Small extracellular vesicles secreted from TGF-β1-licensed mesenchymal stromal cells reduce inflammation-associated injury following corneal alkali burn.

IF 7.1 2区 医学 Q1 CELL & TISSUE ENGINEERING
Ellen Donohoe, Aoife Canning, Eanna Johnston, Seyedmohammad Moosavizadeh, Jiemin Wang, Martin Leahy, Oliver Treacy, Aideen E Ryan, Thomas Ritter
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引用次数: 0

Abstract

Background: It is well established that the mesenchymal stromal cell (MSC) therapeutic potency can be enhanced by cytokine pre-activation or licensing. However, its effects on therapeutic efficacy of small extracellular vesicles (MSC-sEV) have not yet been well established. Here we report on two different cytokine licensing strategies, using either a pro-inflammatory or anti-inflammatory cytokine and evaluate their therapeutic potency in vitro and in a preclinical model of corneal chemical burn.

Methods: BALB/c MSCs were cultured with no supplement, recombinant IFNγ, or recombinant TGFβ1 for 72 h. sEV, sEVIFNγ, and sEVTGFβ were then isolated from conditioned medium of parental cells by a combination of ultrafiltration and size exclusion chromatography. Following isolation MSC-sEV were thoroughly characterized for size, marker expression and therapeutic efficacy. To evaluate their immunomodulatory capacity, both naïve and licensed MSC-sEV were tested in in vitro macrophage and T cell assays and in a preclinical corneal injury model.

Results: Relative to unlicensed sEV, sEVIFNγ exhibited increased expression of MHC I and PD-L1 on their surface, whereas sEVTGFβ expressed higher levels of CD44, CD29, and CD73. For immunomodulatory capacity, only sEVTGFβ was found to reduce macrophage expression of MHC II and CD80 and induced the secretion of anti-inflammatory macrophage cytokines. sEVTGFβ were also found to increase Treg expansion and FOXP3 expression. Given the superior efficacy observed of sEVTGFβ in vitro, this product was evaluated in a preclinical mouse model of corneal chemical burn. sEVTGFβ were applied either topically (day 0, 1, and 3) or subconjunctivally (day 0, and 3), and mice were monitored for 14 days. sEVTGFβ ameliorated burn-induced structural damage and accelerated restoration of normal corneal thickness, compared to PBS-treated controls. sEVTGFβ also resulted in reduced inflammatory mediators (IL-1β, iNOS) and minimised levels of fibrosis-associated collagen in the cornea. Mice that received subconjunctival, but not topical, administration of sEVTGFβ exhibited regulatory immune cell profiles with reduced pro-inflammatory- macrophages, increased anti-inflammatory macrophages, and restored Treg function and balance of the Treg/Th17 axis.

Conclusions: Overall, IFNγ and TGFβ licensing strategies were found to yield unique MSC-sEV phenotypes that can modulate inflammation differentially in vitro and in a corneal chemical burn model. This work found sEVTGFβ to represent a promising cell-free therapy for the treatment of corneal chemical burns.

TGF-β1许可间充质间质细胞分泌的细胞外小泡减少角膜碱烧伤后炎症相关损伤。
背景:间充质间质细胞(MSC)的治疗效力可以通过细胞因子的预激活或许可来增强。然而,其对小细胞外囊泡(MSC-sEV)治疗效果的影响尚未得到很好的证实。在这里,我们报告了两种不同的细胞因子许可策略,使用促炎或抗炎细胞因子,并评估了它们在体外和角膜化学烧伤的临床前模型中的治疗效力。方法:BALB/c MSCs在不添加任何补充的情况下培养,重组IFNγ或重组tgf - β1培养72 h,然后通过超滤和隔层析相结合的方法从亲代细胞的条件培养基中分离sEV、sEVIFNγ和sevtgf - β。分离后,对MSC-sEV的大小、标志物表达和治疗效果进行了全面的表征。为了评估它们的免疫调节能力,naïve和许可的MSC-sEV在体外巨噬细胞和T细胞检测以及临床前角膜损伤模型中进行了测试。结果:相对于未经许可的sEV, sEVIFNγ表面MHC I和PD-L1的表达增加,而sEVTGFβ表面表达更高水平的CD44、CD29和CD73。在免疫调节能力方面,只有sEVTGFβ能降低巨噬细胞MHC II和CD80的表达,并诱导巨噬细胞分泌抗炎细胞因子。sEVTGFβ也增加Treg扩增和FOXP3表达。鉴于体外观察到的优越疗效,我们在临床前小鼠角膜化学烧伤模型中对该产品进行了评估。将sEVTGFβ局部(第0、1和3天)或结膜下(第0和3天)施用,并对小鼠进行14天的监测。与pbs处理的对照组相比,sEVTGFβ改善了烧伤引起的结构损伤,加速了正常角膜厚度的恢复。sEVTGFβ也导致炎症介质(IL-1β, iNOS)的减少和角膜中纤维化相关胶原蛋白的最低水平。接受结膜下而非局部给药的小鼠显示出调节性免疫细胞谱,促炎巨噬细胞减少,抗炎巨噬细胞增加,Treg功能和Treg/Th17轴平衡恢复。结论:总体而言,IFNγ和TGFβ许可策略被发现产生独特的MSC-sEV表型,可以在体外和角膜化学烧伤模型中不同地调节炎症。这项工作发现sEVTGFβ代表了一种治疗角膜化学烧伤的有前途的无细胞疗法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Stem Cell Research & Therapy
Stem Cell Research & Therapy CELL BIOLOGY-MEDICINE, RESEARCH & EXPERIMENTAL
CiteScore
13.20
自引率
8.00%
发文量
525
审稿时长
1 months
期刊介绍: Stem Cell Research & Therapy serves as a leading platform for translational research in stem cell therapies. This international, peer-reviewed journal publishes high-quality open-access research articles, with a focus on basic, translational, and clinical research in stem cell therapeutics and regenerative therapies. Coverage includes animal models and clinical trials. Additionally, the journal offers reviews, viewpoints, commentaries, and reports.
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