Session Information
Session Title: Rheumatoid Arthritis - Animal Models I
Session Type: Abstract Submissions (ACR)
Background/Purpose: An important step in the optimal personalized treatment of rheumatoid arthritis (RA) patients, is the accurate assessment of disease activity, disease progression and therapy response. The quantification of biomarkers has been proposed to monitor these processes, but no single biomarker has proven to be sufficiently reliable for this purpose. Instead of measuring the quantity of a biomarker, we focus on the biomarker promoter activity to take into account the complex interplay between stimulating and inhibiting factors.
The TNF stimulated gene 6 (TSG-6) encodes a 35-kDa secretory glycoprotein that is induced in fibroblasts, chondrocytes, synovial cells and mononuclear cells by proinflammatory cytokines. Large amounts of TSG-6 protein were found in synovial fluid of patients with RA. In this study, we set out to use the TSG-6 promoter to develop a promoter-reporter construct to monitor disease activity in RA.
Methods: Arthritis was induced in DBA1/J mice by immunization with bovine collagen-type II (CIA), or by intra-articular injection of 5 µg Streptococcal Cell Wall (SCW). Microarray experiments were performed on 15 CIA samples, 20 human end-point RA synovium samples and 6 control synovium samples. The TSG-6 promoter (-499 to +63) was cloned in a lentiviral vector (LV) encoding firefly luciferase. Murine 3T3 cells were cultured and stimulated with 1% mouse serum or 10% human serum for 6 hours. Luciferase activity was measured using BrightGlo reagent.
Results: TSG-6 was upregulated in arthritic CIA joints and expression correlated with disease severity. It was also upregulated ~6-fold in 20 human end-point RA synovium samples, compared to 6 non-arthritic synovium samples (P=0.01).
Subsequently, we studied the disease-inducibility of the TSG-6 promoter by developing a lentiviral construct in which the isolated TSG-6 promoter controls the expression of a luciferase reporter transgene (TSG6-luc). We infected mice with the TSG6-luc promoter reporter and induced SCW arthritis. The expression peaked at day 7 (19-fold compared to naive mice), indicating the disease-responsiveness at full blown synovitis.
Next, we transduced murine 3T3 cells with the LV TSG6-luc construct and incubated the cells with serum from mice with increasing severity of collagen induced arthritis. The response of the promoter correlated significantly with the macroscopically assessed disease severity (R2 0.47, P<0.0001) and with their circulating levels of the neutrophil chemokine KC (R2 0.31, P=0.002), the functional homologue of human IL-8. In addition, serum from human RA patients showed a significantly higher activation of the TSG6-luc construct, compared to serum from healthy patients (P=0.001).
An important characteristic of a successful reporter is a reduced signal after successful treatment. We tested this by comparing the response to serum from diseased arthritic mice to serum from mice that were successfully treated with Enbrel. Serum from Enbrel treated mice showed a significantly lower activation of the TSG-6 promoter, compared to serum from untreated mice.
Conclusion: The TSG-6 promoter reporter might be a valuable tool to monitor disease progression in RA patients.
Disclosure:
M. G. A. Broeren,
None;
E. A. Vermeij,
None;
O. J. Arntz,
None;
M. B. Bennink,
None;
E. Sterken,
None;
C. Popa,
None;
T. L. Jansen,
None;
W. B. van den Berg,
None;
F. A. J. Van de Loo,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-tumor-necrosis-factor-stimulated-gene-6-promoter-reporter-can-monitor-the-disease-activity-in-rheumatoid-arthritis/