Background/Purpose: Rheumatoid Arthritis (RA) is a progressive and systemic autoimmune disorder characterized by chronic and destructive joint inflammation. Key features of RA include synovial cell proliferation, angiogenesis and extensive immune cell infiltration in the synovium.  In vitro approaches that replicate the complex cell interactions within RA synovial tissue are essential for preclinical and translational research. These models enhance our understanding of RA pathophysiology and help in the development of new diagnostic and therapeutic strategies. Recently, we developed a novel RA synovial tissue spheroid-based model that integrates RA fibroblast-like-synoviocytes (RAFLS), endothelial cells (ECs), and macrophages, allowing for the study of both angiogenesis and inflammatory processes. 

In this study, we examined the potential of the 3D model, incorporating either inflammatory “M1”-like or anti-inflammatory “M2”-like macrophages, to modulate key RA inflammatory processes. We used established anti-TNF therapies and novel small molecule inhibitors of the NF-κB signaling pathways.

Methods: We created spheroids composed of RAFLS, ECs and either M1 or M2 macrophages derived from monocytes. These spheroids were cultured in a collagen-based matrix and exposed to various conditions: pro-angiogenic factors (VEGF/bFGF), the pro-inflammatory cytokine TNFα, or RA synovial fluid (SF). The impact of macrophage phenotype on spheroid outgrowth, macrophage containment, and cytokine production was quantified using confocal imaging and digital image analysis by machine learning, and ELISA for measuring pro-inflammatory soluble factors in culture supernatants. Finally, therapeutic compounds were tested in the 3D model with M1 macrophages in the presence of GF, SF or TNF using the same readouts.

Results: M2 macrophages exhibited greater migration away from the spheroid core compared to M1 macrophages in all conditions. SF significantly enhanced  the containment of M1 macrophages within the spheroid core, a result not observed with the M2 macrophages. The inhibitor of κB Kinase (IKKβi) reduced spheroid outgrowth (~50%) in the model with M1 macrophages under GF stimulation, while the NF-κB-inducing kinase (NIK) inhibitor fully prevented spheroid outgrowth compared to the unstimulated condition. Both TNF inhibitors, etanercept (ETA) and certolizumab pegol (CP), reduced interleukin (IL)-6 production to levels comparable to unstimulated conditions upon GF stimulation. TNF stimulation significantly increased IL-6 production, which was strongly inhibited by IKKβi, ETA and CP. None of the compounds affected macrophage containment within the core compared to SF-only conditions.

Conclusion: Our 3D RA synovial tissue effectively replicates alterations in cellular interactions based on macrophage phenotype and demonstrates the inhibitory effects of therapeutic compounds on spheroid outgrowth and soluble mediator production. This model shows the potential for testing the effect of various therapeutic applications on RA synovial inflammation and angiogenesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/modulating-inflammation-and-angiogenesis-in-an-advanced-3d-rheumatoid-arthritis-synovial-tissue-model/