Next Generation Sequencing to understand the pathogenic role of synovial fibroblasts in experimental models of arthritis

The goal of this study is to define the pathophysiology of synovial fibroblasts in chroninc joint inflammation using NGS-derived synovial transcriptome profiling (RNA-seq) Methods: Synovial fibroblasts were isolated by FACs sorting [CD45-CD31-Popdoplanin+] from the synovium of healthy mice and mice undergoing experimental Collagen-Induced Arthritis (12 weeks old)(CIA). Methods: Whole Transcriptome Profiling of healthy and CIA synovial fibroblasts were generated by deep sequencing, in triplicate, using Illumina NextSeq™ 500 platform. Libraries were prepared using polyA selection (TruSeq stranded mRNA kit). Methods: The sequence reads that passed quality filters were aligned to mouse reference genome (GRCM38) using Hisat2 version 2.1.0. we mapped about 30 million sequence reads per sample (75 bp, paired-end) Methods: Featurecounts version 1.4.6 was used to quantify reads counts. Data quality control, non-expressed gene filtering, median ratio normalization (MRN) implemented in DESeq2 package and identification of differentially expressed (DE) genes were done using the R bioconductor project DEbrowser. Results: Results: We detected several differentially expressed (DE) genes in CIA synovial fibroblasts compared to naïve cells. These included 298 up-regulated genes and 88 down-regulated (>2 fold, adjp <0.05). DE genes reflected the reflecting the hyperplasia and activation observed during chronic joint disease. Pathways associated to DE genes were cell cycle and cell division and inflammatory response. Conclusions: our study shows pathophysiological changes associated to inflammatory synovial fibroblasts in a model of rheumatoid arthritis. Cells were directly isolated from fresh tissue, providing a valuable tool to study stromal inflammation. Synovial fibroblast mRNA profiles of naïve healthy DBA1J and CIA mice.本研究旨在借助下一代测序（NGS）来源的滑膜转录组谱分析（RNA-seq），明确慢性关节炎症中滑膜成纤维细胞的病理生理学机制。 方法：通过荧光激活细胞分选（FACS）以[CD45⁻CD31⁻Podoplanin⁺]表型，从健康小鼠及12周龄实验性胶原诱导性关节炎（CIA）模型小鼠的滑膜组织中分离滑膜成纤维细胞。 方法：采用Illumina NextSeq™ 500平台，对健康小鼠与CIA模型小鼠的滑膜成纤维细胞进行三次重复深度测序，以获取全转录组表达谱。文库构建采用PolyA富集法（TruSeq stranded mRNA kit）。 方法：将通过质量过滤的序列读段，使用Hisat2 v2.1.0版本比对至小鼠参考基因组（GRCM38）。每个样本约获得3000万条75bp双端测序序列读段。 方法：使用FeatureCounts v1.4.6版本完成测序读段计数。随后借助R语言Bioconductor项目的DEbrowser工具，完成数据质量控制、非表达基因过滤、DESeq2包内置的中位数比归一化（MRN）分析，以及差异表达（DE）基因的鉴定。 结果：与未致敏（naïve）健康细胞相比，本研究在CIA模型小鼠的滑膜成纤维细胞中检测到多个差异表达基因，其中298个基因上调、88个基因下调（差异倍数>2，校正后P值<0.05）。差异表达基因的特征反映了慢性关节疾病过程中观察到的细胞增生与活化状态。与差异表达基因相关的通路涵盖细胞周期、细胞分裂及炎症应答。 结论：本研究揭示了类风湿关节炎模型中炎症性滑膜成纤维细胞相关的病理生理学改变。本研究直接从新鲜组织中分离细胞，为基质炎症的研究提供了极具价值的工具。本数据集包含未致敏健康DBA/1J小鼠与CIA模型小鼠的滑膜成纤维细胞mRNA表达谱。