1. Systemic Sclerosis Dermal Fibroblasts Suppress Th1 Cytokine Production via Galectin- 9 Overproduction due to Fli1 Deficiency 
Ryosuke Saigusa, Yoshihide Asano, Kouki Nakamura, Megumi Hirabayashi, Shunsuke Miura, Takashi Yamashita, Takashi Taniguchi, Yohei Ichimura, Takehiro Takahashi, Ayumi Yoshizaki, Tomomitsu Miyagaki, Makoto Sugaya, Shinichi Sato 
Journal of Investigative Dermatology 
Volume 137, Issue 9, Pages (September 2017)
DOI: /j.jid
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2. Figure 1
Galectin-9 is up-regulated in SSc dermal fibroblasts in vivo and in vitro. (a) Representative images of galectin-9 staining in human skin samples from healthy control (HC) and systemic sclerosis (SSc) subjects. Right panels (original magnification ×400, scale bar = 50 μm) show the images with higher magnification, corresponding to the areas shown with dotted squares in left panels (original magnification ×100, scale bar = 200 μm). (b, c) LGALS9 mRNA levels in (b) the bulk skin and (c) cultivated dermal fibroblasts from HC and SSc subjects were assessed by quantitative real-time reverse transcriptase–PCR (n = 5 for each group). In the graphs, the relative value compared with HC is expressed as mean ± standard error of the mean. AU, arbitrary unit.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
Clinical correlation of galectin-9 levels in the skin and sera in systemic sclerosis patients. Serum galectin-9 levels were measured by a specific ELISA. LGALS9 mRNA levels were determined in the skin samples by quantitative real-time reverse transcriptase–PCR. (a) Serum galectin-9 levels were compared among diffuse cutaneous systemic sclerosis (dcSSc) patients (n = 39), limited cutaneous systemic sclerosis (lcSSc) patients (n = 26), and healthy control subjects (HC) (n = 20). (b) Serum galectin-9 levels were positively correlated with mRSS in systemic sclerosis patients (r = 0.36, P = 0.01). (c) There was a positive correlation between LGALS9 mRNA expression and mRSS in SSc patients (r = 0.43, P = 0.024). (d) Serum galectin-9 levels were positively correlated with ground-glass opacity score in systemic sclerosis patients with interstitial lung disease (r = 0.35, P = ). The solid lines represent the regression lines in b–d. AU, arbitrary unit; mRSS, modified Rodnan total skin thickness score.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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4. Figure 3
Galectin-9 is up-regulated by autocrine endothelin stimulation in activated dermal fibroblasts. (a, c) Lgals9 mRNA levels in the skin of (a) C57BL/6 wild-type mice treated with PBS or BLM for 4 weeks (n = 10 for each group) and in the skin of (c) BLM-treated C57BL/6 wild-type mice under daily administration of PBS or bosentan (20 mg/kg) (n = 10 for each group) were assessed by quantitative real-time reverse transcriptase–PCR. In the graphs, the relative value compared with control group is expressed as mean ± standard error of the mean. (b, d) Under the same conditions as in a and c, galection-9 expression was assessed by immunohistochemistry. Representative images were shown (original magnification ×400, scale bar = 50 μm). (e) Galectin-9 expression was evaluated by immunoblotting in systemic sclerosis dermal fibroblasts treated with the indicated concentration of bosentan for 48 hours. A representative result of three independent experiments is shown. AU, arbitrary unit; BLM, bleomycin; BOS, bosentan; M, mol/L; PBS, phosphate buffered saline; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
Fli1 regulates galectin-9 expression in dermal fibroblasts. (a) LGALS9 and FLI1 mRNA levels in human normal dermal fibroblasts treated with scrambled nonsilencing RNA (SCR) or FLI1 siRNA were assessed by quantitative real-time reverse transcriptase–PCR (n = 6). (b) Chromatin was isolated from normal dermal fibroblasts, and immunoprecipitation was conducted with rabbit anti-Fli1 antibody or rabbit IgG. PCR amplification was carried out using LGALS9 promoter-specific primers. (c) Lgals9 mRNA levels in the back skin of wild-type (WT) and Fli1+/– mice and in dermal fibroblasts from those mice were assessed by quantitative real-time reverse transcriptase–PCR (bulk skin, n = 8 for WT mice and n = 7 for Fli1+/– mice; dermal fibroblasts, n = 10 for each group). (d) Galectin-9 protein expression was evaluated by immunoblotting in WT and Fli1+/– dermal fibroblasts in cell culture (n = 7). Band intensity was quantified with densitometry, and galectin-9 expression levels were normalized by loading controls. Representative blots are shown. In the graphs, the relative value compared with control group is expressed as mean ± standard error of the mean. AU, arbitrary unit; siRNA, small interfering RNA.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
Galectin-9 suppresses IFN-γ production of CD4+ T cells in cell culture and BLM-treated mice. (a) Percentages of CD4+ T cells expressing IFN-γ, IL-4, and IL-17A co-cultured with dermal fibroblasts from wild-type (WT) and Fli1+/– mice (n = 6). (b) Percentages of IFN-γ, IL-4, and IL-17A–expressing CD4+ T cells co-cultured with dermal fibroblasts from Fli1+/– mice treated with scrambled nonsilencing RNA (SCR) or Lgals9 siRNA (n = 6). (c) Mean fluorescence intensity (MFI) of IFN-γ, IL-4, and IL-17A in CD4+ T cells co-cultured with dermal fibroblasts from Fli1+/– mice treated with SCR or Lgals9 siRNA (n = 6). (d) Lgals9 mRNA expression in dermal fibroblasts from Fli1+/– mice treated with SCR or Lgals9 siRNA (n = 6). (e, f) C57BL/6 wild-type mice were subcutaneously injected with Lgals9 siRNA or SCR mixed with atelocollagen, followed by BLM injection. (e) MFI of IFN-γ, IL-4, and IL-17A in skin-infiltrating CD4+ T cells was determined 1 week after injection by flow cytometry (n = 4). (f) Collagen deposition in the lesional skin was assessed by hydroxyproline assay 4 weeks after injection (n = 4). For flow cytometry, representative plots are shown. In the graphs, the relative value compared with control group is expressed as mean ± standard error of the mean. AU, arbitrary unit; BLM, bleomycin; siRNA, small interfering RNA.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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7. Figure 6
Bosentan suppresses the expression of galectin-9 in systemic sclerosis dermal fibroblasts in vivo as well as circulating galectin-9 levels. (a) Immunostaining for galectin-9 was carried out in three systemic sclerosis patients treated with bosentan and three systemic sclerosis patients not treated with bosentan. Representative images are shown. Original magnification ×400, scale bar = 50 μm. (b, c) Serum galectin-9 levels were measured by a specific ELISA in (b) six systemic sclerosis patients treated with bosentan and in (c) seven patients treated with intravenous cyclophosphamide pulse (IVCY). The levels were compared before versus after the treatments.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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