regulates MHC class II trafficking and BCR-driven antigen presentation. J Cell Biol. 2007;176(7):1007-19.

126. van Ree R, Hummelshoj L, Plantinga M, Poulsen LK, Swindle E: Allergic sensitization: host-immune factors. Clin Transl Allergy 2014;4(1):12-7022-4-12.

127. Park SR, Zan H, Pal Z, Zhang J, Al-Qahtani A, Pone EJ, Xu Z, Mai T, Casali P. HoxC4 binds to the promoter of the cytidine deaminase AID gene to induce AID expression, class-switch DNA recombination and somatic hypermutation. Nat Immunol. 2009;10(5):540-50.

128. Geha RS, Jabara HH, Brodeur SR. The regulation of immunoglobulin E class-switch recombination. Nat Rev Immunol. 2003;3(9):721-32.

129. Wu LC, Scheerens H. Targeting IgE production in mice and humans. Curr Opin Immunol. 2014;31:8-15.

130. Takai T, Kato T, Ota M, Yasueda H, Kuhara T, Okumura K, Ogawa H. Recombinant Der p 1 and Der f 1 with in vitro enzymatic activity to cleave human CD23, CD25 and alpha1-antitrypsin, and in vivo IgE-eliciting activity in mice. Int Arch Allergy Immunol. 2005;137(3):194-200.

131. Gough L, Schulz O, Sewell HF, Shakib F. The cysteine protease activity of the major dust mite allergen Der p 1 selectively enhances the immunoglobulin E antibody response. J Exp Med. 1999;190(12):1897-902.

132. Mayer RJ, Bolognese RJ, Al-Mahdi N, Cook RM, Flamberg PL, Hansbury MJ, Khandekar S, Appelbaum E, Faller A, Marshall LA. Inhibition of CD23 processing correlates with inhibition of IL-4-stimulated IgE production in human PBL and hu-PBL-reconstituted SCID mice. Clin Exp Allergy. 2000;30(5):719-27.

133. Cubells-Baeza N, Verhoeckx KCM, Larre C, Denery-Papini S, Gavrovic-Jankulovic M, Diaz Perales A. Applicability of epithelial models in protein permeability/transport studies and food allergy. Drug Discovery Today: Disease Models. 2015;17-18:13-21.

134. Vickery BP, Burks AW. Immunotherapy in the treatment of food allergy: focus on oral tolerance. Curr Opin Allergy Clin Immunol. 2009;9(4):364-70.

135. Noval Rivas M, Burton OT, Wise P, Charbonnier LM, Georgiev P, Oettgen HC, Rachid R, Chatila TA. Regulatory T cell reprogramming toward a Th2-cell-like lineage impairs oral tolerance and promotes food allergy. Immunity. 2015;42(3):512-23.

136. Schmitt EG, Haribhai D, Williams JB, Aggarwal P, Jia S, Charbonnier LM, Yan K, Lorier R, Turner A, Ziegelbauer J, Georgiev P, Simpson P, Salzman NH, Hessner MJ, Broeckel U, Chatila TA, Williams CB. IL-10 produced by induced regulatory T cells (iTregs) controls colitis and pathogenic exiTregs during immunotherapy. J Immunol. 2012;189(12):5638-48.

137. Berin MC, Shreffler WG. Mechanisms underlying induction of tolerance to foods. Immunol Allergy Clin North Am. 2016;36(1):87-102.

138. Buyuktiryaki B, Sahiner UM, Girgin G, Birben E, Soyer OU, Cavkaytar O, Cetin C, Arik Yilmaz E, Yavuz ST, Kalayci O, Baydar T, Sackesen C. Low indoleamine 2,3-dioxygenase activity in persistent food allergy in children. Allergy. 2016;71(2):258-66.

139. Chinthrajah RS, Hernandez JD, Boyd SD, Galli SJ, Nadeau KC. Molecular and cellular mechanisms of food allergy and food tolerance. J Allergy Clin Immunol. 2016;137(4):984-97.

140. Raker VK, Domogalla MP, Steinbrink K. Tolerogenic dendritic cells for regulatory T cell induction in man. Front Immunol. 2015;6:569.

141. Scott CL, Aumeunier AM, Mowat AM. Intestinal CD103⁺ dendritic cells: master regulators of tolerance? Trends Immunol. 2011;32(9):412-9.

142. Hadis U, Wahl B, Schulz O, Hardtke-Wolenski M, Schippers A, Wagner N, Muller W, Sparwasser T, Forster R, Pabst O. Intestinal tolerance requires gut homing and expansion of FoxP3⁺ regulatory T cells in the lamina propria. Immunity. 2011;34(2):237-46.
143. Shiokawa A, Tanabe K, Tsuji NM, Sato R, Hachimura S. IL-10 and IL-27 producing dendritic cells capable of enhancing IL-10 production of T cells are induced in oral tolerance. Immunol Lett. 2009;125(1):7-14.

144. Schmitt EG, Williams CB. Generation and function of induced regulatory T cells. Front Immunol. 2013;4:152.

145. MaruYama T. TGF-beta-induced IκB-zeta controls Foxp3 gene expression. Biochem Biophys Res Commun. 2015;464(2):586-9.

146. Wohlfert EA, Grainger JR, Bouladoux N, Konkel JE, Oldenhove G, Ribeiro CH, Hall JA, Yagi R, Naik S, Bhairavabhotla R, Paul WE, Bosselut R, Wei G, Zhao K, Oukka M, Zhu J, Belkaid Y. GATA3 controls Foxp3(+) regulatory T cell fate during inflammation in mice. J Clin Investig. 2011;121(11):4503-15.

147. Linden J, Cekic C. Regulation of lymphocyte function by adenosine. Arterioscler Thromb Vasc Biol. 2012;32(9):2097-103.

148. van de Veen W, Stanic B, Wirz OF, Jansen K, Globinska A, Akdis M. Role of regulatory B cells in immune tolerance to allergens and beyond. J Allergy Clin Immunol. 2016;138(3):654-65.

149. Noh J, Lee JH, Noh G, Bang SY, Kim HS, Choi WS, Cho S, Lee SS. Characterisation of allergen-specific responses of IL-10-producing regulatory B cells (Br 1) in Cow Milk Allergy. Cell Immunol. 2010;264(2):143-9.

150. Noh G, Lee JH. Regulatory B cells and allergic diseases. Allergy Asthma Immunol Res. 2011;3(3):168-77.

151. Liu ZQ, Wu Y, Song JP, Liu X, Liu Z, Zheng PY, Yang PC. Tolerogenic CX3CR1⁺ B cells suppress food allergy-induced intestinal inflammation in mice. Allergy. 2013;68(10):1241-8.

152. Saluja R, Khan M, Church MK, Maurer M. The role of IL-33 and mast cells in allergy and inflammation. Clin Transl Allergy 2015;5:33-015-0076-5 (eCollection 2015).

153. Asokananthan N, Graham PT, Stewart DJ, Bakker AJ, Eidne KA, Thompson PJ, Stewart GA. House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease-activated receptor (PAR)-2 and inactivates PAR-1. J Immunol. 2002;169(8):4572-8.

154. Palomares O. The role of regulatory T cells in IgE-mediated food allergy. J Investig Allergol Clin Immunol 2013;23(6):371-82 (quiz 2 p preceding 382).

Submit your next manuscript to BioMed Central
and we will help you at every step:

* We accept pre-submission inquiries

* Our selector tool helps you to find the most relevant journal

* We provide round the clock customer support

* Convenient online submission

* Thorough peer review

* Inclusion in PubMed and all major indexing services

* Maximum visibility for your research

Submit your manuscript at
www.biomedcentral.com/submit

BioMed Central