Background: Previously, a cDNA phage display library, constructed from the hip synovium of three axial spondyloarthritis (axSpA) patients was screened, resulting in the identification of antibodies reactive to double homeobox 4 (DUX4) protein in the plasma of a subset of axSpA and rheumatoid arthritis (RA) patients [1]. DUX4 is a transcription factor only expressed during early embryogenesis and epigenetically silenced in most adult somatic tissues, except the thymus and testes. DUX4 re-expression has been linked with cell death and inflammation in facioscapulohumeral dystrophy (FSHD) and immune evasion in cancer. Interestingly, we found that DUX4 protein was detected in the synovial lining layer of patients with axSpA and RA but not in healthy controls. More specifically, DUX4 was found in fibroblast-like synoviocytes (FLS), which are mesenchymal stromal cells that drive inflammation and joint damage in arthritis.

Objectives: This study aims to investigate DUX4 re-expression in both tissue-derived and synovial fluid-derived FLS (SF-FLS) from different types of arthritis, and to study the cellular effects upon overexpression.

Methods: DUX4 expression in tissue-derived FLS from RA (n=4) and osteoarthritis (n=17) patients was investigated using a nested RT-PCR approach, and the genetic origin of the transcripts was confirmed by Sanger sequencing. The induction of embryonic DUX4 target gene expression was measured by RT-qPCR. In order to study overexpression of DUX4, SW982 synovial sarcoma cells and SF-FLS were transfected with the pCI-Neo-DUX4 or pCI-Neo-control plasmid, using lipofection. Therefore, SF-FLS were isolated from the synovial fluid of RA and spondyloarthritis patients. The purity of SF-FLS cultures was assessed via flow cytometry by confirming the expression of FLS markers, CD90 and podoplanin (PDPN), and the absence of immune cell markers (CD45, CD14). Functional responses of SF-FLS to 48 hours of stimulation with pro-inflammatory cytokines TNFα and IL-1β were evaluated at the mRNA and protein levels. Live-cell imaging, MTT assay and RT-qPCR were used to evaluate the cellular effect of DUX4 overexpression, 48 hours after transfection.

Results: Using nested PCR, DUX4 mRNA transcripts were detected and confirmed by sequencing in 2/4 (50%) of RA tissue-derived FLS and 0/17 of the OA tissue-derived FLS. Additionally, DUX4-positive RA-FLS showed a strongly increased expression of embryonic DUX4 target genes. In SW982 cells, transfection of the pCI-Neo-DUX4 significantly (p<0.001) induced DUX4 target gene expression, confirming the presence of functionally active DUX4 protein post-transfection (Figure 1). In contrast to observations in DUX4-associated muscular disease FSHD, DUX4 overexpression did not induce apoptotic and oxidative stress pathways. This resistance to DUX4 toxicity in SW982 cells was confirmed using the MTT assay and live-cell imaging, as no significant decrease in metabolic cell activity (p>0.6) and cell growth (p>0.8) was observed (Figure 1). SF-FLS were successfully isolated from the synovial fluid of RA and SpA patients. Flow cytometry confirmed the presence of both CD90 and PDPN on >95% of SF-FLS. Upon stimulation with pro-inflammatory cytokines, SF-FLS showed increased metabolic cell activity as well as MMP-1, MMP-3, and IL-6 expression. However, DUX4 overexpression in RA and SpA SF-FLS did not induce cell death, as metabolic cell activity and cell growth remained stable compared to control.

Conclusion: DUX4 re-expression, and the associated increases in target gene expression, were detected in 50% of the tested RA tissue-derived FLS, indicating the activation of this embryonic pathway in a subset of patients with arthritis. In contrast to other DUX4-driven pathologies, DUX4 did not induce apoptosis or oxidative stress in SW982 cells or SF-FLS. Functionally active FLS can be isolated from the synovial fluid of RA and SpA patients. These findings implicate a resistance to DUX4-mediated cytotoxicity in synovial fibroblasts, as well as highlighting the potential of SF-FLS in arthritis research. Future research should explore whether this DUX4-induced embryonic pathway affects the inflammatory state of FLS and contributes to the disease process of arthritis.

REFERENCES: [1] Quaden D, Vandormael P, Ruytinx P. Antibodies Against Three Novel Peptides in Early Axial Spondyloarthritis Patients From Two Independent Cohorts. Arthritis Rheumatology 2020; 72; 2094-2105.

Figure 1.

Synovial sarcoma SW982 cells are resistant to DUX4-induced cell death. A ) embryonic DUX4 target genes (n=4) and B ) oxidative stress and apoptosis genes (n=4) were measured using RT-qPCR, 48h after DUX4 overexpression in SW982 cells. C ) Cell activity was assessed 48h post-transfection in SW982 cells with the MTT assay (n=4). Bars show mean ± SEM. **** p≤0.0001; ns = not significant

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.