Further information is available in Supplemental Methods.
C57BL/6 (wild-type) mice were obtained from Janvier. RorcGFP/GFP (in text referred to as Rorc–/–), Tcrβδ–/–, Rag1–/–, and Rag2–/–Il2rg–/– mice were purchased from The Jackson Laboratory. Tcrb–/– and Tcrd–/– mice were bred from Tcrβδ–/– mice. Rorc-Cre crossed with Rosa26-stop-eYFP mice (called Rorc-Cre × EYFP mice herein) were provided by A. Diefenbach (University of Freiburg, Freiburg, Germany), Il17a–/– mice by Y. Iwakura (University of Tokyo, Tokyo, Japan), Il17f–/– mice by Merck Serono S.A., Il22–/– mice by J.C. Renauld (Université Catholique de Louvain, Yvoir, Belgium), and Il15ra–/– mice by S. Bulfone-Paus (University of Giessen, Giessen, Germany).
The mouse back was shaved, and a daily dose of 50 mg of Aldara (5% IMQ cream; 3M Pharmaceuticals) or control vehicle cream (Soft Kreme KA, Kantonsapotheke Zürich) was applied on the back and 5 mg to each ear for 5–7 days. Anti–IL-12/23p40 treatment was performed by injection of the mice with 200 μg/mouse rat anti–mouse IL-12/23p40 mAb (C17.8) and respective IgG2A isotype control antibody (2A3, both Bio X Cell) on day 2 of Aldara application.
Skin and ears were cut into small pieces and digested with 1 mg/ml collagenase type IA and 100 mg/ml DNase (Sigma-Aldrich) for 60 minutes at 37°C. Isolation of leukocytes from the LN involved teasing the organs apart. Both were followed by filtering through 70-μm cell strainers to obtain single-cell suspensions.
Cells were incubated with antibodies for 20 minutes at 4°C. For intracellular cytokine staining, cells were stimulated with PMA (Applichem) and ionomycin (Invitrogen) and treated with GolgiStop (BD) for 3 hours. After surface staining, cells were permeabilized according to the manufacturer’s (BD) recommendations and stained intracellularly. Stainings were analyzed with a FACS LSRII Fortessa (BD). Post-acquisition analysis was performed with FlowJo (Tree Star) software.
Tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin. Deparaffinized sections were stained using H&E. Immunohistological staining for MPO (1:100, rabbit, Thermo Scientific) was performed by a Ventana Benchmark XT automated staining system according to the manufacturer’s guidelines.
For disease severity, differences between groups were evaluated by 2-way ANOVA with Bonferroni’s post hoc test. For analysis of scatter plots of maximum thickness comparing ≥3 groups of mice, 1-way ANOVA with Bonferroni’s post-test was used. Differences between two sets of data were evaluated by 2-tailed Student’s t test. Data represent mean ± SEM. P ≤ 0.05 was considered statistically significant. All statistics were done using GraphPad Prism (GraphPad Software).
All animal experiments were approved by the Swiss Cantonal Veterinary Office (33/2010; Zurich, Switzerland).