Background/Purpose:
During the last years, there was a shift in systemic sclerosis (SSc)-related causes of death, indicating cardiac
involvement and inflammatory dilated cardiomyopathy (iDCM)
as one of the major cause of death in these patients. Insight from iDCM models revealed that bone marrow derived cells serve
as a main source of pathological myofibroblasts.
Nevertheless, despite the high unmet clinical need, so far little is known
about the etiology of iDCM
and the mechanisms leading to heart dysfunction in SSc
patients. We hypothesized that the inflammatory myeloid cell compartment might
be involved in post-inflammatory cellular remodelling in SSc
patients.

Methods: CD14⁺ monocytes, isolated from peripheral
blood of SSc patients and healthy subjects, were differentiated
towards a myofibroblast phenotype by 7-day
stimulation with TGF-β1/IL-1β/IL-4/IL-10/IL-13 with or without inhibitors
of TGF-β receptor I, Smad2/3 or canonical Wnt signalling. We established a co-culture system
allowing co-culture of monocytes with human fibroblasts or activated human T
cells in order to analyse the role of direct and indirect
cell-to-cell-interaction on differentiation potentials. Moreover, an endomyocardial biopsy from a SSc patient, who underwent heart transplantation, was
screened with immunohistochemistry.

Results:
CD14⁺ monocytes up-regulated myofibroblast
markers (αSMA, fibronectin, collagen I), the transcription
factor Fra-2 and different Wnts in cytokine-enriched
monoculture. In direct co-cultures: a) with fibroblasts (in the presence of TGF-β1,
n=7) or b) with T cells stimulated with IL-2/IL-7/IL-15/IL-21 (activation of common
γ-chain receptor, n=3), or c) with CD3/CD28 activated T-cells (activation
of T cell receptor, n=2) monocytes differentiated towards a myofibroblast
phenotype. Interestingly, activation of the common γ-chain receptor in T
cells up-regulated mostly αSMA gene expression, while activation of T cell
receptor up-regulated αSMA, fibronectin and collagen
I gene expression in the fibroblasts. Inhibition of TGF-β receptor I or the
canonical Smad2/3-dependent pathway, and inactivation of extracellular Wnt or TCF/β-catenin-mediated transcription
significantly abrogated the myofibroblast
differentiation of CD14⁺ monocytes. Moreover, human myocardium of a SSc patient with iDCM revealed the presence of CD14⁺/Fra-2⁺
monocyte-derived fibroblast-like cells in the fibrotic myocardium.

Conclusion:
We showed here that TGF-β/Wnt/Fra-2 signalling
axis mediates the differentiation of circulating monocytes towards a myofibroblast phenotype. Therefore, these cells might be
considered as a potential cellular source for pathological myofibroblasts
in SSc.