Department of Ophthalmology, Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, United States.
Department of Ophthalmology, Harvard Medical School, Schepens Eye Research Institute/Massachusetts Eye and Ear Infirmary, Boston, MA, United States.
Front Immunol. 2020 Feb 21;11:39. doi: 10.3389/fimmu.2020.00039. eCollection 2020.
Multiphoton intravital microscopy (MP-IVM) is a powerful tool to image cells . Its application in immunology research has opened new horizons, allowing intravital imaging of leukocytes at the single-cell level. A transparent cornea is vital to retain vision. As an immune privileged site, a rapid innate response to foreign antigens is crucial in clearing opportunistic bacterial and viral pathogens, and minimizing collateral structural damage to the cornea. Furthermore, dissecting the mechanisms and preventing the immunological rejection process after corneal transplantation is imperative to retain sight. Therefore, understanding the underlying mechanisms behind corneal immunity, specifically the process of antigen presentation and adaptive immunity in the mandibular draining lymph nodes (dLNs) , is crucial. Attempts of intravital imaging of mandibular dLNs have yielded little success to date, due to breathing artifacts and the location that is difficult to access. Herein, we present the first MP-IVM mouse model of the mandibular dLNs, utilizing transgenic mice in which CD11c cells are fluorescently labeled. Furthermore, we demonstrate that CD11c-YFP cells are localized mainly in the parafollicular cortex (T cell zone) and subcapsular area and are sparsely distributed in the follicular region (B cell zone) of mandibular dLNs during steady state. A significant increase in host CD11c-YFP cell density is noted at 14 and 21 days following allogeneic corneal transplantation, compared to steady state ( < 0.05). Moreover, allogeneic corneal transplantation results in increased host-derived CD11c-YFP cell mean speed and displacement in mandibular dLNs, compared to steady state ( < 0.001). The meandering index, an index for directionality, is significantly increased after allogeneic corneal transplantation at both 14 and 21 days, compared to steady state ( < 0.001). Taken together, our study demonstrates the necessary methodology required for intravital multiphoton imaging of the mandibular dLNs, allowing visualization of spatiotemporal kinetics of immune cells , and provides a window into the corneal immune reflex arc. This technique will be a powerful tool to investigate the pathogenesis of ocular immune and inflammatory diseases.
多光子活体显微镜（MP-IVM）是一种强大的成像细胞的工具。它在免疫学研究中的应用开辟了新的视野，使得能够在单细胞水平上对白细胞进行活体成像。透明的角膜对于保持视力至关重要。作为一个免疫特惠部位，快速的先天反应对于清除机会性细菌和病毒病原体，以及最大限度地减少对角膜的结构损伤至关重要。此外，剖析角膜移植后免疫排斥过程的机制并加以预防对于保持视力至关重要。因此，了解角膜免疫的潜在机制，特别是下颌引流淋巴结（dLNs）中的抗原呈递和适应性免疫过程，至关重要。迄今为止，由于呼吸伪影和难以接近的位置，尝试对下颌 dLNs 进行活体成像的效果甚微。在此，我们介绍了第一个利用荧光标记 CD11c 细胞的转基因小鼠模型进行下颌 dLNs 的多光子活体显微镜成像。此外，我们证明，在稳态下，CD11c-YFP 细胞主要位于副皮质区（T 细胞区）和被膜下区，稀疏分布于下颌 dLNs 的滤泡区（B 细胞区）。与稳态相比，同种异体角膜移植后 14 天和 21 天，宿主 CD11c-YFP 细胞密度显著增加（<0.05）。此外，与稳态相比，同种异体角膜移植导致下颌 dLNs 中宿主来源的 CD11c-YFP 细胞平均速度和位移增加（<0.001）。与稳态相比，在同种异体角膜移植后 14 天和 21 天，方向性指标——曲折指数显著增加（<0.001）。总之，我们的研究证明了进行下颌 dLNs 活体多光子成像所需的必要方法学，使得能够可视化免疫细胞的时空动力学，并为角膜免疫反射弧提供了一个窗口。这项技术将成为研究眼部免疫和炎症性疾病发病机制的有力工具。
