The intrinsic physical properties of food allergens

The average daily intake of protein in the US diet is approximately 1 g per kilogram of body weight, and is derived from a great variety of mammals, birds, fish, fungi, and plants. Yet amidst this wide range, only a relatively small number of foods cause the vast majority of food allergies. This fact provides an important clue to the underlying pathophysiology of the disease and suggests that these main food allergens—milk, egg, wheat, soy, peanut, tree nuts, fish, and crustacea—though diverse in origin, share common characteristics that confer allergenicity. These characteristics include (1) a relatively small molecular weight, generally less than 70 kD; (2) an abundant source of the relevant allergen (eg, the seed storage proteins in nuts that are required to sustain plant growth); (3) glycosylation residues; (4) water solubility; and (5) resistance to heat and digestion. This combination of characteristics is likely unique to food allergens, which unlike inhaled or contact allergens, must pass through the harsh environment of the digestive system, beginning immediately upon entry into the oropharynx. Following ingestion, dietary proteins undergo digestion by enzymes in the saliva and stomach as well as by gastric acid. This processing results in reduced protein immunogenicity, likely by the destruction of conformational epitopes, ie, antigenic regions formed when noncontiguous amino acids are brought together by the tertiary folded structure of the protein. However, proteins that display these physicochemical properties resist this processing and thus have allergenic potential upon reaching the small intestine. Additional factors that disrupt normal digestion, such as coadministration of antacids, have been shown in animal models to result in a breakdown in oral tolerance induction.

Because food antigens are nonself proteins, they are recognized as such by the MALT, and as a result all normal individuals mount immune responses to ingested food proteins. However, once putative allergens survive the digestion process relatively intact, they must initiate a T _H 2 response to result in IgE production and disease expression. There has been a great deal of recent research interest in understanding the cellular and molecular basis of allergenicity, and what intrinsic signals derived from the proteins themselves lead to allergic priming. Whereas the operative allergenic motif has long been considered to be the protein or peptide epitope owing to its central importance in T-cell stimulation, recent studies have highlighted the emerging importance of the carbohydrate residues that decorate proteins and influence T _H 2 polarization. Studies of inhalant allergens have also identified the intrinsic protease activity of the proteins as well as their molecular resemblance to Toll-like receptor (TLR) ligands, but glycosylation appears to play a key role in allergenicity of dietary protein. This is not entirely surprising, given that the mucosal IgE system evolved to defend the host from intestinal metazoan parasites, organisms that are themselves heavily glycosylated.

Shreffler and colleagues demonstrated that the cell-surface receptor Dendritic Cell–Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), also known as CD209 DC-SIGN, mediates recognition of the major peanut allergen Ara h 1 by human DCs in vitro. DC-SIGN is a c-type lectin expressed on APCs that identifies conserved carbohydrate residues on pathogens and is generally considered to be a component of the innate immune system. Thus, the recognition of Ara h 1 by DC-SIGN is dependent on the carbohydrates in the peanut allergens, and furthermore this interaction is sufficient for DC activation and T _H 2 skewing of naïve human T cells. This may be one example of a broader effect, because other lectins besides DC-SIGN, such as the mannose receptor and Dectin-1 and Dectin-2, appear to play important roles in recognition of allergens and activation of APCs in murine systems. Interestingly, peanut allergens subjected to high-heat preparation (ie, dry roasting) undergo a nonenzymatic glycosylation reaction called the Maillard reaction. This increases the binding of IgE from peanut-allergic subjects and has been hypothesized to partially account for the disproportionate increase in peanut allergy in Westernized societies that consume roasted peanuts, as compared with other cultures with different preparation methods. Other major allergens, including those from egg, shrimp, milk, and red meats (as well as dust mite and other inhalant allergens), are also known to be heavily glycosylated.

The importance of timing and dose

Mucosal responses to soluble protein antigens early in life tend to be T _H 2 biased, which has led to the general idea that this occurs by default in both animals and humans. Genetics plays a clear role in mouse models, in which certain strains have exaggerated T _H 2 bias, whereas others tend to be resistant to sensitization, and although family studies suggest a strong genetic component in human food allergy, efforts have largely failed to identify risk alleles. More recent evidence supports that impairment in T-reg induction and innate immunity may also contribute to T _H 2 polarization in early life. Although these findings might be expected in atopic infants, prospective birth cohort studies have shown that IgE production to egg, milk, and peanut commonly occur, even in healthy infants. Importantly, although there remains some controversy, the inability of recent studies to identify a committed allergen-specific memory T _H 2 response early in life suggests that allergic responses develop postnatally. Several studies claimed to present evidence that allergic priming could occur in humans in utero, but these studies have largely been irreproducible, and methodologic flaws in their approach have been identified.

In nonallergic individuals, the T _H 2 bias appears to be transient, and IgE levels fall, possibly through a counterbalancing induction of antigen-specific T _H 1 responses (ie, interferon [IFN]-γ); in contrast, these T _H 2 responses consolidate and strengthen in allergic children, perhaps through induction of IL-4 signaling. IL-4 is known to be a key cytokine that acts as a critical step in the allergic cascade, signaling B cells to undergo class-switch recombination and begin producing IgE. A number of cell types produce IL-4, including T _H 2 cells, natural killer T (NKT) cells, and basophils; recent mouse studies have implicated basophils as a likely contributor of early IL-4 after allergen exposure. Interestingly, in a large cohort of children at high risk for allergy, CD25+ cells expressed significant amounts of IL-4 after stimulation with food allergens, but did not express significant amounts of the T _H 2-specific transcription factor GATA-3. This indirectly suggests that in humans, a CD25+ non–T cell (ie, a basophil) may play a role in early allergic sensitization. Identifying the source of IL-4 early in human allergen priming is a key research question.

In mouse models, high-dose exposure to antigen in early life, even a single isolated dose, can produce lymphocyte anergy, whereas low-dose exposure, especially when repeated, induces tolerance through T-reg development. Interestingly though, the differences in the actual dosing in these studies is quite small. Emerging evidence in human disease suggests that exposure to the proper dose of antigen during this critical period in early life is important for the shaping of the appropriate immune response to foods. Several epidemiologic studies have implicated delayed weaning patterns in the increased prevalence of peanut allergy. Similarly, there is evidence that delayed introduction of cereals is associated with a higher risk of wheat allergy, although methodologic limitations in retrospective studies make definitive conclusions difficult. Recently, European and American guidelines for the introduction of potentially allergenic solid foods were revised to reflect the position that insufficient high-quality evidence exists to support delayed weaning as a preventive (ie, tolerogenic) strategy. However, early introduction is not automatically better, because mature immune regulation may require time. In a set of classic experiments, Strobel and Ferguson showed that immunologic priming and allergic sensitization are enhanced in neonatal mouse pups fed the common experimental egg allergen ovalbumin in the first few days of life, whereas tolerance develops only after waiting 7 to 10 days to introduce antigen; if and how this “window” of priming versus tolerance translates to humans is unknown. Cow’s milk is typically the first potentially allergenic exposure, often occurring early in life, and yet cow’s milk is by far the most common food to which children are allergic. Although oral tolerance has been shown to occur across a range of doses, frequent or continuous exposure to relatively low doses typically results in robust oral tolerance induction. Defining the most appropriate time and dose for tolerance induction in humans is a great research need. Interventional studies are under way to investigate the importance of early life oral exposure in tolerance development.

The importance of timing and dose

Mucosal responses to soluble protein antigens early in life tend to be T _H 2 biased, which has led to the general idea that this occurs by default in both animals and humans. Genetics plays a clear role in mouse models, in which certain strains have exaggerated T _H 2 bias, whereas others tend to be resistant to sensitization, and although family studies suggest a strong genetic component in human food allergy, efforts have largely failed to identify risk alleles. More recent evidence supports that impairment in T-reg induction and innate immunity may also contribute to T _H 2 polarization in early life. Although these findings might be expected in atopic infants, prospective birth cohort studies have shown that IgE production to egg, milk, and peanut commonly occur, even in healthy infants. Importantly, although there remains some controversy, the inability of recent studies to identify a committed allergen-specific memory T _H 2 response early in life suggests that allergic responses develop postnatally. Several studies claimed to present evidence that allergic priming could occur in humans in utero, but these studies have largely been irreproducible, and methodologic flaws in their approach have been identified.

In nonallergic individuals, the T _H 2 bias appears to be transient, and IgE levels fall, possibly through a counterbalancing induction of antigen-specific T _H 1 responses (ie, interferon [IFN]-γ); in contrast, these T _H 2 responses consolidate and strengthen in allergic children, perhaps through induction of IL-4 signaling. IL-4 is known to be a key cytokine that acts as a critical step in the allergic cascade, signaling B cells to undergo class-switch recombination and begin producing IgE. A number of cell types produce IL-4, including T _H 2 cells, natural killer T (NKT) cells, and basophils; recent mouse studies have implicated basophils as a likely contributor of early IL-4 after allergen exposure. Interestingly, in a large cohort of children at high risk for allergy, CD25+ cells expressed significant amounts of IL-4 after stimulation with food allergens, but did not express significant amounts of the T _H 2-specific transcription factor GATA-3. This indirectly suggests that in humans, a CD25+ non–T cell (ie, a basophil) may play a role in early allergic sensitization. Identifying the source of IL-4 early in human allergen priming is a key research question.

In mouse models, high-dose exposure to antigen in early life, even a single isolated dose, can produce lymphocyte anergy, whereas low-dose exposure, especially when repeated, induces tolerance through T-reg development. Interestingly though, the differences in the actual dosing in these studies is quite small. Emerging evidence in human disease suggests that exposure to the proper dose of antigen during this critical period in early life is important for the shaping of the appropriate immune response to foods. Several epidemiologic studies have implicated delayed weaning patterns in the increased prevalence of peanut allergy. Similarly, there is evidence that delayed introduction of cereals is associated with a higher risk of wheat allergy, although methodologic limitations in retrospective studies make definitive conclusions difficult. Recently, European and American guidelines for the introduction of potentially allergenic solid foods were revised to reflect the position that insufficient high-quality evidence exists to support delayed weaning as a preventive (ie, tolerogenic) strategy. However, early introduction is not automatically better, because mature immune regulation may require time. In a set of classic experiments, Strobel and Ferguson showed that immunologic priming and allergic sensitization are enhanced in neonatal mouse pups fed the common experimental egg allergen ovalbumin in the first few days of life, whereas tolerance develops only after waiting 7 to 10 days to introduce antigen; if and how this “window” of priming versus tolerance translates to humans is unknown. Cow’s milk is typically the first potentially allergenic exposure, often occurring early in life, and yet cow’s milk is by far the most common food to which children are allergic. Although oral tolerance has been shown to occur across a range of doses, frequent or continuous exposure to relatively low doses typically results in robust oral tolerance induction. Defining the most appropriate time and dose for tolerance induction in humans is a great research need. Interventional studies are under way to investigate the importance of early life oral exposure in tolerance development.

Eluding the defenses

As we have seen, specific characteristics intrinsic to food proteins and the details of their exposure are important in determining the potential for inducing a deleterious allergic immune response. However, robust mechanisms exist within the host intestine to prevent would-be allergens from causing harm. The “first-line” features of mucosal defense serve to prevent luminal antigens from interacting with the MALT entirely. These include a hydrophobic layer of mucin oligosaccharides, which trap antigen, and both constitutive and inducible antimicrobial peptides. Secretory IgA has generally been considered to provide important tolerogenic function by binding to luminal antigens and preventing absorption (ie, “immune exclusion”), although its specific importance has been controversial. A recent study showed that mice deficient in the receptor that secretes IgA and IgM into the intestinal lumen are hypersensitive to IgG-mediated anaphylaxis; nonetheless, they can be tolerized by an oral feed before systemic priming. In this model, tolerance was transferrable by CD4+CD25+ splenocytes, suggesting that cellular mechanisms can compensate for an impaired immune exclusion mechanism. However, a recent case-control study from a larger placebo-controlled trial examining probiotics for allergy prevention in high-risk infants showed that the risk of atopy was inversely correlated with fecal IgA levels. These data serve as one example of the complex and complementary forces that act to suppress immunity in the gut.

If a potential allergen penetrates these first few physical factors, the intestinal epithelium itself acts as a barrier to sequester luminal antigens from the MALT, and leakiness of this barrier has been postulated to result in allergic sensitization. Structural integrity of the intestinal epithelium is conferred by epithelial junction complexes, also called adherens junctions, and tight junctions. However, it may take years for complete developmental maturation of the gut barrier in healthy children. In mice, the permeability of this barrier is further influenced by exposures to microbial pathogens such as viruses, alcohol, nonsteroidal anti-inflammatory drugs (NSAIDs), and other toxins, as well as cytokines such as IL-9, immune cells, and apoptotic pathways. These environmental exposures ultimately result in changes in gene expression and phosphorylation of tight junction proteins such as occludins, claudins, and JAM-ZO1 proteins, which in turn are associated with changes in intestinal mast cells and allergic sensitization.

Interestingly, intestinal permeability was assessed in food-allergic infants by examining the lactulose/mannitol ratio in urine, and these infants were noted to have increased intestinal permeability when compared with healthy young children. Investigators examined this ratio in children who had been on an allergen-free diet for at least 6 months and determined that intestinal permeability remained increased in food-allergic children, despite the absence of food allergen stimulation. Further evidence linking intestinal epithelial barrier dysfunction and food allergy comes from studies in immunosuppressed humans, who, after solid-organ transplantation, developed food allergy while on calcineurin inhibitors. Initially, investigators assumed this allergy was the result of transfer of sensitized donor lymphocytes. However, it is now theorized that medication-induced decreases in cellular adenosine triphosphate (ATP) levels altered the integrity of junctional complexes, resulting in increased intestinal permeability. Mutations in the gene encoding filaggrin also lead to profound epidermal barrier dysfunction and are highly prevalent in patients with atopic dermatitis, which is in turn associated with an increased prevalence of food allergy. Acquired barrier defects associated with decreased filaggrin expression have been observed in the esophagus of patients with eosinophilic esophagitis, and are thought to be downregulated secondary to IL-13. However, no studies to date have examined the mechanistic relationship of filaggrin mutations to IgE priming in the gut or clinical food allergy.

Increasing evidence suggests that the mucosal epithelium is likely to play an active role in determining the host response to food allergens, which goes far beyond simply acting as an inert physical barrier. Epithelial cells are known to express major histocompatibility complex (MHC) class II molecules on their basolateral membranes and thus may act as nonprofessional APCs that do not express conventional costimulatory molecules, favoring anergy or tolerance. In addition, factors derived from the gut epithelium are generally believed to condition the DCs in the stroma, dampening immune responses and promoting gut homeostasis. One such factor, constitutively expressed by the gut epithelium, is thymic stromal lymphopoetin (TSLP). TSLP is an IL-7–like cytokine that has been shown to activate expression of OX40L on dendritic cells and drive T _H 2 differentiation. Thus, TSLP is a critical mediator of allergic inflammation in the lung and skin. By contrast, in the gut, TSLP appears to play a regulatory role, limiting deleterious T _H 1 and T _H 17 inflammation in models of helminth infection and colitis. Although incompletely understood, this regulation may occur at the level of the DC, which expresses the TSLP receptor and has been shown to develop tolerogenic properties after TSLP exposure. Interestingly, regulatory responses to dietary allergens are evidently normal in TSLP receptor-deficient animals. These findings suggest that although allergic sensitization in the gut may be mediated or regulated via TSLP, it is not required for oral tolerance. Little is known about the role of TSLP in food sensitization in humans; however, a recent study identified TSLP gene expression in the esophagus of patients with eosinophilic esophagitis and showed that genetic variants in TSLP and the TSLP receptor are associated with the disease.