Center for Neurobiology and Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA.
Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center, Los Angeles, California, USA.
mBio. 2024 Oct 16;15(10):e0145424. doi: 10.1128/mbio.01454-24. Epub 2024 Sep 9.
Periodic reactivation of herpes simplex virus type 1 (HSV-1) triggers immune responses that result in corneal scarring (CS), known as herpes stromal keratitis (HSK). Despite considerable research, fully understanding HSK and eliminating it remains challenging due to a lack of comprehensive analysis of HSV-1-infected immune cells in both corneas and trigeminal ganglia (TG). We engineered a recombinant HSV-1 expressing green fluorescent protein (GFP) in the virulent McKrae virus strain that does not require corneal scarification for efficient virus replication (GFP-McKrae). Next-generation sequencing (NGS) analysis, along with and assays, showed that GFP-McKrae virus was similar to WT-McKrae virus. Furthermore, corneal cells infected with GFP-McKrae were quantitatively analyzed using image mass cytometry (IMC). The single-cell reconstruction data generated cellular maps of corneas based on the expression of 25 immune cell markers in GFP-McKrae-infected mice. Corneas from mock control mice showed the presence of T cells and macrophages, whereas corneas from GFP-McKrae-infected mice on days 3 and 5 post-infection (PI) exhibited increased immune cells. Notably, on day 3 PI, increased GFP expression was observed in closely situated clusters of DCs, macrophages, and epithelial cells. By day 5 PI, macrophages and T cells became prominent. Finally, immunostaining methods detected HSV-1 or GFP and gD proteins in latently infected TG. This study presents a valuable strategy for identifying cellular spatial associations in viral pathogenesis and holds promise for future therapeutic applications.IMPORTANCEThe goal of this study was to establish quantitative approaches to analyze immune cell markers in HSV-1-infected intact corneas and trigeminal ganglia from primary and latently infected mice. This allowed us to define spatial and temporal interactions between specific immune cells and their potential roles in virus replication and latency. To accomplish this important goal, we took advantage of the utility of GFP-McKrae virus as a valuable research tool while also highlighting its potential to uncover previously unrecognized cell types that play pivotal roles in HSV-1 replication and latency. Such insights will pave the way for developing targeted therapeutic approaches to tackle HSV-1 infections more effectively.
单纯疱疹病毒 1 型（HSV-1）的周期性再激活引发免疫反应，导致角膜瘢痕（CS），即所谓的疱疹性基质角膜炎（HSK）。尽管进行了大量研究，但由于缺乏对感染 HSV-1 的角膜和三叉神经节（TG）免疫细胞的全面分析，全面了解 HSK 并消除它仍然具有挑战性。我们构建了一种表达绿色荧光蛋白（GFP）的重组 HSV-1，该病毒在强毒 McKrae 病毒株中表达，不需要角膜划痕即可实现高效病毒复制（GFP-McKrae）。下一代测序（NGS）分析以及 和 测定表明，GFP-McKrae 病毒与 WT-McKrae 病毒相似。此外，使用图像质谱细胞术（IMC）对感染 GFP-McKrae 的角膜细胞进行了定量分析。基于 GFP-McKrae 感染小鼠中 25 种免疫细胞标志物的表达，单细胞重建数据生成了角膜的细胞图谱。模拟对照小鼠的角膜显示存在 T 细胞和巨噬细胞，而在感染后第 3 和第 5 天（PI）的 GFP-McKrae 感染小鼠的角膜中观察到免疫细胞增加。值得注意的是，在 PI 第 3 天，在 DC、巨噬细胞和上皮细胞紧密相邻的簇中观察到 GFP 表达增加。在 PI 第 5 天，巨噬细胞和 T 细胞变得突出。最后，免疫染色方法在潜伏感染的 TG 中检测到 HSV-1 或 GFP 和 gD 蛋白。这项研究提出了一种用于鉴定病毒发病机制中细胞空间关联的有价值策略，并为未来的治疗应用提供了前景。
重要性
本研究的目的是建立定量方法来分析原发性和潜伏性感染小鼠的 HSV-1 感染完整角膜和三叉神经节中的免疫细胞标记物。这使我们能够定义特定免疫细胞之间的空间和时间相互作用及其在病毒复制和潜伏中的潜在作用。为了实现这一重要目标，我们利用 GFP-McKrae 病毒作为一种有价值的研究工具，同时也强调了它在揭示以前未被认识到的在 HSV-1 复制和潜伏中起关键作用的细胞类型方面的潜力。这种见解将为开发更有效地治疗 HSV-1 感染的靶向治疗方法铺平道路。
