Correlation Study Of IL-35 In Pathogenesis With Chronic Spontaneous Urticaria

Objective:Interleukin-35（IL-35）is a new member of the IL-12 family.It is a newly discovered anti-inflammatory cytokine in recent years.It has been found that IL-35 plays an anti-inflammatory and immunosuppressive role in a variety of inflammatory and autoimmune diseases.Chronic spontaneous urticaria（CSU）is an inflammatory and allergic skin disease caused by degranulation of mast cells in skin and mucosa,its etiology and pathogenesis are unknown.The purpose of this study was to investigate the relationship between IL-35 and chronic spontaneous urticaria and its mechanism in chronic spontaneous urticaria.Methods:Serum samples from patients with chronic spontaneous urticaria（before and after treatment）,atopic dermatitis patients and healthy people were collected.The expression of IL-35 in serum of each group was detected by Enzyme-Linked Immunosorbnent Assay（ELISA）,and the expression of IL-35 receptor in mast cell line（HMC-1）was detected by Polymerase Chain Reaction（PCR）.Mast cell lines were treated with IL-35 recombinant human protein and PBS respectively,then use CCK8 to detect the proliferation of mast cell in each group.The mast cell lines were pretreated with IL-35recombinant human protein and PBS respectively,then activated by PMA and Ca²⁺,IL-35+PMA+Ca²⁺as experimental group,PMA+Ca²as control group and PBS as blank control group,the proliferation of mast cell was detected by CCK-8 too.Mast cell supernatants of IL-35+PMA+Ca²⁺group,PMA+Ca²⁺group and blank control group were collected,the histamine concentration in each group was detected by ELISA.Total RNA of mast cells in IL-35+PMA+Ca²⁺group,PMA+Ca²⁺group and blank control group were collected and retranscribed to cDNA.The expression of IL-17,IFN-gamma,IL-6,TNF-alpha and IL-4 in mast cells of each group was detected by RT-PCR.Total mast cell protein was extracted from IL-35+PMA+Ca²⁺group,PMA+Ca²⁺group and blank control group,we use Western blot to detecte the phosphorylation of ERK,P38 and JNK.Results:Serum test results showed that the expression of IL-35 in patients with CSU was significantly lower than that in normal persons and patients with atopic dermatitis.After treatment,the expression of IL-35 was significantly higher than that before treatment.Mast cells can express IL-35 receptors;the proliferation of inactivated mast cell lines is not affected by IL-35,while the proliferation of activated mast cell lines is inhibited by IL-35;The content of histamine in the supernatant from activated mast cells after IL-35 treatment was significantly decreased;the expression of cytokines IL-17 and IL-6 secreted by activated mast cells after IL-35 treatment was decreased,while the expression of IFN-γ,TNF-αand IL-4 remained unchanged;IL-35 could inhibit the phosphorylation of ERK,P38 and JNK in activated mast cells.Conclusion:In this study,we show firstly that IL-35 as a new type of immunosuppressive cytokine,participates in the pathogenesis of chronic spontaneous urticaria;IL-35 can decrease the proliferation of activated mast cells,and block the degranulation of histamine and the expression of IL-17 and IL-6.IL-35 may inhibit mast cell activation by interfering with ERK,P38 and JNK signaling pathway phosphorylation.IL-35 is expected to become a new target for the treatment of patients with CSU in the future.