Xiao-Yin-Fang Therapy Alleviates Psoriasis-like Skin Inflammation Through Suppressing γδT17 Cell Polarization


doi: 10.3389/fphar.2021.629513.


eCollection 2021.

Affiliations

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Xilin Zhang et al.


Front Pharmacol.


.

Abstract

Psoriasis is an immune-mediated chronic inflammatory skin disease primarily mediated by the activation of interleukin (IL)-17-producing T cells. Traditional Chinese Medicine (TCM) represents one of the most effective complementary and alternative medicine (CAM) agents for psoriasis, which provides treasured sources for the development of anti-psoriasis medications. Xiao-Yin-Fang (XYF) is an empirically developed TCM formula that has been used to treat psoriasis patients in Shanghai Changhai Hospital for over three decades. Imiquimod (IMQ)-induced psoriasis-like dermatitis mouse model was utilized to investigate the therapeutic effects of XYF by the assessment of disease severity and skin thickness. Flow cytometric assay was performed to explore the influence of XYF on skin-related immunocytes, primarily T cells. And, RNA sequencing analysis was employed to determine the alternation in gene expression upon XYF therapy. We discovered that XYF alleviated psoriasis-like skin inflammation mainly through suppressing dermal and draining lymph-node IL-17-producing γδT (γδT17) cell polarization. Moreover, XYF therapy ameliorated the relapse of psoriasis-like dermatitis and prohibited dermal γδT cell reactivation. Transcriptional analysis suggested that XYF might regulate various inflammatory signaling pathways and metabolic processes. In conclusion, our results clarified the therapeutic efficacy and inner mechanism of XYF therapy in psoriasis, which might promote its clinical application in psoriasis patients and facilitate the development of novel anti-psoriasis drugs based on the bioactive components of XYF.


Keywords:

Xiao-Yin-Fang therapy; interleukin-17; psoriasis; relapse; traditional Chinese medicine; γδT cells.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures


FIGURE 1



FIGURE 1

UPLC-Q/TOF-MS analysis of Xiao-Yin-Fang. Base Peak Chromatograms of Xiao-Yin-Fang (XYF) in ESI-negative mode (A) and ESI-positive mode (B) together with the chemical structures and ultraviolet (UV) chromatograms of seven components (1, (R, S)-goitrin; 2, baicalin; 3, salvianolic acid B; 4, emodin; 5, matrine; 6, oxymatrine; 7, tanshinone II a).


FIGURE 2



FIGURE 2

High-dose Xiao-Yin-Fang alleviated imiquimod-induced psoriasis-like skin inflammation on mouse back. (A) Schematic of the experimental procedures. Mice were given different-dose Xiao-Yin-Fang (XYF), multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) solution or distilled water (H2O) by gavage twice daily from day 0 to day 9, and received imiquimod (IMQ) or Vaseline (VAS) application on shaved back skin once a day from day 5 to day 9. (B) Representative pictures of mouse skin lesions (XYF-L, low-dose XYL; XYF-M, medium-dose XYF; XYF-H, high-dose XYF). (C–E) Evaluation of PASI score (C), skin thickness (D) and body weight (E) of mice. The statistic differences between IMQ + XYF-H group and IMQ + H2O group were annotated (n = 54, three independent experiments). (F) H&E staining and calculated epidermal acanthosis (G) and papillomatosis index (H) (x200; bar = 100 μm; n = 36, three independent experiments). (I) Ki-67 staining and assessment of its H-score of epidermal fields (J) (x200; bar = 100 μm; n = 36, three independent experiments). The data were presented as mean ± s.e.m.


FIGURE 3



FIGURE 3

Xiao-Yin-Fang therapy prevented lymphnode γδT cell secretion of IL-17. Mice were treated as in Figure 2. Skin-draining axillary, brachial and inguinal lymph nodes (LN) were harvested on day 10. Freshly isolated LN cells were in vitro cultured in the presence of PMA, ionomycin and Brefeldin A for 4 h. LN cells were then stained with anti-CD45, TCRβ, γδTCR, CD11b, and IL-17 antibodies, which were analyzed by flow cytometry. Representative scatter plots, the ratios, and numbers of LN γδT cells (A) and IL-17+ γδT cells (B). n = 31, three independent experiments. The data were presented as mean ± s.e.m.


FIGURE 4



FIGURE 4

Xiao-Yin-Fang lessened imiquimod-induced psoriasis-like dermatitis on mouse ears. (A) Schematic of the experimental procedures. Mice were given XYF or H2O by gavage twice daily from day 0 to day 9, and received IMQ or VAS application on mouse ears once a day from day 5 to day 9 (B) Representative pictures of mouse ear lesions. (C, D) Evaluation of PASI score (C) and skin swelling (D) of mouse ears (n = 27, three independent experiments) (E) H&E staining and calculated epidermal acanthosis (F) (x200; bar = 50 μm; n = 18, three independent experiments). (G) Ki-67 staining and assessment of its H-score of epidermal fields (H) (x200; bar = 50 μm; n = 18, three independent experiments). (I–K) Freshly isolated dermal cells were obtained and stained with anti-CD45, MHC-II, CD11b, F4/80, CD11c, Ly6C, and Ly6G antibodies, which were analyzed by flow cytometry. (I) Representative scatter plots of dermal neutrophils and inflammatory monocytes. The ratios and cell numbers of dermal neutrophils (J) and inflammatory monocytes (K) (n = 16, three independent experiments). The data were presented as mean ± s.e.m.


FIGURE 5



FIGURE 5

Xiao-Yin-Fang suppressed dermal γδT17 cell polarization. Mice were treated as in Figure 4, and the dermal cells of mouse ears were harvested. (A–C) Freshly isolated dermal cells were in vitro cultured in the presence of PMA, ionomycin and Brefeldin A for 4 h. Cultured dermal cells were stained with anti-CD45, TCRβ, γδTCR, and CD11b antibodies (n = 24, four independent experiments) (A) Representative scatter plots of dermal γδint T cells and γδhigh T cells. (B) The ratios and numbers of dermal γδint T cells. (C) Uncultured dermal cells were stained with anti-CD45, TCRβ, γδTCR, CD11b and Ki-67 antibodies. Representative scatter plots and the ratios of dermal Ki-67+ γδint T cells (n = 17, three independent experiments). (D) Uncultured dermal cells were stained with anti-CD45, TCRβ, γδTCR, CD11b and Annexin-V antibodies. Representative scatter plots and the ratios of dermal Annexin-V+ γδint T cells (n = 18, three independent experiments). (F) Cultured dermal cells were stained with anti-CD45, TCRβ, γδTCR, CD11b, and IL-17 antibodies. Representative scatter plots, the ratios and numbers of dermal IL-17+ γδint T cells (n = 24, three independent experiments). (G) Uncultured dermal cells were stained with anti-CD45, TCRβ, γδTCR, CD11b, and ROR-γt antibodies. Representative scatter plots and the ratios of dermal ROR-γt+ γδint T cells (n = 17, three independent experiments). The data were presented as mean ± s.e.m.


FIGURE 6



FIGURE 6

Xiao-Yin-Fang alleviated the relapse of psoriasis-like dermatitis and prohibited dermal γδint T cell reactivation. Mice were treated as in Supplementary Figure S7. (A) Representative pictures of mouse right ear lesions (1st: the initial occurrence of psoriasis-like dermatitis; 2nd: the recurrence of psoriasis-like dermatitis). (B, C) Evaluation of PASI score (B) and skin swelling (C) of mouse right ears (n = 36, two independent experiments). (D) H&E staining and calculated epidermal acanthosis (E) (x200; bar = 50 μm; n = 30, two independent experiments). (F) Ki-67 staining and assessment of its H-score of epidermal fields (G) (x200; bar = 50 μm; n = 30, two independent experiments). (H-K) Freshly isolated dermal cells were in vitro cultured in the presence of PMA, ionomycin and Brefeldin A for 4 h. Cultured dermal cells were stained with anti-CD45, TCRβ, γδTCR, CD11b, and IL-17 antibodies (n = 30, two independent experiments). (H) Representative scatter plots of dermal γδint T cells and γδhigh T cells. (I) The ratios and numbers of dermal γδint T cells. (J) Representative scatter plots of dermal IL-17+ γδint T cells. (K) The ratios and numbers of dermal IL-17+ γδint T cells. The data were presented as mean ± s.e.m.


FIGURE 7



FIGURE 7

Transcriptional analysis of Xiao-Yin-Fang therapeutic effects. Epidermal and dermal sheets of mouse ears were freshly obtained from VAS + H2O, IMQ + H2O and IMQ + XYF group (n = 3 for each group), which underwent RNA sequencing analysis. (A, B) Heatmap clustering analysis of epidermal and dermal differentially expressed genes (DEG). (C) Venn diagrams showed the numbers of overlapping epidermal DEGs (upper panel) and dermal DEGs (lower panel) between VAS + H2O vs. IMQ + H2O and IMQ + H2O vs. IMQ + XYF. (D-G) KEGG pathway analysis of DEGs.

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