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Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, fibrosis, and immune dysregulation. In SSc pathogenesis, circulating monocytes can be recruited to the skin and differentiate into macrophages. This possibly contributes to the activation and conversion of resident fibroblasts into myofibroblasts, driving fibrosis and skin thickness. However, how these cells exert their functions and via which fibroinflammatory mediator(s) is not fully understood. Objectives: To investigate the role of monocytes in mediating myofibroblast contraction and activation using an innovative 3D collagen hydrogel model. Methods: For our 3D skin model we cocultured human primary dermal fibroblasts with either peripheral blood mononuclear cells (PBMCs) or MACS-sorted CD14+ monocytes in a 3D collagen type 1 hydrogel. Subsequently, monocyte-driven tissue contraction was measured over time. We performed immunostainings for CD68, fibroblast activation protein (FAP), and alpha-smooth muscle actin (α-SMA) to evaluate monocyte/macrophage and fibroblast activation. To investigate the signaling pathways involved in the observed myofibroblast activation, we measured transcription factor-driven luciferase production using reporter constructs in the same dermal fibroblasts. The activity of the following reporter constructs was determined after 24 hours: Sis-Inducible Element (SIE); SMAD-Binding Element (SBE); Nuclear Factor of Activated T-cells 5 Response Element (NFAT-5), and NFκB Response Element (NFκB). Results: After 60 hours of co-culture, hydrogel plugs containing fibroblasts + monocytes displayed a strong spontaneous contraction, seen by a (approx. 80-90%) decrease in the area of the plugs. Hydrogels containing only fibroblasts did not contract. Hydrogels with fibroblasts + monocytes contracted as fast as fibroblasts + PBMCs, but depletion of CD14+ cells from PBMCs slowed down contraction, showing that monocytes strongly activated fibroblasts. The expression of FAP and α-SMA by fibroblasts increased in monocyte-containing hydrogels, compared to fibroblasts cultured without monocytes. Furthermore, CD68 expression also increased in co-culture, indicating enhanced monocyte differentiation/activation into macrophages by fibroblasts. Evaluating which intracellular pathway lead to fibroblast activation, we observed that SIE and NFκB reporter fibroblast constructs were strongly elevated in the presence of monocytes. However, we could not observed enhanced TGF-β activity using the SBE reporter. Together, our results suggest that inflammatory mediators like IL-6, IL-1 and S100A8/9 may contribute to the monocytes-driving myofibroblast contraction. Conclusion: This study highlights the importance of monocytes and the SIE and NFκB intracellular signaling pathways in myofibroblast contraction in a 3D skin model, contributing to understanding the basic mechanisms of these cells in SSc skin fibrosis and thickness. REFERENCES: 1 van Caam, Arjan et al. "Unraveling SSc Pathophysiology; The Myofibroblast." Frontiers in immunology vol. 9 2452. 13 Nov. 2018, doi:10.3389/fimmu.2018.02452. 2 Al-Adwi, Yehya et al. "Macrophages as determinants and regulators of fibrosis in systemic sclerosis." Rheumatology (Oxford) vol. 62,2 (2023): 535-545. Acknowledgements: Grant #2023/04897-7, São Paulo Research Foundation (FAPESP). Disclosure of Interests: None declared.
Zanin-Silva et al. (Sat,) studied this question.