SUPPLEMENTARY INFORMATION Involvement of IL-21 in the epidermal hyperplasia of psoriasis Roberta Caruso 1, Elisabetta Botti 2, Massimiliano Sarra 1, Maria Esposito 2, Carmine Stolfi 1, Laura Diluvio 2, Maria Laura Giustizieri 2, Valentina Pacciani 2, Annamaria Mazzotta 2, Elena Campione 2, Thomas T MacDonald 3, Sergio Chimenti 2, Francesco Pallone 1, Antonio Costanzo 2, Giovanni Monteleone 1. Gastroenterology 1 and Dermatology 2 Units, Internal Medicine Department, University of Rome Tor Vergata, Rome, Italy; 3 Institute of Cell and Molecular Science, Bart s and the London School of Medicine and Dentistry, London, UK.
Caruso et al. 2 METHODS Patients This study received ethical approval by the local Committee. Biopsies were collected from the lesional and non-lesional skin of 34 patients with psoriasis, 15 normal controls, four patients with lichen planus, five patients with contact dermatitis, five patients with atopic dermatitis, and 3 patients with LES. Cell culture PBMC were isolated by Ficoll gradients, and used to assess cytokines, CLA, CD4, and CD161, or to purify CD4 +, CD8 +, CD20 +, CD56 + cells, by specific magnetic beads (Miltenyi Biotec). PBMC of psoriatic patients were also resuspended in RPMI 1640 supplemented with 10% inactivated fetal bovine serum, penicillin (100 U ml 1 ), streptomycin (100 µg ml 1 ) (complete medium) (Life Technologies-GibcoCRL), and activating CD3 and CD28 beads (Miltenyi) for 48 hours, and then used in the human psoriasis-xenograft mouse model. Skin biopsies of psoriatic patients were cultured in dispase solution (2 U ml 1 ; Roche) overnight at 4 C, to peel off the epidermis. The dermal layer was minced and resuspended in complete medium for 72 hours at 37 C. The resulting dermal cells and circulating CD4 + cells were stimulated with Phorbol 12-myristate 13-acetate (80 pm), ionomycin (1 mg ml 1 ), and monensin (2 µm, from ebioscience) for 5 hours, and analyzed by flow-cytometry. The epidermis of non-lesional skin of psoriatic patients and controls was minced and incubated in the presence of trypsin for 25 minutes at 37 C. The resulting keratinocytes were either used to assess IL-21R or stimulated with IL-21 (50 200 ng ml 1, Biosource) or EGF (20 ng ml 1, Peprotech). Cells were also pre-incubated with PD98059 (50 µm, Inalco) for 1 hour prior to adding IL-21. After 48 hours, cell proliferation and apoptosis were assessed by flow-cytometry. The
Caruso et al. 3 growth of keratinocytes was also assessed by measuring the incorporation of 5-bromo-2'- deoxyuridine (BrdU) into DNA. The level of BrdU-positive cells was assessed by ELISA. Flow-cytometry The following anti-human antibodies were used: IL-21-PE, IL-17-APC (ebioscience), IFNγ-FITC, IL-21R PE, CD4-FITC, CD161-APC, and CLA-FITC (from Becton Dickinson), CD56- APC (Biolegend), CD4-APC, CD8-APC (Miltenyi Biotec), CD20-FITC (Immunotools), and isotype control IgGs (Becton Dickinson). To track the proliferation, keratinocytes were incubated in 0.2 µm CFSE (Invitrogen) at 37 C for 30 minutes, and then stimulated as described above. After 48 hours, CFSE fluorescence was evaluated. Annexin V and propidium iodide-positive cells were examined by a commercially available kit (Beckman Coulter). Real-time PCR The following conditions were used: denaturation 1 minute at 95 C, annealing 30 seconds at 58 C for human IL-21, human IFN-γ, mouse IL-22, mouse KRT6 and mouse KRT 16, at 62 C for mouse IL-21, followed by 30 seconds of extension at 72 C. Primers sequence was as follows: human IL-21, FWD: 5 -GGAGAGGATTGTCATCTGTC-3 ; REV: 5 - CACAGTTTGTCTCTACATCTTC-3 ; human IFN-γ, FWD: 5 - TGGAGACCATCAAGGAAGAC-3 ; REV: 5 - GCGTTGGACATTCAAGTCAG-3 ; mouse IL- 21, FWD: 5 -CCTCCTGATTAGACTTCGTCAC-3 ; REV: 5 - GGTTTGATGGCTTGAGTTTGGC-3 ; mouse IL-22 FWD: 5 - GACCAAACTCAGCAATCAGC-3 ; REV: 5 -ATCTCTCCACTCTCTCCAAG-3 ; mouse KRT 6, FWD: 5 -TGAAGGAGTACCAGGAACTC-3 ; REV: 5 -CACCACAGAGATGTTGACTG-3 ; mouse KRT16, FWD: 5 -AAGACTACAGCCCCTACTTC-3 ; REV: 5 - CATTCTCGTACTTGGTCCTG-3. Human IL-7 and IL-15 were evaluated using TaqMan probes
Caruso et al. 4 (Applied Biosystems). Primers for mouse IFN-γ, mouse IL-17A, human IL-17A, mouse β-actin, and human β-actin were described elsewhere 1, 2. ELISA IL-21 was evaluated using a commercially available ELISA kit (ebioscience) Western blotting Blots were incubated with p-erk1/2-specific antibody (Santa Cruz Biotechnology) followed by a horse-radish peroxidase-conjugated secondary antibody. After detection, blots were stripped and incubated with a total ERK1/2 antibody (Santa Cruz Biotechnology). Injection of IL-21 into the skin of mice Balb/c mice were injected intradermally with IL-21 (500 ng), IL-23 (500 ng; R&D Systems) or vehicle (saline). Injections were performed daily, and mice were killed at day 4. In parallel experiments, mice were treated intra-peritoneally with a neutralizing IL-22 (200 µg per mouse, R&D Systems ) or control antibody one day prior the first IL-21 or IL-23 injection. Human psoriasis-xenograft mouse model The transplantation procedure was conducted as described elsewhere with minor modifications 3. Keratome biopsies were taken from clinically symptomless skin on the lower back of psoriatic patients. Skin xenografts were orthotopically transplanted onto the back of SCID mice, followed 2 weeks later by intradermal injection of activated PBMC. One week later, mice were randomly allocated into three groups and either left untreated, or treated intra-peritoneally with a neutralizing human IL-21 or control antibody (1 mg twice weekly intra-peritoneally) for 4 weeks.
Caruso et al. 5 Histopathological analysis Paraffin-embedded sections were stained with H&E, and epidermal thickness was measured as previously described 4. Sections were also stained with mouse CD3-specific antibody (Santa Cruz Biotechnology) followed by incubation in a dilution of a secondary antibody using a highly sensitive alkaline phosphatase technique (Dako). Data analysis Difference between groups will be compared using either the Mann-Whitney U test or the Student's t test.
Caruso et al. 6 References 1. Fantini, M.C., et al. Eur. J. Immunol. 37, 3155 3163 (2007). 2. Fina, D. et al. Gastroenterology 134, 1038 1048 (2008). 3. Wrone-Smith, T. & Nickoloff, B.J. J. Clin. Invest. 98, 1878 1887 (1996). 4. Chan, J.R. et al. J. Exp. Med. 203, 2577 2587 (2006).
Caruso et al. 7 Supplementary figures Figure 1. Expression of IL-7 and IL-15 in psoriasis. Real time PCR for IL-15 (a) and IL-7 (b) transcripts in biopsies taken from lesional skin of nine psoriatic patients, and from 15 normal controls. Levels are normalized to β-actin. Values are mean ± SD of all experiments. *, P < 0.001.
Caruso et al. 8 Figure 2. Characterization of IL-21R in psoriasis. a. Percentages of CD4 +, CD8 +, CD20 +, CD56 + cells, and primary keratinocytes expressing IL-21R. Cells were isolated from lesional skin of four psoriatic patients and evaluated by flow-cytometry. Data indicate mean ± SD of all experiments. Right Insets. A representative dot-plot showing IL-21R staining in primary keratinocytes isolated from non-lesional skin of psoriatic patients. Numbers in quadrants indicate the percentage of IL- 21R-positive cells. Staining with a control IgG is also shown. b. Percentages of blood CD4 +, CD8 +, CD20 +, and CD56 + cells expressing IL-21R. PBMC were isolated from five psoriatic patients and simultaneously evaluated for IL-21R, CD4, CD8, CD20, and CD56 by flow-cytometry. Data indicate mean ± SD of all experiments.
Caruso et al. 9 Figure 3. IL-21 enhances the growth of primary keratinocytes isolated from the skin of normal donors. Keratinocytes were either left unstimulated (Unst) or stimulated with IL-21 (100 ng ml 1 )or EGF (20 ng ml 1 ) for 48 hours. Cell proliferation was determined by measuring the incorporation of 5-bromo-2'-deoxyuridine into DNA. Results are reported as the mean ± standard deviation and expressed in arbitrary units (a.u.); * unst vs IL-21 or vs EGF, P < 0.01.
Caruso et al. 10 Figure 4. IL-21 enhances keratins (KRT) 6 and KRT16 expression. Real-time PCR for KRT6 (a) and KRT16 (b) in samples prepared from skin biopsies of mice treated with IL-21 or PBS daily for 4 days. Levels are normalized to β-actin. Values are mean ± SD of all experiments. *, P < 0.001.