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

Abstract

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, 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.