1. IL-36γ Induced by the TLR3-SLUG-VDR Axis Promotes Wound Healing via REG3A 
Ziwei Jiang, Yuanqi Liu, Changwei Li, Leilei Chang, Wang Wang, Zhenhua Wang, Xiaoguang Gao, Bernhard Ryffel, Yelin Wu, Yuping Lai 
Journal of Investigative Dermatology 
Volume 137, Issue 12, Pages (December 2017)
DOI: /j.jid
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2. Figure 1
IL-36 cytokines are increased in epidermal keratinocytes after skin injury. (a) IL-36α, (b) IL-36β, and (c) IL-36γ mRNA expression of 2 mm of skin surrounding the wound edge after aseptic injury. (d) Immunoblot of IL-36γ in skin extracts from mouse wound edges treated as in a. (e) Immunofluorescent staining of IL-36γ in mouse skin wounds treated as in a. Long scale bars = 0.2 mm, short scale bars = 0.05 mm. Rectangles designate region of original magnification ×400 shown in insets. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < P-values were determined by one-way analysis of variance. Data are mean ± standard error of the mean and are representative of two independent experiments with n = 3. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
TLR3 is required for IL-36γ expression in keratinocytes. (a) IL-36γ expression in day 3 skin wounds of wild-type (n = 10; black bars), Tlr3–/– (n = 8; grey bars), or Trif–/– (n = 8; white bars) mice. (b, c) IL-36γ expression in NHEKs stimulated with damaged cells after (b) TLR3 silencing or (c) treatment with nucleases. (d–f) IL-36γ or TLR3 production in NHEKs stimulated with different doses of (d) poly(I:C) for 24 hours, (e) 5μg/ml poly(I:C) for indicated times, or (f) 5μg/ml poly(I:C) for 24 hours after TLR3 silencing. (g) IL-36γ production in wild-type and Tlr3–/– murine keratinocytes stimulated with poly(I:C) for 24 hours. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < P-values were determined by (b, d, e) one-way analysis of variance or (a, c, f, g) two-way analysis of variance. Data are mean ± standard error of the mean and are representative of three independent experiments. h, hour; NHEK, neonatal human epidermal keratinocyte; n.s., not significant; poly(I:C), polyinosinic:polycytidylic acid; si, small interfering; TLR, toll-like receptor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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4. Figure 3
TLR3-mediated IL-36γ is negatively regulated by VDR. (a) VDR production in NHEKs stimulated with 5μg/ml poly(I:C) or (d) 1μmol/L MC903. (b, c) IL-36γ production in NHEKs stimulated with poly(I:C) after (b) VDR silencing or (c) overexpression. (e) IL-36γ and VDR production in NHEKs stimulated with poly(I:C) and MC903 for 24 hours. (f) PCR amplification of VDR binding sites on IL-36γ promoter after CHIP assay. (g–i) Normalized IL-36γ luciferase activity in NHEKs transfected with IL-36γ promoter and stimulated with (g) poly(I:C) and MC903 or promoter with the (h) truncation or (i) mutation in VDR binding sites. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < P-values were determined by (a, d, g-i) one-way analysis of variance or (b, c, e) two-way analysis of variance. Data are mean ± standard error of the mean and are representative of three independent experiments with n = 3. BP, binding position; CHIP, chromatin immunoprecipitation; EV, empty vector; FL, full length; h, hour; mut, mutated; NHEK, neonatal human epidermal keratinocyte; poly(I:C), polyinosinic:polycytidylic acid; sh, short hairpin; TLR, toll-like receptor; VDR, vitamin D receptor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
TLR3-induced SLUG inhibits VDR. (a) SLUG and (b) VDR mRNA expression in primary wild-type and Tlr3–/– murine keratinocytes treated with different doses of poly(I:C) for 24 hours. (c) SLUG production in NHEKs stimulated with 5μg/ml poly(I:C) for indicated times. (d, e) The production of IL-36γ, VDR, and SLUG in NHEKs stimulated with 5μg/ml poly(I:C) before and after (d) SLUG overexpression or (e) silencing. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < P-values were determined by (c) one-way analysis of variance or (a, b, d, e) two-way analysis of variance. Data are mean ± standard error of the mean and are representative of three independent experiments with n = 3. EV, empty vector; h, hour; NHEK, neonatal human epidermal keratinocyte; poly(I:C), polyinosinic:polycytidylic acid; sh, short hairpin; TLR, toll-like receptor; VDR, vitamin D receptor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
IL-36γ induces REG3A in keratinocytes. (a–c) REG3A production in NHEKs stimulated with (a) 100ng/ml IL-36γ or (b) 10μg/ml poly(I:C) for indicated times or (c) different doses of poly(I:C) for 24 hours. (d) RegIIIγ prodcution in primary murine keratinocytes stimulated with 10μg/ml poly(I:C) for 24 hours. (e) RegIIIγ mRNA expression in day 3 skin wounds of wild-type (n = 15) and Tlr3–/– (n = 8) mice. (f–h) REG3A expression in NHEKs stimulated with 10μg/ml poly(I:C) for 24 hours after (f) SLUG silencing or (g) VDR overexpression or (h) IL-36γ silencing. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < P-values were determined by (a–c, f, g) one-way analysis of variance or (d, e, h) two-way analysis of variance. Data are mean ± standard error of the mean and are representative of three independent experiments. EV, empty vector; h, hour; poly(I:C), polyinosinic:polycytidylic acid; sh, short hairpin; VDR, vitamin D receptor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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7. Figure 6
TLR3-mediated IL-36γ promotes wound healing via the induction of RegIIIγ. (a, c) Loricrin and (b) filaggrin expression in NHEKs stimulated with 100ng/ml IL-36γ and/or 1.6 mmol/L calcium (c) for 24 hours. (d, e) Wound healing after (d) REG3A silencing and (e) mitomycin C treatment. (f) Wound healing of wild-type (n = 4) and Il36r–/– mice (n = 6). (g–i) RegIIIγ expression in skin wounds of (g) WT and Il36r–/– mice or (h) WT and Tlr3–/– mice or (i) 24 hours after intradermal injectiton with IL-36γ. (j, k) Splinting wound healing of wild-type and Tlr3–/– mice treated with IL-36γ (n = 3). Scale bars = 0.05 mm. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < P-values were determined by two-way analysis of variance. Data are mean ± standard error of the mean and are representative of three independent experiments. h, hour; NHEK, neonatal human epidermal keratinocyte; n.s., not significant; sh, short hairpin; TLR, toll-like receptor; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions