Murine ifn γ

Human epithelial cell line HeLa were maintained in DMEM containing 4.5 g x l^-1 glucose, 4 mM L-glutamine and sodium pyruvate (Biochrom) supplemented with 10% fetal calf serum (FCS) in an atmosphere of 5% CO₂ and 90% humidity at 37°C. The murine macrophage cell line RAW264.7 (ATCC no. TIB-71) were cultured in DMEM containing 4.5 g x l⁻¹ glucose and 4 mM stable glutamine (Biochrom) supplemented with 6% FCS. For activation of RAW264.7, cells were cultured in medium with 5 ng x ml^-1 murine IFN-γ (R&D Systems) for 24 h prior to infection. The efficacy of activation was routinely confirmed by analyses of macrophages oxidative burst.

The IMQ mouse model was carried out as described (van der Fits et al., 2009 (link)). All mouse experiments were performed under the auspices of a UK Home Office license and with permission from the University of Glasgow Ethics Committee. Six- to 8-week-old WT (from Charles River Laboratories, Elphinstone, UK) and ACKR2-deficient (Jamieson et al., 2005 (link)) mice on a C57BL/6J background were used throughout the study. A minimum of six mice were used per group in each of the experiments. IFN-γ–treated mice received murine IFN-γ (R&D Systems, Abingdon, UK) 10,000 or 20,000U either intraperitoneally or subcutaneously twice daily (as indicated in figures). IFN-γ was reconstituted in 0.01% sterile BSA in phosphate buffered saline (Sigma-Aldrich, Dorset, UK). Mice were assessed daily, and skin inflammation was scored using a modified Psoriasis Area Severity Index (PASI) (see Supplementary Figure S2). Ear thickness was measured using digital calipers on day of cull.
All tissues for RNA extraction were stored in RNAlater (Life Technologies, Paisley, UK) for 24 hours at 4 °C. For long-term storage, RNAlater-treated tissues were stored at –80 °C. Tissues for histology and immunohistochemistry were fixed in 10% neutral buffered formaldehyde (Sigma-Aldrich).

Human and mouse cytokines were measured in kidney homogenates using species‐specific enzyme‐linked immunosorbent assays (ELISA) for human IL‐10 (Cat# D1000B) human indoleamine 2,3‐dioxygenase (IDO; Cat# DY6030‐05), murine IL‐10 (Cat# M1000B) and murine IFNγ (Cat# MIF00) (R&D Systems, Minneapolis, MN). Antibodies were demonstrated by the manufacturer to be free of interspecies cross‐reactivity (ie. the human and mouse IL‐10 antibodies detected only IL‐10 from that species). ELISAs were loaded with tissue homogenates at a concentration of 2 mg/mL and developed according to manufacturer protocols. Western blotting was performed by loading 20 μg of total protein were resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis on a 4%‐12% polyacrylamide gel under reduced conditions. Proteins were transferred onto nitrocellulose membranes, blocked in 5% bovine serum albumin and hybridized with primary antibodies overnight at 4°C. Primary antibodies (dilutions in parentheses) included human IL‐10 (1:500), murine IL‐10 (1:500) (eBioscience, San Diego, CA), or β‐actin (1:5000) (Abcam, Cambridge, MA). Species‐appropriate and fluorescently labelled secondary antibodies (dilutions ranging from 1:1000 to 1:10000) (Abcam) were incubated for 1 hour at room temperature. Blots were imaged using a ChemiDoc MP system (Bio‐Rad, Hercules, CA).