Allergic sensitization can be induced via multiple physiologic routes in an adjuvant-dependent manner

Abstract

Background: Oral exposure to food allergens may be limited in infancy, and the initial site of antigen exposure likely plays an important role in food allergy induction.

Objective: To examine the impact of different routes of exposure by using milk allergens, with and without adjuvant, on sensitization.

Methods: C3H/HeJ mice were repeatedly exposed to the milk allergen α-lactalbumin (ALA), with or without cholera toxin (CT). Sensitization routes used were intragastric, cutaneous, intranasal, and sublingual. Anaphylaxis severity was assessed by symptoms and body temperature in response to oral challenge. Antigen-specific serum antibodies were measured by ELISA. The mechanism of adjuvant activity of cutaneous CT was also determined.

Results: Sensitization to ALA as measured by allergen-specific IgE occurred by all routes of sensitization and was maximal in response to cutaneous exposure. Sensitization was dependent on CT and did not occur to antigen alone by any route. Mucosal, but not cutaneous, exposure resulted in a robust allergen-specific IgA response. Anaphylaxis occurred in all sensitized groups when orally challenged with ALA. Topical CT induced migration of langerin(neg) dermal dendritic cells to the lymph node, resulting in enhanced proliferation and T(H)2 cytokine production from responder T cells.

Conclusions: Sensitization can occur via all physiologic routes when adjuvant is present. The skin is a potent and likely important physiologic route of sensitization whereby adjuvant induces an efflux of antigen-bearing dermal dendritic cells to the lymph node that generate a proallergic T(H)2 response.

Figure 1. Antigen-specific serum antibody profile

C3H/HeJ mice were exposed to ALA plus CT by the intragastric (oral) (n=9), sublingual (SL) (n=9), intranasal (IN) (n=5) or cutaneous (skin) (n=5) route for 6 weeks prior to measurement of antigen-specific antibodies in the serum by ELISA. *p<.05, **p<.01, ***p<.001 vs. none, ^¶p<.05 vs oral, IN, SL

Figure 2. Symptom score and body temperature

C3H/HeJ mice were exposed to ALA ± CT by the intragastric (oral), sublingual (SL), intranasal (IN) or cutaneous (skin) route. Mice were then challenged with progressively increasing oral ALA and 30 min after the first dose eliciting symptoms (A) and a decrease in body temperature (B), a symptom score was assigned and body temperature measured. *p<.05, **p<.01, ***p<.001 vs. non-exposed (None). (n are the same as in fig 1)

Figure 3. Effect of CT on skin-draining DCs

A: Effect of CT on migratory DCs and their subsets in the draining lymph node. n=6 B: Effect of CT on migration of FITC-dextran-positive DCs to the draining lymph nodes. n=3 experiments of 3 Balb/c mice/group C: Median fluorescent intensity (MFI) of MHC class II of FITC-dextran-positive DCs. n=3 experiments of 3 Balb/c mice/group D: Representative flow cytometry plot showing the phenotype of FITC-dextran-positive DCs (lower panel) compared to the total migratory DC population (upper panel). n=3, *p<.05, **p<.01

Figure 4. Effect of CT on the antigen-specific T cell response

A representative histogram (A) and quantification (B) of proliferation of DO11.10 T cells in the skin-draining lymph node after exposure to PBS, OVA, or OVA + CT. n=5 C: Cytokine secretion from transferred OVA-specific T cells after in vivo exposure to PBS, OVA, or OVA+CT after re-stimulation with OVA peptide and subsequent stimulation with anti-CD3/CD28. n=3 experiments of 3 Balb/c mice/group *p<.05, **p<.01, ***p<.001

Figure 5. Effect of in vivo treatment with CT on the immunogenicity of DCs

DCs were purified from the inguinal lymph node of Balb/c mice treated topically with PBS, OVA, or OVA+CT and co-cultured with DO11.10 cells. Secreted cytokines were measured by ELISA after re-stimulation with OVA peptide and subsequent stimulation with anti-CD3/CD28. n=2 experiments of 3 mice/group, **p<.01, ***p<.001