TSLP acts on infiltrating effector T cells to drive allergic skin inflammation

Abstract

Thymic stromal lymphopoietin (TSLP) is a cytokine expressed by epithelial cells, including keratinocytes, and is important in allergic inflammation. Allergic skin inflammation elicited by epicutaneous immunization of mice with ovalbumin (OVA), a potential model of atopic dermatitis, was severely impaired in TSLPR(-/-) mice, as evidenced by decreased infiltration of eosinophils and decreased local expression of T helper 2 (Th2) cytokines. However, secretion of Th2 cytokines by splenocytes from epicutaneous sensitized TSLPR(-/-) mice in response to OVA was normal. Skin dendritic cells from TSLPR(-/-) mice were normal in their ability to migrate to draining lymph nodes, express activation markers, and induce proliferation and Th2 cytokine production by naïve T cells. CD4(+) T cells from TSLPR(-/-) mice expressed the skin homing receptor E-selectin ligand normally, and homed to the skin normally, but failed to transfer allergic skin inflammation to WT recipients. TSLP enhanced Th2 cytokine secretion in vitro by targeting TSLPR on antigen specific T cells. Intradermal injection of anti-TSLP blocked the development of allergic skin inflammation after cutaneous antigen challenge of OVA immunized WT mice. These findings suggest that TSLP is essential for antigen driven Th2 cytokine secretion by skin infiltrating effector T cells and could be a therapeutic target in allergic skin inflammation.

Fig. 1.

Decreased allergic skin inflammation in EC-sensitized TSLPR^−/− mice. (A) H&E staining of sections from saline- and OVA-sensitized skin at ×200 magnification. Further magnification of the red bordered box shows the presence of multiple eosinophils (arrows). (B) Skin infiltration of eosinophils and CD4⁺ T cells. (C) Quantitative PCR analysis of mRNA levels of IL-4 and IL-13 in sensitized skin expressed as fold induction compared with saline-sensitized skin sites. (D) IL-4, IL-13, and IFN-γ secretion by cells from skin DLNs after stimulation for 4 days with OVA. Columns and error bars represent the mean ± SEM (n = 5–7 per group). *, P < 0.05, **, P < 0.01, ***, P < 0.0001. N.D., not detectable.

Fig. 2.

Systemic immune response in EC-sensitized TSLPR^−/− mice. Proliferation (A) and secretion of IL-4, IL-13, and IFN-γ (B) by splenocytes from OVA EC-sensitized WT and TSLPR^−/− mice after in vitro OVA stimulation. (C) Serum levels of OVA-specific IgE, IgG1, and IgG2a in EC-sensitized WT and TSLPR^−/− mice. Columns and error bars represent the mean ± SEM (n = 6 per group), and similar results were obtained in two other independent experiments with 4–6 mice per group. *, P < 0.05, **, P < 0.01, ***, P < 0.0001.

Fig. 3.

Skin DCs migrate, express activation markers, and present antigen normally in TSLPR^−/− mice. Representative FACS analysis of percentage (A) and numbers (B) of CD11c⁺FITC⁺ cells in DLNs from WT and TSLPR^−/− mice 24 h after FITC application to shaved skin. (C) Representative FACS analysis of expression of MHC class II, CD40, CD86, CD80, and OX40L on CD11c⁺FITC⁺ DCs of WT and TSLPR^−/− mice. (D) Proliferation and secretion of IL-4, IL-13, and IFN-γ by DO11.10 CD4⁺ T cells stimulated with OVA_323–339 peptide in the presence of skin-derived DCs from WT or TSLPR^−/− mice. Data are representative of three experiments.

Fig. 4.

TSLPR^−/− T cells express skin homing receptors and traffic to skin normally. Percentages and numbers of donor-derived CD4⁺KJ1–26⁺ cells (A) and CD4⁺KJ1–26⁺E-lig⁺ cells (B) in the DLNs of WT recipient mice challenged with OVA or control. (C) Percentages and numbers of donor-derived KJ1–26⁺ CD4⁺ T cells in the total cells extracted from the challenged ears of WT recipient mice. Columns and error bars represent the mean ± SEM (n = 3 per group).

Fig. 5.

CD4⁺ T cells from TSLPR^−/− mice fail to transfer allergic skin inflammation. Number of infiltrating CD4⁺ cells (A) and expression of mRNA for IL-4 and IL-13 (B) in OVA-challenged skin of WT or TSLPR^−/− recipient mice that received CD4⁺ T cells from EC-sensitized WT or TSLPR^−/− donor mice. Columns and error bars represent the mean ± SEM of two experiments (n = 3–5 recipients per group). *, P < 0.05; **, P < 0.01.

Fig. 6.

TSLP amplifies Th2 cytokine secretion by engaging TSLPR on T cells. (A) Secretion of IL-4, IL-13, and IFN-γ and proliferation by splenocytes from EC-sensitized mice with OVA after in vitro OVA stimulation with or without TSLP. (B) Secretion of IL-4, IL-13, and IFN-γ and proliferation by DO11.10 CD4⁺ T cells stimulated with OVA_323–339 peptide with or without TSLP in the presence of DCs from the spleens of WT or TSLPR^−/− mice. Number of infiltrating eosinophils and CD4⁺ cells (C) and expression of mRNA for IL-4 and IL-13 (D) in OVA-challenged skin of i.p. immunized mice treated with neutralizing antibodies to TSLP or isotype control. Columns and error bars represent the mean ± SEM of 5 mice per group in A, C, and D, and mean ± SEM of four experiments in B. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.