Innovative translational models to study human synovial pathology: Target validation and preclinical imaging

Abstract

Purpose: Many experiments to study inflammation, hyperplasia, and fibrosis in the synovium have been performed in animal models of RA and OA. However, the predictive value of these models for the screening of potential drugs in RA is variable and for OA, none were sufficiently effective in clinical trials. Translational arthritis research with human cells is often performed in monolayer culture where the absence of extracellular matrix and other cell types results in alterations of cell functions and loss of phenotype. To improve the predictive value of preclinical arthritis research by developing and optimizing innovative translational models to study human synovial pathology in vitro and in vivo. Methods: Synovial biopsies from arthritis patients were obtained during joint replacement surgery and processed for either (1) explant cultures, (2) 3D-synovial micromasses, (3) RA-SCID transplantation studies, and/or (4) a biobank for corresponding mRNA and IHC profiling. For explant culture, 3mm biopsies were cultured for 24 hours w/o various inhibitors, and cytokine production was analyzed by Luminex. 3D-micromasses were generated from primary RA FLS and CD14+ PBMCs, stimulated for 3 weeks with 10 ng/ml TNFα or TGFβ and analyzed by histology, IHC and QPCR. For target validation and preclinical imaging, 6mm biopsies were engrafted into SCID mice. Radio- and fluorescently-labelled anti-FcγRI antibodies were injected intravenously, and targeting was determined by biodistribution, μSPECT/CT and IVIS imaging analysis. Results: Our first assay with human arthritis synovium explants demonstrated to be highly suitable to test the therapeutic efficacy of inhibitors for TNFα, TLR4, p38 and the JAK-pathway, resulting in significantly reduced production of proinflammatory cytokines and chemokines at 24 hours of culture (1) . In contrast to the explant cultures, our 3D synovial micromasses could be followed for weeks. In this second translational model, lining formation was observed at day 7 and the micromasses could be stimulated to mimic RA- or OA-like features of synovial hyperplasia or fibrosis respectively. Long-term exposure to the RA-related cytokine TNFα lead to hyperplasia of the lining and an altered macrophage phenotype characterized by reduced CD163 expression. Conversely, the repair-related growth factor TGFβ induced fibrosis-like changes in the micromass lining, a hallmark of OA. This was accompanied by an increased expression of PLOD2, alpha-SMA, and collagen type 1 (2) Our third preclinical model, the human arthritis synovium SCID mouse, was previously validated using adalimumab, secukinumab, and rituximab (3), and was now used to study FcγRI as a potential marker to image synovitis. Gene expression of FCGRI in synovial explants from RA patients was shown to correlate positively and significantly to gene expression of pro-inflammatory factors IL1B, TNFA, IL8, S100A8 and S100A9 while gene expression of FCGRI in synovial explants from OA patients significantly correlated with S100A9. Interestingly, dual-labelled [111In]In-DTPA-IrDye800CW-anti-FcγRI antibody showed high uptake in the synovial transplants of the engrafted SCID mice, and specifically visualized the subcutaneous synovial grafts by both uSPECT/CT and IVIS imaging. Conclusions: The development of these translational models allows us to bridge the gap between preclinical and clinical drug development research. Whereas our synovial explant assay is ideal for short-term interventions, and the RA-SCID mouse a valuable translational model for in vivo preclinical studies, donor variability and access to sufficient tissue may be challenging. In such cases, the 3D synovial micromass model may be an excellent alternative. Especially in combination, these 3 translational approaches will improve target validation, lead optimization and preclinical development of novel anti-rheumatic drugs. (1) Abdollahi S., J Clin Invest. 2008; 118(1): 205-216. (2) Broeren M. et al., ALTEX. 2019; 36(1):18-28 (3) Koenders et al., Arthritis Rheum. 2012 Jun;64(6):1762-70