Intestinal protein uptake and IgE-mediated food allergy

Food allergy is affecting 5 – 8% of young children and 2 – 4% of adults and seems to be increasing in prevalence. The cause of the increase in food allergy is largely unknown but proposed to be influenced by both environmental and lifestyle factors. Changes in intestinal barrier functions and increased uptake of dietary proteins have been suggested to have a great impact on food allergy. In this review, we aim to give an overview of the gastrointestinal digestion and intestinal barrier function and provide a more detailed description of intestinal protein uptake, including the various routes of epithelial transport, how it may be affected by both intrinsic and extrinsic factors, and the relation to food allergy. Further, we give an overview of in vitro , ex vivo and in vivo techniques available for evaluation of intestinal protein uptake and gut permeability in general. Proteins are digested by gastric, pancreatic and integral brush border enzymes in order to allow for sufficient nutritional uptake. Absorption and transport of dietary proteins across the epithelial layer is known to be dependent on the physicochemical properties of the proteins and their digestion fragments themselves, such as size, solubility and aggregation status. It is believed, that the greater an amount of intact protein or larger peptide fragments that is transported through the epithelial layer, and thus encountered by the mucosal immune system in the gut, the greater is the risk of inducing an adverse allergic response. Proteins may be absorbed across the epithelial barrier by means of various mechanisms, and studies have shown that a transcellular facilitated transport route unique for food allergic individuals are at play for transport of allergens, and that upon mediator release from mast cells an enhanced allergen transport via the paracellular route occurs. This is in contrast to healthy individuals where transcytosis through the enterocytes is the main route of protein uptake. Thus, knowledge on factors affecting intestinal barrier functions and methods for the determination of their impact on protein uptake may be useful in future allergenicity assessments and for development of future preventive and treatment strategies.

the Western countries during the last decades, and relate to changes in both environmental and lifestyle parameters, such as increased pollution, decreased physical activity, increased hygiene, increased use of drugs, increased psychological stress and changes in the diet (Granum et al., 2020;Lozano-Ojalvo et al., 2019b;Marshall, 2004;Platts-Mills, 2015).Why and how these external factors contribute to the increased prevalence of food allergy remains to be fully resolved, but may be attributed to a direct effect on the host or an indirect effect on the host triggered by changes in the microbiota composition (Platts-Mills, 2015;Reitsma et al., 2014).
As the gastrointestinal (GI) tract constitutes the largest surface of our body exposed to the external environment, it has a crucial role in not only digesting and absorbing the nutrients, but also in maintaining immune homeostasis to protect the body from adverse foreign matter (Farré et al., 2020).Hence, the GI tract has a delicate and pivotal role in inducing tolerance to harmless food components, commensals and selfantigens, while defending the body from pathogenic attack, and upon failure people may become sensitised to the otherwise harmless dietary proteins and develop food allergy (Yu et al., 2016).
Environmental and lifestyle factors, suggested to play a role in the increased prevalence of food allergy, may have a profound role in shaping the GI health; impacting on the digestion of the dietary proteins, the transport of the proteins and their peptide fragments across the GI mucosa and the immune responses directed against the proteins and their digestive fragments, and thereby impact on the faith of the dietary proteins in the GI tract (Farré et al., 2020;Gilani et al., 2005).However, the effect that external factors have on the GI tract, digestion, uptake and immune regulation as well as their complex interplay, is only poorly understood, and may either be a direct effect or an indirect effect exerted through the modulation of the gut microbiota (Alemao et al., 2021;Farré et al., 2020;Platts-Mills, 2015;Reitsma et al., 2014) (Fig. 1).
Especially the uptake of dietary proteins and their transport through the epithelial layer is suggested to play a pivotal role in both sensitisation and elicitation of food allergy, where an increased intestinal permeability has been shown to correlate with food allergy (Järvinen et al., 2013).However, whether increased intestinal permeability predispose people to become allergic or whether it is a result of having food allergy remains to be fully resolved (Järvinen et al., 2013;Perrier & Corthésy, 2011).Yet, understanding the fate of dietary proteins from the moment they reach the mouth until they are taken up and presented to the immune system in the GI tract is fundamental in food allergy, and is a necessity to develop new and improved strategies for preventing and treating food allergy targeting the mechanisms responsible for the faith of the dietary protein in the GI tract.Thus, with this review we provide an overview of digestion and uptake of dietary proteins in the GI tract, how these are affected by external factors, and the impact on food allergy.

Food allergy
As an immune-mediated disease, food allergy can be divided into immunoglobulin E (IgE)-mediated and non-IgE-mediated food allergies, which are caused by distinct underlying mechanisms (Abonia & Rothenberg, 2012;Boyce et al., 2010;Caubet & Nowak-Węgrzyn, 2011;Cetinkaya et al., 2021;Cianferoni, 2020).IgE-mediated food allergy, which is responsible for the majority of food allergic reactions (Sicherer & Sampson, 2018), is characterised by the presence of IgE towards specific proteins in food, so-called allergens (Broekman et al., 2015), and is the focus of this review.
The exact prevalence of food allergy is unknown, due to lack of worldwide epidemiologic studies and the great variation between different countries and study methodologies (Dunlop & Keet, 2018).Yet, the prevalence of food allergy is estimated to 5-8 % in young children and 2-4 % in adults (Sicherer & Sampson, 2014, 2018), and different epidemiological studies from various parts of the world have demonstrated food allergy to be an increasing problem in at least Australia (Loke et al., 2016;Osborne et al., 2011;Sasaki et al., 2018), Europe and the United States (Kotz et al., 2011;McWilliam et al., 2015).The most common food allergies arise from peanut, tree nuts, cow's milk, hen's egg, fish, shellfish, wheat, soy and seeds (Sicherer & Sampson, 2018), where the likelihood of resolution of the allergy depends on the specific culprit food (Sicherer & Sampson, 2018), as well as which parts of the allergens, the so-called epitopes, that the IgE reacts toward (Kim et al., 2011;Peters et al., 2014).
To predict potential allergenicity of new or modified foods and to develop new and improved strategies for food allergy prevention and treatment, great focus has been directed on identifying features that confer dietary proteins their allergenicity.In the search for answering the question "what makes a food protein a food allergen?"several physicochemical characteristics have been suggested, such as a size between 10 and 70 kDa, high water-solubility, and resistance towards enzymatic degradation and heat treatment (Bannon, 2004;Costa et al., 2022a,b).
As no cure exists for food allergy, and as only one drug has been approved for treatment of specifically peanut allergy (Chaplin, 2021), to date, the most viable management option for food allergy is strict avoidance of the offending food.However, this approach can be challenging and may have a huge impact on the quality of life for food allergy sufferers and their families due to the constant fear of accidental exposure and severe adverse reactions (DunnGalvin et al., 2017;Lau et al., 2014;Muraro et al., 2014).Therefore, investigating factors impacting on food allergy are crucial to increase our knowledge and assure improved strategies for prevention and treatment in the future.
The pathogenesis of IgE-mediated food allergy is composed of two phases; a sensitisation phase and an elicitation phase (Fig. 2).To evoke an immune reaction, allergens must access our body and immune system.It is well-accepted that food allergy may develop through both oral and skin route of exposure and to a lesser extent through the respiratory route (Asero & Antonicelli, 2011;Dunkin et al., 2011;Ramirez & Bahna, 2009;Tordesillas et al., 2014).Hence, sensitisation may occur after ingestion and involves a primary exposure to the dietary protein or their digested peptide fragments that are transported though the gut lumen for uptake and presentation by innate immune cells to cells of the adaptive immune system leading to a cascade of events resulting in the production of specific IgE by plasma cells, as outlined in Fig. 2. Elicitation involves re-exposure to the culprit dietary protein or a cross- reacting protein, which binds and cross-links FcԑRI-bound IgEs on the surface of mast cells or basophils, leading to a degranulation response and the release of an array of mediators, such as histamine, proteases, cytokines, leukotrienes and prostaglandins, which are responsible for the symptoms characterising IgE-mediated food allergy (Stone et al., 2010) (Fig. 2).Symptoms can occur within minutes to hours and involve one or more organs.The most common site of food allergy manifestation is the skin (urticaria) followed by the gastrointestinal tract (vomiting, diarrhoea, abdominal pain) and the respiratory tract (wheezing, coughing) (Ahrens et al., 2012).In severe cases several organs are involved, and may result in the systemic reaction anaphylaxis which may be fatal (Fiocchi et al., 2010).

The gastrointestinal tract
Even though the GI tract is found inside the body, the GI epithelial layer is the largest surface of our body exposed to the outside environment.The major part of the GI tract is the around 20 µm thick epithelial cell monolayer.Underneath the epithelial cell layer is the loose connective tissue, lamina propria, containing blood vessels and the mucosa-  (Divekar & Kita, 2015;Hussain et al., 2018;Tordesillas et al., 2017;Yu et al., 2016).The differentiated Th2 cell further activates a naïve B cell via CD40L-CD40 ligation which leads to proliferation and differentiation of the naïve B cell into plasma cells secreting high amounts of allergen-specific IgE (Stone et al., 2010).Specific IgE binds to their high affinity receptors FcԑRI on mast cells resident in tissue or basophils present in the blood.This phase is designated as sensitisation (Sutton & Gould, 1993).Upon re-exposure to allergens, cross-linking of FcԑRI-bound IgE on the effector cells leads to mediator release, which gives rise to the symptoms characterising food allergy, and is defined as the elicitation phase (Stone et al., 2010).Figure created with BioRender.com.Fig. 3.The gastrointestinal tract and digestion of dietary proteins.Degradation of dietary proteins starts in the mouth and continues in the stomach where they are exposed to gastric proteases.Subsequently, degradation continues in the small intestine with pancreatic proteases and peptidases as well as brush border peptidases.The majority of the dietary proteins are degraded to free amino acids and di-and tripeptides readily available for absorption through the intestinal mucosa.Figure created with BioRender.com.associated lymphoid tissue (MALT), and beneath the lamina propria is a layer of smooth muscles (Jaladanki & Wang, 2011).In the small intestine, the surface is increased by formation of villi and crypts, covered by the epithelial monolayer, composed by a heterogeneous population of cells (Umar, 2010;Van Der Flier & Clevers, 2009).Enterocytes, the most abundant cells type in the epithelial monolayer, are columnar cells located at the upper end of the crypts and throughout the entire villi, having a highly folded apical surface forming a microvilli structure, the so-called brush border (Reitsma et al., 2014) (Fig. 3).The overall purpose of the GI tract is to maintain gut homeostasis, which relies on two main functions; the breakdown and absorption of nutrients, minerals and water, and the defence of the body against pathogens and undesirable products in the lumen (Farré et al., 2020).

Gastrointestinal protein digestion
Ingested food undergoes a complex series of degradative processes in the GI tract in order to ensure extraction of nutrients required for maintenance of health.Understanding the fate of dietary proteins, from they reach the mouth until they are taken up by immune cells in the GI tract is essential in food allergy, as the state in which the dietary proteins are absorbed from the lumen impact on their allergenic potential.Thus, protein denaturation and digestibility are major determinants of allergenicity.It is believed, that the longer the protein survives the digestion process as intact protein or as large peptide fragments, the larger is the allergenic potency and thereby the capacity to sensitise or elicit an allergic reaction (Bøgh & Madsen, 2016).
Degradation of dietary proteins starts in the mouth by mechanical breakdown carried out by mastication and saliva wetting, whereas proteolysis of the proteins starts in the stomach, where the proteins are exposed to an acidic environment with a pH of around 1.2 to 3.0, that causes the proteins to denature, rendering them more susceptible for proteolysis by the gastric protease pepsin (van Lieshout et al., 2020).The gastric transit time may vary, but it is estimated in average to take around 1 to 2 h before the gastric content is released into the duodenum of the small intestine, which provides a neutral environment with a pH of around 6.5 (Marciani et al., 2001).In the duodenum, the dietary proteins are subjected to the endopeptidases trypsin, chymotrypsin and elastase, and the exopeptidases carboxypeptidase A and B, produced by the pancreas (Bhagavan & Ha, 2015).The brush border also plays an important role in the digestion of dietary proteins, where neutral endopeptidases are anchored into the apical side of the epithelial cells as integral membrane proteins with their active site projecting into the lumen.Exopeptidases are also present in the brush border, with the most abundant being the aminopeptidases, in addition to di-and tripeptidases, which are either secreted onto the brush border or found inside the epithelial cells (Nilius et al., 2015) (Fig. 3).
The overall goal of the gastric, pancreatic and brush border proteases is to degrade the majority of dietary proteins to a mixture of free amino acids, dipeptides, and tripeptides, making them available to the various carrier-transporters of the brush border membrane for rapid and efficient absorption through the intestinal mucosa to serve as nutrients (Fig. 3).However, the digestibility of dietary proteins varies greatly and larger immunologically active peptides, even intact proteins, may survive the digestion process, to be absorbed and presented to the immune system of the intestine (Bøgh & Madsen, 2016;Verhoeckx et al., 2019).Several factors may impact the digestion of dietary proteins, these being intrinsic features of the dietary proteins themselves, factors conferred by the individual's digestive system or external factors originating from the outside environment.For example, a huge variability in susceptibility to digestion exists among dietary proteins, which is moreover influenced by food processing as well as the matrix in which the proteins are composed (Bøgh & Madsen, 2016;Costa et al., 2022a,b;Dallas et al., 2017;Verhoeckx et al., 2019).Further, the extent of digestion significantly vary from person to person, affected by factors such as gastric pH, enzymatic activity, host surfactant, age, health and GI microbial composition (Dallas et al., 2017;Gass et al., 2007;Moreno et al., 2005;Oliphant & Allen-Vercoe, 2019;Untersmayr & Jensen-Jarolim, 2008;Verhoeckx et al., 2019).Environmental and lifestyle factors may additionally affect the digestion of dietary proteins, where for example other dietary components, alcohol, exercise and tobacco have been shown to impact the proteolysis of proteins (Gilani et al., 2005;Hall & Crowe, 2011;van Wijck et al., 2013).

Intestinal epithelial layer
The intestinal epithelial layer plays a central role in maintaining gut homeostasis, by guaranteeing the delicate balance between limiting the entrance of dietary allergens yet ensuring sampling of the dietary antigens for establishment of tolerance.It consists of a single layer of various types of epithelial cells covering the lamina propria.The most abundant cell type is enterocytes that form an effective barrier and control nutrient absorption.Scattered between the enterocytes are goblet cells, enteroendocrine cells, Paneth cells and crypt base stem cells (Umar, 2010;Van Der Flier & Clevers, 2009) (Fig. 4).Goblet cells are secretory cells located both in the crypts and in the villi.The goblet cells contain various storage granules possessing mucin proteins that are slowly but constantly released into the lumen (Kim & Ho, 2010).Enteroendocrine cells are a secretory cell type within the epithelium.They produce GI hormones, peptides and neurotransmitters (Gribble & Reimann, 2016).In the crypts, Paneth cells are found.These cells are highly specialised epithelial cells specific for the small intestine, and are responsible for secretion of anti-microbial products such as peptidoglycans, defensins and lysozymes, regulating microbiota homeostasis (Clevers & Bevins, 2013;Elphick & Mahida, 2005).Stem cells are located at the base of the crypt and give rise to the above mentioned four types of epithelial cells, most of which migrate upwards and populate the villi (Umar, 2010).
In the organised lymphoid follicular tissue of Peyer's patches (PPs), another type of specialised epithelial cells is found, designated microfold (M) cells (Fig. 4).M cells have reduced microvilli and hence an irregular brush border.They inhere a unique ability to sample antigens from the lumen of the small intestine and are highly involved in antigen transport to the underneath gut lymphoid tissue (Kobayashi et al., 2019).PPs contain a high amount of B cells, T cells, dendritic cells (DCs) and macrophages resident in the dome region between the intestinal epithelium and the follicle structure, which is connected to the gut draining lymph nodes, the mesenteric lymph nodes (mLNs), through the lymphatic vessels (Booth & Toapanta, 2021).

Intestinal barrier function
The intestine is constantly in contact with the external environment, thus a strong barrier function to protect against foreign entities is needed.The intestinal barrier is composed of two important layers; the outer physical layer and the inner immunological barrier (Fig. 4).A main part of the physical barrier is the GI microbiota.The intestinal commensal microbiota resides in close and mutualistic proximity with the host and inhibit colonisation of the luminal surface with pathogenic bacteria.The commensal microbiota also contribute to shaping the intestinal immune system, e.g. by release of microbe-associated molecular patterns (MAMPs) or by induction of secretory IgA (sIgA) (Schuijt et al., 2013).sIgA also serves as a first line of defence, protecting the luminal surface from colonisation with pathogenic bacteria and promotes clearance of antigens from the intestinal lumen (Mantis et al., 2011).Beneath the microbiota, a strong component of the physical barrier is the mucus.The mucus is mainly composed of glycoproteins, mucins, constituting a hydrated gel to protect the barrier.Mucus consists of two layers, an outer highly moveable layer and an inner layer densely packed and attached to the epithelium (Johansson et al., 2008;Lock et al., 2018).Mucus is secreted from the specialised goblet cells, and is found throughout the GI tract, from the stomach throughout the large intestine, with the highest density in the colon (Atuma et al., 2001).The main function of the mucus is to prevent large particles, mainly bacteria, from interacting with the intestinal epithelium (Johansson et al., 2008).
Another part of the physical barrier function lies within the epithelial monolayer itself.In the presence of a healthy intact epithelial cell layer, transport of molecules across the monolayer between the intestinal epithelial cells, the paracellular pathway, is highly regulated by cell junctional complexes and restricted to small molecules (Steed et al., 2010), which will be further explored in section 4.
Besides the physical barrier, the immunological barrier involves various immune cells resident in the intestinal epithelium and subepithelial area.These cells comprise cells from both the innate and the adaptive immune system and are able to distinguish between, for example, commensal and pathogenic bacteria.Cells of the innate immune system constitute the first line of immunological defence in the intestine, comprised of e.g., DCs, macrophages, neutrophils, mast cells and eosinophils.The response of the innate immune cells is triggered by activation of pattern-recognition receptors (PRRs) such as Toll-like receptors (TLRs) that gets alarmed by conserved pathogen-associated molecular patterns (PAMPs), MAMPs, and damage-associated molecular patterns (DAMPs) on the surface of pathogenic bacteria.TLR activation, for example, leads to activation of innate immune cells and their release of antimicrobial compounds, inflammatory mediators and enzymes (Santaolalla & Abreu, 2012).When the response of the innate immune system is insufficient for avoidance of antigen penetration, the adaptive immune response is activated.The adaptive immune response is, opposite to activation by conserved patterns of molecules, characterised by its immunological memory, and is primarily mediated by lymphocytes.In the intestine, two main adaptive anti-inflammatory mechanisms exist; the immune exclusion executed by the plasma cell secreted sIgA (as described above) and oral tolerance to harmless antigens executed primarily by regulatory T cells (Brandtzaeg, 2011).

Oral tolerance
Oral tolerance is the default response to ingested proteins in healthy individuals.Dietary antigens entering the intestinal subepithelial area are taken up and processed by antigen-presenting cells such as DCs.A subset of regulatory DCs expressing CD103+ is responsible for delivery of the dietary antigens to the mLNs.In response to antigen presentation, naïve T cells differentiate to regulatory T cells upon activation and proliferation, through a mechanism dependent on transforming growth factor-beta (TGF-β) and retinoic acid, promoting the oral tolerance (Lozano-Ojalvo et al., 2019b;Tordesillas & Berin, 2018).However, when oral tolerance is not established, sensitisation to dietary proteins can occur.

Intestinal protein uptake
Dietary proteins are digested by gastric and pancreatic as well as by integral brush border enzymes, with the purpose of degrading the proteins into small peptides and amino acids.Yet, larger peptides and even intact proteins may survive the digestion process in the GI tract (Bøgh & Madsen, 2016).Absorption and transport of dietary proteins across the epithelial layer are greatly affected by the digestibility of the proteins, as absorption and transport are dependent on physicochemical properties of the proteins and peptides and is defined by their size, solubility, polarity, structure and aggregation status (Costa et al., 2022b;Reitsma et al., 2014).The absorption takes place through two main routes of transport, the transcellular and the paracellular route, where the transcellular route refers to the transport of proteins and peptides across the enterocytes and the paracellular route refers to the transport of proteins and peptides via the intercellular space between the epithelial cells (Fig. 5).

Transcellular transport
Transcellular transport across the enterocytes covers a wide range of ways that the dietary proteins and peptides can be absorbed and may occur by simple diffusion, carrier transport mediated diffusion, transcytosis or by active transport (Price et al., 2013).
Simple passive diffusion is normally restricted to transport of amino acids as well as di-and tripeptides and is negligible for larger peptides and proteins due to their size and most often hydrophilic nature.Hence, simple diffusion across the lipid bilayer of the epithelial cell membrane is largely dependent on size, solubility and concentration gradient, and therefore varies between the different amino acids and di-and tripeptides, with hydrophobic amino acids diffusing more readily across the epithelial layer than hydrophilic amino acids, with the concentration gradient being the driving force (Tauqir, 2016;Webb, 1990).Diffusion may also be mediated by carrier transporters relating to the passive transport of amino acids, di-and tripeptides without the use of energy via membrane bound transport molecules, with the concentration gradient as the driving force (Friedman & Amidon, 1990;Grassl, 2001).Transport via membrane bound carrier proteins can also be facilitated with the utilisation of energy in the form of conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP).The ATP dependent transport of amino acids mainly rely on the co-transport of sodium, whereas the transport of di-and tripeptides mainly rely on the co-transport of protons, allowing a transport against the concentration gradient (Price et al., 2013).
The majority of larger proteins and peptides that reaches the intestine for absorption through the epithelial layer are transported across the enterocytes by transcytosis (Garcia-Castillo et al., 2017;Rojas & Apodaca, 2002).The dietary proteins or peptides are engulfed and internalised via endocytosis of the cell membrane for transport within small vesicles, the endosomes, or within larger phagosomes inside the cell from the apical to the basolateral surface, where the endosomes or phagosomes fuse with the cell membrane for release of their content by exocytosis.Transcytosis may occur by different mechanisms, such as clathrin-mediated endocytosis or caveolin-mediated endocytosis (Apodaca, 2001), where transport of proteins and peptides may occur by means of two main ways (Apodaca, 2001;Reitsma et al., 2014;Yu, 2012).Most often the proteins or peptides are endocytosed with subsequent fusion of the endosomes with lysosomes containing enzymes, allowing for endolysosomal degradation of the proteins or peptides before release at the basolateral side (Cuervo & Dice, 1998;Dunn, 1994).However, protected transcytosis may also occur, where the dietary proteins or peptides are transported without any further degradation and released in their intact form at the basolateral side of the enterocytes (Ziv & Bendayan, 2000).Studies investigating transcytosis of dietary proteins and peptides stress that by far most proteins and peptides undergo degradation inside the enterocytes and that only small amount are transported without further degradation (Laiping So et al., 2000;Warshaw et al., 1974).A study by Heyman et al. (Heyman et al., 1982), demonstrated that around 97 % of the proteins were degraded during the transport through the epithelial layer, while Ménard et al.A.-S.R. Ballegaard and K.L. Bøgh (Ménard et al., 2010), described that around 10 % were transported across the enterocytes in intact form, around 40 % were degraded to smaller peptide fragments, and around 50 % were degraded into amino acids.However, the exact amount of proteins or peptides undergoing endolysosomal degradation remains unknown and probably depend on several factors such as the physicochemical properties of the proteins or peptides themselves, the matrix in which they are embedded as well as factors related to the host.
Dietary proteins and peptides may also be transported across the enterocytes by facilitated transport.One mean of facilitated transport is related to receptor-mediated transport where peptides or proteins bind to MHC II receptors on the surface of the enterocytes, for transportation through the epithelial cell to the basolateral side where small membrane vesicles, the exosomes, are released (Heuberger et al., 2020;Van Niel et al., 2003).This has been demonstrated to allow for protected transport with a direct and highly efficient antigen presentation to downstream immune cells (Mallegol et al., 2007).Conflicts in the literature exist on whether MHC II transport and subsequent exosome release for antigen presentation promotes tolerance or not (Heuberger et al., 2020;Reitsma et al., 2014).
Another mean of facilitated transport is related to Ig-mediated transepithelial transport, where Ig bound to its receptor, e.g.FcRn for IgG or polymeric Ig receptor (pIgR) for IgA, is transported forth and back from the basolateral membrane (Garcia-Castillo et al., 2017;Rojas & Apodaca, 2002).Dimeric IgA is the main type of immunoglobulins found in the gut mucosa, produced by local plasma cells, and transported from the basolateral site of the enterocytes to the apical site for release into the lumen, where the primary role is to protect the gut from pathogenic attack and avert microbial and dietary antigens from being taken up (Rojas & Apodaca, 2002;Tuma & Hubbard, 2003).However, more recently it has been suggested that sIgA may undergo retrotransport from the apical to the basolateral site of the enterocytes allowing for facilitated transport of luminal dietary proteins and peptides across the epithelial layer (Mathias et al., 2014;Ménard et al., 2010).For example, in patients with celiac disease, an abnormal protected transport of peptides bound to IgA is facilitated by the transferrin receptor CD71, where transferrin has been shown as an activator of the interaction between the CD71 receptor and IgA, and promoting retrospective transport from the apical to the basolateral site (Lebreton et al., 2012;Matysiak-Budnik et al., 2008;Moura et al., 2001).
In addition, retrospective transport of IgA has been reported to take place through M cells (Corthesy, 2007;Rochereau et al., 2015), via mechanisms not fully elucidated but is suggested to take part in sampling of antigens for general training of the gut immune system (Rochereau et al., 2021).For example, in Crohn's disease retrospective transport of IgA has been shown in situations of deficiencies of the intracellular PRR NOD2 (Nucleotide-binding oligomerisation domaincontaining protein 2), which is known to contribute to the regulation of the intestinal barrier integrity (Rochereau et al., 2021).
M cells are specialised epithelial cells known to deliver proteins or peptides directly to the underneath lymphoid tissue, thus transport through M cells may play a fundamental role in the immunological response towards the proteins and peptides.The M cells form pockets where B and T cells, macrophages and DCs may be present and are important for mounting an immune response (Farstad, 1994;Iwasaki & Kelsall, 2001).M cells contain fewer lysozymes, compared with enterocytes, thus degradation of proteins is only seen to a low extent when transported through M cells.It is the perception that M cells are specialised in the uptake of particular materials, such as aggregated proteins and peptides (Graversen et al., 2020b;Roth-Walter et al., 2008).
Transport through goblet cells is only scarcely described, yet it has been observed that small peptides may be taken up by goblet cells in healthy individuals (McDole et al., 2012).
DCs are antigen-presenting cells capable of extending their dendrites in between juxtaposed epithelial cells by opening cell junction complexes and sample peptides directly from the intestinal lumen (Rescigno et al., 2001;Schulz et al., 2009).Similar to M cells, DCs seem to have a preference for sampling of larger particles (Farache et al., 2013;Graversen et al., 2020b).

Paracellular transport
Paracellular transport refers to the transport of molecules via the intercellular space between the epithelial cells.This route of transport is highly regulated by cell junctional complexes, often referred to as tight junction complexes (Chelakkot et al., 2018).Cell junctional complexes are composed of tight junctions, adherence junctions, desmosomes and gap junctions.Tight junctions are responsible for sealing the intercellular space and regulating ion transport.Tight junctions are the most apical cell complexes forming a continuous belt-like ring around the epithelial cells at the border between the apical and lateral membrane region (Tsukita et al., 2001).Tight junctions consist of integral membrane proteins, such as claudins and occludins that are composed of a branching network of sealing strands (Fig. 5).Claudins and occludins connect with the intracellular peripheral membrane zonula occludens (ZO) proteins, which anchors the claudin and occludin strands to intracellular actin filaments (Furuse et al., 1993;Schneeberger & Lynch, 1992).Adherence junctions maintain cellular proximity and intercellular communication.Adherence junctions are composed of two types of integral membrane protein families anchored directly to the intracellular actin filaments, namely cadherins and catenins (Ivanov & Naydenov, 2013;Perez-Moreno & Fuchs, 2006).The adherence junctions often form a continuous belt just below the tight junctions (Hartsock & Nelson, 2008).Desmosomes provide a strong adhesion between the cells for tissue integrity maintenance (Kowalczyk & Green, 2013).Desmosomes consist of dense cytoplasmic plaque proteins anchored to intracellular keratin filaments and connected to intercellular transmembrane proteins of the cadherin family (Garrod & Chidgey, 2008).Lastly, gap junctions are clusters of intercellular channels that allow direct diffusion of ions and small molecules between the adjacent cells.The channels are formed by the proteins termed connexions (Goodenough et al., 1996).
Transport through the paracellular route is defined in terms of size and charge and is driven by water movement due to a transepithelial osmotic gradient.Under normal conditions only passage of small hydrophilic molecules up to 0.6 kDa is allowed (Heyman, 2005;Kondoh et al., 2012;Ménard et al., 2010).In contrast to the transcellular pathway, peptides are not exposed to lysozymes and thereby allowed crossing the epithelial layer without degradation (Shimizu, 2004).However, the intercellular space between epithelial cells may in some situations form a leaky pathway, a route allowing for transport of small quantities of larger peptides between 0.5 and 1.5 kDa (Perrier & Corthésy, 2011;Shen et al., 2011).Transport through the leaky pathway may be increased by inflammatory cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) (F.Wang et al., 2005;Watson et al., 2005).

Transport of larger peptides and proteins
It is now well-accepted that proteins may survive the digestion process and be transported in their intact form across the intestinal barrier, as intact proteins have been found in the blood, the lymph, as well as in breastmilk (Husby et al., 1985).Several studies have investigated the amount of dietary proteins absorbed in their intact form, where e.g.Warshaw et al. (Warshaw et al., 1974), found that 2 % of bovine serum albumin (BSA) was transported from the intestinal lumen into the lymph and blood as intact proteins.Yet, the absorption of intact dietary proteins may differ from one situation to another, and Warshaw et al. (Warshaw et al., 1971), e.g.found that BSA was 100 to 1000 times more readily absorbed than horseradish peroxidase (HRP).Further, it is well-acknowledged that infants with their immature GI tract allow more intact dietary proteins cross the epithelial layer than adults with a healthy and mature intestinal barrier (Weström et al., 2020).

Food allergy and protein uptake
The intestinal barrier function and protein uptake through the epithelial layer have been demonstrated to differ between healthy individuals and food allergic patients (Perrier & Corthésy, 2011), stressing an imbalance in the intestinal barrier function associated with food allergic diseases.It still remains to be fully resolved whether increased intestinal permeability is a risk factor in the development of food allergy and/or whether food allergy causes increased intestinal permeability.In studies investigating the intestinal permeability in food allergic individuals, it was shown that the permeability increased after allergen challenge, but returned to normal after strict avoidance of the culprit food (Andre et al., 1987;Dupont et al., 1989), indicating that the increased permeability is a results of the allergic reaction.However, in another study increased permeability was seen even after six months of strict avoidance of the offending food (Ventura et al., 2006), suggesting that other mechanisms than those exerted by the allergic reaction are at play.Not only the overall intestinal permeability, but also the exact route of protein uptake may have a role in food allergy, as it has been shown that the specific transport route may impact on proteins' sensitising as well as eliciting capacity (Graversen et al., 2020b;Knoop et el., 2013;Perrier & Corthésy, 2011;Roth-Walter et al., 2008).
To induce an allergic reaction, intact dietary proteins or their peptide fragment derivatives, large enough to cross-link FcԑRI-bound IgE on the surface of tissue mast cells or blood basophils, must cross the intestinal epithelial layer.The route of allergen uptake in allergic individuals has been extensively studied, especially with the help of in vitro and in vivo animal studies (Perrier & Corthésy, 2011;Yu, 2009).In contrast to healthy individuals, where most proteins and peptide fragments are transcytosed through enterocytes via the endolysosomal pathway, entailing degradation processes (Laiping So et al., 2000;Warshaw et al., 1974), enhanced uptake devoid of breakdown were seen in allergic mice and rats (Berin et al., 1997;Bevilacqua et al., 2004;Yang et al., 2000).This uptake was demonstrated to be specific and exclusively transcellular, occurring within minutes of the allergen exposure and facilitated by the IgE/CD23 complex (Berin et al., 1997;Yang et al., 2000;Yu et al., 2001).In allergic individuals an upregulation of the IgE receptor CD23 (FcεRII) was seen in intestinal epithelial cells, regulated by the Th2 cytokine IL-4 (Yu et al., 2001), and upon exposure to the culprit dietary allergens these can bind to the IgE/CD23 complex for protected transport across the epithelial layer to the subepithelial area.This phase of the enhanced transport seen in allergic individuals is designated phase I (Perrier & Corthésy, 2011;Yu, 2009;Yu et al., 2001) (Fig. 6).Once in the subepithelial area, the lamina propria, the dietary allergens may come in contact with tissue resident mast cells and cross-link their FcεRI-bound IgE inducing a degranulation response.Mediators released upon mast cell degranulation, such as histamines, prostaglandins and proteases affect the cell junctional complexes.For example, the release of tryptase from mast cells has been shown to cause re-localisation of tight junction proteins and thereby increased protein uptake through the paracellular route (Berin et al., 1998;Jacob et al., 2005).The mast cellinduced increased permeability and consequential enhanced protein uptake seen in allergic individuals, is designated phase II (Perrier & Corthésy, 2011;Yu, 2009) (Fig. 6).

Techniques for measuring intestinal permeability and protein uptake
Assessing intestinal permeability and specific protein transport may be of great value and an important tool when evaluating the protein physicochemical properties, as well as environmental and lifestyle factors, affecting transport of dietary proteins, including allergens.Different techniques are available and used depending on the purpose of the given study.In the following paragraphs, these different techniques will be explained and discussed according to in vitro, ex vivo and in vivo methods, particularly in relation to allergen uptake (Fig. 7).

In vitro methods
In the 1970s a collection of cell lines were established from human colonic carcinoma with the aim of performing studies on cancer mechanisms and related therapies (Fogh et al., 1977).One of these cell lines, the Caco-2 cell line, was later shown to undergo a process of spontaneous differentiation leading to formation of a monolayer of cells expressing morphological and functional characteristics of mature enterocytes (Pinto, 1983).Cultivation of Caco-2 cells for around 3 weeks leads to formation of a tight monolayer and an apical brush border, with expression of typical metabolic enzymes and efflux transporters such as P-glycoprotein (P-gp) (Hidalgo et al., 1989;Hilgers et al., 1990).Due to these properties, Caco-2 cell culture studies have been widely used for prediction of drug absorption and intestinal transport (Sambuy et al., 2005).Another tumour cell line is the HT-29 cell line that contains both absorptive cells and mucus-producing cells (Fogh et al., 1977).Differentiated HT-29 cells express brush border-associated hydrolases that are Fig. 6.Mechanisms of increased protein uptake in allergic individuals.In allergic individuals the IgE receptor CD23 (FcεRII) is upregulated in enterocytes.Upon exposure of the culprit dietary allergens, they bind to the IgE/CD23 complex for protected transport across the epithelial layer.This phase is designated phase 1.Once the dietary allergens come in contact with tissue mast cells and cross-link their FcεRI-bound IgE, it induces a degranulation response.Mediators released by the mast cell affect the cell junctional complex resulting in increased paracellular space between juxtaposed enterocytes, which may lead to increased protein uptake through the paracellular route, designated phase 2. Figure created with BioR ender.com.
A.-S.R. Ballegaard and K.L. Bøgh typical for the small intestine (Lesuffleur et al., 1990(Lesuffleur et al., , 1991)).HT-29 cells are often treated with methotrexate to get mature goblet cells, and are then referred to as HT-29-MTX.These cells show fewer and shorter microvilli than Caco-2 cells (Pontier et al., 2001).HT-29-MTX cells are often co-cultured with Caco-2 cells in order to closely mimic the human intestinal barrier (Béduneau et al., 2014;Lozoya-Agullo et al., 2017).A different cell line derived from human colon carcinoma is the T84 cell line (Dharmsathaphorn et al., 1984).Fully differentiated T84 cells express functional proteins regulating paracellular transport and have been used as a model system for studying electrolyte transport processes (Madara, 1987;Matthews et al., 1992).Besides the human colonic carcinoma derived cell lines, a cell line used for intestinal investigations is the porcine derived IPEC-J2 cell line isolated from jejunum epithelial cells of neonatal piglets.This cell line is unique since it derives from the small intestine and not the colon as Caco-2, HT-29 and T84 cells (Berschneider, 1989;Kandil et al., 1995).This cell line has been used as a model system for studying interactions of animal and human pathogens with the intestinal epithelium (Brosnahan & Brown, 2012).
In general, for all cell lines, cultivation in transwells allows for investigation of the cell monolayer integrity as well as apical to basolateral transport with a readout of the barrier function and transport of a compound of interest, e.g. an allergen, or a tracer molecule.Intestinal barrier integrity can be assessed by measuring the transepithelial electrical resistance (TEER) with e.g.use of chopstick electrodes on each side of the monolayer measuring ohmic resistance across the cell monolayer (Srinivasan et al., 2015).Transport of allergens or tracer molecules through the monolayer can be assessed by measuring the amount and rate of absorption through the monolayer by adding the allergen or tracer molecule to the apical compartment and collect aliquots from the basolateral compartment at different time points for analysis (Turco et al., 2011), e.g. by enzyme-linked immunosorbent assay (ELISA), if assays or antibodies for detection of the specific allergen or tracer molecule are available, or by detection of the allergens or tracer molecule by liquid chromatography (LC) coupled to mass spectrometry (MS) (Fig. 7).Examples of such detection of allergen transport through Caco-2 monolayer relates to the cow's milk allergen, β-lactoglobulin (BLG) also designated Bos d 5, where transport of BLG was measured upon sampling at the basolateral side of monolayer after adding it to the apical site of the monolayer.The impact of processing (Graversen et al., 2020b), as well as the impact of other dietary components such as the kiwi allergen Act d 1 (Nešić et al., 2019a) or quinoa saponins (Ballegaard et al., 2021) on the transport of BLG has also been investigated, stressing that various factors may indeed impact on the amount and kinetics of allergen transported through the Caco-2 cell monolayer as will be further elaborated in section 7. Effects of allergens on cell barrier integrity as well as their transport through a cell monolayer may also be assessed using HRP-labelled protein, Lucifer Yellow, fluorescein isothiocyanate (FITC)labelled protein or FITC-dextran (Diesner et al., 2012;Perusko et al., Fig. 7. Overview of methods for measurement of intestinal permeability and protein uptake.Different techniques and model organisms can be used for measurement of intestinal permeability and protein uptake according to the purpose of the given study.The methods can be divided into in vitro, ex vivo and in vivo methods containing both methods from animals and humans.In vitro methods may consist of cell line experiments with ability to measure intestinal barrier function by transepithelial electrical resistance (TEER) or measure transport of molecules through the cell layer.A more recent developed method is the gut-on-a-chip, where intestinal cells can obtain a 3D-cellular structure which allows for a more physiological relevant investigation.An ex vivo method used for measurement of intestinal permeability is the Ussing chamber technique.Fresh intestinal tissue is mounted in the Ussing chamber, and barrier integrity and intestinal transport can be assessed.In vivo methods can consist of both animal and human methods, where in animals, intestinal permeability can be assessed within the intestine, in blood or in faecal samples.In humans, intestinal permeability is most often assessed using urine, blood and faecal samples.Intestinal permeability and protein uptake can be measured using techniques such as enzyme-linked immunosorbent assay (ELISA), histology, gene expression profiling and mass spectrometry (MS).Figure created with BioR ender.com.2018; Price et al., 2014).Examples of such detection of BLG transport across the Caco-2 monolayer has been reported with the use of FITClabelling, where studies of the impact of processing was also investigated (Perusko et al., 2018;Stojadinovic et al., 2014).
In order to explore cellular or molecular changes of the cell monolayer posed by allergens or other components suspected to impact on the intestinal integrity, immunohistochemistry, immunofluorescence, Western blotting or gene expression analyses can be performed (Drago et al., 2006;Grozdanovic et al., 2016;Nešić et al., 2019b;Price et al., 2014).
In general, one of the major advantages of cell cultures is the relative low cost and easy handling whereas a major drawback is the lack of the physiological relevant environment.Development of in vitro techniques that better reflects in vivo intestinal epithelium is constantly progressing.Recently, novel techniques with microfluidic organ-on-a-chip models and organoids have been developed and used when studying intestinal epithelial layer (Puschhof et al., 2021) (Fig. 7).Gut-on-a-chip technology has been developed based on Caco-2 cells forming 3D leak-tight polarised tubules after only four days of culture (Beaurivage et al., 2019;Trietsch et al., 2017).Other cell types, such as primary cells or stem cells can also be used to populate chip devices.Furthermore, the gut-on-a-chip technique enables features such as luminal flow and peristalsis-like movement (Kim, Huh, Hamilton, & Ingber, 2012).Organoids are derived from intestinal epithelial stem cells, either from adult stem cells isolated from intestinal crypts or from pluripotent stem cells, which can form a 3D self-renewing cellular structure with villuslike structures and crypt-like proliferative zones (Clevers, 2016;Kretzschmar & Clevers, 2016).These organoids consist of a fluid-filled central space that can be microinjected with for example microbes (Puschhof et al., 2021).Gut-on-a-chip has been used to investigate intestinal barrier integrity by measurement of TEER and effect of drugs on intestinal permeability using Lucifer Yellow and FITC-dextran (Imura et al., 2009;Trietsch et al., 2017).This technique could further accommodate studies assessing allergen uptake in a more complex setup than allowed by the more widely used cell lines.The biggest challenge is still to fully incorporate physiologically relevant cells and increase the life span of the cells on the chip (Ashammakhi et al., 2020).

Ex vivo methods
As a bridge between in vitro and in vivo studies, ex vivo techniques are often used to study intestinal barrier integrity and protein transport.The most common ex vivo method is the Ussing chamber technique (Thomson et al., 2019).This technique requires small sections of fresh intestinal tissue, most often dissected from mice or rats, or in some cases from humans through biopsy samples (Clarke, 2009;Wallon et al., 2005).If whole intestinal sections are dissected, the intestine is opened longitudinally and a segment of the intestine is mounted into the Ussing chamber (Fig. 7).The tissue can be accessed from both the apical and the basolateral side.The chambers on each side of the tissue are typically filled with Krebs solution, and in each chamber electrodes are attached which enables measurement of TEER (Clarke, 2009;Ussing & Zerahn, 1951).The system also allows the investigation of intestinal transport across tissue samples from either allergic sensitised or non-sensitised animals using allergens or tracer molecules.For example, intestinal transport was investigated in a study by Castan et al. (Castan et al., 2018), with tissue from allergic sensitised animals and measured by HRP flux through the intestine or in a study by Oscarsson et al. (Oscarsson et al., 2020), with tissue from non-sensitised animals stimulated in the chamber by food components suspected to influence barrier integrity with intestinal transport measured by the tracer molecule radiolabelled mannitol.
A less used ex vivo technique is the everted sac method, where an intestinal section is everted and both ends are tied after filling the sac with buffer.Inserting the sac in a chamber with buffer and the allergen alone or together with a tracer molecule allows for assessment of transport through the intestinal sac (Barthe et al., 1998).One of the main advantages of harvesting tissue for ex vivo experiments is the opportunity to gather samples containing different cell types, however, a major drawback is the requirement of fresh tissue from laboratory animals or human biopsies (Plumb et al., 1987).

In vivo methods
Using in vivo techniques increases the physiological relevance of the intestinal integrity and transport studies as all types of intestinal epithelial cells, immune cells as well as blood circulation are present.Assessment of intestinal barrier functions may be invasive, thus animal models as alternatives to human studies, allow for thorough investigations and answers to more mechanistic questions (González-González et al., 2019).

Animal studies
A method for investigation of intestinal permeability in animal models is the measurement of transported allergens or tracer molecules, such as FITC-labelled proteins or FITC-dextran, across the intestinal barrier.FITC-dextran with a size of 4 kDa has for example been orally gavaged in fasted animals and blood sampled 1-3 h post gavage with subsequent measurement of the fluorescence intensity (Cani et al., 2008;Christensen et al., 2014;Tulstrup et al., 2015).This allows for studies in both allergic sensitised and non-sensitised animals and thus evaluation of the effects of allergens or other food components, lifestyle or environmental factors on intestinal permeability.
In a similar way, intestinal permeability has been assessed in animals with determination of the transport of the cow's milk allergen BLG in different intestinal compartments or in serum using ELISAs (Ballegaard et al., 2021;Graversen et al., 2020b;Nešić et al., 2019b), and has been used to investigate the effects of processing and other food components on the transport of BLG.On a more molecular level, the effect of allergens on barrier integrity can be examined by gene expression or Western blotting analyses for assessment of cell junctional complex proteins (González-González et al., 2019;Nešić et al., 2019a), and morphological changes may be investigated by histology and immunohistochemistry (Ballegaard et al., 2021;Mazzon et al., 2002).Thus, in animal experiments it is possible to explore more parameters at the same time than in the simpler in vitro and ex vivo experiments, however animal studies are more resource demanding and may be limited by ethical constrains.

Human studies
A widely used technique to investigate intestinal permeability in humans is the differential sugar absorption lactulose/mannitol (L/M) test.Lactulose is a larger sugar than mannitol and will be transported through the intestinal barrier to a lesser extent than mannitol in healthy individuals.However, a higher amount of lactulose will be transported across the epithelial layer when intestinal barrier defects are present.Thus, oral ingestion of the two sugars allows for detection and quantification of the sugars in urine samples and the ratio between the two sugar molecules is considered a marker of intestinal permeability (Cobden et al., 1978;Menzies et al., 1979;Pearson et al., 1982).Analysis of the urine sample is usually performed by means of HPLC or LC-MS (Bischoff et al., 2014).Other biomarkers based on serological samples include intestinal fatty acid binding proteins (I-FABP), zonulin, and bacterial lipopolysaccharide (LPS), whereas biomarkers from faecal samples include calprotectin and defensins (Bischoff et al., 2014;Campeotto et al., 2010;Karhu et al., 2017).Biomarkers of intestinal permeability are typically detected by ELISA or LC-MS (Bischoff et al., 2014).In addition to measurement of intestinal permeability, assessment of specific allergens transported through the intestine can be determined in human serum.This has been assessed in non-sensitised individuals using ELISA and the CAP-FEIA system (JanssenDuijghuijsen et al., 2017;Pahlow Mose et al., 2020;Untersmayr et al., 2007).

Factors affecting intestinal integrity and food allergy
The intestine normally constitutes some permeability allowing for uptake of nutrition and sampling of antigens for maintenance of health, however the intestinal barrier may be compromised giving rise to increased intestinal permeability.Increased intestinal permeability, also sometimes referred to as leaky gut, is increased fluid exchange between the lumen and the intestinal tissue and may results in inadequate separation of the content in the lumen and the intestinal tissue.Mechanisms of leaky gut are not fully elucidated and may be manifold, involving disruption of one or more components of the intestinal barrier function, including inadequate interaction between epithelial cells both in terms of alterations of cell junctional complexes and intercellular connections (Camilleri, 2019;González-González et al., 2019).Transport through the intestinal epithelial layer may rely on the physicochemical properties of the proteins and peptide fragments themselves as well as the matrix in which they are embedded, but can also be influenced by environmental and lifestyle factors, such as exposure to environmental chemical substances, other dietary components, intake of alcohol, exercise and medications, or indirectly through the changes that the environmental and lifestyle factors exert on the gut microbiota (Groschwitz & Hogan, 2009;Reitsma et al., 2014) (Fig. 1).Yet, knowledge on how these factors impact on the specific route of protein uptake and the consequential impact on food allergy is very limited.

Protein physicochemical properties
Several physicochemical characteristics of the proteins or peptide fragments themselves impact the amount and route by which they are transported across the epithelial layer, which will again have an impact on the immunological response mounted.As previously described, the size, solubility, structure and aggregation status of the proteins and peptide fragments may be decisive for the route of uptake, while in order to induce an allergic response the proteins and peptide fragments absorbed are generally required to have a molecular size of at least 3.5 kDa (Bøgh et al., 2013).
It has been proposed that dietary proteins may themselves contain physicochemical properties conferring them adjuvant capacity that enable them efficient transport across the epithelial layer and that to a certain extent may account for their allergenic potential (Price et al., 2014).Indeed, an efficient and protected transport across Caco-2 cells by means of transcytosis have been shown for the peanut allergens Ara h 1 and Ara h 2 (Price et al., 2014), the Brazil nut allergen Ber e 1, the sesame allergen Ses i 1 (Moreno et al., 2006), the wheat allergens Tri a 14 and Tri a 19 (Bodinier et al., 2007), and the peach allergen Pru p 3 (Tordesillas et al., 2013), where the allergens were transported from the apical to the basolateral site in their immunologically active form.In the study of Tordesillas et al. (Tordesillas et al., 2013), the transport of the peach ns-LTP allergen Pru p 3 was compared with that of a nonallergenic LTP, which revealed that the transport of the allergenic LTP was significantly higher than that of the non-allergenic LTP, stressing that allergens may indeed possess some properties favouring their transport through the epithelial layer.Especially peanut allergens have been shown to affect the epithelial barrier integrity promoting their transport across the epithelial layer.For example, Teodorowicz et al. (Teodorowicz et al., 2013) demonstrated a significant anti-proliferative activity of Ara h 1 on Caco-2 cells and speculated that Ara h 1 possessed cytotoxic properties.Further, Price et al. (Price et al., 2014) demonstrated that the peanut allergens Ara h 1 and Ara h 2 were capable of interacting with the membrane of Caco-2 cells, to decrease the TEER, and promote changes in tight junction protein localisation, suggesting the induction of disrupted barrier integrity, and demonstrated that the allergens were transported through the Caco-2 cell monolayer by transcytosis.Later they showed that the transcytosis of the peanut allergens occurred by endocytotic pathways via engulfment and internalisation of the allergens into early endosomes, trafficked to late endosomes, from where they were either released into the lamina propria or trafficked into lysosomes for endolysosomal degradation (Price et al., 2017).In both studies they observed different capacity of the Ara h 1 and Ara h 2 allergens to cross the epithelial monolayer (Price et al., 2014(Price et al., , 2017)).In fact, they demonstrated that while Ara h 1 was mainly transported by clatherin-mediated endocytosis and to a lesser degree by macropinocytosis, Ara h 2 was transported by caveolaemediated endocytosis and macropinocytosis (Price et al., 2017).This highlights that allergens may be transported by different mechanisms dependent on their physicochemical properties.
Processing of foods by heat treatment, such as baking, cooking, roasting and pasteurisation can affect the properties of the proteins and thereby affect the uptake (Besler et al., 2001).Heat treatment may for example cause conformational changes, such as denaturation and aggregate formation, affecting their digestibility and route of uptake (Graversen et al., 2020b;Rahaman et al., 2016).Graversen et al. (Graversen et al., 2020b), showed that denaturation and aggregation induced by heat treatment of cow's whey affected the route of uptake in an animal model with less whey being absorbed through the epithelial layer covering the lamina propria and more being absorbed through the PPs.This change in uptake was suggested to contribute to the reduction in allergenicity seen upon the heat treatment (Graversen et al., 2020b).Similarly, heat treatment of the hen's egg allergens Gal d 1 and Gal d 2 was shown to prevent their transport across the epithelial cells (Martos et al., 2011).Processing of foods by heat treatment may also lead to induction of Maillard reactions, resulting in formation of glycated proteins, which may also affect protein digestibility and route of uptake (Perusko et al., 2018).For example, in a study by Perusko et al. (Perusko et al., 2018), it was found that glycation of the cow's milk allergen BLG reduced its transport through epithelial cells and increased their endolysosomal degradation, however, whether changes in epithelial transport and degradation were a result of the structural changes or the chemical changes in BLG, or a combination of these can only be speculated.This clearly shows that the structural or chemical modifications induced upon heat treatment may prevent the transport across the epithelial cells, probably redirecting the uptake to PPs and modifying the degradation of the allergens inside the epithelial cells, correlating well with the reduced allergenicity most often seen after heat treatment of hen's egg and cow's milk (Lemon-Mulé et al., 2008;Lieberman et al., 2012).
Non-thermal processing techniques may also be used to obtain desirable techno-functional properties of food, where for example enzymatic cross-linking of proteins has been exploited as an alternative to conventional processing techniques (Ahmed et al., 2021a).Enzymatic cross-linking modifies the structural and biochemical properties of proteins and induces formation of high MW polymers, altering the allergenicity of food proteins.However, while some studies demonstrated reduced allergenicity upon enzymatic cross-linking (Ahmed et al., 2020(Ahmed et al., , 2021b) ) another study revealed an increase in allergenicity (Stojadinovic et al., 2014).The impact of enzymatic cross-linking on the epithelial uptake has been explored in a few studies.For example, in a study by Stojadinovic et al. (Stojadinovic et al. 2014), it was shown that aggregation of the cow's milk allergen BLG by means of protein crosslinking promoted the uptake by PPs while reducing the uptake through epithelial cells and further revealed changed susceptibility of BLG to endolysosomal degradation.In another study, it was shown that the epithelial transport of allergens could be increased by enzymatic cross-linking but that the degree was dependent on the type of enzymatic cross-linking and the allergens being enzymatically cross-linked (Radosavljevic et al., 2014).
Generally, we do not eat pure proteins, and the matrix in which the proteins are embedded may also have an impact on the absorption of proteins across the epithelial layer (Schulten et al., 2011) and is an important aspect to investigate.Lipids may affect the allergen transport across the intestinal barrier (Bublin et al., 2014), which have been explored in a few studies.For example, in a study by Angelina et al. (Angelina et al. 2016), it was shown that the binding of the peanut allergen Ara h 1 and the mustard allergen Sin a 2 to lipids reduced their uptake by DCs as well as their susceptibility to endolysosomal degradation, suggesting that the lipid-binding capacity of these allergens contributes to their allergenic potency.Similarly, in an animal study, it was found that addition of lipids to the soybean allergen Gly Bd 30 K, promoted increased allergen absorption, which was suggested to be a result of enhanced uptake by fat carrier-mediated transport (Weangsripanaval et al., 2005).
Intestinal permeability and protein uptake are not only affected by the proteins or peptides themselves as well as the matrix in which they are embedded, but environmental and lifestyle factors may as well promote a more susceptible intestinal barrier and influence the uptake of proteins.

Dietary components
An important factor in the regulation of the intestinal barrier function is the diet.During the last decades, the dietary intake of processed foods and premade meals has increased, where additives such as salt, organic acids, surfactants, and fatty acids may be present, which have been shown to promote intestinal barrier disruption and increase the intestinal permeability (Glynn et al., 2017;Lerner & Matthias, 2015).The synthetic surfactants polyoxyethylene stearate and sodium cholate are examples of food additives demonstrated to increase paracellular permeability in Caco-2 cells (Zhu et al., 2009;Yu et al., 2013).Polyoxyethylene stearate was found to inhibit P-gp activity, a membrane protein known to pump xenobiotic back to the intestinal lumen (Zhu et al., 2009), whereas sodium cholate increased intestinal permeability shown by increased transport of FITC and FITC-dextran and disruption of cell junctional complexes such as ZO-1, claudin-1, occludin and Ecadherin (Yu et al., 2013).
Numerous naturally occurring dietary components have been shown to affect the permeability of the intestinal barrier (Lerner & Matthias, 2015;Ulluwishewa et al., 2011).For example, in a study conducted by Hashimoto et al. (Hashimoto et al., 1994), 32 different vegetable extracts were screened in relation to their effect on intestinal permeability using Caco-2 cells.They found that sweet pepper influenced intestinal permeability by decreasing TEER.In line, paprika and cayenne pepper, as well as the active compound in these spices, capsaicin, were found to increase intestinal permeability in HCT-8 cells shown by increased transport of FITC-dextran and disruption of ZO-1, whereas black and green pepper, and the active compound in these spices, piperine, revealed opposite effect in the human cell line and decreased intestinal permeability (Jensen-Jarolim et al., 1998).Furthermore, in a large screening study, 300 dietary substances were evaluated in relation to their effects on intestinal permeability using Caco-2 cells, where extracts of galangal, marigold, Nikko maples and hops were shown to increase intestinal permeability by decreasing TEER and increase transport of Lucifer Yellow (Konishi, 2003).
A group of compounds has been extensively studied in relation to their health related effects, namely the polyphenols (Zhang, 2015).Polyphenols consist of phenolic acids and flavonoids and are present in many plants such as cereal grains, legumes, and vegetables as well as in tea and wine (Manach et al., 2004).As part of the health-related effects, polyphenols have in some studies been shown to cause an effect on intestinal permeability.For example, it was shown that the polyphenols gallic acid and epigallocatechin gallate, usually found in some vegetables, grapes, pomegranates and in green tea, in combination with xanthan-chitosan nanofibers, increased intestinal permeability in Caco-2 cells (Faralli et al. 2019).In contrast, it has been shown that aronia berry powder, known to contain high amount of polyphenols, prevented disruption of the barrier functions in Caco-2 cells when evaluated by TEER measurements, transport of FITC-dextran and Lucifer Yellow, and changes of ZO-1 and occludin (Valdez et al., 2020).Overall, several studies have demonstrated effect of polyphenols on intestinal permeability, however, with conflicting results (Kosińska & Andlauer, 2013).
Other naturally occurring substances that have been investigated for their membrane disrupting effect are saponins, which are found in high amounts in foods such as ginseng, soybean, lentils and quinoa seeds (Oleszek & Oleszek, 2020).Saponins were shown to increase intestinal permeability in rat intestinal tissue when studied by the everted sac method (Gee et al., 1989(Gee et al., , 1993;;Johnson et al., 1986), and saponins derived from quinoa seeds were also shown to increase intestinal permeability in rats after oral exposure evaluated by i.a.intestinal transport of BLG (Ballegaard et al., 2021).Saponins derived from for example quinoa seeds and quillaja have been found to possess adjuvant capacity, probably due to the ability to increase intestinal permeability (Estrada et al., 1998;Verza et al., 2012).
Food cysteine proteases, such as the kiwifruit allergen actinidin, have been reported to disrupt the intestinal barrier function and increase intestinal permeability through degradation of tight junction proteins, in both mouse models, human HEK-293 cells and Caco-2 cells.The intestinal disruption capacity has been suggested to be the cause of their high sensitising capacity (Grozdanovic et al., 2016;Nešić et al., 2019a,b).Disruption of the intestinal barrier by food proteases may additionally facilitate sensitisation towards other allergens.

Chemical substances
Humans are daily exposed to a wide range of chemical substances from the environment, which may affect epithelial barriers such as the skin, the respiratory or the intestinal barrier.For example, the chemical surfactant and antibacterial agent, benzalkonium, found in cosmetics and soaps, has been shown to disrupt the barrier function and increase paracellular permeability, demonstrated by its capacity to decrease TEER and disrupt the actin filament structure in the human intestinal Caco-2 cells (Narai et al., 1997).Examples of chemicals migrating from plastics into food and beverages have been described in relation to allergy (Benjamin et al., 2017;Kwak et al., 2010;Yu et al., 2018).An example is bisphenol A (BPA) which is mainly studied in relation to endocrine disrupting effects, but has also been found to increase intestinal permeability (Feng et al., 2018;Misme-Aucouturier et al., 2022), and significantly reduce TEER in Caco-2 cells (Nanayakkara et al., 2021), as well as interrupt development of oral tolerance in mice (Ménard et al., 2014;Nygaard et al., 2015) and to promote elevated IgE levels and anaphylaxis in mice (Misme-Aucouturier et al., 2022).Increased usage of plastics has raised the risk of microplastics entering the food chain, either as primary microplastics in personal products as e. g. toothpaste, or as secondary microplastics in e.g.seafood, where fish and shellfish have ingested microplastics from the environment (Cole et al., 2011;van Wezel et al., 2016).A study including in vitro analyses with Caco-2 cells and THP-1 macrophages, as well as an in vivo study using mice revealed that a minor fraction of microplastic particles are taken up by intestinal cells (Stock et al., 2019).Further, several studies have shown that microplastics can increase intestinal permeability, shown by reduction in cell junctional complex protein expression or reduced mucus secretion (de Souza-Silva et al., 2022).However, the influence on the intestinal cells has not yet been elucidated in humans.

Exercise
It is well-acknowledged that exercise increases intestinal permeability (Chantler et al., 2021;Karhu et al., 2017), with the responsible underlying mechanisms being manifold, including sympathetic nervous system activation and decreased gut perfusion, contributing to epithelial injury and alterations in cell junctional complexes (Chantler et al., 2021;van Wijck et al., 2012).A study by Karhu et al. (Karhu et al., 2017), found that running significantly increased intestinal permeability and serum I-FABP concentration unaffected of whether the athletes had previously experienced GI symptoms during training.An example of a known disorder, where it has been hypothesised that increased intestinal permeability caused by exercise is a major contributor, is wheatdependent exercise-induced anaphylaxis (WDEIA) (Scherf et al., 2016).As the name indicate, WDEIA usually develops after intake of wheat followed by physical activity.Besides exercise, alcohol, medication and stress have as well been described in relation to increased intestinal permeability and as co-factors in food allergic reactions (Muñoz-Cano et al., 2021;Wölbing et al., 2013).

Alcohol
Chronic alcohol consumption has been shown to be associated with increased intestinal permeability (Beck & Dinda, 1981;Parlesak et al., 2000).The effects of alcohol have in many cases been shown to be a consequence of alcohol metabolised by-products such as acetaldehyde and stimulation of inducible nitric oxide synthase (Atkinson & Rao, 2001;Forsyth et al., 2011;Rao, 1998;Zakhari, 2006).For example, acetaldehyde has been shown to affect paracellular permeability in Caco-2 cells measured by reduced TEER and elevated transport of mannitol (Rao, 1998).As described in section 7.4., alcohol is a co-factor suggested to play a role in food allergy (Muñoz-Cano et al., 2021), and have been shown to promote allergic reactions in humans (Alcoceba Borràs et al., 2007;Versluis et al., 2019).

Medication
Drugs such as antacids, proton pump inhibitors and H 2 blockers, increase the gastric pH and hence may reduce protein digestion (Dallas et al., 2017).This will consequently impact on the size and shape of the proteins and peptide fragments available for uptake through the epithelial layer, and in the end the amount and route by which they are absorbed.Use of drugs that increases gastric pH, has been shown to have an effect on food allergy development as described by Untersmayr et al. (Untersmayr et al., 2003), where anti-ulcer drugs turned digestion-labile fish proteins into potent food allergy elicitors.This has additionally been addressed in a human cohort, where patients revealed changes in the food-specific IgE antibody pattern after three months of treatment with anti-ulcer drugs (Untersmayr et al., 2005).Drugs such as acetylsalicylic acid and other non-steroidal anti-inflammatory drugs (NSAIDs) have been described as co-factors in relation to food-dependent exerciseinduced anaphylaxis (FDEIA) (Harada et al., 2001;Scherf et al., 2016).Acetylsalicylic acid affects the cell junctional complexes and may thereby lead to increased intestinal permeability (Lambert et al., 2001).

Microbiota
Several studies have suggested that the above mentioned factors may not only exert a direct effect on intestinal permeability and protein uptake but may also have an indirect effect exerted through changes in the community of the gut microbiota (Hussain et al., 2019;Moreira et al., 2012).It is well-acknowledge that the microbiota composition plays an important role in the establishment and maintenance of the gut integrity homeostasis.In an animal study, the gut microbial community was disrupted in rats by the antibiotic amoxicillin, which induced an increased intestinal permeability demonstrated with a higher protein uptake of BLG in the lamina propria and in serum when compared with rats dosed with a saline solution (Graversen et al., 2020a).In another study, the impact of the microbiota composition on the intestinal integrity was investigated in a mouse model of food allergy, where a high Clostridia-containing microbiota induced IL-22 expression in lamina propria lymphocytes which reinforced the epithelial barrier and thereby decreased the intestinal permeability.This was measured by uptake of the peanut allergens Ara h 2 and Ara h 6, where Clostridiacolonised mice had lower levels of circulating allergens than germ free mice and B. uniformis, colonised mice (Stefka et al., 2014).
A key player in the colonic mucus-associated microbiota, Akkermansia mucinphila, which colonises the gut of a considerable part of the human population (Derrien et al., 2008), has been found to be involved in gut mucosa homeostasis and improvement of barrier integrity by increasing TEER in Caco-2 cells (Ottman et al., 2017).Another example of a protective effect of bacteria, is the probiotic Lactobacillus rhamnosus GG (LGG) originally isolated from the intestinal tract of a healthy subject.
LGG was found to possess a protective role on intestinal barrier function based on investigation in organoids incubated with faecal supernatants from irritable bowel syndrome patients.Supplementation with the probiotic was seen to normalise tight junction protein gene expression compared with organoids not supplemented with the probiotic (Han et al., 2019).
The microbial-derived metabolites, short-chain fatty acids (SCFA), have been found to influence the mucosal immune function and increase intestinal barrier function (Morrison & Preston, 2016;Wang et al., 2012).The SCFA butyrate was for example demonstrated to improve the intestinal barrier in relation to mucin production and tight junction reassembly (Ma et al., 2012;Miao et al., 2016;Willemsen et al., 2003).Thus, different bacteria and their metabolites may exert various effects on intestinal integrity and the microbiota composition therefore plays a pivotal role in maintaining gut homeostasis.
Several factors have been shown to induce an indirect effect on the intestinal barrier and protein uptake exerted through the changes they promote on the intestinal microbiota community.For example, Hussain et al. (Hussain et al., 2019), showed that a high-fat diet induced a decrease in gut microbiota diversity, which again promoted reduced barrier integrity measured by increased transport of FITC-dextran and changes in tight junction proteins in a mouse model of food allergy.These changes were suggested to increase the susceptibility to food allergy (Hussain et al., 2019).Also, dietary fibres have an impact on the gut microbiota that may lead to changes in the intestinal barrier (Alemao et al., 2021).It has for example been shown that dietary fibre deficiency results in the enrichment of mucus-degrading bacteria with mucus glycoproteins being a nutritional source instead of the dietary fibres, with consequential erosion of the mucus barrier leading to intestinal barrier dysfunction (Desai et al., 2016).Opposite, high-fibre intake and increased SFCA production promote a healthy intestinal barrier (Alemao et al., 2021), where it for example was shown with the use of Caco-2 cells that SCFA stimulated tight junction protein formation and increased TEER, resulting in increased epithelial barrier function (Feng et al., 2018).
Other examples relate to environmental factors, where a study on BPA and nanoparticle exposure in zebrafish demonstrated that exposure to these environmental substances induced a shift in the microbiota community in a dose-dependent manner, which led to increased intestinal permeability (Chen et al., 2018).Further, in a mouse study investigating the impact of exposure to air pollution particular matter, it was found that the inhalant exposure caused changes in gut microbiota with concomitant increase in intestinal permeability associated with disruption of tight junction proteins.It was suggested that in addition to a direct effect of the particular matter on intestinal permeability, also an indirect effect via the altered gut microbiota was a contributing factor in the increased intestinal permeability (Mutlu et al., 2018).Similarly, in another mouse study it was shown that air pollution particulate matter ingested via contaminated food could alter gut microbiota community and function, which was coherent with changes in the intestinal barrier function (Kish et al., 2013;Salim et al., 2013).

Discussion
In recent decades a great increase in allergic diseases, including food allergy, has been described, in particular, in Western countries (Kotz et al., 2011;Loke et al., 2016;McWilliam et al., 2015;Osborne et al., 2011;Sasaki et al., 2018).The 'barrier hypothesis' described by Pothoven and Schleimer, asserts that epithelial barrier dysfunction, which may result in excess exposure to the environment, can promote allergic sensitisation and the development of allergic diseases (Pothoven & Schleimer, 2017).An extension of this hypothesis, described by Akdis, argues that environmental changes caused by industrialisation, urbanisation and an altered lifestyle in Western countries affect the intestinal integrity and underlie the increase in allergic diseases (Akdis, 2021).That disruption of the intestinal barrier integrity, with a consequential increase in allergen transport through the gut epithelial layer, is a contributing factor in the observed increase in food allergy prevalence, and that the intestinal barrier integrity is affected by environmental and lifestyle factors correlate well with the outcome of the present review.Indeed, this review suggests that an increased intestinal permeability and an effective transport of immunologically active allergens are important factors in food allergy development and that a great variety of environmental and lifestyle factors attributed to Western countries may affect both the intestinal integrity and allergen uptake.
Intestinal integrity and protein absorption across the epithelial layer may undoubtedly be affected at several levels, as a broad array of mechanisms contribute to a healthy intestinal barrier function, including components in the physical barrier, constituted by the cells of the epithelial layer, their interconnection, and the molecules that they secrete, and components in the immunological barrier, constituted by various immune cells and the molecules that they secrete.Hence, it is a complex and multifaceted interplay between various components in the intestine that safeguard the delicate balance between sampling of immunological active proteins and peptide fragments for maturation and continued training of the immune system, including induction of oral tolerance, and the avoidance of allergen entrance for prevention of sensitisation and elicitation of allergic reactions.Yet, how intestinal permeability and allergen uptake exactly affect this delicate balance seem underexplored and warrant further investigations.
Indeed, increased intestinal permeability has been associated with food allergy, and mechanisms involved in the decreased intestinal barrier integrity seen in food allergic individuals upon elicitation of allergic reactions, have been studied, demonstrating that both facilitated transport via the IgE/CD23 complex and increased paracellular transport are mechanisms involved (Perrier & Corthésy, 2011;Yu, 2009).However, if increased intestinal permeability is a prerequisite for food allergy sensitisation through the GI tract and if certain specific mechanisms are involved remains to be fully established.In other diseases where the intestinal barrier integrity is compromised, several mechanisms of disrupted intestinal barrier have been demonstrated to be contributing factors of the disease pathogenesis.For example, in inflammatory bowel disease (IBD), increased epithelial cell dead, alterations in tight junctions, modified mucus configuration and changed expression of microbial peptides are all components involved (Holmberg et al., 2018).As the disease pathogenesis in food allergy may not only relate to the intestinal integrity as such, but rather to the transport of immunological active allergens or peptide fragments, how the allergens are transported across the epithelial layer is of particular interest for future studies.
As the majority of proteins and peptide fragments large enough to induce an immunological response are transported across the epithelial layer by transcytosis, a better understanding of the association between the specific mechanisms involved in the transcytosis of allergens and the risk of sensitisation is of great importance.Especially, it remains to be fully resolved if allergens possess specific properties allowing them for protected transport without or with limited endolysosomal degradation as only few studies are currently available.If facilitated transport plays a role in the development of food allergy remains to be explored.As transport facilitated by the IgE/CD23 complex should be irrelevant without prior sensitisation, facilitated transport by means of other mechanisms should be explored.As evidence has emerged on facilitated transport mediated by IgA, allowing retrotransport of antigens, and its involvement in diseases associated with intestinal disorders (Corthesy, 2007;Mathias et al., 2014;Matysiak-Budnik et al., 2008;Ménard et al., 2010;Moura et al., 2001;Rochereau et al., 2015Rochereau et al., , 2021)), the role of IgA in food allergy beyond immune exclusion in the intestine would be highly relevant to explore.Not only the mechanisms of allergen uptake but also the role of the specific cells by which the allergens are transported may be of importance and require more knowledge.Indeed, conflicting evidence exist if e.g.transport through M cells and facilitated transport by MHC II promote oral tolerance or sensitisation (Graversen et al., 2020b;Reitsma et al., 2014;Roth-Walter et al., 2008;Heuberger et al., 2020).
Investigating the role of intestinal allergen absorption in food allergy sensitisation and oral tolerance induction, and especially the impact of route and mechanisms of uptake, may create a better foundation for developing new and improved strategies for prevention and treatment of food allergy.This may allow for strategies targeting components involved in intestinal barrier integrity, for either prevention or restoration of intestinal barrier disruption, as well as targeting allergen transport.Whereas studies investigating therapeutic strategies targeting prevention or restoration of intestinal barrier disruption are few, most studies in therapeutic strategies targeting intestinal barrier integrity, have contrarily concerned intestinal disruption for drug delivery (Akdis, 2021;Artursson & Knight, 2015;Bischoff et al., 2014).
More knowledge on the impact of intestinal allergen transport on the development of food allergy may not only be of importance for new prophylactic and therapeutic strategies but may also be used in the prediction of food protein allergenicity.With the great focus on introducing new and sustainable foods and alternative proteins to alleviate climate changes and to meet the growing world population, it is particularly pertinent to involve tools allowing assessment of downstream events of food sensitisation, enabling the prediction of the capacity of new food proteins to induce de novo sensitisation (Mullins et al., 2022).Indeed, recently an adverse outcome pathway (AOP) framework for food allergy sensitisation was proposed allowing the identification of major initiating and key events underlying food allergy sensitisation, where the first events relate to allergen absorption through the intestinal barrier.It is proposed to investigate the activation of the epithelial cells, including tight junction disruption, receptor-mediated endocytosis and unspecific endocytosis (Lozano-Ojalvo et al., 2019a;van Bilsen et al., 2017).Hence, assays allowing for investigation of the impact of allergens on epithelial integrity and allergen transport may be used in future assessments for prediction of allergenic potential of new foods before entering the market.However, before an AOP for food allergy sensitisation can be applied in practice more knowledge on allergen absorbance in the intestine and its association to sensitisation is required, with subsequent validation of methods allowing for investigation of the key events related to the impact of allergens in the intestinal integrity as well as allergen uptake.
Techniques for assessment of intestinal integrity and allergen absorption have been employed for the last four decades.Yet, most studies have been based on simple cell lines derived from human colonic carcinoma that do not resemble the physiological relevant complex environment of the intestine constituted by various epithelial cells, immune cells as well as blood circulation contrary to ex vivo and in vivo techniques.Whereas the use of in vitro as well as ex vivo methods do not allow for a direct comparison and linking of the barrier integrity and allergen absorption to the development of food allergy or allergic reactions, studies in animal models and humans allow for such association.However, more studies investigating a direct link between intestinal permeability and allergen uptake with food allergy sensitisation in settings resembling the complex environment of the intestine calls for development and validation of more advanced in vitro techniques.More sophisticated in vitro techniques, devoid use of animals, are currently being investigated, such as microfluidic gut-on-a-chip and organoids (Puschhof et al., 2021), which may accommodate future studies in barrier function, intestinal permeability, and protein uptake, and their impact on food allergy.
What are the factors underlying the increased intestinal permeability, suggested by Akdis (Akdis, 2021) to result from changes in A.-S.R. Ballegaard and K.L. Bøgh environmental changes, due to industrialisation and urbanisation, and an altered lifestyle attributed to Western countries?As demonstrated by present review these factors are manifold.We have presented a nonexhaustive list and description of factors that, beside properties of the allergens themselves, may impact on the intestinal barrier and/or uptake of immunologically active food allergens, ranging from food processing as well as matrix and other dietary components, such as surfactants, saponins and polyphenols, to chemical substances form the environment, such as BPA and microplastics, and lifestyle related factors, such as exercise and alcohol.In addition to the direct effect of environmental and lifestyle factors on intestinal barrier integrity and allergen uptake, also a direct or indirect effect may be exerted by the gut microbiota.This combines very well the hypothesis by Akdis (Akdis, 2021), with previous hypotheses relating the increase in food allergy to changes in the gut microbiota composition (Haahtela et al., 2013;Rook et al., 2004;Strachan, 1989).Even though many environmental and lifestyle factors have been found to affect intestinal permeability and allergen uptake, we have yet only scratched the surface.There is a great need to investigate a broader panel of environmental and lifestyle factors that may be suspected to impact on the intestinal barrier function and hence allergen uptake, to understand to which extent, at which exposure levels and by which mechanisms these factors contribute to an intestinal barrier dysfunction and the development of food allergy.Further, it seems that studies so far have merely investigated the effect attributed by individual environmental and lifestyle factors rather than investigated the cocktail effect.As we are not only exposed to a single environmental or lifestyle factor at the time, there is an unmet need to investigate mixtures for defining the impact of a cocktail effect.

Conclusion
With the present review we provided an overview of digestion, intestinal barrier function and dietary protein uptake in the GI tract, how this may be affected by various intrinsic and extrinsic factors, and the impact on food allergy.The faith of the dietary proteins from they reach the mouth until they are taken up and transported across the intestinal epithelial layer may be manifold, and is dependent on the complex interplay between digestion, intestinal barrier function, protein uptake and the immune regulation (Fig. 1).This complex interplay is affected by various stimuli originating from the host, the proteins themselves, or from the external environment, and will impact on a potential food allergic response mounted after allergen exposure.Whereas the effect on the allergy sensitisation phase is only scarcely described, more is known about the implication it may have on the allergy elicitation phase.Yet, as no curative treatment is available for food allergic individuals, and as immunotherapy is only available for peanut allergy, the only viable management option is presently strict allergen avoidance.However, as our understanding of the mechanisms at play in transport of proteins and peptides across the intestinal epithelial layer increases, new prophylactic and therapeutic approaches may emerge.To this end, the topic of the review warrants further research and attention.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.Illustration of factors affecting food allergy development.Lifestyle and environmental factors, such as diet, exercise, alcohol, pollution, tobacco or drugs may impact on digestion, protein uptake and immune regulation, as well as their complex interplay, and hence influence food allergy development.The effect of lifestyle and environmental factors may either be a direct effect (solid arrows), or an indirect effect exerted through the modulation of the gut microbiota (stippled arrows).Figure created with BioRender.com.

Fig. 2 .
Fig. 2. The pathogenesis of IgE-mediated food allergy divided into the sensitisation and elicitation phases.A dendritic cell internalises and processes the allergen for presentation via major histocompatibility complex (MHC) class II to the T cell receptor (TCR) of a naïve T cell.The naïve T cell differentiate into an active T helper (Th) 2 cell after proper signals from CD80/86 ligation of CD28 and co-stimulatory molecules such as interleukin (IL)-4, thymic stromal lymphopoietin (TSLP), IL-33 and IL-25.The activated T cell contributes to the Th2 environment by secreting IL-4, IL-5 and IL-13(Divekar & Kita, 2015;Hussain et al., 2018;Tordesillas et al., 2017;Yu et al., 2016).The differentiated Th2 cell further activates a naïve B cell via CD40L-CD40 ligation which leads to proliferation and differentiation of the naïve B cell into plasma cells secreting high amounts of allergen-specific IgE(Stone et al., 2010).Specific IgE binds to their high affinity receptors FcԑRI on mast cells resident in tissue or basophils present in the blood.This phase is designated as sensitisation(Sutton & Gould, 1993).Upon re-exposure to allergens, cross-linking of FcԑRI-bound IgE on the effector cells leads to mediator release, which gives rise to the symptoms characterising food allergy, and is defined as the elicitation phase(Stone et al., 2010).Figure created with BioRender.com.

Fig. 4 .
Fig. 4. Intestinal epithelial layer.Intestinal epithelial cells serve as a physical barrier that segregates the internal milieu from the luminal content, such as the microbiota.A single layer of different epithelial cells covers the lamina propria, where the most abundant cell type is enterocytes.Scattered between the enterocytes are mucus-producing goblet cells, hormone-releasing enteroendocrine cells, and in the crypts are the Paneth cells producing anti-microbial peptides and the base stem cells giving rise to the other epithelial cell types.Throughout the small intestine, lymphoid follicles are present as Peyer's patches.Specialised epithelial microfold (M) cells are located in the Peyer's patches and play a pivotal role in antigen uptake.The immunological barrier involves various immune cells resident in the intestinal epithelium and subepithelial area, such as dendritic cells (DC), T cells and B cells.Secretory IgA (sIgA) is the main isotype of immunoglobulins found in the gut mucosa and exerts a crucial role in protecting the intestine from pathogenic attack and averts antigens from being taken up.Figure created with BioR ender.com.

Fig. 5 .
Fig.5.Routes of intestinal protein uptake.Proteins and peptide fragments are transported through the epithelial layer by means of several routes; either being transcellular or paracellular.Proteins and peptides may cross the epithelial layer by different routes through the enterocytes.Amino acids, di-and tripeptides may cross the epithelial layer by simple diffusion (A) or carrier-mediated (B) transport, either in an energy dependent or independent way.Transcytosis may occur via endosomes, either in a protected way releasing the proteins and peptides in the same condition as they are taken up (C), or by the endolysosomal pathway where the proteins or peptides are degraded to smaller peptides or amino acids during the transport (D).Proteins and peptides may also cross the epithelial layer by facilitated transport, mediated either by the binding to MHC II (E) or Ig (F).Transcellular transport can also include uptake by M cells located in the Peyer's patches (G), which are specialised in sampling of larger particular substances, or through goblet cells (H) or by dendritic cells (I) sampling antigens directly from the lumen.Small molecules can pass between the epithelial cells via the paracellular route (J).The space between epithelial cells is highly regulated by cell junctional complexes, composed of tight junctions (claudin, occluding and ZO), adherence junctions (cadherin and catenins), desmosomes and gap junctions (connectin), restricting the pathway in healthy individuals.Figure created with BioRender.com.