Soluble Fas ligand blocks destructive corneal inflammation in mouse models of corneal epithelial debridement and LPS induced keratitis
Soluble Fas ligand blocks destructive corneal inflammation in mouse models of corneal epithelial debridement and LPS induced keratitis
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DOI：
10.1016/j.exer.2018.10.013
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发表时间：
2019-02-01
影响因子：
3.4
通讯作者：
Ksander, Bruce R.
中科院分区：
文献类型：
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作者：
Gregory-Ksander, Meredith;Perez, Victor L.;Ksander, Bruce R.
中性粒细胞介导的炎症在角膜损伤或感染后的损伤中起关键作用。先前的研究表明，膜结合FasL （mFasL）诱导中性粒细胞趋化因子的产生。然而，mFasL的细胞外结构域通常被基质金属蛋白酶切割，释放出可溶性形式的FasL (sFasL)， sFasL可拮抗mFasL介导的趋化因子的产生。因此，我们假设sFasL可用于预防与损伤和细菌性角膜炎相关的中性粒细胞介导的角膜炎症。为了验证这一假设，我们通过质粒DNA或腺病毒载体（AV）在C57BL/6小鼠角膜基质中表达GFP-only、sFasL- gfp和mFasL- gfp，并在角膜损伤和lps诱导的角膜炎模型中检测mFasL和sFasL在角膜炎症中的作用。我们的工作解决了FasL领域中一个重要的分歧领域，即FasL调节眼部炎症的机制。在此，我们证明，在角膜基质内注射GFP-only、sFasL-GFP或mFasL-GFP质粒DNA可导致整个角膜基质中GFP表达长达两周，在GFP-only组和sFasL-GFP组中几乎没有炎症迹象，而在mFasL-GFP组中角膜炎症轻微。同样，在上皮清创后，仅表达gfp或sFasL-GFP的角膜没有明显的角膜炎症迹象，在清创后15天角膜清晰。相比之下，表达mFasL-GFP的角膜上皮清创引发了持续的角膜炎症和角膜中央混浊的发展，而sFasL阻断了这种炎症。与mFasL-GFP质粒DNA类似，基质内注射mFasL-GFP AV会引发轻微的角膜炎症，但这种炎症是短暂的，并在第10天消退，注射后30天角膜保持清澈。然而，mFasL-GFP AV在基质内的表达加重了lps诱导的角膜炎、角膜混浊和新生血管的形成，而sFasL-GFP AV的表达阻止了lps诱导的角膜炎，导致角膜清晰。lps诱导角膜炎角膜的组织学分析显示，巨噬细胞和中性粒细胞大量浸润，sFasL的表达特异性地阻断了中性粒细胞的内流。总的来说，我们的数据表明mFasL的间质表达具有炎症性，而sFasL无炎症性，并且在上皮清创和lps诱导的角膜炎小鼠模型中与mFasL的作用相反。这些数据表明sFasL和mFasL之间的微妙平衡调节眼部炎症。本研究进一步确定sFasL是中性粒细胞介导的角膜损伤的有效抑制剂，并支持sFasL在中性粒细胞介导的角膜炎治疗中的潜在应用。这些结果有力地支持了这样的假设，即在眼睛的免疫特权环境中，FasL的异构体调节免疫特权并决定炎症的程度：mFasL促进炎症，sFasL阻断炎症。
Neutrophil-mediated inflammation plays a critical role in corneal damage following injury or infection. Previous studies demonstrated that membrane-bound FasL (mFasL) induces neutrophil chemokine production. However, the extracellular domain of mFasL is normally cleaved by matrix metalloproteinases to release a soluble form of FasL (sFasL) and sFasL antagonizes mFasL-mediated chemokine production. Therefore, we hypothesized that sFasL could be used to prevent neutrophil-mediated corneal inflammation associated with injury and bacterial keratitis. To test this hypothesis, GFP-only, sFasL-GFP, or mFasL-GFP were expressed in the corneal stroma of C57BL/6 mice, using infra-stromal injections of plasmid DNA or adenoviral vectors (AV) and the role of mFasL and sFasL in corneal inflammation was examined in models of corneal injury and LPS-induced keratitis. Our work addresses an important area of disagreement in the field of FasL, with regard to the mechanism by which sFasL regulates ocular inflammation. Herein, we demonstrate that an intrastromal injection of GFP-only, sFasL-GFP, or mFasL-GFP plasmid DNA resulted in GFP expression throughout the corneal stroma for up to two weeks with little to no evidence of inflammation in the GFP-only and sFasL-GFP groups and mild corneal inflammation in the mFasL-GFP group. Similarly, following epithelial debridement, corneas expressing GFP-only or sFasL-GFP showed no significant signs of corneal inflammation, with clear corneas at 15 days post debridement. By contrast, epithelial debridement of corneas expressing mFasL-GFP triggered persistent corneal inflammation and the development of central corneal opacities that was blocked by sFasL. Similar to the mFasL-GFP plasmid DNA, intrastromal injection of mFasL-GFP AV triggered mild corneal inflammation, but it was transient and resolved by day 10 with corneas remaining clear out to 30 days post injection. Nevertheless, intrastromal expression of mFasL-GFP AV exacerbated LPS-induced keratitis, corneal opacity, and neovascularization, while sFasL-GFP AV expression prevented LPS-induced keratitis, resulting in a clear cornea. Histological analysis of corneas with LPS-induced keratitis revealed a robust infiltration of macrophages and neutrophils and sFasL expression specifically blocked the neutrophil influx. Overall, our data demonstrate that stromal expression of mFasL is inflammatory, while sFasL is non-inflammatory, and opposes the effects of mFasL in mouse models of epithelial debridement and LPS-induced keratitis. These data demonstrate that a delicate balance between sFasL and mFasL regulates ocular inflammation. This study further identifies sFasL as a potent inhibitor of neutrophil-mediated corneal damage, and supports the potential use of sFasL in the treatment of neutrophil-mediated keratitis. These results strongly support the hypothesis that, in the immune privileged environment of the eye, the isoform of FasL regulates immune privilege and determines the extent of inflammation: mFasL promotes inflammation and sFasL blocks inflammation.