Cardiac (myo)fibroblasts modulate the migration of monocyte subsets

Figures

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  Figure 1. IFN-γ reduces TGF-ß1-induced transdifferentiation of cardiac fibroblasts. Data show the gene expression of the extracellular matrix proteins Col1a1 (A) and Col3a1 (B) and myofibroblast markers TGF-ß1 (E) and α-SMA (F) after 6 and 24 h incubation with or without TGF-ß1 and/or IFN-γ. Additionally, representative western blots (C,D) and quantification of Collagen I in cell lysate (C) or supernatant (D) after 72 h stimulation are depicted. (G) α-SMA marked by a Cy3-conjugated streptavidin antibody (red) and DAPI for nuclear staining (blue). Recording of the images was done in a 100× magnification (scale bar = 100 μm). To characterize collagen crosslinking, the gene expression of the crosslinking enzymes (H) Lox-1 and (I) LoxL-2 were measured. Bar graphs represent the mean ± SEM of 3 independent experiments after normalization to the corresponding control fibroblasts. Statistical analysis was performed by One way-ANOVA or Kruskal Wallis test (#p < 0.05 vs co, *p < 0.05 vs TGF-β1, §p < 0.05 vs IFN-γ).

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  Figure 2. IFN-γ modulates TGF-ß1-induced cytokine and adhesion molecule expression of cardiac fibroblasts. To determine the inflammatory phenotype after stimulation with TGF-ß1, IFN-γ, or both, the mRNA expression of (A) TNF-α, (B) IL-6, (C) ICAM-1, and (D) VCAM-1 was investigated. Bar graphs represent the mean ± SEM of 3 independent experiments after normalization to the corresponding control fibroblasts. Statistical analysis was performed by One way-ANOVA or Kruskal Wallis test (#p < 0.05 vs co, *p < 0.05 vs TGF-β1, §p < 0.05 vs IFN-γ).

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  Figure 3. IFN-γ- and TGF-ß1-induced chemokine secretion and -modulated CCL2/Cx3CL1 and CCL2/Cx3CL1 protein ratio. (A–C) Protein levels of CCL2 (pg/ml), CCL7 (pg/ml), and Cx3CL1 (ng/ml) and (D,E) corresponding CCL2/Cx3CL1 and CCL2/Cx3CL ratio’s of control, TGF-ß1-, IFN-γ-, or TGF-β1 + IFN-γ-stimulated C4 fibroblasts after 72 h incubation time. In agreement, changes in secreted (F) CCL2 (pg/ml), (G) CCL7 (pg/ml) and (H) Cx3CL1 (ng/ml) and (I,J) CCL2/Cx3CL1 and CCL2/Cx3CL ratio’s from control, TGF-ß1-, IFN-γ-, or TGF-β1 + IFN-γ-stimulated cardiac fibroblasts were observed. Data are depicted as mean ± SEM from 3 independent experiments. Statistical analysis was performed by One way-ANOVA or Kruskal Wallis test (#p < 0.05 vs co, *p < 0.05 vs TGF-β1, §p < 0.05 vs IFN-γ).

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  Figure 4. IFN-γ reduces TGF-ß1-induced expression and activity of MMP-2 in cardiac fibroblasts. Gene expression of CCL7 (A) and MMP-2 (B) after 6 and 24 h incubation time. To investigate the MMP-2 activity, zymography (C) of the cell culture supernatant was performed followed by quantification of proMMP-2 (D) and MMP-2 (E). Data are depicted as mean ± SEM from 3 independent experiments. Statistical analysis was performed by One way-ANOVA or Kruskal Wallis test (#p < 0.05 vs co, *p < 0.05 vs TGF-β1, §p < 0.05 vs IFN-γ).

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  Figure 5. IFN-γ- and TGF-ß1-induced monocyte migration. (A) Flow cytometry analysis of CD11b+CD115+Ly6C+ stained cells shows differences between the ratios of Ly6Clow/Ly6Chigh monocytes versus the different conditioned media in C4 fibroblasts. (B) Indication for enhanced migration of Ly6Clow monocytes towards TGF-ß1-conditioned media of cardiac fibroblasts, whereas co-stimulation with TGF-β1 + IFN-γ increases the number of migrated Ly6Chigh monocytes. (C) Gating strategy to define the amount of CD11b+CD115+Ly6C+ cells. (D) Representative dot blots for the migrated monocytes of the different conditioned media of stimulated C4 fibroblasts (n = 6/condition). (E) Representative dot blots for the migrated monocytes of the different conditioned media of stimulated cardiac fibroblasts (n = 3/condition). Data are shown as ratio of Ly6Clow/Ly6Chigh monocytes of 3 independent experiments. Statistical analysis was performed by One way-ANOVA or Kruskal Wallis test (#p < 0.05 vs co, *p < 0.05 vs TGF-β1, §p < 0.05 vs IFN-γ).

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  Figure 6. Hypothetical scheme representing how TGF-ß1 and IFN-γ affect the chemokine expression and subsequent monocyte migration of cardiac fibroblasts. The Figure illustrates the transdifferentiation process of fibroblasts towards myofibroblasts under TGF-ß1 and TGF-ß1 + IFN-γ conditions. Depending on the stimulus, the specific chemokine profile (CCL2, CCL7, and Cx3CL1) of the myofibroblasts results in a more pronounced attraction of Ly6Clow monocytes (TGF-ß1 stimulation) or of Ly6Chigh monocytes (TGF-ß1 + IFN-γ stimulation). The Figure uses elements previously published by Van Linthout et al.2 Stands for Ly6C.