A novel model of sensitization and oral tolerance to peanut protein

Abstract

The prevalence of food allergic diseases is rising and poses an increasing clinical problem. Peanut allergy affects around 1% of the population and is a common food allergy associated with severe clinical manifestations. The exact route of primary sensitization is unknown although the gastrointestinal immune system is likely to play an important role. Exposure of the gastrointestinal tract to soluble antigens normally leads to a state of antigen-specific systemic hyporesponsiveness (oral tolerance). A deviation from this process is thought to be responsible for food-allergic diseases. In this study, we have developed a murine model to investigate immunoregulatory processes after ingestion of peanut protein and compared this to a model of oral tolerance to chicken egg ovalbumin (OVA). We demonstrate that oral tolerance induction is highly dose dependent and differs for the allergenic proteins peanut and OVA. Tolerance to peanut requires a significantly higher oral dose than tolerance to OVA. Low doses of peanut are more likely to induce oral sensitization and increased production of interleukin-4 and specific immunoglobulin E upon challenge. When tolerance is induced both T helper 1 and 2 responses are suppressed. These results show that oral tolerance to peanut can be induced experimentally but that peanut proteins have a potent sensitizing effect. This model can now be used to define regulatory mechanisms following oral exposure to allergenic proteins on local, mucosal and systemic immunity and to investigate the immunomodulating effects of non-oral routes of allergen exposure on the development of allergic sensitization to peanut and other food allergens.

Figure 1

DTH response to peanut protein and OVA. Animals were fed peanut protein or OVA at doses of 0·02 mg (a), 0·2 mg (b), 2 mg (c), 20 mg (d) or 100 mg (e) and DTH responses were measured following immunizations. Results are expressed as mean footpad increment + 1 SEM (n ≥ 6). Individual experiments were repeated between 2 and 10 times with similar results. SPP: saline-fed, immunized with peanut in CFA, challenged with peanut; PPP: peanut-fed, immunized with peanut in CFA, challenged with peanut; SOO: saline-fed, immunized with OVA in CFA, challenged with OVA; OOO: OVA fed, immunized with OVA in CFA, challenged with OVA.

Figure 2

Antigen-specific T-cell proliferation. Peanut-specific (left column) and OVA-specific (right column) proliferation by PLN T cells were measured from animals fed peanut protein or OVA at doses of 0·02 mg (a), 0·2 mg (b), 2 mg (c), 20 mg (d) or 100 mg (e) prior to tailbase immunization with peanut protein or OVA in CFA and challenge. Specific T-cell proliferation was measured after 90 hr in vitro reactivation with peanut or OVA. Cells were pooled from n ≥ 6 mice per group and results are expressed as the mean c.p.m. of triplicate cultures ± 1 SEM. Background proliferation when no antigen was present has been subtracted. Squares represent proliferation to a control antigen. All groups proliferated with similar c.p.m. to the mitogen Con A. Individual experiments were repeated between 2 and 10 times with near identical results. ▴: Saline-fed, immunized with peanut in CFA, challenged with peanut; ▵: peanut-fed, immunized with peanut in CFA, challenged with peanut; •: saline-fed, immunized with OVA in CFA, challenged with OVA; ○: OVA fed, immunized with OVA in CFA, challenged with OVA.

Figure 3

Peanut-specific IFN-γ, IL-4, IL-10 and TGF-β secretion. IFN-γ (a), IL-4 (b), IL-10 (c) and TGF-β (d) production by PLN T cells from animals fed saline, 0·2 mg peanut protein or 100 mg peanut protein followed by immunization with peanut protein in CFA and challenge with peanut. Cytokine production was analysed following reactivation with peanut protein in vitro between 24 and 120 hr (left column) or routinely for 96 hr (right column). Cells were pooled from n ≥ 6 mice per group and analysed in triplicate. Results are expressed as the mean cytokine secretion + 1 SEM of two (PPP 0·2 mg) to seven (four for TGF-β) (SPP and PPP 100 mg) individual experiments. The horizontal dashed line indicates the limit of detection for the cytokine assays, though this is close to the x-axis in (a, c). None of the cytokines were released in response to a control antigen or when cells were not reactivated. SPP (•): Saline-fed, immunized with peanut in CFA, challenged with peanut; PPP 0·2 mg (□): Fed 0·2 mg peanut, immunized with peanut in CFA, challenged with peanut; PPP 100 mg (▵): Fed 100 mg peanut, immunized with peanut in CFA, challenged with peanut.

Figure 4

Antibody response to peanut. Peanut-specific IgG, IgG1 and IgG2a as well as total IgE were measured in serum from animals fed peanut protein at doses of 0·02 mg (a), 0·2 mg (b), 2 mg (c), 20 mg (d) and 100 mg (e) prior to immunizations with peanut protein. Sera were diluted 1000 times for peanut-specific IgG and IgG1, 200 times for IgG2a and 10 times for total IgE measurements. Results are expressed as mean antibody levels + 1 SEM of n ≥ 6 animals per group. The individual experiments were repeated between 2 and 10 times with identical antibody patterns between the saline- and peanut-fed groups. SPP: saline-fed, immunized with peanut in CFA, challenged with peanut; PPP: peanut-fed, immunized with peanut in CFA, challenged with peanut.

Figure 5

Antibody response to OVA. OVA-specific IgG, IgG1 and IgG2a as well as total IgE were measured in serum from animals fed OVA at doses of 0·02 mg (a), 0·2 mg (b), 2 mg (c), 20 mg (d) and 100 mg (e) prior to immunizations with OVA. Sera were diluted 1000 times for OVA-specific IgG and IgG1, 200 times for IgG2a and 10 times for total IgE measurements. Results are expressed as the mean antibody levels + 1 SEM of n ≥ 6 animals per group. The individual experiments were repeated between one and three times with similar results. SOO: saline-fed, immunized with OVA in CFA, challenged with OVA; OOO: OVA fed, immunized with OVA in CFA, challenged with OVA.

Figure 6

Effect of feeding peanut protein or OVA on subsequent peanut-specific responses. Animals (n = 6/group) were fed saline, 100 mg peanut protein or 100 mg OVA prior to immunization with peanut protein in CFA and challenge with peanut. (a) DTH responses 24 hr after challenge in the footpad. Results are expressed as the mean footpad increments + 1 SEM. (b) Peanut-specific T-cell proliferation in PLN cell cultures after 90 hr in vitro reactivation with peanut protein. Cells were pooled from six mice per group and results are expressed as the mean c.p.m. of triplicate culture ± 1 SEM. Background proliferation without antigen has been subtracted. Squares show proliferation to a control antigen. All groups proliferated with similar (c.p.m.) to Con A stimulation. (c) Peanut-specific IgG, IgG1, IgG2a and IgE levels in serum 7 days after challenge. Sera were diluted 1000 times for IgG and IgG1, 200 times for IgG2a and 10 times for IgE measurements. Results are expressed as the mean antibody levels + 1 SEM of six individual animals per group. SPP (•): Saline-fed, peanut immunized and peanut challenged; PPP (▵): Peanut-fed, peanut immunized and peanut challenged; OPP (◊): OVA fed, peanut immunized and peanut challenged.