High-throughput RNA-sequencing identifies mesenchymal stem cell-induced immunological signature in a rat model of corneal allograft rejection
High-throughput RNA-sequencing identifies mesenchymal stem cell-induced immunological signature in a rat model of corneal allograft rejection
复制标题
高通量 RNA 测序鉴定了角膜同种异体移植排斥大鼠模型中间充质干细胞诱导的免疫特征
DOI：
10.1371/journal.pone.0222515
复制
发表时间：
2019-09-23
期刊：
影响因子：
3.7
通讯作者：
Zhao, Shaozhen
中科院分区：
文献类型：
--
作者：
Lu, Xiaoxiao;Chu, Chenchen;Zhao, Shaozhen
目的CD4+T细胞和抗原提呈巨噬细胞介导的免疫排斥反应是角膜移植失败的主要原因。骨髓间充质干细胞(BM-MSCs)具有强大的免疫调节能力，已被我们和其他人证明可以促进同种异体角膜移植物的存活。然而，BM-MSCs保护作用的免疫学机制尚不清楚。因此，本研究在BM-MSC治疗大鼠同种异体角膜移植排斥反应模型上对其机制进行了研究，以期为寻找新的干预靶点治疗同种异体角膜移植排斥反应提供帮助。方法采用大鼠角膜移植模型，分别于术后即刻和术后第3天结膜下注射骨髓间充质干细胞(BM-MSCs)(2×10~6个/100μL骨髓基质)。对照组注射等体积的PBS。术后定期评估异体角膜移植的临床参数，包括混浊、水肿和新生血管。术后第10天收集角膜移植物，进行流式细胞术和高通量RNA测序(RNA-seq)。分析了GO的富集途径和KEGG途径。用实时定量聚合酶链式反应(QPCR)和免疫组织化学(IHC)分别在转录水平和蛋白水平验证所选目的基因的表达。结果BM-MSC结膜下注射可延长角膜移植物存活时间，减少混浊，减轻水肿，减少新生血管形成。流式细胞术显示，与PBS组相比，经BM-MSC治疗的同种异体角膜移植物中的CD4+T细胞和CD68+巨噬细胞减少，调节性T细胞(Treg)增加。此外，RNA-seq和qPCR结果显示，与PBS组相比，BM-MSC组细胞毒性T淋巴细胞相关蛋白4(CTLA4)、蛋白酪氨酸磷酸酶、C型受体(Ptprc)和C-X-C基序趋化因子配体9(Cxcl9)基因的转录丰度增加，而热休克蛋白A家族(Hsp70)成员8(Hspa8)基因的表达下调。IHC从蛋白水平证实了这些基因的表达。结论结膜下注射BM-MSCs可提高同种异体角膜移植物的存活率，减少CD4+和CD68+细胞的渗入，丰富角膜移植物中的Treg细胞。BM-MSC诱导的CTLA4、Ptprc、Cxcl9基因上调和Hspa8基因下调可能参与了BM-MSCs的保护作用，为同种异体角膜移植排斥反应提供了潜在的干预靶点。
Objective The immune rejection mediated by CD4+ T cell and antigen presenting macrophages is the leading cause of corneal transplantation failure. Bone marrow-derived mesenchymal stem cells (BM-MSCs) possess robust immunomodulatory potentials, and have been shown by us and others to promote corneal allograft survival. However, the immunological mechanism underlying the protective effects of BM-MSCs remains unclear. Therefore, in the current study, this mechanism was investigated in a BM-MSC-treated rat model of corneal allograft rejection, in the hope to facilitate the search for novel interventional targets to corneal allograft rejection. Methods Lewis rats were subjected to corneal transplantation and then received subconjunctival injections of BM-MSCs (2×106 cells / 100 μl PBS) immediately and at day 3 post-transplantation. The control group received the injections of PBS with the same volume. The clinical parameters of the corneal allografts, including opacity, edema, and neovascularization, were regularly evaluated after transplantation. On day 10 post-transplantation, the corneal allografts were collected and subjected to flow cytometry and high-throughput RNA sequencing (RNA-seq). GO enrichment and KEGG pathways were analyzed. The quantitative realtime PCR (qPCR) and immunohistochemistry (IHC) were employed to validate the expression of the selected target genes at transcript and protein levels, respectively. Results BM-MSC subconjunctival administration prolonged the corneal allograft survival, with reduced opacity, alleviated edema, and diminished neovascularization. Flow cytometry showed reduced CD4+ T cells and CD68+ macrophages as well as boosted regulatory T cells (Tregs) in the BM-MSC-treated corneal allografts as compared with the PBS-treated counterparts. Moreover, the RNA-seq and qPCR results demonstrated that the transcript abundance of Cytotoxic T-Lymphocyte Associated Protein 4 (Ctla4), Protein Tyrosine Phosphatase, Receptor Type C (Ptprc), and C-X-C Motif Chemokine Ligand 9 (Cxcl9) genes were increased in the allografts of BM-MSC group compared with PBS group; whereas the expression of Heat Shock Protein Family A (Hsp70) Member 8 (Hspa8) gene was downregulated. The expression of these genes was confirmed by IHC at protein level. Conclusion Subconjunctival injections of BM-MSCs promoted corneal allograft survival, reduced CD4+ and CD68+ cell infiltration, and enriched Treg population in the allografts. The BM-MSC-induced upregulation of Ctla4, Ptprc, Cxcl9 genes and downregulation of Hspa8 gene might contribute to the protective effects of BM-MSCs and subserve the potential interventional targets to corneal allograft rejection.