Background Disease activity in rheumatoid arthritis (RA) patients is commonly determined by calculating the DAS28 score, incorporating the number of swollen and tender joints, along with a measurement of the acute phase response (APR). Because the joint count relies on a personal assessment, many effort is being put into finding biomarkers that can improve an objective disease assessment. However, no single biomarker has been found that can sufficiently reflect disease activity.
A novel approach for a bioassay can be through disease-inducible promoter-reporters, consisting of a promoter sensitive to RA-related disease factors that can drive the expression of a reporter gene. After incubation with patient serum, the signal from the reporter assay is a direct measurement of the disease activity.
Objectives This study aims to develop promoter-reporter constructs that are sensitive to serum from RA patients for the development of a novel bioassay.
Methods Microarrays on joint tissue of 20 RA patients and 7 controls were analysed to find upregulated genes during active disease. The upregulation was validated by qPCR in Pam3Cys stimulated human THP-1 monocytes.
The promoters of the upregulated genes were isolated from human cDNA and cloned into a lentiviral vector containing the firefly luciferase reporter gene. THP-1 cells were transduced with the lentiviral constructs and stimulated for 6 hours with pro-inflammatory stimuli, 64 RA patient sera and 34 control sera to determine the inducibility of the promoters.
Results The microarray analysis resulted in a list of 8 genes that were upregulated ≥4-fold in RA synovium. The increased expression was confirmed by qPCR.
To mimic the inflammation in RA patients in vitro, THP-1 monocytes were stimulated by Pam3Cys. 6/8 genes were upregulated, showing the validity of the selected genes and the applicability of the THP-1 cells.
After transduction of THP-1 cells with the promoter reporter constructs, Pam3Cys stimulation resulted in a 15.5-fold upregulation of the CXCL10 promoter reporter and a 26.5-fold upregulation of the IL-8 promoter reporter, indicating that the isolated promoter sequences contain the important regulatory binding sites. In addition, a promoter that contained 4 repeated binding sites for NF-κB was induced 68.5-fold by Pam3Cys.
Next, the transduced THP-1 cells were stimulated with human sera. The promoter constructs with CXCL10, IL-8 and 4x NF-κB could distinguish between RA and healthy serum (P=0.017, P=0.036 and P=0.023 respectively). Finally, we compared the “assay-positive” RA sera that induced a signal of at least 1x standard deviation above the healthy control average in at least one reporter construct and found that these sera had a significantly higher erythrocyte sedimentation rate (P=0.0022), an acute phase response marker often incorporated in the DAS28, compared to “assay-negative” RA sera. Furthermore, the differences between the binding sites present on the promoters and the differences in RA sera that were tested positive between the promoters indicates that the different reporters might reflect different disease processes.
Conclusions Promoter-reporter constructs can respond to RA-related disease factors in the serum of RA patients, indicating their potential for the development of a novel disease characterizing assay.
Disclosure of Interest None declared
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