The Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA, USA.
Department of Ophthalmology, Duke University School of Medicine, Durham, NC, USA.
Exp Eye Res. 2019 Feb;179:47-54. doi: 10.1016/j.exer.2018.10.013. Epub 2018 Oct 23.
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 intra-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.
中性粒细胞介导的炎症在损伤或感染后角膜损伤中起关键作用。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，释放可溶性 FasL（sFasL），并且 sFasL 拮抗 mFasL 介导的趋化因子产生。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，我们使用质粒 DNA 或腺病毒载体（AV）在 C57BL/6 小鼠的角膜基质中表达 GFP 仅、sFasL-GFP 或 mFasL-GFP，并在角膜损伤和 LPS 诱导的角膜炎模型中检查 mFasL 和 sFasL 在角膜炎症中的作用。我们的工作解决了 FasL 领域一个重要的分歧领域，涉及 sFasL 调节眼内炎症的机制。在此，我们证明，GFP 仅、sFasL-GFP 或 mFasL-GFP 质粒 DNA 的基质内注射导致角膜基质中 GFP 的表达长达两周，GFP 仅和 sFasL-GFP 组几乎没有炎症证据，mFasL-GFP 组有轻度角膜炎症。同样，在上皮清创后，表达 GFP 仅或 sFasL-GFP 的角膜没有明显的角膜炎症迹象，清创后 15 天角膜清晰。相比之下，mFasL-GFP 的上皮清创角膜引发持续的角膜炎症和中央角膜混浊的发展，这被 sFasL 阻断。与 mFasL-GFP 质粒 DNA 相似，基质内注射 mFasL-GFP AV 引发轻度的角膜炎症，但炎症是短暂的，到第 10 天就消退了，到第 30 天注射后角膜仍保持清晰。然而，基质内表达 mFasL-GFP AV 加剧了 LPS 诱导的角膜炎、角膜混浊和新生血管形成，而 sFasL-GFP AV 的表达则阻止了 LPS 诱导的角膜炎，使角膜保持清晰。用 LPS 诱导角膜炎的角膜组织学分析显示，巨噬细胞和中性粒细胞大量浸润，sFasL 表达特异性阻断中性粒细胞浸润。总的来说，我们的数据表明基质表达 mFasL 是炎症性的，而 sFasL 是非炎症性的，并拮抗 mFasL 在小鼠上皮清创和 LPS 诱导的角膜炎模型中的作用。这些数据表明，sFasL 和 mFasL 之间的微妙平衡调节眼内炎症。这项研究进一步确定 sFasL 是一种有效的中性粒细胞介导的角膜损伤抑制剂，并支持在治疗中性粒细胞介导的角膜炎中使用 sFasL。这些结果强烈支持这样的假设，即在眼睛的免疫特权环境中，FasL 的同种型调节免疫特权并决定炎症的程度：mFasL 促进炎症，而 sFasL 阻止炎症。
