Langerhans cells are critical in epicutaneous sensitization with protein antigen via thymic stromal lymphopoietin receptor signaling

Abstract

Background: The clarification of cutaneous dendritic cell subset and the role of thymic stromal lymphopoietin (TSLP) signaling in epicutaneous sensitization with protein antigens, as in the development of atopic dermatitis, is a crucial issue.

Objectives: Because TSLP is highly expressed in the vicinity of Langerhans cells (LCs), we sought to clarify our hypothesis that LCs play an essential role in epicutaneous sensitization with protein antigens through TSLP signaling.

Methods: By using Langerin-diphtheria toxin receptor knock-in mice and human Langerin-diphtheria toxin A transgenic mice, we prepared mice deficient in LCs. We also prepared mice deficient in TSLP receptors in LCs by using TSLP receptor-deficient mice with bone marrow chimeric technique. We applied these mice to an ovalbumin (OVA)-induced epicutaneous sensitization model.

Results: Upon the epicutaneous application of OVA, conditional LC depletion attenuated the development of clinical manifestations as well as serum OVA-specific IgE increase, OVA-specific T-cell proliferation, and IL-4 mRNA expression in the draining lymph nodes. Consistently, even in the steady state, permanent LC depletion resulted in decreased serum IgE levels, suggesting that LCs mediate the T(H)2 local environment. In addition, mice deficient in TSLP receptors on LCs abrogated the induction of OVA-specific IgE levels upon epicutaneous OVA sensitization.

Conclusion: LCs initiate epicutaneous sensitization with protein antigens and induce T(H)2-type immune responses via TSLP signaling.

FIG 1

LCs are crucial for epicutaneous sensitization with OVA. A, Total clinical severity scores (left panel) and total histology scores (right panel) of LC–non-depleted (LC⁺) and LC-depleted (LC⁻) mice (n = 5 mice per group). B, Serum OVA-specific antibodies as determined by ELISA. OD values for IgE, IgG1, and IgG2a levels were measured at a wavelength of 450 nm. *P < .05.

FIG 2

LCs are critical for antigen-specific T-cell proliferation. Mice in the presence or absence of LCs (LC⁺ and LC⁻, respectively) were treated with OVA and transplanted with CFSE-labeled OT-II T cells (n = 5 mice per group). Skin-draining LNs were analyzed for OVA-specific T-cell proliferation (A and B) and mRNA expression levels for IFN-γ and IL-4 (C). Boxes in (A) demarcate divided cells (left) and undivided cells (right). *P < .05. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; ND, not detected.

FIG 3

LCs are essential for IgE production. The serum IgE levels (A) and IgE expression levels (B) on peritoneal mast cells (indicated by MFI) of WT and Langerin-DTA mice on FVB (left panel) and B6 (right panel) backgrounds. Mast cells were also preincubated with IgE (labeled with pre-IgE) in vitro before the measurement of IgE expression (Fig 3, B). Each symbol represents an individual animal. *P < .05. MFI, Mean fluorescence intensity.

FIG 4

TSLPR on LCs is a responsible target of TSLP upon epicutaneous OVA sensitization. Epidermal cell suspensions from B6 (WT) mice with (sensitized) or without (nonsensitized) epidermal application of OVA were stained with TSLPR antibody. TSLPR expressions of MHC class II⁺ CD11c⁺ LCs was analyzed by flow cytometry (left, histogram; right, average ± SD of MFI). n = 3 per group. *P < .05. MFI, Mean fluorescence intensity.

FIG 5

An essential target of TSLP for IgE induction is TSLPR on LCs. A, B6 (Ly45.2) mice were irradiated and transplanted with BM cells from B6 (Ly45.1) mice. The epidermis and the dermis of BMC mice were separated, and single-cell suspensions were stained and analyzed by flow cytometry. B, Total clinical severity scores (left panel) and histology scores (right panel) of TSLPR^+/+ BMC, LC-TSLPR^−/− BMC, and TSLPR^−/− BMC mice (n = 5 mice per group). C, Serum OVA-specific antibodies as determined by ELISA. OD values for IgE, IgG1, and IgG2a levels were measured at a wavelength of 450 nm. *P < .05.

FIG 6

TSLPR on LCs are vital for T_H2 induction. TSLPR^+/+ BMC, LC-TSLPR^−/− BMC, and TSLPR^−/− BMC mice were treated with OVA or saline and transplanted with CFSE-labeled OT-II T cells. Skin-draining LNs were analyzed for OVA-specific T-cell proliferation (A and B) and cytokine mRNA expression levels for IFN-γ and IL-4 (C). Boxes in (A) demarcate divided cells (left) and undivided cells (right). n = 5 mice per group. *P < .05. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; ND, not detected.