Abstract
Cardiac fibroblasts play an important role in the regulation of the extracellular matrix and are newly recognized as inflammatory supporter cells. Interferon (IFN)-γ is known to counteract transforming growth factor (TGF)-ß1-induced myofibroblast differentiation. This study aims at investigating in vitro how IFN-γ affects TGF-ß1-induced monocyte attraction. Therefore, C4 fibroblasts and fibroblasts obtained by outgrowth culture from the left ventricle (LV) of male C57BL6/j mice were stimulated with TGF-β1, IFN-γ and TGF-β1 + IFN-γ. Confirming previous studies, IFN-γ decreased the TGF-ß1-induced myofibroblast differentiation, as obviated by lower collagen I, III, α-smooth muscle actin (α-SMA), lysyl oxidase (Lox)-1 and lysyl oxidase-like (LoxL)-2 levels in TGF-β1 + IFN-γ- versus TGF-ß1-stimulated cardiac fibroblasts. TGF-β1 + IFN-γ-stimulated C4 and cardiac fibroblasts displayed a higher CC-chemokine ligand (CCL) 2, CCL7 and chemokine C-X3-C motif ligand (Cx3CL1) release versus sole TGF-ß1-stimulated fibroblasts. Analysis of migrated monocyte subsets towards the different conditioned media further revealed that sole TGF-β1- and IFN-γ-conditioned media particularly attracted Ly6Clow and Ly6Chigh monocytes, respectively, as compared to control media. In line with theses findings, TGF-β1 + IFN-γ-conditioned media led to a lower Ly6Clow/Ly6Chigh monocyte migration ratio compared to sole TGF-ß1 treatment. These differences in monocyte migration reflect the complex interplay of pro-inflammatory cytokines and pro-fibrotic factors in cardiac remodelling and inflammation.
Highlights
Cardiac fibroblasts are centrally involved in myocardial remodelling[1]
Confirming previous findings[17, 24] h stimulation of cardiac fibroblasts with transforming growth factor (TGF)-β1 led to a 6.2-fold (p < 0.0001), 4.1-fold (p < 0.0001), 3.9-fold (p < 0.0001) and 6.1-fold (p < 0.0001) higher collagen 1a1, collagen 3a1, TGF-ß1, and α-smooth muscle actin (α-SMA) mRNA expression as compared respectively to control cardiac fibroblasts, which is indicative for myofibroblast differentiation (Fig. 1A,B,E,F)
Further evaluation of the collagen crosslinking enzymes lysyl oxidase (Lox)-1 and lysyl oxidase-like (LoxL)-2 demonstrated that TGF-ß1 induced Lox-1 and LoxL-2 gene expression versus control cardiac fibroblasts (Lox-1: 4.8-fold, p < 0.0001; LoxL-2: 6.1-fold, p < 0.0001) 24 h post stimulation (Fig. 1H,I)
Summary
Cardiac fibroblasts are centrally involved in myocardial remodelling[1] While their main role is traditionally believed to be the regulation of the extracellular matrix proteins, their function as inflammatory supporter cells has attracted recognition[2,3]. We demonstrated that increased levels of IFN-γ and an excessive Th1 reaction after myocardial infarction leads to decreased myofibroblast differentiation with detrimental effects in scar formation and myocardial remodelling[11]. This supports the general consensus that an extended or prolonged inflammation results in inadequate myocardial wound healing and scar formation associated with a suppression of systolic function and chamber dilation[12]. Based on the finding that IFN-γ counteracts TGF-ß1-induced cardiac fibrosis[11] on the one hand, and the relevance of monocyte subpopulations during inflammation and wound healing on the other hand[15], we sought to examine in vitro how IFN-γ can influence the chemokine profile of cardiac (myo)fibroblasts and subsequent attraction of pro-inflammatory versus anti-inflammatory monocytes
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