Immunomodulatory effects of ponesimod on human astrocytes

Ponesimod is a slective S1PR1 antagonist and is approved for the treatment of Multiple Sclerosis by FDA. To determine how ponesimod controls neuroinflammatory responses in astrocytes, single-cell RNA-sequencing (scRNA-seq) was conducted in cytokine mixture-stimulated human primary astrocytes in the presence or absence of ponesimod. Human primary astrocytes (Lonza; Cat# CC-2565) were stimulated with or without cytokine mixture (25 ng/mL each; TNF-a, IFN-g, IL-17, IL-1b, and IL-6) in the presence or absence of ponesimod for 24 h or 48 hrs. Single-cell capture (target capture of 10,000 cells per sample) and library preparation was conducted using Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3 (10x Genomics, Cat # PN-1000075) and Chromium Chip B Single Cell Kit (10X Genomics, Cat # PN-1000074) and according to manufacturer's instruction. Single-nucleus libraries were sequenced on the Illumina NovaSeq 6000 machine at GENEWIZ, Inc. (U.S.) Raw FASTQ files were input into the cellranger count pipeline (Cell Ranger V4.0.0, 10x Genomics) to align reads to the GRCh38 human genome.

波尼莫德（Ponesimod）是一种选择性S1PR1拮抗剂，已获美国食品药品监督管理局（FDA）批准用于多发性硬化症（Multiple Sclerosis）的治疗。为探究波尼莫德如何调控星形胶质细胞的神经炎症应答，研究团队在经细胞因子混合物刺激的人原代星形胶质细胞中分别添加或不添加波尼莫德，开展了单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）实验。本次实验使用的人原代星形胶质细胞购自Lonza，货号为CC-2565；将其分别在添加或不添加细胞因子混合物（每种细胞因子终浓度为25 ng/mL，包括TNF-α、IFN-γ、IL-17、IL-1β及IL-6）的体系中，与波尼莫德共孵育24小时或48小时。单细胞捕获（每个样本目标捕获10000个细胞）与文库制备工作采用Chromium单细胞3' GEM、文库与磁珠试剂盒v3（10x Genomics，货号PN-1000075）以及Chromium Chip B单细胞试剂盒（10x Genomics，货号PN-1000074），并严格遵循制造商的操作说明书进行。单细胞核文库于美国GENEWIZ公司的Illumina NovaSeq 6000测序仪上完成测序。原始FASTQ文件被导入cellranger count分析流程（Cell Ranger V4.0.0，10x Genomics），以将测序reads比对至GRCh38人类参考基因组。