| Background and objects:When encountering the invaded pathogens,epithelium can secret various cytokines and recruit immune cells,then initiate innate immune response via functioning as non-professional antigen-presenting cell.Rencently it has been found that epithelium can also act as a professional antigen-presenting cell and regulate adaptive immune response via transdifferentiation.Our previous work demonstrated that renal tubular epithelial cells(RTECs)could express DC-SIGN via transdifferentiation during renal inflammation,resulting in RTECs to obtain the professional antigen-presenting ability and independently activate specific T cell immune response which is similar to DC,and participate in renal immune injury.Since the expression and regulation mechanism of DC-SIGN in RTECs remains largely unknown,based on our previous work,in this study we discuss(1)the effect of NF-?B-mTOR-RUNX1 pathway on the expression of DC-SIGN in RTECs;(2)the effect of DC-SIGN in RTECs on the inflammatory cytokines production and T cells activation and polarization.We attempt to demonstrate the immune regulation effect of DC-SIGN on renal tubulo-interstitial lesions and it may further provide a potential candidate target for preventing clinical renal diseases.Methods:(1)HK-2 cells were stimulated with 20ng/ml TNF-?for different time points,and the protein levels of DC-SIGN and RUNX1 were detected by Western blot;then HK-2 cells were pretreated with NF-?B inhibitor Bay11-7082,mTOR inhibitor Rapamycin or mTOR siRNA following with TNF-?stimulation,and the protein levels of DC-SIGN and RUNX1 were detected by Western blot.(2)HK-2 cells were pretreated with RUNX1 inhibitor Ro5-3335 or its siRNA following with TNF-?stimulation,and the protein and mRNA levels of DC-SIGN and RUNX1 were detected by RT-qPCR and Western blot analysis;then HK-2 cell lines with stable overexpression of GFP and RUNX1 were constructed,and the protein and mRNA levels of DC-SIGN and RUNX1 in these cells with TNF-?stimulation were examined.(3)The luciferase reporter plasmid containing DC-SIGN promoter was constructed and transfected into human embryonic kidney 293T cells,then pretreating these cells with Bay11-7082,Rapamycin or Ro5-3335following with TNF-?stimulation,and the luciferase values were detected by a dual-luciferase reporter assay;(4)HK-2 cells were transfected with DC-SIGN siRNA following with TNF-?stimulation,and the mRNA levels of TNF-?,IL-6,IL-8 were detected by RT-qPCR;(5)Jurkat cells were cocultured with HK-2 cells that were transfected with DC-SIGN siRNA following with TNF-?stimulation,and the mRNA level of IL-2 in Jurkat cells was detected by RT-qPCR;then naive T cells were cocultured with these HK-2 cells,and the expression levels of IFN-?and IL-4 were analyzed by flow cytometry.Results:(1)The expression levels of DC-SIGN and RUNX1 could be upregulated by TNF-?stimulation in a time-dependent manner in HK-2 cells,and DC-SIGN promoter luciferase activity could be increased by TNF-?stimulation in a dose-dependent manner in293T cells.(2)TNF-?-induced DC-SIGN and RUNX1 upregulation could be inhibited by pretreatment with NF-?B inhibitor,mTOR inhibitor and mTOR siRNA,and TNF-?-induced DC-SIGN promoter luciferase activity could be supressed by pretreatment with NF-?B inhibitor,mTOR inhibitor and RUNX1 inhibitor.(3)Both RUNX1 inhibitor and its siRNA could inhibit TNF-?-induced DC-SIGN upregulation,and DC-SIGN expression could be significantly upregulated in HK-2 cells that overexpressed RUNX1compared with control cells that overexpressed GFP after TNF-?stimulation.(4)The mRNA levels of TNF-?,IL-6 and IL-8 induced by TNF-?stimulation could be alleviated by transfection with DC-SIGN siRNA in HK-2 cells.(5)HK-2 cells transfected with DC-SIGN siRNA could inhibit IL-2 production by Jurkat cells in the coculture systerm,and suppress IFN-?production by naive CD4~+T cells in the mixed lymphocyte reaction and downregulate the ratio of IFN-?/IL-4.Conclusion:Our results demonstrated that NF-?B-mTOR-RUNX1 pathway participates in regulating TNF-?-induced DC-SIGN expression in RTECs,and indicated that RUNX1 may be the potential transcription factor that initiates DC-SIGN expression in RTECs.Furthermore,our results showed that DC-SIGN can regulate TNF-?-induced inflammatory cytokines expression in RTECs,and modulate T cells activation and polarization.Therefore targeting DC-SIGN may serve as a new therapeutic target for clinical renal inflammatory diseases,which needs further study. |
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