Thymic stromal lymphopoietin-mediated epicutaneous inflammation promotes acute diarrhea and anaphylaxis

Abstract

Atopic dermatitis (AD) and food allergy are closely linked; however, the mechanisms that guide the progression of AD to allergic inflammatory responses at other mucosal surfaces, including the gastrointestinal tract, are not well understood. Here, we determined that exposure of mice that have been epicutaneously sensitized with thymic stromal lymphopoietin (TSLP) and antigen to repeated oral doses of the same antigen induced acute diarrhea and anaphylaxis. In this model, loss of TSLP signaling specifically in DCs led to loss of induced allergic diarrhea through lack of sensitization. While TSLP responses were not required during oral allergen challenge, CD4(+) T cells were required and transferred disease when introduced into naive hosts. In addition, oral exposure to the antigen prior to skin sensitization blocked development of allergic disease. Finally, mice lacking the receptor for IL-25 failed to develop acute diarrhea and anaphylaxis, highlighting a role for IL-25 in the initiation of type 2 immunity in the intestine. These results demonstrate a role for TSLP and IL-25 in the atopic march from skin sensitization to food allergic responses and provide a model system for the generation of potential therapeutic interventions.

Figure 9. IL-25 signaling is required to induce GI allergy.

(A) Quantitative RT-PCR for Tslp and Il25 gene expression from the jejunum following oral challenge. (B) Diarrhea occurrence. (C) Diarrhea score. (D) Intracellular cytokine staining of MLN cells isolated from WT mice (upper panels) and IL17rb-deficient mice (lower panels). Plots are gated on CD4⁺CD44^hi cells and representative of 4 mice analyzed. (E) Serum leakage at intestine. Data are representative of 3 independent experiments with 3 to 4 mice per group. Error bars indicate the mean ± SD. **P ≤ 0.01.

Figure 8. Antigen feeding before skin sensitization suppresses GI allergy.

(A) Diarrhea occurrence. (B) Diarrhea score. (C) Symptom scores. (D) Intracellular cytokine staining of MLN cells isolated from mice fed with normal water (upper panels) and 1% OVA in drinking water (lower panels). Plots are gated on CD4⁺CD44^hi cells. (E) MMCP-1 serum levels. (F) OVA-specific serum IgE levels. (G) Serum leakage at skin (upper panels) and intestine (lower panels). Data are representative of 2 independent experiments with 4 mice per group. Error bars indicate the mean ± SD. ***P ≤ 0.001.

Figure 7. CD4⁺ T cells are required for TSLP-mediated GI allergy.

(A and E) Diarrhea occurrence. (B and F) Diarrhea score. (C and G) OVA-specific serum IgE levels. (D and H) MMCP-1 serum levels. Data are representative of 2 independent experiments with 4 to 6 mice per group. Groups of animals were compared using Student’s t tests (G and H). Error bars indicate the mean ± SD. ***P ≤ 0.001.

Figure 6. Basophil depletion attenuates GI allergy.

(A) Diarrhea score. (B) MMCP-1 serum levels. (C) Serum leakage at skin and intestine. Data are representative of 2 independent experiments with 4 mice per group. Error bars indicate the mean ± SD. ***P ≤ 0.001.

Figure 5. Diminished allergen-induced GI allergy in Cd11c-Cre Tslpr^f/f mice.

(A) Diarrhea occurrence. n = 6. (B) Diarrhea score. n = 6. (C) OVA-specific serum IgE levels. (D) Mmcp1 and type 2 cytokine expression levels in the jejunum. (E) Intracellular cytokine staining of MLN cells isolated from a Tslpr^f/f mouse and a Cd11c-Cre Tslpr^f/f mouse. Plots are gated on CD4⁺CD44^hi cells and representative of 6 mice analyzed. For C and D, data are representative of 3 independent experiments with 3 mice per group. Error bars indicate the mean ± SD. ***P ≤ 0.001.

Figure 4. TSLP is dispensable during challenge phase.

(A and E) Diarrhea occurrence. n = 10. (B and F) Diarrhea score. (C and G) OVA-specific serum IgE levels. (D and H) MMCP-1 serum levels. Data are representative of 2 independent experiments with 5 mice per group (B–D and F–H). M+O, MSA+OVA; T+O, TSLP+OVA. Error bars indicate the mean ± SD.

Figure 3. Antigen-driven anaphylaxis in TSLP+OVA-sensitized mice.

(A) Symptom scores. (B) Body temperature responses after antigen challenge. (C) Serum leakage at skin and intestine. Data are representative of 3 independent experiments with 4 mice per group. Error bars indicate the mean ± SD. ***P ≤ 0.001.

Figure 2. Intradermal administration of TSLP promotes antigen-induced diarrhea.

(A) Experimental protocol. (B) Representative cecum and colon from MSA+OVA– and TSLP+OVA–treated mice. (C) Diarrhea occurrence. (D) Diarrhea score. (E) OVA-specific serum IgE levels. (F) Quantitative RT-PCR for Mmcp1 and cytokine gene expression from the jejunum. (G) Intracellular cytokine staining of MLN cells isolated from mice treated with MSA+OVA (upper panels) and TSLP+OVA (lower panels). Plots are gated on CD4⁺CD44^hi cells and representative of 10 mice analyzed. (H) MMCP-1 serum levels. (I) TSLP serum levels. K5-Tslp mice were treated with 1 mg/ml of dox for 3 weeks and used as assay control. ND, <7.8 pg/ml. Data are representative of 3 independent experiments with 5 mice per group. Groups of animals were compared using Student’s t tests (G and I). Error bars indicate the mean ± SD. ***P ≤ 0.001.

Figure 1. Increased TSLP expression in the skin promotes antigen-induced GI allergy.

(A) Experimental protocol. Experimental and control animals were given dietary dox (0.01 mg/ml in drinking water) and intradermal OVA as indicated, followed by a rest and oral challenge. (B) Representative photograph of the cecum and colon from indicated mice. NLC, normal littermate control. (C) Diarrhea occurrence. (D) Diarrhea score. (E) Frequencies of peripheral eosinophils. Cells were gated on CD45⁺ cells, and plots are representative of 10 mice analyzed. (F) OVA-specific serum IgE levels. (G) MMCP-1 serum levels. (H) Mmcp1 and cytokine expression levels in the jejunum. (I) TSLP serum levels. Control K5-Tslp mice were treated with 1 mg/ml of dox for 3 weeks and used as assay control. ND, not detected (<7.8 pg/ml). For C, D, and F–H, data are representative of 3 independent experiments with 5 mice per group. Groups of animals were compared using Student’s t tests. Error bars indicate the mean ± SD. ***P ≤ 0.001.