Forty-one patients with PV, twenty-three patients with Atopic Dermatitis (AD), and thirty-eight age and gender-matched controls were enrolled in this study. The detailed history for each patient was provided in Table 1. The severity of disease in patients with PV or AD was determined using Psoriasis Area and Severity Index (PASI) or scoring AD (SCORAD). Among them, 17 PV patients were treated with topical steroid and tar, and oral compound glycyrrhizin (Each table contains Glycymhizin 25 mg, Monoammonium Glycyrrhizinate 35 mg, Glycine 25 mg, and DL-Methionine 25 mg. The patients were treated with two tables one time, triple daily. Nippon Kayaku, Tokyo, Japan). The serum samples of these patients were collected both before and after treatment. In addition, heparinized blood samples were collected from 16 PV patients. There was not any history of recent viral infections or lymphocytosis or lymphopenia or any other correlating investigation in these patients. All the PV patients were strictly psoriasis vulgaris only, without any other phenotypes overlap.

Serum IL-29 levels in a different group of patients were measured by commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kits (Boster, Wuhan, China) following the manufacturer’s instruction.

PBMCs of PV patients were isolated from collected peripheral blood by gradient density centrifugation using commercially available kits (Tianjin Haoyang Biological Manufacture Co. Ltd., Tianjin, China). The isolated PBMCs from each patient were divided into two groups and cultured in DMEM containing 10% FBS, 100 mg/mL penicillin and streptomycin in a 6-well plate at 1 mL per well. One group of cells was treated with recombinant 100 ng/mL IL-29 (Peprotech, London, UK) for 6 h, and the other group was treated with PBS as control. After treatment, total RNA was extracted by Trizol reagent (Invitrogen Corp, Carlsbad, CA, USA) according to the manufacturer's instructions.

HaCaT cells were cultured in DMEM containing 10% FBS, 100 mg/mL penicillin and streptomycin and treated with recombinant IL-29 (1 ng/mL, 10 ng/mL, 100 ng/mL, 1000 ng/mL) or TNF-α (10 ng/mL) for 2 h. Cells were treated with PBS as control. After treatment, the total RNA of cells was extracted by Trizol reagent according to the manufacturer's instructions.

The mRNA levels of IL-6, TNF-α, IL-17, IL-4, IFN-γ, IL-10, and GAPDH in PBMCs after incubated with IL-29 or PBS and the expression of IL-6, IL-8 and GAPDH in HaCat cells after incubated with IL-29, TNF-α or PBS was determined by real-time quantitative PCR. The cDNA was synthesized from the total RNA by using RT reagent Kit with gDNA Eraser (Takara, Dalian, China). cDNA samples were amplified in a 20 μL reaction volume containing 10 μL of 2 × SYBR Green Master Mix (Takara, Dalian, China), 2 μL of cDNA and 0.25 μM qPCR primers. The following primers were used: IL-4 (P216616, Bioneer, Inc., Daejeon, Korea); IL-6 (P211161, Bioneer, Inc., Daejeon, Korea); IL-10 (P285360, Bioneer, Inc., Daejeon, Korea); IL-17 (P291322, Bioneer, Inc., Daejeon, Korea); IFN-γ (Catalog: HQP009467, GeneCopoeia, USA); TNF-α (P237423, Bioneer, Inc., Daejeon, Korea); GAPDH (5’-CGGAGTCAACGGATTTGGTC-3’ and 5’-CGGTGCCATGGAATTTGCCA-3’). The conditions were 95 °C for 5 min, and 95 °C for 15 s and 60 °C for the 30 s of 40 cycles with a final extension at 72 °C for 5 min. The mRNA levels of IL-6, TNF-α, IL-17, IL-4, IFN-γ, IL-10, and IL-8 were expressed as relative mRNA levels compared with control and determined by the 2-ΔΔCt method.

Each experiment was carried out at least three times. The data were expressed as mean ± SD, Statistical differences between groups were determined according to one-way analysis of variance, t-test or Mann-Whitney U Test. Paired data were compared by Wilcoxon signed-rank test. The association of IL-29 with PASI scores was analyzed by the Spearman test; p < 0.05 was set as statistically significant.

The serum levels of IL-29 in patients with PV and AD and control were detected by ELISA. As shown in Fig. 1A, IL-29 serum levels in patients with PV (41.62 ± 23.26 ng/mL) were significantly higher than those in AD patients (24.35 ± 12.57 ng/mL) and control group (29.83 ± 19.52 ng/mL). Moreover, 17 PV patients had obviously elevated serum IL-29 levels when compared with those after treatment (Fig. 1B); (p = 0.0007). Furthermore, as presented in Fig. 1C, serum IL-29 levels in PV patients were correlated with PASI scores (r = 0.3319, p = 0.034).

Figure 1.

Increased serum IL-29 levels in PV patients. Serum levels of IL-29 in patients with PV and AD and healthy controls were determined by ELISA. (A) Serum IL-29 levels in patients with PV in the acute stage were significantly higher than those in AD patients and control subjects; p-values are based on the Mann-Whitney U Test. (B) Wilcoxon Signed Ranks Test for paired data showed that serum IL-29 levels of 17 patients with PV in the acute stage were significantly higher than those in the convalescent stage. (C) A positive correlation was shown between IL-29 serum levels and PASI scores in PV patients by Spearman tests.

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To explore the role of IL-29 in the pathogenesis of PV, the authors assayed the effects of IL-29 on cytokines mRNA expression (including IL-6, TNF-α, IL-17, IL-4, IFN-γ, and IL-10) in PBMCs from 10 PV patients by real-time quantitative PCR. As presented in Fig. 2A–C, IL-29 caused a markedly increase in the mRNA levels of IL-6, TNF-α and IL-17 in PBMCs from PV patients treated with IL-29 when compared with PBS controls. However, mRNA expression of IL-4, IL10, and IFN-γ were no changes in PBMCs from PV patients after IL-29 incubated (data not shown).

Figure 2.

(A–C) IL-29 induced IL-6, IL-17 and TNF-α mRNA expression in PBMCs from PV patients. PBMCs obtained from 10 PV patients were treated with IL-29 100 ng/mL or PBS for 6 h. After that, mRNA expression of IL-6, IL-17 and TNF-α in PBMCs were measured by Real-time quantitative PCR. IL-29 obviously enhanced IL-6, IL-17 and TNF-α mRNA expression in PBMCs; p-values are based on t-test. n = 10, *p < 0.05, **p < 0.01, compared with control group. (D–E) IL-29 enhanced IL-6 and IL-8 mRNA expression in HaCat cells. HaCaT cells were treated with recombinant IL-29 (1 ng/mL, 10 ng/mL, 100 ng/mL, 1000 ng/mL) or TNF-α (10 ng/mL) for 2 h. After that, mRNA expression of IL-6 and IL-8 in HaCat cells was measured by Real-time quantitative PCR. IL-29 obviously enhanced IL-6 and IL-8 mRNA in HaCat cells; p-values are based on the One-way analysis of variance. n = 10, *p < 0.05, **p < 0.01, compared with control group. All data are expressed as mean ± SD.

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To explore the function of IL-29 in the pathogenesis of PV, the authors also assayed cytokines mRNA expression (IL-6 and IL-8) in Hacat cells after treatment with recombinant IL-29 by real-time quantitative PCR. As presented in Figs. 2D and 2E, IL-29 caused an obvious increase in the mRNA levels of IL-6 and IL-8 in HaCat cells treated with IL-29 when compared with PBS controls.