Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis

Abstract

We have demonstrated that cytokine thymic stromal lymphopoietin (TSLP), whose expression is rapidly induced upon keratinocyte-selective ablation of retinoid X receptors (RXRs) -alpha and -beta in the mouse (RXRalphabeta(ep-/-) mice), plays a key role in initiating a skin and systemic atopic dermatitis-like phenotype. We show here that topical application of the physiologically active ligand [1alpha,25-(OH)(2)D(3); calcitriol] of the vitamin D receptor, or of its low-calcemic analog MC903 (calcipotriol; Dovonex), induces TSLP expression in epidermal keratinocytes, which results in an atopic dermatitis-like syndrome mimicking that seen in RXRalphabeta(ep-/-) mutants and transgenic mice overexpressing TSLP in keratinocytes. Furthermore, topical application of retinoic acid receptor RARgamma-selective agonist BMS961 also induces TSLP expression either on its own or synergistically with 1alpha,25-(OH)(2)D(3). Our data demonstrate that RXR/vitamin D receptor and RXR/retinoic acid receptor-gamma heterodimers and their ligands cell-autonomously control the expression of TSLP in epidermal keratinocytes of the mouse. We propose molecular mechanisms through which vitamin D3 and retinoic acid signalings could be involved in the pathogenesis of atopic diseases.

Fig. 1.

Skin topical application of 1α,25-(OH)₂D₃ and MC903 activates TSLP expression in epidermal keratinocytes. (a) TSLP RNA at day 5 in ears topically treated with NR agonists (4 nmol). (b–e) Induction of TSLP expression is a rapid and local skin effect. TSLP and CYP24A1 RNA levels in an ethanol-treated left ear and MC903-treated right ear (b), TSLP RNA levels in ethanol-treated and MC903-treated dorsal skin at days 2, 3, and 4 (c), and increased serum TSLP levels at days 2, 3, and 4 (d), in contrast to undetectable level (<8 pg/ml) at day 0 (before treatment). Data are representative of three independent experiments. D, day. (e) TSLP RNA levels at day 5 in ear (E), lung (Lu), thymus (Thy), salivary gland (Sa), tongue (To), colon (Co), spleen (Sp), lymph node (LN), and liver (Li) of mice topically treated by ethanol or MC903 on ears. (f) IHC of TSLP (green) at day 4 in sections of ear (Upper) and dorsal skin (Lower) topically treated with ethanol or MC903. The same sections from MC903-treated skin were stained with keratin 1 antibody (K1, red), and overlaid images of TSLP and K1 staining are shown, as indicated. Blue corresponds to DAPI staining of nuclei. The white arrowhead points to autofluorescent erythrocytes, and white arrows point to the dermal/epidermal junction. (Scale bar, 50 μm.)

Fig. 2.

Topical treatment with MC903 triggers an AD-like skin inflammation. (a and b) Appearance of ethanol- and MC903-treated ears at day 17. (c and d) Hematoxylin and eosin-stained ear sections of ethanol- and MC903-treated mice at day 17. Eosinophils displaying cytoplasmic red staining are indicated by yellow arrows in d Inset. (e–n) IHC performed on ear sections from ethanol- or MC903-treated mice at day 17, with antibodies against GR1 (e and f), CD3 (g and h), CD4 (i and j), CD8 (k and l), and CD11c (m and n). Yellow corresponds to staining of antibodies, whereas blue corresponds to DAPI staining of nuclei. (o and p) Toluidin blue (TB) staining of ear sections. Red arrows point to one of the mast cells with intense blue in the dermis. White arrows in c–p point to the dermal/epidermal junction. (q) Cytokine RNA levels in ethanol- and MC903-treated ears at day 17. (r) Serum IgE and IgG levels of ethanol- and MC903-treated mice at day 17. (s) Hematoxylin and eosin-stained sections of ear-draining lymph node and liver of ethanol- and MC903-treated mice at day 17. Yellow arrows point to three of many eosinophils (red cytoplasmic staining) in sections of lymph node and liver of MC903-treated mice. (Scale bars, 50 μm.)

Fig. 3.

Keratinocytic VDR and RXR are required for generation of an AD-like skin inflammation and induction of TSLP expression upon MC903 treatment. Appearance of MC903-treated ears of VDR CT (a) and VDR^ep−/− mice (b) at day 17. White arrow in a points to lesioned skin. (c and d) Hematoxylin and eosin-stained ear sections. Yellow arrows in c Inset point to three of many eosinophils (red cytoplasmic staining) in MC903-treated CT skin. Black arrows point to dermal/epidermal junction. hf, hair follicle; u, utriculi (resulting from hair follicle degeneration in VDR^ep−/− mice). (Scale bar, 50 μm.) (e) TSLP RNA levels at day 4 in ethanol- and MC903-treated ears of VDR CT (lanes 1 and 2), VDR^ep−/− (lanes 3 and 4), and VDR^−/− (lanes 5 and 6) mice. (f) TSLP RNA levels at day 4 in ethanol- and MC903-treated ears of RXRαβ CT (lanes 1 and 2) and RXRαβ^{ep −/−} (lanes 3 and 4) mice.

Fig. 4.

Agonist-liganded VDR and RARγ synergistically induce TSLP expression. (a) Mouse ears were treated with ethanol, BMS961 (4 nmol), 1α,25-(OH)₂D₃ (0.4 nm), or BMS961 (4 nmol) plus 1α,25-(OH)₂D₃ (0.4 nmol), as indicated. TSLP RNA levels in the ears (Left) and serum TSLP levels (Right) were measured at day 4. (b) Putative VDREs (DR3) and RAREs (DR2 and DR1) upstream of the mouse TSLP promoter. (c) Putative VDREs (DR3) and RAREs (DR2 and DR1) upstream of the human TSLP promoter.

Fig. 5.

Schematic model of RXRα(β)/VDR- and RXRα(β)/RAR-mediated regulation of TSLP expression in mouse keratinocytes (see Discussion). As concluded from our study (25), keratinocytic RXRs are shown bound to a non-RA-agonistic ligand.