Monocytes Strongly Induce (MYO)Fibroblast Contraction in a New 3D Skin Model to Understand the Inflammation-Fibrosis Axis in Systemic Sclerosis

ABSTRACT 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 and activation, 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 (myo)fibroblasts activation/contraction in collagen type I hydrogels. We identified that co-culture of fibroblasts with monocytes displayed strong spontaneous hydrogel contraction, coupled with an upregulation of myofibroblasts activation-associated markers, such as α-SMA and fibroblast activation protein. Using transcription-factor reporter constructs and small molecules inhibitors, we demonstrated that monocyte-fibroblast communication was mediated by JAK/STAT3 and TGF-β/Smad2/3 signaling pathways. Flow cytometry analyses revealed that monocytes, after interacting with 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 myofibroblast 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.