Yuna Son, Daniel Korenfeld, Abel Suarez-Fueyo, Melanie Ruzek, Jing Wang, Bohdan Harvey
{"title":"Understanding mechanisms of JAK1 inhibition on synovial fibroblasts using combinatorial approaches of bulk and single cell RNAseq analyses.","authors":"Yuna Son, Daniel Korenfeld, Abel Suarez-Fueyo, Melanie Ruzek, Jing Wang, Bohdan Harvey","doi":"10.55563/clinexprheumatol/s705qn","DOIUrl":null,"url":null,"abstract":"<p><strong>Objectives: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":10274,"journal":{"name":"Clinical and experimental rheumatology","volume":null,"pages":null},"PeriodicalIF":3.4000,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Clinical and experimental rheumatology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.55563/clinexprheumatol/s705qn","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/6/19 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"RHEUMATOLOGY","Score":null,"Total":0}
引用次数: 0
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.
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.