Il 17f

The wound edge was intradermally injected with either rmIL-17A (500 ng in 20 μl of PBS, carrier-free; PeproTech) or PBS control immediately after a 4-mm biopsy. The following cytokine concentrations were used for daily intradermal injections for 7 days in unwounded ear pinnae and compared with the PBS-only vehicle control: IL-17A (500 ng in 20 μl of PBS, carrier-free; PeproTech), IL-17F (1 μg in 20 μl of PBS, carrier-free; PeproTech), or IL-22 (500 ng in 20 μl of PBS, carrier-free; PeproTech).

Primary epithelial cell spheroids derived from wild-type C57BL/6 mice were maintained in L-WRN media. As described above, epithelial cell spheroids were grown for 24 hours in DM with and without the following cytokines: TNF-α, IFN-γ, IL-1β, IL-22, IL-6, IL-33, IL-12, IL-13, IL-17A, and IL-17F (all from Peprotech), and IL-12/23p40, Oncostatin M, IL-23, IL-17C, and IL-17E (from R&D Systems). Cytokines were added at either 20ng/ml or 100ng/ml doses. RNA was extracted and reverse transcription and quantitative PCR conducted as described below to measure the expression of Serpine1 and Plat relative to Gapdh housekeeping gene.

MEFs, HK-2 and HEK293T cells were cultured in α-minimum essential medium (α-MEM; Sigma-Aldrich, St. Louis MO) with L-glutamine, antibiotics and 10–15% FBS. HK-2 cells (ATCC) were cultured in DMEM/F12 (Gibco), antibiotics and 10% FBS. IL-17, IL-17F and TNFα (PeproTech, Rocky Hill, NJ) were used at 200 and 10 ng/ml. Actinomycin D (Sigma-Aldrich) was used at 10 μg/ml.

For macrophage and T cell coculture experiments, bone marrow–derived monocytes (BMDMs) were isolated and differentiated into macrophage as previously described. At day 0, BMDMs were seeded at 60,000 cells/cm² and incubated at 37°C for 7 days in medium supplemented with macrophage colony-stimulating factor (100 ng/ml) (PeproTech) into a 12-well insert of a transwell plate (Corning). Naïve CD4⁺ T cells were isolated from murine lymph nodes at day 4 using a magnetic bead negative selection kit (Miltenyi). Then, T cells were seeded at 1 million cells/ml in 2 ml into six-well plates (Corning) in naïve condition supplemented with IL-2 (100 ng/ml) (PeproTech) or skewed toward the T_H17 phenotype using a commercially available kit (R&D Systems). At day 7, differentiated macrophages were exposed to naïve and T_H17 T cells at 250,000 cells/cm² or IL-17A, IL-17F (PeproTech, 100 ng/ml), and control medium for 72 hours.
Fibroblasts were isolated using previously reported methods (45 ), seeded to T-75 tissue culture plates (Corning), and passaged once before reseeding at 50,000 cells/cm² for 24 hours and exposed to IL-36γ (R&D Systems) or IL-17A (PeproTech) at 100 ng/ml for 24 hours. Cells were lysed using RLT plus buffer (Qiagen) reagent for quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Recombinant human IL-17A (Invitrogen), IL-17F (Tonbo), IL-22 (Peprotech) and TNFα (Tonbo) were reconstituted according to the manufacturers’ instructions in 0.01% bovine serum albumin (BSA). These were used at a range of concentrations as described in the results section and figure legends.