The Roles And Mechanisms Of IL-35 In Psoriasis Vulgaris

Objective:The study aims to further understand the roles and mechanisms of IL-35 in psoriasis vulgaris,and provide more theoretical basis for the clinical transformation of IL-35 in PV.Methods:(1)Case studies:peripheral venous blood was collected from patients with PV(29 cases in progressing stage,16 cases in degenerative stage),16 patients with atopic dermatitis(AD),and 25 healthy volunteers.The isolated serum of each group was detected by ELISA.PASI score was assessed in patients with PV in progressing stage.Pearson correlation analysis was used to evaluate the correlation between serum IL-35 level and PASI score in patients with PV in progressing stage.Magnetic beads and flow cytometry were used to isolate and identify PBMCs and CD4~+T cells from the whole blood of patients with PV in progressing stage.The m RNA expression of P35 subunit and EBI3subunit in PBMCs were detected by RT-PCR.PBMCs and CD4~+T cells were treated with recombinant human IL-35.The m RNA expression of Th1/Th17/Tregs associated cytokines(IL-6,IL-10,TNF-α,IFN-γand IL-17)in PBMCs were detected by RT-PCR.The m RNA expression of IL-6 and IL-17 in CD4~+T cells were detected by RT-PCR.(2)In vivo study:recombinant mouse IL-35 plasmid was constructed and named as p IL-35.The mouse model of psoriasis induced by imiquimod(IMQ)was established.The mice back skin was shaved and divided into three groups.Control group:the back skin of mice was rubbed with Vaseline and injected i.v.with PBS;IMQ group:the back skin of mice was rubbed with IMQ and injected i.v.with the equal amounts of no-load PCDNA3.1;p IL-35 group:the back skin of mice was rubbed with IMQ and injected i.v.with the equal amounts of p IL-35.After treatment,the psoriasis symptoms and the disease severity of the back skin of groups were observed and evaluated by PASI score system.Then,the mice were euthanized and each group of skin samples were taken for testing.HE was used to stain the tissue samples from mice sections,and antibody against CD3~+and MPO~+were used for immunohistochemical observation.The epidermal thickness was measured by Image J software,and the psoriatic lesions in mice were evaluated by Baker pathological score.RT-PCR was used to detect the mice lesions m RNA expression of IL-6 and IL-17.The phosphorylation level of STAT3 protein in mice skin lesions was detected by Western blot.Results:(1)The serum of IL-35 level in patients with PV in progressing stage(161.24±80.85ng/ml)was significantly lower than that in the control group(1397.09±853.04ng/ml)and AD group(1324.78±752.82ng/ml).H value and P value indicated that(H=-34.609,P<0.0001),(H=-33.725,P<0.0001).The serum of IL-35 level in patients with PV in the degenerative stage(282.63±191.62ng/ml)was significantly higher than that in patients with PV in the progressing stage(145.72±54.22ng/ml),(Z=-2.43,P=0.015).There was no significant correlation between serum of IL-35 level and PASI score in patients with PV in progressing stage(r=-0.228,P=0.234).(2)The EBI3 and p35 subunits of PBMCs in patients with PV in progressing stage were significantly decreased.T value and P value indicated that(t=-3.107,P=0.009),(t=-2.895,P=0.013).(3)IL-35 can reduce the m RNA expression of IL-6 and IL-17 in PBMCs in patients with PV in progressing stage.T value and P value indicated that(t=-3.212,P=0.009),(t=-3.011,P=0.009).(4)IL-35 can reduce the m RNA expression of IL-6 and IL-17 in CD4~+T cells in patients with PV in progressing stage.T value and P value indicated that(t=-2.717,P=0.022),(t=-2.661,P=0.021).(5)IL-35 can improve the severity of IMQ-induced psoriatic lesions in mice.(6)IL-35 can decrease the m RNA expression of IL-6 and IL-17in IMQ-induced psoriatic lesions of mice.F value and P value indicated that(F=25.179,P<0.0001),(F=24.08,P<0.0001).(7)IL-35 can reduce the infiltration of CD3~+T cells,MPO~+neutrophils in the IMQ-induced psoriatic lesions in mice.F value and P value indicated that(F=25.430,P=0.011),(F=14.118,P<0.0001).(8)IL-35 can inhibit phosphorylation of STAT3 in the IMQ-induced psoriatic lesions in mice.Conclusion:(1)The serum of IL-35 level in patients with PV decreased significantly in progressing stage and increased significantly in degenerative stage.IL-35 may participate in the pathogenesis of PV.(2)IL-35 may mediate the anti-inflammatory effect of PV by down-regulating IL-6 and IL-17 and inhibiting STAT3 phosphorylation,which is expected to be a new therapeutic target.