Arthritis Research & Therapy · Published 2026-06-23 · DOI 10.1186/s13075-026-03849-z
Eva M.L. Philippon, Lisanne J. E. van Rooijen, Aldo Jongejan, Gary P. Sims, Jeeho Lee, Lichchavi Rajasinghe, Jan Piet van Hamburg, Conny J. van der Laken, Sander W. Tas
Abstract Background In rheumatoid arthritis (RA), interactions between macrophages, fibroblast-like synoviocytes (FLS), and endothelial cells (ECs) drive synovial tissue (ST) inflammation and pathological angiogenesis. Preclinical models reflecting this crosstalk are needed to evaluate novel therapies. We developed a human 3D model of RA ST to assess inflammatory mechanisms and responses to small-molecule kinase inhibitors. Methods Polarized M1-like or M2-like macrophages were co-cultured with RAFLS and ECs to generate spheroids embedded in a collagen-based scaffold. Spheroids were unstimulated or exposed to pro-inflammatory stimuli, including tumor necrosis factor-α (TNF) or RA synovial fluid (SF). Treatment effects of tofacitinib (JAK1/3 inhibitor) and an IKKβ inhibitor (IKKβi; targeting NF-κB signaling) were evaluated. Readouts included spheroid outgrowth and macrophage distribution quantified by semi-automated image analysis (n = 8), cytokine production measured by ELISA/Luminex (n = 8), and RNA sequencing to identify differentially expressed genes (n = 4). Results M1-like macrophages exhibited higher core retention, while M2-like macrophages showed increased outward migration (p < 0.05). M1 spheroids demonstrated elevated expression of M1 markers, chemokines, and EC-activating signatures, whereas M2 spheroids upregulated pathways related to matrix remodeling, migration, and immunoregulation (FDR < 0.05). In M1 spheroids, TNF and SF increased macrophage core density compared with unstimulated conditions (p < 0.01) and exhibited differential effects on spheroid outgrowth. SF induced RA-associated molecular programs that were broadly suppressed by IKKβi, whereas tofacitinib selectively reduced JAK-dependent signaling (FDR < 0.05). IKKβi significantly decreased TNF-induced mediators (IL-8: p < 0.001, M-CSF: p < 0.05, SPP1: p < 0.001), and tofacitinib primarily inhibited IL-6 production (p < 0.01). Conclusions This human 3D multicellular in vitro spheroid model of synovial inflammation recapitulates key RA pathological processes and provides a robust platform for mechanistic studies and therapeutic evaluation.
Abstract from DOAJ. Public domain (CC0 1.0).
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Philippon, E., Rooijen, L., Jongejan, A., et al. (2026). Targeting inflammatory and angiogenic responses by small-molecule kinase inhibitors in a 3D macrophage-containing spheroid model of rheumatoid arthritis synovial tissue. Arthritis Research & Therapy. https://doi.org/10.1186/s13075-026-03849-z