1.1 Construction of an in-vitro psoriatic model
All experimental protocols used in this study were approved and implemented in accordance with the guidelines of the review board of Jining Medical University. Human keratinocyte cell line (HaCaT) cells were maintained in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10 % foetal bovine serum (FBS, Thermo Fisher Scientific), 100 μg/mL streptomycin (Thermo Fisher Scientific), and 100 U/mL penicillin (Thermo Fisher Scientific). Inflammation was induced in HaCaT cells following their stimulation with M5 (mixture of five proinflammatory cytokines including TNF-a, IL-17A, IL-22, IL-1a, and oncostatin-M at 10 ng/mL each; ProSpec, East Brunswick, NJ), thereby mimicking various characteristics of psoriasis (Guilloteau et al., 2010). Briefly, HaCaT cells were seeded in 6-well plates and cultured for 12 h. After 12 h of culture, cells were treated with different concentrations of IL-35 (0, 50, 100, or 200 ng/mL) and stimulated with M5, and the secretion of IL-6 and CXCL8 was determined. To test the levels of secreted IL-6 and CXCL8, culture supernatants were collected at 24, 48, and 72 h post-stimulation. The HaCaT cell line was provided by Professor Jiong Li from the State Key Laboratory of Biotherapy and Cancer Center (Chengdu, China).