RF003-V1: The expression of ERAP2 and LST1 distinguishes between Glucocorticoid-Responders and Non-Responders in Rheumatoid Arthritis

Objective: Glucocorticoids (GC) have been a cornerstone of Rheumatoid arthritis (RA)-therapy for the last decades. However, about a third of RA-patients do not respond adequately. As monocytes and T-cells play an important role in RA-pathogenesis, the differential gene expression of these cells before pulse therapy with methylprednisolone was evaluated in order to find predictors for clinical response to GC. Methods: CD14-positive and CD4-positive cells were isolated by MACS sorting from five GC-Responders meeting the European League against Rheumatism (EULAR) response criteria and five Non-Responders. The clinical response was measured by disease activity score (DAS28) at the end of pulse therapy (3x 1000mg Methylprednisolone) at day 5. Cellular RNA was hybridized to Agilent 4x44K microarray chips and differentially expressed genes were calculated by MAANOVA. False discovery rate (FDR) was used as multiple testing correction. Selected genes were verified by quantitative real-time PCR (qPCR). Results: Eight genes were differentially regulated in CD14-cells and 4 genes in CD4-T-cells of GC-Responders compared to Non-Responders. Up-regulation of ERAP2 in monocytes and CD4-cells and LST1 and FAM26F in CD4-Tcells of GC-Responders was verified by qPCR and correlated with DAS28 at day 5 and (for LST1 and FAM26F) with smoking. Conclusion: As ERAP2 and LST1 are implicated in autoimmune disease, their increased expression in GC-Responders before GC-therapy may constitute not only a new tool for prediction of the clinical response to GC in RA, but also warrants further investigation to elucidate their role in the inflammatory process of RA. PBMC from patients were obtained before therapy by centrifugation of heparinized blood over Ficoll-Hypaque (Amersham Biosciences). CD14+ monocytes and CD4+ T-cells were isolated by positive (CD14-cells) or negative (CD4-cells) selection by AutoMACS (Miltenyi Biotec, Auburn, CA) and the respective isolation kits for CD14- or CD4- isolation (Miltenyi Biotec). Purity of the viable population assessed by flow cytometry was generally >90%. RNA was isolated using the RNeasy Mini Kit from Qiagen (Venlo, The Netherlands). All patient sample cRNAs were labeled with cy5, and co-hybridized with reference cRNA, labeled with cy3 on Human Whole Genome Gene Expression Microarrays v1. Differences between responders (RF-iv-r-) and non-responders (RF-iv-nr-) were analyzed.