Neutrophils Modulate Fibroblast Function and Promote Healing and Scar Formation after Murine Myocardial Infarction †

Figure 1. Validation of neutrophil and mononuclear fractions after isolation. The neutrophil fraction was positive for Ly-6G, but not for CD115 or F4/80 (A). Their phenotype did not change but was slightly shifted after 4 days in culture (B). The viability of the cells was not affected during culturing (C). Mononuclear fraction was positive for CD115 and showed differentiation potential towards macrophage after 4 days in culture (D).

Figure 2. The effect of neutrophil-fraction on extracellular matrix protein synthesis. (A) Fibronectin mRNA expression and (B) collagen mRNA expression in isolated fibroblasts cultured in normoxia (N), hypoxia (H) and co-cultured with neutrophil (Ne) fraction (H+Ne) and mononuclear (Mo) fraction (H+Mo), respectively (n = 9, * p < 0.05, **** p < 0.0001). (C) Characteristic matrix-assisted laser desorption/ionization (MALDI) mass fingerprint-spectrum of tryptic peptides of fibronectin (n = 3). (D) Characteristic MALDI mass fingerprint-spectrum of tryptic peptides of collagen I detected in isolated fibroblasts co-incubated with mononuclear fraction (n = 3). Protein scores from the Mascot database are shown for each identification in right insets (arrow indicates the identified protein). Neutrophils (H+Ne) increased the gene expression of (E) IL-1ß (n = 9), while mononuclear cells (H+Mo) increased the gene expression of (F) PPAR γ (n = 9). (* p < 0.05; ** p < 0.01; **** p < 0.0001).

Figure 3. Validation of neutrophil-depletion treatment. Mice undergoing neutrophil depletion showed a significant reduction in blood neutrophils (A) and some inflammatory monocytes (B). However, while neutrophil infiltration was significantly reduced one day after myocardial infarction (MI) (C) (scale bar 50 µm), the macrophages were not affected by the treatment at later time points after MI (D) (scale bar 100 µm).

Figure 4. The effect of neutrophils on TGF-β1 dynamics during MI. (A) TGF-β1 mRNA expression in fibroblasts (–TGF-β1) and myofibroblasts (+TGF-β1) co-incubated under hypoxic conditions without/with neutrophil and mononuclear fractions, respectively (n = 4–8, ** p < 0.01). (B) Characteristic MALDI mass fingerprint-spectrum of tryptic peptides of TGF-β1 in fibroblast lysates after co-incubation with neutrophil fraction. Protein score from MASCOT database is shown in the right inset. (C) Time-dependent myocardial mRNA expression of TGF-β1 after MI (n = 5−6, ** p < 0.01). (D) TGF-β1 staining in myocardium by immunofluorescence (green) at different MI set points in control and in neutrophil-depleted mice (n = 5−6). Double immunofluorescence of TGF-β1 (green), smooth alpha actin (red) and overlay (yellow) at different MI set points is shown in insets (scale bar 50 µm). (E) Time-dependent myocardial mRNA expression of IL-6 after MI (n = 6, ** p < 0.01, n.d. not detected) in mice without (black columns) and with (white columns) neutrophil depletion. (F) Representative double immunofluorescence of IL-6 (green) at different MI set points is shown in insets (scale bar 50 µm).

Figure 5. Sketch of healing after MI based on neutrophil-modulated fibroblast function. MI is followed by complex cellular and molecular events, in which fibroblasts proliferate, producing an initial provisional matrix (green fibers). In the inflammatory phase (red panel), the recruited neutrophils assist fibroblasts with provisional fibrin/fibronectin matrix formation (green fibers). TGF-β1 (green capsules) expressed from monocytes and neutrophil-stimulated fibroblasts leads to fibroblast differentiation, collagen synthesis (blue fibers) and macrophage polarization, respectively. Reaching an elevated expression, a self-regulatory negative feedback is activated, reducing TGF-β1 expression, allowing the healing phase to complete the mature scar.