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).