Prevention Of Autoimmune Uveitis Through Targeting IL-23 With Small Molecule Polypeptide

PurposeUveitis is one of the most common autoimmune diseases in ophthalmology.The incidence of uveitis in China remains very high.It has been reported that 10% of irreversible blindness is caused by uveitis,for this reason,the treatment of uveitis has always been the key for the prevention of blindness in China.Recent studies have shown that the IL-23/Th17 axis plays an important role in the pathogenesis of uveitis.IL-23 is considered to be a key cytokine for the pathogenesis of uveitis.The c-Rel/IL-23/IL-17 pathway was reported to be tightly associated with the development and progression of uveitis and other autoimmune diseases.The aim of this study is synthesizing to prevent uveitis with a small polypeptide inhibitor,which can specifically block the binding site of the transcription factor c-Rel on the IL-23p19 gene promoter,and consequently inhibit the expression of IL-23p19 and reduce the number of pathogenic Th17 cells.The finish of this study will provide new insights into the treatment of autommune uveitis and other autoimmune diseases.Methods1.To investigate whether the small polypeptide inhibitor can inhibit the activity of IL-23p19 promoter,vector containing IL-23p19 promoter was constructed and the promoter activity was examined by dual luciferase assay.2.To investigate whether the small polypeptide inhibitor can affect the expression of IL-23p19,BMDC was treated with or without small polypeptide inhibitor and the expression of IL-23 was detected by ELISA and RT-qPCR.3.To investigate whether small polypeptide inhibitor can specifically affect the expression of IL-23p19,BMDC,BMDM and T cells were treated with or without small polypeptide inhibitor and the expression of inflammatory cytokines IL-12,IL-6,INF-?,TNF-?,IL-1? and IL-2 was detected by ELISA.4.To investigate whether the small polypeptide inhibitor influence cell viability,BMDC,BMDM,293 T,Raw 634.7,B16 and ?-TC-6 cells were treated with or without small polypeptide viability was detected by MTS.5.To investigate whether the small polypeptide inhibitor can enter into cell nucleus,Raw 264.7 cell was treated with small polypeptide inhibitor and was stained by acridine orange.The location of the small polypeptide inhibitor was detected by fluorescent microscope.6.To investigate whether the small polypeptide inhibitor can prevent autoimmune uveitis,mice EAU models were established and mice were treated with small polypeptide inhibitor or DMSO.The model was successful established based on the paraffin section of the eyeball.The expression of inflammatory cytokines IL-17 A and IL-23 were detected by ELISA after stimulating cells with anti-CD3+ anti-CD28 or LPS.Result1.By constructing the plasmid containing IL-23p19 promoter,the dual luciferase assay showed that small polypeptide inhibitor could inhibit the activity of IL-23p19 promoter in vitro.The difference was statistically significant(**P<0.01).2.The expression of IL-23p19 mRNA level was significantly reduced by BMDC treated with small polypeptide inhibitor(**P< 0.01,*** P < 0.001).The expression of IL-23 protein was also significantly reduced(** P< 0.01).3.The expression of IL-12,IL-6,INF-?,TNF-?,IL-1? and IL-2 showed no significant difference between the small polypeptide inhibitor group and control group(P>0.05).4.There was no significant difference in cell viability between the small polypeptide inhibitor group and control group(P>0.05).5.Fluorescent microscope analysis showed that small polypeptide inhibitor could enter into the nucleus.6.Results from the EAU model showed that the score of retinal pathology of the small peptide inhibitor group was lower,and the difference between two groups was statistically significant(*P<0.05).The level of cytokines IL-17 A and IL-23 in the supernatant of splenocytes and eye-derived cells was lower in the small molecule peptide inhibitor group compared with those in DMSO group,and the difference was statistically significant(* P<0.05,**P<0.01).Conclusion1.Small polypeptide inhibitor significantly inhibits the expression of IL-23p19.2.Small polypeptide inhibitor does not affect the expression of other cytokines exceptIL23-p19.3.Small polypeptide inhibitor treatment has no obvious toxic effect on cell growth.4.Small polypeptide inhibitor can prevent the development of uveitis through targetingIL-23p19.