Lentivirus-mediated IL-10-expressing Bone Marrow Mesenchymal Stem Cells promote corneal allograft survival via upregulating lncRNA 003946 in a rat model of corneal allograft rejection
Lentivirus-mediated IL-10-expressing Bone Marrow Mesenchymal Stem Cells promote corneal allograft survival via upregulating lncRNA 003946 in a rat model of corneal allograft rejection
复制标题
慢病毒介导的表达 IL-10 的骨髓间充质干细胞通过上调角膜同种异体移植排斥大鼠模型中的 lncRNA 003946 促进角膜同种异体移植物的存活
DOI：
10.7150/thno.31711
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发表时间：
2020-01-01
期刊：
影响因子：
12.4
通讯作者：
Zhao, Shaozhen
中科院分区：
文献类型：
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作者：
Lu, Xiaoxiao;Ru, Yusha;Zhao, Shaozhen
角膜移植是治疗角膜盲的有效方法。然而，免疫排斥反应危及角膜移植物的存活。目前迫切需要一种介入治疗的方法来抑制免疫排斥反应，促进同种异体移植物的存活。在我们之前的研究中，结膜下注射骨髓间充质干细胞（BM-MSCs）到大鼠角膜移植排斥模型中可延长移植物存活2天。在这项研究中，我们试图产生IL-10过表达的BM-MSCs，旨在提高BM-MSCs对角膜移植物的存活促进作用，并探讨增强保护的分子和细胞机制。方法：通过慢病毒转染和流式细胞仪纯化获得IL-10高表达的BM-MSCs（命名为IL-10-BM-MSCs）。IL-10-BM-MSCs的自我更新、多向分化和免疫抑制能力通过常规测定进行检测。将IL-10-BM-MSCs结膜下注射到角膜移植排斥模型中，并每天监测同种异体移植物。长链非编码RNA（lncRNA）在同种异体移植物中的表达谱通过RNA测序和实时定量PCR验证。通过RNAscope原位杂交鉴定主要上调lncRNA表达的浸润免疫细胞类型。通过体内和体外的功能丧失和获得实验证明了上调的lncRNA的功能。结果如下：IL-10-BM-MSCs具有较强的免疫抑制能力和较强的自我更新和多向分化潜能。在角膜移植排斥模型中，与IL-10蛋白或普通BM-MSC相比，结膜下注射的IL-10-BM-MSC减少了免疫细胞浸润并使移植物存活时间加倍（20 d）。此外，IL-10-BM-MSC显著上调浸润角膜同种异体移植物的CD 68+巨噬细胞中的lncRNA 003946表达。在巨噬细胞培养物中沉默和过表达lncRNA 003946分别消除和模拟IL-10-BM-MSC对巨噬细胞抗原呈递的抑制作用。同时，敲低和过表达lncRNA分别消除和模拟IL-10-BM-MSCs对角膜移植物的存活促进作用。结论：IL-10-BM-MSCs的显著保护作用支持将其进一步发展为对抗角膜移植排斥反应的有效干预方式。IL-10-BM-MSCs主要通过上调移植物浸润的CD 68+巨噬细胞中新的lncRNA表达来促进角膜移植物存活。LncRNA首次整合到IL-10-BM-MSC驱动的免疫调节轴中，以对抗角膜移植物的免疫排斥反应。
Rationale: Corneal transplantation is an effective treatment to corneal blindness. However, the immune rejection imperils corneal allograft survival. An interventional modality is urgently needed to inhibit immune rejection and promote allograft survival. In our previous study, subconjunctival injections of bone marrow-derived mesenchymal stem cells (BM-MSCs) into a rat model of corneal allograft rejection extended allograft survival for 2 d. In this study, we sought to generate IL-10-overexpressing BM-MSCs, aiming to boost the survival-promoting effects of BM-MSCs on corneal allografts and explore the molecular and cellular mechanisms underlying augmented protection. Methods: A population of IL-10-overexpressing BM-MSCs (designated as IL-10-BM-MSCs) were generated by lentivirus transduction and FACS purification. The self-renewal, multi-differentiation, and immunoinhibitory capabilities of IL-10-BM-MSCs were examined by conventional assays. The IL-10-BM-MSCs were subconjunctivally injected into the model of corneal allograft rejection, and the allografts were monitored on a daily basis. The expression profiling of long noncoding RNA (lncRNA) in the allografts was revealed by RNA sequencing and verified by quantitative real-time PCR. The infiltrating immune cell type predominantly upregulating the lncRNA expression was identified by RNAscope in situ hybridization. The function of the upregulated lncRNA was proved by loss- and gain-of-function experiments both in vivo and in vitro. Results: The IL-10-BM-MSCs possessed an enhanced immunoinhibitory capability and unabated self-renewal and multi-differentiation potentials as compared to plain BM-MSCs. The subconjunctivally injected IL-10-BM-MSCs reduced immune cell infiltration and doubled allograft survival time (20 d) as compared to IL-10 protein or plain BM-MSCs in the corneal allograft rejection model. Further, IL-10-BM-MSCs significantly upregulated lncRNA 003946 expression in CD68+ macrophages infiltrating corneal allografts. Silencing and overexpressing lncRNA 003946 in macrophage cultures abolished and mimicked the IL-10-BM-MSCs' suppressing effects on the macrophages' antigen presentation, respectively. In parallel, knocking down and overexpressing the lncRNA in vivo abrogated and simulated the survival-promoting effects of IL-10-BM-MSCs on corneal allografts, respectively. Conclusion: The remarkable protective effects of IL-10-BM-MSCs support further developing them into an effective interventional modality against corneal allograft rejection. IL-10-BM-MSCs promote corneal allograft survival mainly through upregulating a novel lncRNA expression in graft-infiltrating CD68+ macrophages. LncRNA, for the first time, is integrated into an IL-10-BM-MSC-driven immunomodulatory axis against the immune rejection to corneal allograft.