Immune Response to Dietary Antigens (Oral Tolerance)

6. Immune Response to Dietary Antigens (Oral Tolerance)

Immunological Basis for Oral Tolerance

Foreign dietary antigens interact with the intestinal immune system in such a way as to prevent unnecessary and detrimental immune reactions to them. In so doing, systemic immunity is rendered effectively unresponsive if the same antigen reaches the systemic circulation. This absence of reactivity to orally administered antigens is termed oral tolerance. Oral tolerance is generated in an antigen-specific and active manner that involves the induction of an atypical immune response.

Peyer’s patches are the primary inductive area of the intestinal immune system. The specialized M-cells within the epithelium overlying the lymphoid follicles sample, unspecifically or by receptor-mediated uptake, particulate and insoluble antigens, and whole microorganisms (Brandtzaeg, 2001). Antigens and organisms are then transported to leukocytes that reside within basal membrane invaginations, namely B-cells, macrophages, and dendritic cells. In the normal intestine these antigen presenting cells (APCs) lack co-stimulatory molecules such as CD80 and CD86. Antigens processed by these "un-activated" APCs are then presented to naïve B and T cells within the follicle, which then proliferate poorly. This occurs within a local microenvironment that differs from other sites in the body and results in induction of hyporesponsive, Th3 or Th2 biased T cells (Kellermann & McEvoy, 2001). Activated cells then leave via lymphatics and pass via the mesenteric lymph nodes into the systemic circulation. They will then exit at mucosal sites via engagement of cell adhesion molecules (CAMs) specifically expressed by the high-endothelial venules of mucosal tissues. Thus activated or memory B and T lymphocytes enter the lamina propria to await a secondary encounter with their specific antigen (Figure 11).

Figure 11. Activation and re-homing of intestinal lymphocytes.

The activated cells may secrete cytokines, but full differentiation into effector T cells or plasma cells may not occur without secondary exposure. For both cell types to be re-exposed to antigen, intact antigens must reach the lamina propria. Intestinal epithelial cells are responsible for the absorption of antigen, release to professional APCs, and limited antigen presentation to cells within the mucosa on MHC class II. In the normal intestine, these secondary APCs will, like the primary presenters, lack co-stimulatory molecule expression and further add to the toleragenic environment. The effector T cell clones resident in the normal intestine secrete a bias towards Th2 and Th3 cytokines, in particular IL-10 and TGF-β, thus directing B-cell isotype switching to produce IgA-secreting plasma cells, whilst inhibiting the development of Th1 lymphocytes and IgG production.

It is important that the immune system reserves the ability to rapidly respond to pathogens. This ability to recognize pathogenicity is based on the engagement of PAMP receptors such as TLRs, producing "danger signals".

Predictably, expression of TLR-2 and TLR-4 is low to non-existent in the mucosal cells of the normal human intestine, but they can be rapidly expressed in response to inflammatory cytokines (Abreu et al, 2001). The absence of these "danger signals" results in relatively inefficient antigen processing by intestinal APCs, markedly reduced or absent TNF-α / IL-1 / IL-12 production, and the absence of CD80/86 co-stimulatory molecule expression. T cells activated by such an APC, will divide less with most clones undergoing early deletion by apoptosis, whilst the surviving memory cells will tend to secrete IL-10, TGF-β, or no cytokines (Jenkins et al, 2001). This combination of apoptosis, functional defects in surviving clones, and T cells secreting the anti-inflammatory and IgA-supporting cytokines, is the general basis for immunological tolerance to luminal antigens (Figure 12).

Figure 12. The general basis for immunological tolerance to luminal antigens.

Thus oral tolerance is composed of a delicate balance between induction of IgA, T cell deletion, anergy, and immunosuppression; and the retention of antigen-specific lymphocytes capable of responding to invasive pathogens though antibody isotype switching to IgM, IgE, or IgG, and the production of inflammatory cytokines such as IFN-γ, IL-12, and IL-6.

Loss of Tolerance to Dietary Antigens

Loss of tolerance to dietary antigens will produce a conventional but detrimental immune response against the dietary antigen. Such an inappropriate response may produce inflammation locally, or at another anatomical site. The response will be characterized by one or a combination of:
- Local cell mediated inflammation: the resulting chronic stimulus may lead to lymphocytic intestinal infiltrates characteristic of inflammatory bowel disease.
- Local antibody production of isotypes other than IgA: the production of IgE will lead to mast cell priming and intestinal hypersensitivity, i.e., food allergy with gastrointestinal signs (vomiting and/or diarrhea).
- Systemic antibody production: circulating IgE will lead to priming of mast cells at sites distal to the intestine such as dermal hypersensitivity, i.e., food allergy with pruritus as the clinical sign.

The initiating events that lead to loss of oral tolerance, or prevent it from developing have not been described in cats, and remain poorly understood in any species. Suggested mechanisms include:
- Increased mucosal permeability: e.g., following mucosal injury, or the neonatal intestine
- Co-administration of a mucosal adjuvant: that activates and changes the phenotype of intestinal dendritic cells e.g., bacterial enterotoxins
- Parasitism: intestinal parasitism in cats leads to an exaggerated systemic humoral response that includes increased production of IgE (Gilbert & Halliwell 2005).

Currently, there is speculation as to the importance of infections that stimulate a Th-1-biased immune response in preventing Type-1 hypersensitivity reactions in people. This has been termed the ""”, which states that a lack of maturation of the infant immune system from a Th-2 to a Th-1 type of immune response may be caused by less microbial stimulation in Western societies (Romagnani, 2004). It is proposed that bacterial and viral infections during early life promotes a net shift of the maturing immune system towards Th-1 biased responses, and reduce potentially allergenic Th-2 biased responses. The assumed reduction in the overall microbial burden is supposed to allow the natural Th-2 bias of neonates to persist and allow an increase in allergy.

The special role of parasites in modulating allergic responses to food and other allergens has been debated for half a century. Several older reports suggested that, similar to cats, parasitized humans are more likely to suffer from allergic diseases (Warrell et al, 1975; Carswell et al, 1977; Kayhan et al, 1978). In contrast to that is the higher incidence of allergic disease in Western populations, and the growing incidence of allergic disease in developing nations. Elevations of anti-inflammatory cytokines, such as interleukin-10, that occur during long-term helminth infections have been shown to be inversely correlated with allergy. It has recently been suggested that the host’s response to the parasite determines their predisposition to develop allergic diseases, and that the induction of a robust anti-inflammatory regulatory response (e.g., IL-10) induced by persistent immune challenge offers a unifying explanation for the observed inverse association of many infections with allergic disorders (Yazdanbakhsh et al, 2002). In cats, the role parasitism and other infections that would fall within the hygiene hypothesis have yet to be defined in determining the development of food hypersensitivity. Since the immunological mechanism for the majority of food sensitivities may not be IgE-mediated, the story may be even more complicated.

Food Immunogenicity

Adverse reactions to food are surprisingly common in cats: they have been reported to be present in up to 29% of all cases of chronic gastrointestinal disease in cats (Guilford et al, 2001).

In addition, inflammatory bowel disease is the single most common cause of chronic gastrointestinal disease in cats, and novel antigen and hydrolysed protein diets are commonly reported to be effective in its management (Guilford & Matz, 2003; Nelson et al, 1984). However, although the involvement of immunological mechanisms in a proportion of these adverse reactions is suspected, it is unproven. Indeed, the normal immunological response to ingested dietary antigens in cats has only recently been partially described (Cave & Marks, 2004). Surprisingly, cats develop robust serum IgG and IgA responses to dietary proteins when fed as either aqueous suspensions or as part of canned diets.

The relatively short intestinal tract of the cat suggests that they may be poorly suited to poorly digestible diets (Morris, 2002). It is well established that the commercial canning process decreases protein digestibility and that this has biologically significant effects in cats (Kim et al, 1996).

In rodents and rabbits, intact particulate and insoluble antigens are preferentially absorbed across the intestine through M-cells overlying the Peyer’s patches (Frey et al, 1996). Classically, such antigens tend to invoke active immunity appropriate for microorganisms. In contrast, soluble antigens have been found to be associated with oral tolerance (Wikingsson & Sjoholm, 2002). It has also been shown that oral tolerance can be abrogated when soluble proteins are fed in oil-in-water emulsions, resulting in robust systemic humoral responses (Kaneko et al, 2000). This effect may also have relevance to the pet-food industry where interactions between dietary proteins and lipids in canned or extruded diets during the cooking and the manufacturing process could feasibly result in novel interactions not present in their native states.

In stark contrast to rodents is the intestinal response in chickens, where particulate antigens induce tolerance, whilst soluble antigens provoke active immunity (Klipper et al, 2001). If the physical nature of the proteins within the natural diet of a species dictates how the intestinal immune system has evolved, this might have special relevance to species that are commonly fed diets different from their ancestors.

Commercial pet foods are subjected to significant heating during the manufacturing process. The effect of heat treatment on proteins is mostly to change the 3-dimensional conformation of the protein. Although this may disrupt some antigens, it may equally uncover previously hidden antigenic determinants, or create new ones. Other reactions occurring at high temperatures include the Maillard reactions, which involve the reactions between certain amino acids and reducing sugars to produce less digestible compounds called melanoidins, which give a characteristic brown color. Melanoidins tend to be less digestible, less soluble, and certain melanoidins have been shown to be more "allergenic" than the original uncooked protein (Maleki et al, 2000; 2003).

As obligate carnivores, felids have evolved on a highly digestible diet. (© Y. Lanceau/Royal Canin).

The effect of heating during the canning process on the immunogenicity of dietary proteins has been evaluated in cats (Cave & Marks 2004). Using soy and casein proteins, the canning process resulted in the creation of new antigens not present in the uncooked product. In addition, a product of heated casein induced a salivary IgA response that was not induced by the raw product. Thus commercial food processing can qualitatively and quantitatively alter the immunogenicity of food proteins. Although the significance of this finding is uncertain at present, it emphasizes the need for feeding highly digestible proteins sources, or perhaps even hydrolysed proteins, when enteritis is present.