Targeting inflammatory and angiogenic responses by small-molecule kinase inhibitors in a 3D macrophage-containing spheroid model of rheumatoid arthritis synovial tissue

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.