MT-7117 synthesized at Mitsubishi Tanabe Pharma Corporation was dissolved in dimethylsulfoxide (DMSO) for in vitro assays or suspended in 0.5% methylcellulose solution for in vivo experiments.
BLM-induced skin fibrosis and lung inflammation—prophylactic evaluation
Ten-week-old female C3H/HeNCrlCrlj mice (Charles River Laboratories Japan, Inc.) were used, and all animal experiments were conducted in accordance with the Guidelines for Animal Experimentation of Mitsubishi Tanabe Pharma Corporation (approval number: BJ14-0699). The backs of mice were shaved, and the middle of the backs was marked with oil-based red ink. Phosphate-buffered saline (PBS) or BLM (Nippon Kayaku, Tokyo, Japan) (0.15 mg/0.1 mL per animal) was subcutaneously injected at the marked site once daily from day 0 to day 25 (since repeated BLM injections reduced the body weights of the mice remarkably, BLM injection was suspended several times for all groups). MT-7117 solutions at 0.1 mL/10 g of body weight were orally administered once daily (approximately 2 h before the BLM injection) for 29 consecutive days from day 0 until the day before the end of the evaluation (day 28). On day 29, mice were euthanized by inhalation anesthesia with isoflurane, and whole blood and back skin samples were collected. The left lung was dissected, weighed, and immersed in RNAlater solution (Qiagen, Hilden, Germany). The collected blood was separated into serum. The injection site of the skin was excised, weighed (wet weight), and immersed in 0.5 mol/L acetic acid solution containing 0.3 mg/mL pepsin to solubilize collagen. The collagen content of the solubilized sample was explored using the QuickZyme Soluble Collagen Assay kit (QuickZyme Biosciences, Leiden, Netherlands), and absorbance was determined by a microplate reader (VERSAmax, Molecular Devices, Sunnyvale, CA, USA). Serum levels of surfactant protein D (SP-D) were assayed using the Rat/Mouse SP-D kit YAMASA EIA (Yamasa Corporation, Choshi, Japan), and absorbance was determined by a microplate reader (VERSAmax). Total RNA of lungs was isolated using RNeasy 96 Universal Tissue Kit (Qiagen) according to the manufacturer’s instructions. Real-time PCR was performed using the One Step SYBR PrimeScript PLUS RT-PCR Kit (Takara Bio Inc., Kusatsu, Japan) and ABI PRISM 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Takara perfect real-time primer pairs (Takara Bio Inc.) were used to analyze the gene expression of chemokine CC ligand-2 (Ccl2, also known as monocyte chemotactic protein-1 (Mcp-1), MA066003), interleukin-6 (Il-6, MA039013), and hypoxanthine phosphoribosyltransferase (Hprt, MA031262). Quantification was performed using the comparative CT (cycle threshold) method employing Hprt as the housekeeping gene.
Pharmacokinetic study of MT-7117
A pharmacokinetic study of MT-7117 was performed using the BLM-induced SSc model (prophylactic evaluation) described above. After repeated oral administrations of MT-7117 once daily for 29 days, whole blood was collected from individual mice at each sampling point (1, 3, 6, and 24 h after the last dose) and serum was separated from blood. Serum concentration of MT-7117 was measured by an API 5000 MS/MS system (AB Sciex) coupled with Agilent 1200 HPLC (Agilent Technologies, Santa Clara, CA, USA).
Pre-established BLM-induced skin fibrosis—therapeutic evaluation
Evaluation of the therapeutic effect of MT-7117 using a pre-established BLM-induced skin fibrosis model was performed as previously described  and is briefly described as follows. Six to 7-week-old female C57BL/6 mice (Laboratory Animal Service Center, University of Zurich) were used, and all animal experiments were conducted in accordance with the European Community Council Directive for Care and Use of Laboratory Animals and the Swiss law (approval number: ZH002/17). BLM (Baxter AG, Deerfield, IL, USA) was dissolved in saline and subcutaneously administered every other day (0.1 mg/0.1 mL per animal) in defined areas of the upper back for 6 weeks (day 1 to 42). During the last 3 weeks of the experimental period (day 22 to 42), the mice were orally administered MT-7117, imatinib (Selleckchem, Huston, TX, USA), or vehicle once daily. On day 43, the mice were euthanized by CO2 inhalation, and back skin and serum samples were collected.
Mouse skin samples were fixed in 4% formalin and embedded in paraffin. Hematoxylin and eosin (HE) staining was performed following standard procedures. Skin thickness was determined by measuring the thickness of HE-stained skin samples using a microscope with an image analysis program (Zeiss Imager Z1, Carl Zeiss AG, Oberkochen, Germany). Skin thickness was measured by randomly selecting 3 records of non-overlapping pictures taken at 100× magnification of each piece of HE-stained mouse skin sample (in total, 6 pictures and 18 measurements per sample). Analyses were performed by two independent examiners in a blinded manner. Each value of skin thickness was calculated as an average of 36 measurements.
α-smooth muscle actin (αSMA, encoded by ACTA2) staining was performed for skin sections. After deparaffinization and rehydration, 10% goat serum (Vector Laboratories, Burlingame, CA, USA) was used to prevent unspecific antibody binding. The samples were incubated with primary antibody (1:750, monoclonal mouse anti-αSMA, clone 1A4, Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature, followed by incubation with alkaline phosphatase-labeled goat anti-mouse secondary antibody (Dako, Glostrup, Denmark) for 30 min at room temperature. The numbers of αSMA-positive myofibroblasts in the skin were determined manually using a microscope with an image analysis program (Zeiss Imager Z1). Quantification of αSMA-positive myofibroblasts in mouse skin was performed for 6 slides by recording 6 randomly selected, non-overlapping areas (172.5 × 218 μm, 37605 μm2) per slide of mouse skin at 400× magnification. Two independent examiners performed the analyses in a blinded manner. The number of αSMA-positive myofibroblasts in an area of 37605 μm2 was calculated as an average of 12 values.
Masson’s trichrome staining of skin sections was performed following standard procedures. Masson’s trichrome-stained sections were observed using a microscope with an image analysis program (Zeiss Imager Z1) at 100× magnification.
Microarray-based gene expression analysis
Gene expression profiling with Agilent Expression Array
Total RNA of mouse lung tissue samples of PBS_vehicle (control group without disease), BLM_vehicle (control group with disease), and BLM_MT-7117 (0.3 mg/kg) obtained from the BLM-induced SSc model (prophylactic evaluation) was used for analysis. The purity, concentration, and quality of the RNA samples were confirmed with NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 bioanalyzer (Agilent Technologies). The RNA was then Cy3-labeled using a Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies). Labeled cRNA samples were fragmented and then hybridized with the Agilent SurePrint G3 Mouse GE v2 8 × 60 K Microarray (Agilent Technologies) using a Gene Expression Hybridization Kit (Agilent Technologies). After hybridization, the microarray was washed with the Gene Expression Wash Buffers Pack (Agilent Technologies) and imaged with a scanner to detect the signals. Signal intensities were evaluated with Agilent Feature Extraction software 12.0.
Bioinformatics analysis in this study was performed with software, including GeneSpring GX 14.9.1, Ingenuity Pathways Analysis (IPA) 2019 autumn, and Microsoft Excel 2010. Genes that fluctuated ≥ 2 fold in the BLM_vehicle group in comparison with those of the PBS_vehicle group and that satisfied a p-value < 0.05 of the moderated t-test were defined as “differentially expressed genes (DEGs) in the disease model.” Among DEGs, the genes in the MT-7117 treatment group that fluctuated ≥ 1.5 fold in the opposite direction and met a p-value < 0.05 of the moderated t-test in comparison with those of the BLM_vehicle group were defined as “DEGs by MT-7117 treatment.” We inputted DEGs into IPA and performed two types of calculations: “Canonical pathways” and “Diseases or functions.” In this report, “Canonical pathways” is described as “pathways” and “Diseases or functions” as “category.” The former contains a list for a series of gene interactions and the latter is a list related to diseases and biological functions. These datasets are registered in the IPA software. Initially, Fisher’s exact test was used to calculate the probability of overlap between two sets of genes. Thereafter, downstream effect analysis (DSEA)  was used to calculate the activation z-score. DSEA is a technique for predicting the direction of change in expression patterns (either activation or inhibition) based on the expected causal effects between genes and functions. Based on the results of the DSEA analysis, information about the cell type (macrophages, neutrophils, mononuclear leukocytes, T-lymphocytes, B-lymphocytes, smooth muscle cells, endothelial cells, epithelial cells, and fibroblasts) that may be involved in the pathogenesis of SSc was extracted. Subsequently, we extracted information regarding biological functions and molecular signaling pathways, such as inflammation/immune abnormality, vascular dysfunction, and fibrosis, involved in the pathogenesis of SSc.
Serum protein profiling
Serum samples were obtained from mice of the BLM-induced SSc model (therapeutic evaluation). Samples from the saline_vehicle (control group without disease), BLM_vehicle (control group with disease), BLM_MT-7117 (10 mg/kg), and BLM_imatinib (150 mg/kg) groups were used for analysis. Using Luminex® assays (R&D systems, Minneapolis, MN, USA), 110 proteins were investigated (supplementary Table S1). Proteins satisfying any of the following criteria were analyzed: (i) all samples were adequately measured within the quantitative range; (ii) more than half of the samples in one or more groups were quantified, although some samples were out of the quantitative range. The values of samples that were out of the quantitative range were substituted by those within the lower and upper limits.
Skin biopsy samples and fibroblast isolation (fibroblast assay)
Full-thickness skin biopsies were obtained from clinically involved skin of patients affected by dcSSc, who were recruited from the Scleroderma clinic within the Leeds Institute of Rheumatic and Musculoskeletal Medicine (UK). Control skin samples were from healthy donors. All patients with dcSSc and healthy donors provided written informed consent through the stratification for risk of progression in scleroderma (STRIKE SSC), which had been approved by the North–East Research Ethics committee (number 15/NE/0211). Patients with SSc fulfilled the 2013 ACR/EULAR classification criteria for SSc  and were classified as having dcSSc according to the LeRoy and Medsger criteria .
Dermal fibroblasts were isolated from the skin biopsy samples of healthy donors and SSc patients. The skin biopsy samples were minced with a scalpel, placed in plastic culture dishes, and covered with Dulbecco’s modified Eagle medium (DMEM) with 4.5g/L glucose (Thermo Fisher Scientific) supplemented with 20% fetal calf serum (FCS, Thermo Fisher Scientific), 1% penicillin and streptomycin (Merck, Darmstadt, Germany), and 1 μg/ml of amphotericin (Thermo Fisher Scientific) and cultured at 37°C in a humidified atmosphere of 5% CO2. Additional growth medium without amphotericin was added after 3 days. After 7 days, the culture medium was replaced with a medium containing 10% FCS and subsequently replenished every 2 to 3 days. When a visible outgrowth of cells was obtained, the fibroblasts were passaged. Briefly, cells were incubated with PBS containing 0.1% ethylene diamine tetra-acetic acid (EDTA) for 5 min and then detached with trypsin–EDTA solution (Merck) and cultured until 70–80% confluent in DMEM (1 g/L glucose) supplemented with 10% FCS and 1% penicillin and streptomycin. After a subsequent passage, primary cells were retrovirally immortalized using human telomerase reverse transcriptase as previously described .
Treatment of fibroblasts
Cells were grown to confluence in six-well culture plates, then serum starved in DMEM 1% FCS for 24 h, followed by a 24-h incubation in the presence or absence of transforming growth factor-β1 (TGF-β1, 10 ng/mL) (Sigma-Aldrich). MT-7117 or αMSH (Peptide Institute, Osaka, Japan) was added to the culture medium during TGF-β1 stimulation. DMSO (0.1%) was added to all non-MT-7117-containing wells to normalize for the DMSO-induced effects on cells.
RNA isolation and real-time PCR analysis (fibroblast assay)
Total RNA was extracted using the Zymo quick RNA mini prep kit according to the manufacturer’s instructions (Zymo Research Corporation, Irvine, CA, USA). First-strand cDNA was synthesized using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). Quantitative RT-PCR was performed in triplicates using SYBR Green RT-PCR Mastermix Kit (Thermo Fisher Scientific) and the ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems). Quantification was performed using the comparative CT method employing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene. Primer sequences used in qPCR analyses were as follows: GAPDH forward 5′-ACC CAC TCC TCC ACC ACC TTT GA-3′, reverse 5′-CTG TTG CTG TAG CCA AAT TCG T-3′; ACTA2 forward 5′-TGT ATG TGG CTA TCC AGG CG-3′, reverse 5′-AGA GTC CAG CAC GAT GCC AG-3′; and collagen type I alpha 1 (COL1A1) forward 5′-GCT CCG ACC CTG CCG ATG TG-3′, reverse 5′-CAT CAG GCG CAG GAA GGT CAG C-3′.
Skin biopsy samples (immunohistochemical analysis of MC1R)
Full-thickness skin biopsies were obtained from the clinically involved skin of patients affected by dcSSc (n = 50) or limited cutaneous SSc (lcSSc, n = 10) who were recruited from the University of Erlangen–Nuremberg (Germany). Control skin samples (n = 30) were from healthy donors. All patients with SSc and healthy donors provided written informed consent as approved by the institutional ethics committee. Patients with SSc fulfilled the 2013 ACR/EULAR classification criteria for SSc  and were classified as having dcSSc according to the LeRoy and Medsger criteria . The study was approved by the ethical review board of the Medical Faculty of the University of Erlangen–Nuremberg (number 3766).
Immunohistochemical analyses of MC1R
Immunohistochemical analysis of paraffin-embedded sections was performed as previously described [34, 35].
Protein expression of MC1R in skin biopsy samples was detected by incubation with an anti-MC1R monoclonal antibody (1:50, clone EPR6530; Abcam, Cambridge, UK). Peroxidase-labeled goat anti-rabbit secondary antibody (Dako) was used as the secondary antibody. Isotype-matched antibodies were used as controls. Staining was visualized with 3,3′-diaminobenzidine (Sigma-Aldrich) and peroxidase substrate solution (Sigma-Aldrich). A nuclear counterstain was performed using Meyer’s hematoxylin (J.T. Baker, Phillipsburg, NJ, USA). For permanent mounting, the sections were dehydrated and dried, followed by mounting using Pertex (Histolab, Gothenburg, Sweden). A qualitative assessment was performed by assigning a score based on staining intensity after identifying each stained cell type and tissue element (the number of positive cells was not taken into account for the scoring). The staining intensity scale used for the evaluation is as follows: no staining was scored as 0, faint staining was scored 0.5, light staining was scored as 1, moderate staining was scored as 2, and dark staining was scored as 3.
To identify the cell types in the skin that were positive for MC1R, double staining for cell types of interest was performed on nine or ten samples randomly selected from those positive for single MC1R staining. The anti-MC1R monoclonal antibody was reacted in the same manner as described above. Alexa Fluor 488-labeled donkey anti-rabbit secondary antibody (Life Technologies, Carlsbad, CA, USA) was used as the secondary antibody. Subsequently, double staining was performed using the following antibodies against cell-specific markers: anti-prolyl-4-hydroxylase β antibodies (1:50, Acris Antibodies, Herford, Germany) for fibroblasts, anti-CD68 antibodies (1:200, Biolegend, San Diego, CA) for monocytes/macrophages, anti-CD66b antibodies (1:250, Biolegend) for neutrophils, and anti-CD31 antibodies (1:50, R&D Systems) for endothelial cells. Antibodies labeled with Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. For nuclear counterstaining, 4′,6′-diamino-2-phenylindole (Sigma-Aldrich) was used. Immunofluorescence-stained tissue sections were analyzed using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan).
All quantitative pharmacological data were expressed as mean ± standard error of the mean (SEM). Statistical differences between groups in each study were assessed as described below. All analyses were performed using the SAS system, and tests were two-tailed with a significance level of < 0.05 or one-tailed with a significance level of < 0.025.
BLM-induced SSc murine model—prophylactic evaluation
Differences between PBS_vehicle (control group without disease) and BLM_vehicle (control group with disease) groups were analyzed using Student’s t-test. Differences between the BLM_vehicle and MT-7117-treated groups were analyzed using Williams’ test. According to repeated measures analysis of variance (group effect and interaction effect; group × time point), body weights were significantly different (p < 0.01) in the groups mentioned above during the 29-day course of the experiment. Therefore, differences in body weight at each time point were analyzed between the PBS_vehicle and BLM_vehicle groups using Student’s t-test. Differences between the BLM_vehicle and MT-7117-treated groups were analyzed using Williams’ test.
BLM-induced SSc murine model—therapeutic evaluation
Differences between the saline_vehicle (control group without disease) and BLM_vehicle (control group with disease) groups and between the BLM_vehicle and imatinib-treated groups were analyzed using the Wilcoxon test (two-sided). Differences between the BLM_vehicle and MT-7117-treated groups were analyzed using Shirley–Williams’ multiple comparison test (one-sided).
Analysis of serum protein profiling
Differences between the saline_vehicle and BLM_vehicle groups, between the BLM_vehicle and MT-7117-treated groups, and between the BLM_vehicle and imatinib-treated groups were analyzed using Student’s t-test.