The Role And Mechanism Of Dectin-1 Signaling In Promoting DCs Expression Of IL-33

Objective:Malignant tumor is one of the most serious diseases threatening human health and life.Immunotherapy is another of the most promising cancer treatment methods,which is different from the traditional treatment methods(surgery,radiotherapy and chemotherapy).Dendritic cells(DCs)are the important antigen presenting cells(Antigen-presenting cells,APCs)in the body,and DCs is closely related to the occurrence and development of tumor.DCs play an important role in inducing the body's anti-tumor immunity.DCs is considered to be the most powerful APC that has been found at present.In vivo anti-tumor immunity includes cellular and humoral immunity,in which cellular immunity plays a vital role in mediating the anti tumor effect of DC vaccine.Nearly 20 years since 2010,people constantly research vaccines based on DCs in the treatment of various human tumors,DCs based vaccine(sipuleucel-T)has achieved significant clinical efficacy in the treatment of prostate cancer,and by the United States Food and Drug Administration(Food and Drug Administration,FDA)as approved the first DC vaccine for the clinical application of.In 2012,a clinical trial in human ovarian cancer showed that the DC vaccine was able to mobilize the anti tumor immune response in the patients with ovarian cancer.These studies show that DC vaccines has a broad research prospect and the research value is high in clinical,but the clinical curative effect of DC tumor vaccine is stilllow,which limits the clinical research and application.Therefore,elucidate the antitumor mechanism of DCs and DC vaccine and further improve its clinical efficacy has become an urgent problem to be solved in the field of tumor immunotherapy.We have a thorough and systematic study of the anti-tumor effects and mechanisms of Dectin-1-DCs.Our previous studies found that Dectin-1-DCs as a powerful inducer of Th9 cells and antitumor immunity.To study the mechanism that Dectin-1-DCs promoted antitumor Th9 cells differentiation,we analyzed the microarray gene expression data of Cur DCs and BMDCs.We found increased IL-33 expression in Cur DCs compared to BMDCs.However,the roles and mechanisms of high expression of IL-33 by Dectin-1-DCs remain unclear.Part I: The roles of Dectin-1-activated DCs promote the expression of IL-33Methods:1.Na?ve CD4+ T cells were cocultured with Cur DCs under T9-polarizing conditions with or without the addition of ?ST2 or IL-33,followed by testing the expression of Th9/IL-9.2.B16-OVA tumor-bearing OT-II mice were immunized by BMDCs,Cur DCs with IL-33,followed by testing the classification of tumor-infltrating lymphocytes.3.B16-OVA tumor-bearing OT-II mice were immunized by BMDCs,Cur DCs and IL-33,followed by testing the proliferation and phenotype of tumor-infltrating lymphocytes.4.MPC-11 myeloma tumor models were used to examine the role of IL-33 in the antitumor efcacy induced by Cur DCs.Results:1.Dectin-1-DCs potently promote Th9 cell differentiation,?ST2 treatment moderately decreased Cur DCs-induced Th9 cell development and IL-9 expression,while the addition of IL-33 stimulated Cur DCs-induced Th9 cell development and increased IL-9 expression.2.Mice immunized with Cur DCs plus IL-33 had higher frequencies of IL-9+CD4+?IFN-?+CD4+,and Gzm B+CD4+ T cells.3.IL-33 increased Ki67 expression in CD4+ T cells from Cur DCs-treated mice,IL-33 had effects on T cell expression of PD-1 in Cur DCs immunized mice,and had no effects on IL-2 expression.4.Cur DCs plus IL-33 induced potent inhibition on tumor growth.Conclusion:1.IL-33 promotes the differentiation of Th9 cells induced by Dectin-1-DCs in vitro.2.IL-33 promotes the differentiation of Th9 cells induced by Dectin-1-DCs in vivo.3.IL-33 enhances the proliferation of Th9 cells induced by Dectin-1-DCs in vivo.4.IL-33 promotes the antitumor efficacy of Dectin-1-DCs.Part II: The mechanisms of high expression of IL-33 by Dectin-1-DCs Methods:1.DCs were treated by TNF?/IL-1?,Curdlan,GM-CSF,Poly I:C or LPS,followed by testing IL-33 m RNA levels.Syk specific inhibitor Piceatannol and Raf-1specific inhibitor GW5074 or their specific si RNAs were added in the process of DCs maturation,followed by testing IL-33 m RNA and protein levels in DCs.2.NF-?B inhibitor bortizomib was added in the process of DCs maturation,followed by testing IL-33 m RNA and protein levels in DCs.Luciferase reporter system was used to detect whether NF-?B transcription factor could directly bind to IL-33 promoter and activate its expression.3.The gene expression profiles of Cur DCs and BMDCs were analyzed,then confirmed the expression of IRF1,IRF4 and IRF7.DCs was generated from Dectin-1-/-mice,followed by testing the expression of IRF4 and IRF7.4.Irf4(si-Irf4)si RNA was added in the process of DCs maturation,followed by testing IL-33 m RNA and protein levels in DCs.Il33 si RNA(si-Il33)or Irf4 si RNA(si-Irf4)was added in the process of Th9 induction by Cur DCs,followed by testing Il9 m RNA level in T cells.5.Syk,Raf1,NF-?B inhibitors or Syk(si-Syk),Raf1(si-Raf1)si RNAs were used in the maturation of DCs,followed by testing IRF4 m RNA and protein levels in DCs.Il33 si RNA(si-Il33)was added to exam Il33 and Irf4 m RNA levels in DCs.Results:1.Curdlan promoted IL-33 expression in DCs.Inhibition or knockdown of either Syk or Raf1 in DCs reduced IL-33 expression induced by Curdlan.2.Inhibiting NF-?B largely abrogated IL-33 expression in Cur DCs.Only Rel B was detected to slightly bind to and activate Il33 promoter in NF-?B transcription factors.3.Cur DCs expressed high level of IRF4.The upregulation of IRF4 expression was completely abolished in Cur DCs generated from dectin-1-/-mice.4.Knockdown of Irf4 in DCs inhibited IL-33 expression induced by Curdlan.Th9 cells induced by si-Irf4 or si-Il33-treated Cur DCs expressed lower levels of Il9 than those induced by control Cur DCs.5.The inhibition of Syk,Raf-1 or NF-?B in DCs significantly decreased IRF4 expression induced by Curdlan.Knockdown of Il33 in DCs does not affect IRF4 expression in Cur DCs.Conclusion:1.Dectin-1 stimulates IL-33 expression via Syk and Raf-1.2.NF-?B is indirectly involved in Dectin-1-induced IL-33 expression.3.Dectin-1 stimulates DCs to express IRF4.4.Dectin-1 signaling increases IL-33 expression via IRF4.5.Dectin-1 induced IRF4 expression through Syk,Raf1 and NF-?B signaling pathways.