Ocular Surface Center, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States.
Departments of Anatomy, Regenerative Biology and Ophthalmology, The George Washington University Medical School and Health Sciences, Washington, DC, United States.
Invest Ophthalmol Vis Sci. 2024 Oct 1;65(12):29. doi: 10.1167/iovs.65.12.29.
This study aimed to define the heterogeneity, spatial localization, and functional roles of immune cells in the mouse cornea using single-cell RNA sequencing (scRNA-seq) and immunofluorescent staining.
Enriched mouse corneal immune cells (C57BL/6 strain, age 16-20 weeks) underwent single-cell RNA sequencing library preparation, sequencing, and analysis with Seurat, Monocle 3, and CellChat packages in R. Pathway analysis used Qiagen Ingenuity Pathway Analysis software. Immunostaining confirmed cell distribution.
We identified 14 distinct immune cell clusters (56% myeloid and 44% lymphoid). Myeloid populations included resident macrophages, conventional dendritic cells (cDC2s), Langerhans cells, neutrophils, monocytes, and mast cells. Additionally, lymphocyte subsets (B, CD8, CD4, γδT, natural killer, natural killer T, and group 2 innate lymphoid cells) were found. We also found three new subtypes of resident macrophages in the cornea. Trajectory analysis suggested a differentiation pathway from monocytes to conventional dendritic cells, resident macrophages, and LCs. Intercellular communication network analysis using cord diagram identified amyloid precursor protein, chemokine (C-C motif) ligands (2, 3, 4, 6, 7, 9, and 12), Cxcl2, Mif, Tnf, Tgfb1, Igf1, and Il10 as prominent ligands involved in these interactions. Sexually dimorphic gene expression patterns were observed, with male myeloid cells expressing genes linked to immune regulation (Egr1, Foxp1, Mrc1, and Il1rn) and females showing higher expression of antigen presentation genes (Clic1, Psmb8, and Psmb9). Finally, immunostaining confirmed the spatial distribution of these cell populations within the cornea.
This study unveils a diverse immune landscape in the mouse cornea, with evidence for cell differentiation and sex-based differences. Immunostaining validates the spatial distribution of these populations, furthering our knowledge of corneal immune function.
本研究旨在使用单细胞 RNA 测序（scRNA-seq）和免疫荧光染色来定义小鼠角膜中的免疫细胞异质性、空间定位和功能角色。
用 Seurat、Monocle 3 和 CellChat 软件包对从小鼠角膜中富集的免疫细胞（C57BL/6 品系，年龄 16-20 周）进行单细胞 RNA 测序文库制备、测序和分析。通路分析使用 Qiagen Ingenuity Pathway Analysis 软件。免疫染色证实了细胞的分布。
我们鉴定出 14 种不同的免疫细胞簇（56%为髓系，44%为淋巴系）。髓系群体包括常驻巨噬细胞、传统树突状细胞（cDC2）、朗格汉斯细胞、中性粒细胞、单核细胞和肥大细胞。此外，还发现了淋巴细胞亚群（B、CD8、CD4、γδT、自然杀伤细胞、自然杀伤 T 细胞和 2 型固有淋巴细胞）。我们还在角膜中发现了三种新的常驻巨噬细胞亚型。轨迹分析表明，单核细胞向传统树突状细胞、常驻巨噬细胞和 LCs 的分化途径。使用 cord diagram 进行细胞间通讯网络分析，确定了淀粉样前体蛋白、趋化因子（C-C 基序）配体（2、3、4、6、7、9 和 12）、Cxcl2、Mif、Tnf、Tgfb1、Igf1 和 Il10 作为这些相互作用中的重要配体。观察到性别二态性基因表达模式，雄性髓系细胞表达与免疫调节相关的基因（Egr1、Foxp1、Mrc1 和 Il1rn），而雌性细胞表现出更高的抗原呈递基因（Clic1、Psmb8 和 Psmb9）表达。最后，免疫染色证实了这些细胞群体在角膜中的空间分布。
本研究揭示了小鼠角膜中的多样化免疫景观，并有证据表明存在细胞分化和性别差异。免疫染色验证了这些细胞群体的空间分布，进一步加深了我们对角膜免疫功能的认识。
