IL-12b Polymorphism Regulates IL-12/23p40 Expression And The Therapeutic Efficacy Of The Immunoproteasome Inhibitor In Experimental Colitis

In the intestine,aberrant inflammatory responses can cause development of inflammatory bowel disease(IBD),which is characterized by two main clinical forms,Crohn's disease(CD)and ulcerative colitis(UC).The incidence and prevalence of IBD are increasing in China.The study includes two parts.First,genome-wide association studies identified that the single nucleotide polymorphism(SNP)in the promoter region of IL-12b[rs41292470composed of the long allele(LA)and/or short allele(SA)]has been reported to associate with susceptibility to UC,infectious diseases,and additional autoimmune disorders.How these genetic variants impact IL-12b expression at the molecular level was unclear.Therefore,we initiated the present study to probe into the mechanistic basis of the molecular mode in which this polymorphism operates.Second,it has been reported that both CD and UC patients have a high abundance of immunoproteasome,suggesting that it could be a potential target for inflammatory diseases.In Part II,we assessed the therapeutic efficacy of the novel immunoproteasome inhibitor DPLG3 in Dextran sulfate sodium(DSS)induced acute colitis mouse models.In Part I,we previous found that Poly(ADP-ribose)polymerase 1(PARP-1)was a crucial binding protein to IL-12b promoter SNPs by DNA pull-down assays coupled with mass spectrometry analyses.Next,bone marrow derived macrophages(BMDM)from PARP-1-deficient mice had decreased p40 expression at both m RNA and protein levels.Furthermore,selective PARP-1 inhibitors resulted in impaired production of IL-12p40and IL-23 in PBMC.Chromatin immunoprecipitation assay revealed that PARP-1 could bind specifically to IL-12b in LPS-stimulated macrophages.Our study opens the way for further elucidating the molecular mechanism whereby allele-specific immune responses to foreign and self-antigens mediated by IL-12/IL-23 are controlled in an individually variable manner.In Part II,we assessed the therapeutic efficacy of the selective i-20S?5i inhibitors DPLG3 in the dextran sulfate sodium(DSS)-induced acute colitis mouse model.In the DSS colitis model,inhibition of?5i by DPLG3 strongly reduced pathological symptoms compared with vehicle(DMSO)treated mice.Production of numerous inflammatory cytokines such as IL-1?,TNF-?,IL-6,IL-12p35,IL-17A and IFN-?in DPLG3-treated mice was suppressed,resulting in reduced inflammation and tissue pathology.Flow cytometry analysis of the infiltrating cell populations showed the percentage of Tregs in the mesenteric lymph node cells of the DPLG3-treated recipients were significantly increased compared with vehicle-treated controls on Day 13;We observed a decreased in the percentage of IL-17A~+CD4~+T cells,and an increase in the percentage of IL-22~+CD4~+T in lamina propria mononuclear cells in DPLG3 treated mice.Further,the impact of DPLG3 on NF-?B activation was assessed in BMDM,we found that p50 was involved in the DPLG3-reduced IL-1?and TNF-?expression.These findings demonstrate that DPLG3 is a novel immunosuppressive compound with excellent potential for the treatment of intestinal inflammation.It holds great promise for the modulation of immune responses and suppression of autoimmune diseases such as IBD.