Antigen sampling by M cells is selective. Typically, this is the only means by which luminal antigens and macromolecules can cross the intestinal epithelia and are subject to processing by the gut immune system. Antigens which are often insoluble and/or aggregated are endocytosed only via M cells [21]. Here, antigens encounter less protective mechanisms such as mucous and microvilli to be transported easier. Antigens absorbed by M cells are transported directly to the underlying- immune cell rich- lamina propria, with little to no antigen alteration before presentation to a professional antigen presenting cell.

Despite this, some macromolecules have shown the ability to cross the intestinal epithelium without M cell passage, including allergens from milk [22], brazil nuts [23], and egg [24], just to name a few. This can occur transcellularly, where antigens are absorbed directly through epithelial cells or paracellularly, where antigens pass through the tight junctions joining IEC (Fig. 2, a). For an in depth explanation of all modes of epithelial transport, Tukker [25] has reviewed the topic in greater detail, which is beyond the scope of this review.

IECs in vivo and in vitro express many elements involved in antigen processing. This indicates a role for IEC in antigen presentation, independent of M cell involvement. HLA-DR was shown to be expressed in fetal intestinal epithelium from 18 weeks gestation, specifically at villi tips. This same study identified interferon gamma (IFN-γ) as the stimulant for HLA-DR expression by HT29 IEC in vitro [26]. IFN-γ has also shown to induce the expression of MHC class II in another IEC line Caco-2, further suggesting the role of IFN-γ secreting T-lymphocytes as the stimuli for MHC-II expression by IEC [27]. More recently, MoS13 IECs treated with IFN-γ were able to stimulate proliferation of CD4⁺ intestinal epithelial lymphocytes after antigen exposure; though not to the capacity of professional antigen presenting cells [28]. Despite this, levels of IFN-γ secreted by the CD4⁺ intestinal epithelial lymphocytes was significantly higher when in the presence of IEC compared to professional antigen presenting cells, where the increase in IFN-γ secretion was unique to intestinal epithelial CD4⁺ lymphocytes and not those found elsewhere in the body [28]. In addition to MHC-II expression and IFN-γ secretion by IEC, many additional antigen processing components have been identified in the IEC line HT29, which were comparable to those expressed in human small intestinal biopsy tissue [29]. Other studies have demonstrated similar results regarding IEC capability to secrete exosomes with antigen presenting components, with the potential capacity for antigen presentation to downstream immune cells [29-31], given that IEC have the capacity to absorb and process antigens. It is of particular interest to summarize any food allergens that have been observed to cross IEC, either paracellularly or transcellularly, independent of M cells.

Paracellular transport is directly related to the integrity of the intestinal epithelium and is controlled primarily by the tight junction (Fig. 2, a). Although transport via this way may not involve antigen processing by the IEC as such, IEC transport may influence the quantity of allergen passing through to the lamina propria. The tight junctions are located on the apicolateral cell membrane, and attach laterally to neighbouring cells. Thus, tight junctions are directly involved in the permeability and polarity of the intestinal epithelium, as their main function is to exclude luminal contents. Thus, any antigen that can cross here will be directly exposed to underlying immune cells in the lamina propria, effectively triggering the allergenic cascade, in an uncontrolled manner.

To explore the role that tight junctions play in disorders that alter intestinal permeability, including food allergy, we must first gain a brief understanding about the tight junction structure (Fig. 3).

The tight junction is comprised of two main units. Transmembrane protein complexes which span the paracellular space, and intracellular protein complexes which bind the structural elements of the cytoskeleton; such as actin. The transmembrane complexes are comprised of occludin [32], junctional adhesion molecule (JAM) [33] and members of the claudin family, which differ with tissue type [34]. Those proteins which are involved in the joining of the transmembrane proteins to the cytoskeleton, include zonula-occludins (ZO)-1 [35, 36], ZO-2 [36, 37], and ZO-3 [38], just to name a few. There are also numerous other proteins, which have been observed to localise to the tight junction, and are involved in cytoskeletal adhesion, but will not be explored in this review. They are however, adequately descried in several recent reviews by Niessen [39] and Gonzalez-Mariscal et al. [40].

The malfunctioning of tight junctions is associated with many diseases, including cancer, jaundice, edema, and colitis [41-44]. The role that the tight junction plays in these conditions is described in a review by Sawada et al. [45]. The function that the tight junction plays in the physiology of food allergy is less defined. Additionally, allergen paracellular IEC transport via tight junctions is scarcely addressed in the literature.

Experimental evidence for allergen transport across the intestinal mucosa is generally limited. However, a 2004 study by Chambers et al. [46], investigated the epithelial transport of peanut protein in BALB/c mice. This group observed both soluble protein and protein bodies in the cytoplasm of M cells within intestinal Peyer's patches. Interestingly, peanut bodies were also observed in the paracellular space between epithelial cells surrounding Peyer's patches but not in epithelial cells that were not adjacent to Peyer's patches [46]. This indicates the potential for peanut allergen paracellular passage, but also illustrates that peanut allergen IEC passage may be restricted to M cells only. Thus, more research is required to properly understand the transport of peanut allergens across the human intestinal mucosa.

The certain qualities of allergens that make them particularly more allergenic, for example heat resistance and digestion resistance, is reiterated with other potentially destructive qualities such as specific protease activity aimed at epithelial destruction. For example, although not a food allergen per se, the major house dust mite allergen Der p 1 effectively compromises the integrity of airway epithelium via tight junction destruction, resulting in the cleavage of occludin and increased paracellular permeability through a proteolytic mechanism [47]. This not only illustrates the invasive potential of successful allergens, such as those from dust mites, but also highlights the importance of the tight junctions in allergic disease.

Trancellular transport, or the movement of materials through epithelial cells, occurs either by simple diffusion (a), diffusion mediated by carrier proteins (b) or vesicular transport either by trancytosis (c), endocytosis (d) or active transport (e). For simple diffusion based transcellular transport, a compound is limited by its lipophilicity, solubility, concentration and absorption area. This could help to explain when the epithelial transport of the egg allergen ovomucoid was studied in Caco-2 cells, they were pre-treated with varying concentrations of food grade surfactants; the added surfactants effectively increased the permeability of the egg allergen when surfactant levels were < 50 µg/well or 25 µg/mL [24].

For more hydrophilic and poorly absorbed materials, carrier mediated transport is typically the main route. Carrier mediated transport can occur via diffusion, where, with the help of membrane bound carrier proteins, luminal particulates can be transported down its concentration gradient. Alternatively, this process can utilise a source of energy such as the conversion of adenosine triphosphate (ATP) to adenosine diphosphate to drive a protein pump, such as the sodium/potassium ATPase pump. This process can operate against a concentration gradient and is typically used for materials in very low external concentrations.

Endocytosis and transcytosis are two additional transport methods where external materials are engulfed by the cell membrane and enclosed and transported within vesicles within the cell. Generally, when analysing the transport method for various materials using cellular based models (i.e., Caco-2 monolayers), the transepithelial resistance (TEER) is used to address the integrity of the monolayer, where a change in TEER denotes alteration to tight junctions and thus relates to paracellular transport. Studies that exclude paracellular modes and thus accept transcellular modes, usually do so based on TEER and other markers for paracellular permeability. For example, purified wheat allergens ω5-gliadin and lipid transfer protein (LTP) were able to cross Caco-2 monolayers and detected by enzyme-linked immunosorbent assay (ELISA). Here, Caco-2 monolayer integrity was not compromised as no increase in permeability was observed via TEER or dextran flux. Also interestingly, digestion of the wheat allergens appeared to enhance their transcellular transport capacity [48], indicating the preferable uptake of digested allergens compared to whole allergens. It is likely that digestion may have rendered the allergen more soluble, allowing easier IEC absorption. Another study by Moreno et al. [23], reports the transcellular transport of purified 2s albumins Ber e 1 and Ses i 1 within Caco-2 monolayers, both of which are major brazil and white sesame seed allergens, respectively. Here, both Ber e 1 and Ses i 1 did not appear to affect permeability as observed with no change to allergen absorption rate, TEER or phenol red absorption. Considering this, we can assume that the peanut 2S albumins, Ara h 2, Ara h 6, and Ara h 7 [49] may have the potential to cross the epithelial barrier by similar means, due to conserved structural conformations. Recently, purified Ara h 2 has been observed to be endocytosed by HT-29 cells and targeted for lysosomal degradation after endosomal fusion, where the ubiquitin E3 ligase A20 is involved in its lysosomal degradation [50]. Also, the transport of the egg allergen ovalbumin is subject to its phosphorylation state, where it was found that apical to basal transport of dephosphorylated ovalbumin is reduced compared to its phosphorylated form, a result that is averted with the protease inhibitor leupeptin, suggesting lysosomal degradation during IEC transport [51]. Both of the above studies highlight the added importance of IEC digestion survival as an added element of allergen potency. Additionally of interest, the major soybean allergen, Gly m 1, has been shown to be endocytosed by IPEC-J2 cells in vitro [52].