Understanding mechanisms of JAK1 inhibition on synovial fibroblasts using combinatorial approaches of bulk and single cell RNAseq analyses.

IF 3.4 4区 医学 Q2 RHEUMATOLOGY Clinical and experimental rheumatology Pub Date : 2024-11-01 Epub Date: 2024-06-19 DOI:10.55563/clinexprheumatol/s705qn
Yuna Son, Daniel Korenfeld, Abel Suarez-Fueyo, Melanie Ruzek, Jing Wang, Bohdan Harvey
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Abstract

Objectives: The aim of these studies was to characterise the molecular effects of a tool JAK1 inhibitor on cultured primary fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) through both total and individual cell analysis.

Methods: RA-FLS cultures from 6 (Bulk RNA-seq) or 4 (ScRNA-seq) donors were pre-treated with various concentrations (100 nM and 1μM) of ABT-317 with/without exposure to 25% SEB-conditioned PBMC medium to mimic the RA inflammatory milieu. Cells were subjected to both bulk RNA-seq (36 libraries) and single cell RNA-seq (scRNA-seq; 24 libraries) to identify biological processes impacted by CM and ABT-317 treatments.

Results: In our bulk RNA-seq analysis, a total of 2,605 differentially expressed genes (DEGs) were identified between CM-stimulation and unstimulated groups, while 1,122 DEGs were found between ABT-317 1μM and DMSO in CM-stimulated groups using thresholds of log2 (fold change) ≥ |0.58| and FDR ≤ 10%. Both bulk and single cell mRNA analysis of RA-FLS treated with a combination of CM and ABT-317 demonstrated the expected changes in inflammatory pathways such as interferon and IL-6 signalling. However, other non-inflammation associated pathways were also altered by ABT-317. In addition, the single cell analysis highlighted that FLS segregate into distinctive clusters upon combination CM and ABT-317 treatment, suggesting JAK inhibition can drive RA-FLS into multiple heterogenous cell populations. Interestingly, one of the unique RA-FLS clusters that emerged from the CM and ABT-317 treatment showed matrix metalloproteinase-3 (MMP3)high expression as well as several gene signatures that are not found in any other ABT-317 derived clusters.

Conclusions: JAK inhibition with ABT-317 is effective at globally inhibiting CM-induced pro- and non-inflammatory pathways in FLS cultures, but also results in several distinct fibroblast populations with unique gene-associated pathways. This study advances the molecular understanding of JAK1 inhibitor effects on fibroblasts that may contribute to clinical efficacy.

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利用体细胞和单细胞 RNAseq 分析的组合方法了解 JAK1 对滑膜成纤维细胞的抑制机制。
研究目的这些研究旨在通过总体和单个细胞分析,确定一种工具性 JAK1 抑制剂对类风湿性关节炎(RA)患者培养的原代成纤维细胞样滑膜细胞(FLS)的分子影响:用不同浓度(100 nM 和 1μM)的 ABT-317 预处理来自 6 名(大量 RNA-seq)或 4 名(ScRNA-seq)供体的 RA-FLS 培养物,同时/不暴露于 25% SEB 调节的 PBMC 培养基,以模拟 RA 炎症环境。对细胞进行大量 RNA-seq(36 个文库)和单细胞 RNA-seq(scRNA-seq,24 个文库)分析,以确定 CM 和 ABT-317 处理对生物过程的影响:在大量 RNA-seq 分析中,我们在 CM 刺激组和非刺激组之间共鉴定出 2,605 个差异表达基因 (DEGs),而在 CM 刺激组 ABT-317 1μM 和 DMSO 之间发现了 1,122 个 DEGs,阈值为 log2(折叠变化)≥ |0.58|,FDR ≤ 10%。用 CM 和 ABT-317 联合治疗 RA-FLS 的大量和单细胞 mRNA 分析表明,炎症通路(如干扰素和 IL-6 信号)发生了预期的变化。不过,ABT-317 也改变了其他非炎症相关通路。此外,单细胞分析突出显示,FLS 在 CM 和 ABT-317 联合治疗后会分离成不同的细胞群,这表明 JAK 抑制可将 RA-FLS 驱动成多个异源细胞群。有趣的是,在CM和ABT-317处理后出现的独特RA-FLS集群中,有一个集群显示出基质金属蛋白酶-3(MMP3)的高表达以及多个基因特征,而这些特征在其他ABT-317衍生集群中均未发现:结论:ABT-317抑制JAK能有效抑制FLS培养物中CM诱导的促炎症和非炎症通路,但同时也会产生几个具有独特基因相关通路的不同成纤维细胞群。这项研究加深了人们对 JAK1 抑制剂对成纤维细胞影响的分子认识,这可能有助于提高临床疗效。
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来源期刊
CiteScore
6.10
自引率
18.90%
发文量
377
审稿时长
3-6 weeks
期刊介绍: Clinical and Experimental Rheumatology is a bi-monthly international peer-reviewed journal which has been covering all clinical, experimental and translational aspects of musculoskeletal, arthritic and connective tissue diseases since 1983.
期刊最新文献
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