母体炎性暴露对子代小鼠受IRBP抗原诱导的葡萄膜炎的影响

Impact of embryonic uveitis exposure on response of mouse offspring tointerphotoreceptor retinoid-binding protein-induced experimentalautoimmune uveitis

目的 探讨母体葡萄膜炎暴露对子代小鼠受光感受器间视黄醇结合蛋白(interphotoreceptor retinoid-binding protein, IRBP)诱导的实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis, EAU)免疫效应的影响。方法 从本课题组前期获得的受双亲EAU暴露的子代C57BL/6J小鼠眼球的RNA测序转录组数据中,筛选免疫相关差异表达基因(differentially expressed genes, DEGs)。利用基因本体论(Gene Ontology, GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)的富集分析,确定这些免疫相关的差异表达基因生物学途径。通过蛋白质互作网络(protein-protein interaction networks, PPI)分析交集基因以此鉴定出枢纽基因。进一步通过荧光定量聚合酶链反应(quantitative real-time PCR,qPCR)确定筛选出的异常表达的枢纽基因是否也在受母体葡萄膜炎影响的子代小鼠中差异表达,最后通过视网膜抗原IRBP_(651-670)诱导受母体活动期炎症影响的子代以评价母体葡萄膜炎对子代患葡萄膜炎的易感性以及相关免疫效应的影响。结果 从受双亲葡萄膜炎影响的子代小鼠眼球组织中鉴定出72个免疫相关差异基因。这些基因主要富集参与Toll样受体信号通路、MAPK信号通路以及B细胞受体信号通路等。进一步进行PPI网络互作分析,筛选出proteasome 26S subunit, ATPase 5 (Psmc5)、proteasome 26S subunit, ATPase 3 (Psmc3)、proteasome 26S subunit ubiquitin receptor, non-ATPase 4 (Psmd4)和proteasome 26S subunit, non-ATPase 8 (Psmd8)这4个枢纽基因。qPCR检测证实受母体葡萄膜炎影响的子代小鼠中也上调表达这4个枢纽基因。而且与健康子代相比,150μg IRBP抗原诱导可增加受活动期炎症影响的子代EAU临床表现的严重程度和病理组织学损伤(P=0.0087,P=0.0410);另外,受母体活动期炎症影响的子代在IRBP诱导的EAU中脾T细胞增殖功能增强,其血清中产生的细胞因子IL-17和IL-6增加(P=0.0450,P=0.0300)。结论 母体活动性炎症暴露加剧子代小鼠受IRBP诱导的EAU疾病的严重性,增强抗原特异性T细胞增殖以及血清中IL-17、IL-6的产生,可能与受影响的子代上调表达的4个枢纽基因Psmc5、Psmc3、Psmd4、Psmd8相关。

Objective To investigate the effect of embryonic inflammatory exposure on the response of mouse offspring to interphotoreceptor retinoid-binding protein(IRBP)-induced experimental autoimmune uveitis(EAU).Methods RNA transcriptome sequencing data from eyeballs of C57BL/6J mouse offspring born to mothers with active EAU were used to screen immune-associated differentially expressed genes in the eyes of the exposed offspring.Gene fragments overlapping in the two datasets were screened using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses to identify biological pathways associated with the gene fragments.Hub genes were identified from these intersecting genes by protein-protein interaction network analysis.EAU models of maternal uveitis were established by immunization with IRBP 651-670,and expression levels of the pivotal genes in the offspring exposed to inflammation by maternal uveitis were examined by fluorescence quantitative polymerase chain reaction.EAU severity,T lymphocyte proliferation,and serum cytokines were detected to investigate the immune effect in offspring from mothers with an active inflammation response to IRBP induction.Results Microarray analysis identified 72 immune-related differentially expressed genes in exposed samples compared with the findings in control samples.These genes were mainly enriched in Toll-like receptor signaling,mitogen-activated protein kinase signaling,and B cell receptor signaling pathways.Protein-protein interaction network interaction analysis screened out four hub genes,Psmc5,Psmc3,Psmd4,and Psmd8,and mRNA levels of these four genes were increased in the adult offspring from mothers with active uveitis compared with the findings in healthy offspring.In addition,the group induced with 150μg IRBP showed an increase in the severity of clinical and pathological outcomes in offspring with EAU affected by active inflammation,compared with the healthy offspring group(P=0.0087,P=0.0410).Meanwhile,T cell proliferation in the offspring was enhanced during the inflammatory activity stage and secretion of the inflammatory cytokines interleukin(IL)-17 and IL-6 was increased(P=0.0450,P=0.0300).Conclusions Psmc5,Psmc3,Psmd4,and Psmd8 may be important genes exacerbating uveitis in offspring of mothers with active uveitis,associated with increased T cell proliferation and production of IL-17 and IL-6.