Background: An increasing number of studies reveal that synovial fibroblasts (SFs) play a crucial role in the pathogenesis of rheumatoid arthritis (RA), even in the absence of immune cells. Additionally, despite advancements in treatments targeting immune responses, many patients fail to respond adequately, which could be due to the lack of therapies directly targeting SFs. RA is a complex disease, where several determinants, such as epigenetic changes, and genetic predisposition contribute to disease development. Genome-wide association studies have revealed that 80% of genetic risk variants for RA lie in non-coding regions, particularly in distal regulatory elements such as enhancers. Linking these variants to their target genes is context-dependent and influenced mostly by the cell type.

Objectives: We aimed to integrate genetic and epigenetic data to identify and validate potential therapeutic target genes in RA SFs.

Methods: We utilized a multi-omics approach, incorporating a previously published genomic atlas of SFs generated by our group [1]. We integrated DNA methylation data from both published [2] and unpublished sources (15 healthy and 11 RA SFs) using the minfi and limma R packages to identify epigenetic alterations. To better represent the expression profile of the synovium, we utilized a publicly available RNA-seq dataset, which highlights changes in RA SF expression profiles under eight different cytokine stimulations (TGF-β1, IL-18, IL-17, IL-6, IFN-α, IFN-γ, IL-1β, and TNF-α) compared to non-stimulated condition [3]. The DisGeNET database and the NCBI literature search engine were used to assess whether the identified genes were associated with rheumatoid arthritis in the field. For the preliminary characterization of the genes, we employed the following workflow: conventional PCR to determine transcript (and alternative transcripts) expression, qPCR for differential expression following cytokine stimulations, and pyrosequencing for differential methylation analysis. To determine whether a gene was differentially expressed in SFs within tissue in RA compared to healthy controls, we utilized single cell RNA sequencing (scRNA-seq) data previously generated and analyzed in our laboratory, which included samples from 6 healthy individuals and 13 RA patients.

Results: Previously, we have identified 369 potentially pathogenic genetic risk genes in RA [1]. Using publicly available dataset, we found that 243 of these genes showed significant differences across eight different cytokine stimulations in SFs. Out of remaining 126 genes, 95 of them were not expressed in SFs even under stimulatory conditions. Therefore, we were left with 274 genetic risk genes active in SFs that could potentially serve as therapeutic targets. We then conducted a global DNA methylation analysis and identified that 27 genes, within the 274 target genes, had at least one differentially methylated region in their promoters, gene bodies, or untranslated regions (UTRs) in RA SFs when compared to healthy controls (|delta beta| > 0.1, padj < 0.05). Some of these genes, such as CDK4, FOXO1, NR1D1, and NLRP1, have been previously studied and shown to be relevant in the context of RA. However, our analysis using the DisGeNET database and the NCBI literature search revealed that 15 out of 27 identified genes have not yet been investigated in relation to RA. Among these, we prioritized ZFP36L1, an RNA-binding protein encoding gene, due to its significant differential expression and its known role in regulating inflammation-related pathways. Using cultured RA SFs, we confirmed that ZFP36L1 was significantly upregulated upon TNF and IL-1β stimulations (t-test, p<0.1), which was consistent with the cytokine stimulation dataset. We found that while the canonical isoform was highly expressed in SFs, PBMCs, and several immortalized cell lines, the second isoform of ZFP36L1 was exclusively expressed in SFs. Furthermore, using pyrosequencing, we validated hypomethylation in the promoter region of ZFP36L1 (mean of 0.15 beta value decrease). Finally, in scRNA-seq data previously generated and analyzed in our laboratory, we identified this gene as one of the top 25 upregulated genes in RA tissue SFs compared to healthy tissue SFs (padj < 0.05, log2FC > 1).

Conclusion: Through the integration of multi-omics data, we identified 27 genes that were genetically as well as epigenetically modified in RA SFs. Among these, ZFP36L1, an RNA-binding protein, stands out as a potential mediator of inflammation in SFs, influenced by both inflammatory signaling and epigenetic modifications. Future experiments will elucidate the functional role of the ZFP36L1 isoform specifically expressed in SFs and show whether this could be a promising therapeutic target in RA.

REFERENCES: [1] X. Ge et al. , “Functional genomics atlas of synovial fibroblasts defining rheumatoid arthritis heritability.” Genome Biology , vol. 22, no. 1, Aug. 2021, doi: 10.1186/s13059-021-02460-6.

[2] E. Karouzakis et al. , “Analysis of early changes in DNA methylation in synovial fibroblasts of RA patients before diagnosis.” Scientific Reports , vol. 8, no. 1, May 2018, doi: 10.1038/s41598-018-24240-2.

[3] H. Tsuchiya et al. , “Parsing multiomics landscape of activated synovial fibroblasts highlights drug targets linked to genetic risk of rheumatoid arthritis.” Annals of the Rheumatic Diseases , vol. 80, no. 4, pp. 440–450, Nov. 2020, doi: 10.1136/annrheumdis-2020-218189.

Acknowledgements: Department of Rheumatology.

Disclosure of Interests: Ege Ezen: None declared. Alexandra Khmelevskaya: None declared. Oliver Distler OD has/had consultancy relationships with and/or has served as a speaker for the following companies in the area of potential treatments for systemic sclerosis and its complications in the last three calendar years: 4P-Pharma, Abbvie, Acceleron, Acepodia Biotech, Aera, Alcimed, Altavant, Amgen, AnaMar, Anaveon AG, Argenx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Calluna (Arxx), Cantargia AB, Catalyze Capital, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, MSD Merck, Miltenyi Biotec, Mitsubishi Tanabe, Nkarta Inc., Novartis, Orion, Pilan, Prometheus, Quell, Redxpharma, Roivant, EMD Serono, Topadur and UCB. Patent issued “mir-29 for the treatment of systemic sclerosis” (US8247389, EP2331143). Co-founder of CITUS AG, OD has/had consultancy relationships with and/or has served as a speaker for the following companies in the area of potential treatments for systemic sclerosis and its complications in the last three calendar years: 4P-Pharma, Abbvie, Acceleron, Acepodia Biotech, Aera, Alcimed, Altavant, Amgen, AnaMar, Anaveon AG, Argenx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Calluna (Arxx), Cantargia AB, Catalyze Capital, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, MSD Merck, Miltenyi Biotec, Mitsubishi Tanabe, Nkarta Inc., Novartis, Orion, Pilan, Prometheus, Quell, Redxpharma, Roivant, EMD Serono, Topadur and UCB. Research Grants: BI, Kymera, Mitsubishi Tanabe, UCB, Caroline Ospelt: None declared.

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