Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed, paraffin-embedded skin sections with Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) and antibodies against CCL20 (Thermo Fisher Scientific, Waltham, MA, USA), CCR6 (Abcam, Cambridge, UK) and a-smooth muscle actin (a-SMA) (Abcam). Antigen retrieval was performed using Dako Target Retrieval Solution pH 9 (Dako North America, Inc., Carpinteria, CA, USA). Skin samples were obtained from forearms of 6 diffuse cutaneous SSc (dcSSc) patients, 6 limited cutaneous SSc (lcSSc) patients and 5 healthy controls. Horseradish peroxidase activity was detected by 3, 3’-diaminobenzidine. Counterstaining was conducted with methyl green. Arterioles, capillaries and venules were distinguished based on their histological features. Arterioles were determined based on the presence of internal elastic lamina and tunica media and bulged nucleus into the lumen. Capillaries and venules were determined based on their diameter. Blood vessels with diameter almost equal to or less than that of erythrocyte were classified as capillaries, and the others were classified as venules [26]. The determination of blood vessel type was performed by two independent dermatologists (T. Ikawa and Y. Asano).
Gene silencing of FLI1
Human dermal microvascular endothelial cells (HDMECs) (Lonza, Walkersville, MD, USA) were cultured on collagen-coated tissue culture plates in Endothelial Basal Medium-2 supplemented with the Endothelial Cell Growth Medium-2 Bullet Kit (Lonza). Shortly after seeded, HDMECs were transfected with FLI1 siRNA or non-silencing scrambled RNA (SCR) (10 nM, both purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA) mixed with HiPerFect Transfection Reagent (Qiagen, Valencia, CA, USA) for 48 hours. Cells were then collected using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) for RNA isolation.
RNA isolation and quantitative reverse transcription (qRT)-PCR
RNA isolation from HDMECs and qRT-PCR were conducted as described previously [27]. The sequences of primers were as follows: FLI1-forward 5’-GGATGGCAAGGAACTGTGTAA-3’, FLI1-reverse 5’-GGTTGTATAGGCCAGCAG-3’, CCL20-forward 5’- TTGTGCGTCTCCTCAGTAAAAA-3’, CCL20-reverse 5’- GCAAGTGAAACCTCCAACCC-3’; CCR6-forward 5’- GGGGGCTGTCAGTCATCATC-3’, CCR6-reverse 5’- CGTAGAGCACAGGGTTCAGG-3’; GAPDH-forward 5’-ACCCACTCCTCCACCTTTGA-3’, GAPDH-reverse 5’-CATACCAGGAAATGAGCTTGACAA-3’.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was conducted using EpiQuik ChIP kit (Epigentek, Farmingdale, NY, USA) as described previously [27]. Putative FLI1 binding site in the CCR6 promoter was predicted using a web site, JASPAR. The primers that amplify fragments of the CCR6 transcript variant 1 (-252 bp to -39 bp) and transcript variant 2 (-1395 bp to -1186 bp) were as follows: CCR6 transcript variant 1/Forward, 5’- ACTGCCGTATCCCTTGTGC-3’; CCR6 transcript variant 1/Reverse, 5’- TGGGAGAATGGACATTGTGACC-3’, CCR6 transcript variant 2/Forward, 5’- TTCTTTCCAGGCAGGCATTG-3’; CCR6 transcript variant 2/Reverse, 5’- TCCTCCTCATTTCTACCATCGC-3’. The amplified DNA products were resolved by agarose gel electrophoresis.
In vivo local gene silencing of Ccr6 with atelocollagen
Ccr6 siRNA was transfected to murine skin in vivo using atelocollagen (AteloGene® Local Use ‘Quick Gelation’, Koken, Tokyo, Japan). Ten micromolar of Silencer select Ccr6 siRNA or SCR (both from Thermo Fisher scientific) was mixed with atelocollagen, 150 μL of which was subcutaneously given to shaved lower back of wild-type (WT) mice once a week. After the injection of siRNA, 200 mg of BLM was subcutaneously injected to the same place every day for a week or 4 weeks.
Vascular permeability assay
Evans blue dye (0.5%) (Sigma-Aldrich, St. Louis, MO, USA) in 200 µl of 0.9% saline was injected into the tail vein, and mice were sacrificed in half an hour. The presence of vascular leakage was macroscopically evaluated in the skin.
In vitro angiogenesis assay
HDMECs were treated with 20 nM of CCR6 siRNA or SCR (both from Thermo Fisher Scientific) for 48 hours. Then, cells were treated with 10 µg/mL of mitomycin C (Sigma Aldrich) for 2 hours. A 24-well plate was coated with 250 µL of growth factor-reduced Matrigel (BD Biosciences, San Jose, CA, USA). After the gel was solidified, cells were trypsinized and seeded onto the Matrigel at 7 × 104 cells per well and incubated for 24 hours. Cells were treated with calcein AM before observation. Five photographs were taken randomly from each well. The numbers of meshes, tubes and intersections were counted manually.
Statistical analysis
Statistical analysis was conducted with Welch’s t-test to compare two unpaired data. Statistical significance was defined as a P value of <0.05.