Monocytes strongly induce (myo)fibroblast contraction in a 3D skin model to understand inflammation-fibrosis crosstalk

Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive fibrosis and tissue stiffness, in which monocytes and macrophages are increasingly recognized as key contributors to pro-fibrotic myofibroblast formation, although the underlying mechanisms remain incompletely understood. Here, we used a three-dimensional (3D) skin model to study how CD14 + monocytes, M1 and M2-like macrophages induce fibroblasts activation and contraction in collagen type I hydrogels. We identified that the co-culture of dermal (myo)fibroblasts with monocytes displayed strong spontaneous hydrogel contraction, coupled with an upregulation of myofibroblast activation-associated markers, such as alpha-smooth muscle actin (α-SMA). Using transcription factor-responsive reporter constructs and small-molecule inhibitors, we demonstrated that monocytes-(myo)fibroblasts communication was mediated by JAK/STAT3 and TGF-β/Smad2/3 signaling pathways. Flow cytometry analyses revealed that monocytes, after interacting with (myo)fibroblasts, differentiated into a mixed M1/M2 polarization phenotype, characterized by CD163, CD206, CD86, and HLA-DR expression. Both M1 and M2-like macrophages promoted significant (myo)fibroblast contraction, which could be mimicked by supernatant transfer. TGF-β neutralization but not IL-6 blocking abolished this effect. This study demonstrates that monocytes/macrophages can strongly induce (myo)fibroblasts activation/contraction. Together, our work contributes to elucidating pathways and mechanisms associated with skin fibrosis in SSc and paves the way for developing new platforms for targeted therapy testing.