Control of myofibroblast phenotype by paracrine TGF-beta and IL-10 signals in a biomimetic wound healing model

摘要

In dermal wound healing the interplay of many cell types, including fibroblasts and macrophages, regulates tissue regeneration. Various exogenous signals, including extracellular matrix ligands as well as soluble cytokines from neighboring cells, like TGFβ1 and IL10, affect cell behavior. To better dissect the convoluted processes, but still preserving a nearly physiological situation, biomimetic in vitro models are needed to decipher specific signals involved in wound healing. A specific question concerns the regulation of myofibroblast (MFb) phenotype which are involved in increased matrix deposition and tissue contraction, while TGFβ1 is suggested to activate and IL10 to suppress MFb differentiation. We established biophysically and biochemically well-defined 3D matrices based on collagen Ⅰ. The 3D collagen networks were functionalized with human plasma fibronectin (FN) to mimic the in vivo peri-wound. Stimulation with TGFβ1 and IL10 in a combinatorial manner was used to simulate autocrine and paracrine signals. Proliferation and apoptosis analysis, immunofluorescence as well as gene expression and migration analysis were used to evaluate differentiation and de-differentiation of primary human dermal fibroblasts (dFb) into MFb in dependence on the exogenous signals. Furthermore, localized delivery of TGFβ1 or IL10 by microparticles was applied to mimic short-range paracrine gradients of the involved cytokines and to decipher single cell behavior in heterogeneous cell populations. The results indicated distinct dependencies of MFb phenotype on the various stimuli. Migratory capacity of dFb increased in the presence of FN, whereas proliferation decreased. TGFβ1 stimulation induced a MFb phenotype characterized by a higher proliferation, lower migration, expression of MFb markers like α-smooth muscle actin (aSMA), collagen Ⅰ α1 (Coll Ⅰ) and FN with extra-domain A (EDA-FN), as well as immunostaining of aSMA within stress fibers and SMAD2/3 inside the nucleus. Removal of TGFβ1 after 2-4 days leads to a reduction of MFb cell number, suggesting MFb apoptosis. IL10 alone significantly reduced aSMA and matrix production (Coll Ⅰ and EDA-FN), whereas cell number was maintained. In cultures pre-stimulated with TGFβ1, IL10 treatment reversed cell phenotype back to dFb. Simultaneous stimulation with TGFβ1 and IL10 led to a down-regulated proliferation and matrix production, but left aSMA and migration on MFb levels. The short-range paracrine TGFβ1 stimulation revealed the signals to act only over small distances up to 100 μm. In sum our model mimics important phases of wound healing including early fibroblast invasion, differentiation and matrix remodeling as well as later phases of down-regulated matrix remodeling. The results suggest IL10 as an important signal to terminate early wound healing by down-regulation of matrix production and reversing MFb phenotypes, with implications on biomedical applications like reducing scarring or fighting fibrosis.