Bile Acids Improve Psoriasiform Dermatitis through Inhibition of IL-17A Expression and CCL20-CCR6-Mediated Trafficking of T Cells

Abstract

Bile acids (BAs), produced in the liver and further transformed in the gut, are cholesterol-derived molecules involved in essential physiological processes. Recent studies suggest that BAs regulate T helper 17 cell function, but the underlying mechanism of this action and their therapeutic value in disease models remains unclear. Using an IL-23 minicircle DNA-based murine model of psoriasiform dermatitis, we showed that oral administration of secondary BAs, including lithocholic acid (LCA), deoxycholic acid, and 3-oxoLCA, significantly improved psoriasiform dermatitis without inducing apparent hepatotoxicity. Of the BAs tested, LCA possessed the greatest potency in treating psoriasiform dermatitis. Intravenous administration of LCA at a much lower dosage (compared with oral treatment) showed a comparable antipsoriatic effect and markedly suppressed the IL-17A response. Ex vivo experiments revealed that LCA reduced IL-17A production in IL-23-stimulated murine T cells in the absence of BA receptors TGR5 or FXR. Strikingly, BAs inhibited CCL20 expression in keratinocytes, which led to reduced migration of CCR6-expressing Jurkat cells cultured in the conditioned medium of stimulated keratinocytes. Thus, BAs improve psoriasiform dermatitis with minimal toxicity via direct inhibition of IL-17A production and blockade of CCL20-mediated trafficking, supporting the potential use of BAs in psoriasis.

Figure 1.. Oral administration of bile acids ameliorates IL-23 MC-induced psoriasiform dermatitis.

(a) Schematic illustration of experimental protocols. Mice were orally administered PBS vehicle or BAs at a dose of 30 mg/kg/per day for 9 consecutive days beginning the day after MC was delivered. (b) percentage of baseline body weight, (c) serum level of IL-23, (d) representative photographs, (e) time course of ear thickness, (f) image of H&E section (Bar = 50 μm), (g) histological analysis of epidermal thickness, (h) representative immunohistochemical images of Ki-67 (Bar = 50 μm) and (i) absolute numbers of CD45⁺ leukocytes and neutrophils in the ear skin (n = 3–6). Data are presented as mean ± SEM. Data are representative of two independent experiments. * P < 0.05, **P < 0.01. ***P < 0.001, by two-way ANOVA with Bonferroni’s test compared with IL-23 MC + vehicle group in (e) and by one-way ANOVA with Dunnett’s test compared with IL-23 MC + vehicle group in (g, i). BA, bile acid; LCA, lithocholic acid; MC, minicircle DNA.

Figure 2.. Intravenous administration of LCA ameliorates IL-23 MC-induced PsD.

(a) Schematic illustration of experimental protocols. Mice were administered i.v. PBS vehicle or LCA at a dose of 4 mg/kg/per day every other day beginning the following day after MC was delivered. (b) Percentage of baseline body weight, (c) representative photographs, (d) time course of ear thickness, (e) image of H&E section (Bar = 50 μm), (f) histological analysis of epidermal thickness, (g) representative images of Ki-67 immunohistochemistry (Bar = 50 μm) and (h) absolute numbers of CD45⁺ leukocytes and neutrophils in the ear skin. Data are presented as mean ± SEM (n = 3–6). Data are representative of two independent experiments. * P < 0.05, **P < 0.01. ***P < 0.001, by two-way ANOVA with Bonferroni post hoc in (d) and by one-way ANOVA with Dunnett’s test compared with IL-23 MC + vehicle group in (f, h). LCA, lithocholic acid; MC, minicircle DNA.

Figure 3.. LCA suppresses IL-17A response in IL-23 MC-induced PsD.

(a) Representative flow cytometry plots, (b) percentage and absolute numbers of IL-17A–producing T cells in cervical lymph nodes from mice with treatment in Figure 2. (c) Transcripts of Il17a in ear skin and (d) representative immunohistochemical images of p-STAT3 in ear skin (Bar = 50 μm) (n = 3–6). Data are presented as mean ± SEM. Data are representative of two independent experiments. *P < 0.05, **P < 0.01. ***P < 0.001, by one-way ANOVA with Dunnett’s test compared with IL-23 MC + vehicle group in (b, c). LCA, lithocholic acid; LN, lymph node; MC, minicircle DNA.

Figure 4.. LCA inhibits IL-17A production by T cells in vitro.

(a, b) Representative flow cytometry plots of CD3⁺ T cells expressing IL-17A from the cLN of naive mice (a) or IL-23 MC-injected mice (b), after 24 hours incubation in the presence of IL-23 (100 ng/ml) with indicated BAs (50 μM). (c) Total fluorescence intensity of IL-17A in CD3⁺ T cells with particular treatment indicated. (d) Transcripts of Rorc and Il17a in cLN cells, after 3 hours incubation in the presence of IL-23 (100 ng/ml) with indicated BAs (50 μM) (n = 3). (e) Transcripts of Il17a in cLN cells, after 12 hours incubation in the presence of IL-23 (50 ng/ml) and IL-1β (10 ng/ml) with LCA (50 μM). (f) Transcripts of IL17A and RORC analyzed by qRT-PCR (from five individual donors) and (g) positivity of IL-17A analyzed by flow cytometry in healthy adult human CD4⁺CCR6⁺ T cells are inhibited by coculture with LCA overnight in a dose-dependent manner (from three individual donors). Data are presented as mean ± SEM. Data are representative of two independent experiments. *P < 0.05, **P < 0.01. ***P < 0.001. BA, bile acid; cLN, cervical lymph node; DCA, deoxycholic acid; FI, fluorescence intensity; LCA, lithocholic acid; SSC, side scatter; UDCA, ursodeoxycholic acid.

Figure 5.. FXR and TGR5 are not essential for BA-mediated inhibition of IL-17A production.

(a) Total fluorescence intensity of IL-17A on CD3⁺ T cells from cLNs of wild type mice and (b) transcripts of Il17a in the skin-lymph nodes coculture assay, after 24 hours incubation in the presence of IL-23 (100 ng/ml) with INT-747, INT-777, or LCA (50 μM). (c, d) Total fluorescence intensity of IL-17A on CD3⁺ T cells and protein levels of IL-17A in the cLN supernatants from (c) FXR-deficient or (d) TGR5-deficient mice after 24 hours incubation in the presence of IL-23 (100 ng/ml), with or without LCA treatment (50 μM). Data are presented as mean ± SEM. Data are representative of two independent experiments. *P < 0.05, **P < 0.01. ***P < 0.001. BA, bile acid; cLN, cervical lymph node; FI, fluorescence intensity; KO, knockout; LCA, lithocholic acid; TFI, total FI.

Figure 6.. BAs inhibit CCL20 production by keratinocytes and CCL20-CCR6–mediated T-cell chemotaxis.

(a, b) Transcripts of CCL20 and protein levels of CCL20 in the supernatants from keratinocytes stimulated with IL-17A (a) or TNF-α (b), with the treatment of indicated BAs (50 μM). (c) Migration of CCR6+ Jurkat cells toward medium cultured with stimulated keratinocytes plus BAs. (d) Transcripts of Ccl20 in primary keratinocytes from WT, FXR-KO, and TGR5-KO mice, after stimulation with IL-17A in the presence or absence of LCA (50 μM). (e) Immunohistochemical staining for CCL20 in ear skin from mice treated with GFP MC, IL-23 MC + vehicle, or IL-23 MC + LCA. (f) Percentage and absolute numbers of CCR6+, γδ-low T cells in ear skin. Data are presented as mean ± SEM. Data are representative of two independent experiments. *P < 0.05, **P < 0.01. ***P < 0.001. (g) Schematic of a proposed mechanism for improvement of PsD due to BAs. BA, bile acid; DCA, deoxycholic acid; KO, knockout; LCA, lithocholic acid; MC, minicircle DNA; WT, wild-type.