AB0077 RA-Patients Show Differential Responses in A Novel Cell-Based Promoter Reporter Assay

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,