Sinomenine promotes differentiation of induced pluripotent stem cells into immature dendritic cells with high induction of immune tolerance

BACKGROUND Immature dendritic cells(imDCs)play an important role in the induction of donor-specific transplant immunotolerance.However,these cells have limitations,such as rapid maturation and a short lifespan in vivo.In previous studies,induced pluripotent stem cells(iPSCs)differentiated into imDCs,and sinomenine(SN)was used to inhibit the maturation of imDCs.AIM To study the capacity of SN to maintain iPSC-derived imDCs(SN-iPSCs-imDCs)in an immature state and the mechanism by which SN-iPSCs-imDCs induce immunotolerance.METHODS In this study,mouse iPSCs were induced to differentiate into imDCs in culture medium without or with SN(iPSCs-imDCs and SN-iPSCs-imDCs).The imDCrelated surface markers,endocytotic capacity of fluorescein isothiocyanate Dextran and apoptosis were analyzed by flow cytometry.The effects of iPSCs-imDCs and SNiPSCs-imDCs on T-cell stimulatory function,and regulatory T(Treg)cell proliferative function in vitro were analyzed by mixed lymphocyte reaction.Cytokine expression was detected by ELISA.The apoptosis-related proteins of iPSCs-DCs and SN-iPSCs-DCs were analyzed by western blotting.The induced immunotolerance of SN-iPSCs-DCs was evaluated by treating recipient Balb/c skin graft mice.Statistical evaluation of graft survival was performed using Kaplan–Meier curves.RESULTS Both iPSCs-imDCs and SN-iPSCs-imDCs were successfully obtained,and their biological characteristics and ability to induce immunotolerance were compared.SN-iPSCs-imDCs exhibited higher CD11c levels and lower CD80 and CD86 levels compared with iPSCs-imDCs.Reduced major histocompatibility complex II expression,worse T-cell stimulatory function,higher Treg cell proliferative function and stronger endocytotic capacity were observed with SN-iPSCs-imDCs(P<0.05).The levels of interleukin(IL)-2,IL-12,interferon-γin SN-iPSCs-imDCs were lower than those in iPSCs-imDCs,whereas IL-10 and transforming growth factor-βlevels were higher(P<0.05).The apoptosis rate of these cells was significantly higher(P<0.05),and the expression levels of cleaved caspase3,Bax and cleaved poly(ADP-ribose)polymerase were higher after treatment with lipopolysaccharides,but Bcl-2 was reduced.In Balb/c mice recipients immunized with iPSCsimDCs or SN-iPSCs-imDCs 7 d before skin grafting,the SN-iPSCs-imDCs group showed lower ability to inhibit donor-specific CD4+T-cell proliferation(P<0.05)and a higher capacity to induce CD4+CD25+FoxP3+Treg cell proliferation in the spleen(P<0.05).The survival span of C57bl/6 skin grafts was significantly prolonged in immunized Balb/c recipients with a donor-specific pattern.CONCLUSION This study demonstrated that SN-iPSCs-imDCs have potential applications in vitro and in vivo for induction of immunotolerance following organ transplantation.

肾移植术后BK病毒相关性肾病核心基因及免疫微环境的生物信息学分析

目的通过生物信息学方法分析肾移植术后BK病毒相关性肾病(BKVAN)的核心基因及其与浸润的免疫细胞相关性。方法从美国国立生物技术信息中心基因表达综合数据库下载BKVAN相关数据集GSE75693和GSE72925,BK病毒(BKV)血症相关数据集GSE47199。合并GSE75693和GSE72925后筛选差异表达基因(DEGs),然后进行基因本体生物过程(GOBP)以及京都基因与基因组百科全书(KEGG)通路分析,并通过蛋白-蛋白相互作用(PPI)网络进一步筛选核心基因。使用CIBERSORT进行免疫浸润分析,然后计算差异的免疫细胞和核心基因的相关性。最后,在GSE47199数据集筛选BKV血症和BKVAN共同的核心基因,使用基因集富集分析(GSEA)鉴定共同的核心基因分别在BKVAN和BKV血症中的生物过程。所有统计分析及可视化均基于R语言(4.0.2)。P<0.05为差异有统计学意义。结果在合并数据中共筛选出175个上调及70个下调DEGs。在PPI网络中,通过5种方法交集得到9个核心基因,核心基因主要富集在免疫细胞活化与功能相关的进程;在KEGG分析中,核心基因主要富集在病毒蛋白与细胞因子和细胞因子受体间相互作用、细胞因子-细胞因子受体间相互作用以及趋化因子信号通路等。免疫浸润分析表明PTPRC、CCL5、TYROBP、CXCL10、CD2和CXCL9与BKVAN中浸润的免疫细胞相关。CD2是BKVAN和BKV血症的共同核心基因。结论通过生物信息学方法筛选出BKVAN的核心基因,其中PTPRC、CCL5、TYROBP、CXCL10、CD2和CXCL9与BKVAN中浸润的免疫细胞相关,CD2是BKVAN和BKV血症的共同核心基因,这些标志物为肾移植术后BKVAN的诊治提供依据。