Factors Released by Polarized Neutrophil-like Cells Modulate Cardiac Fibroblast Phenotype and Limit the Inflammatory Response After Myocardial Infarction

Figure 1. Impact of cell culture conditions on human cardiac fibroblasts (CFs) morphology and phenotype. (A) Morphology of CFs after 48h in conventional 2D culture (a,c) and 3D culture on ARdH hydrogel (b,d), as determined by F-actin labeling with phalloidin (red) and DAPI nuclear staining (cyan). The scale bar indicates 200 µm for (a,b), and 50 µm for (c,d). (B) Cell viability of CFs after 48 h in 2D and 3D culture, as determined by MTT assay; (C) MCP-1 released in conditioned media by CFs cultured in 2D and 3D conditions determined by ELISA assay; (D) mRNA expression of α-SMA, MCP-1, and IL-1β in CFs isolated from 3D-hydrogel or 2D culture after 48 h. (E) α-SMA protein expression in CFs from aortic root-derived hydrogel (ARdH) or 2D culture, determined by Western Blot; n = 3, * p < 0.05. Data is represented as mean ± SEM.

Figure 2. Profile of inflammatory mediators and of matrix metalloproteinases (MMP) following a 24 h indirect co-culture of human CFs grown on ARdH hydrogel with control granulocytes (CfN), or their pro-inflammatory (N1) or anti-inflammatory (CfN2) subtypes. (A) mRNA level of inflammatory cytokines and chemokines in CFs grown on ARdH hydrogel and indirectly interacted with granulocytes subsets, determined by qPCR—normalized to B2M and represented as fold change compared with C (Cf only); (B) Levels of secreted inflammatory cytokines and chemokines in conditioned media from indirect co-cultures as evaluated by ELISA assay; (C) IL-1β protein level in CFs lysates following indirect interaction with neutrophil-like cell subtypes, as determined by Western Blot assay and normalized to β-actin; (D) Metalloproteases MMP-1, -2, -3, -9 and -13 mRNA expression in CFs indirectly co-cultured with neutrophil-like subtypes; (E) Concentration of soluble MMP-1 and MMP-9 quantified in conditioned media as evaluated by ELISA assay; (F,G) Protein expression of MMP-9/-1 in CFs following indirect interaction, as evaluated by Western Blot and normalized to β-actin; (H) The enzymatic activity of gelatinase MMP-9…

Figure 3. The impact of granulocyte subtypes on the expression of molecules associated with fibrosis in human CFs following 24 h indirect co-culture with neutrophil-like cell subsets. (A) mRNA of fibrotic markers in CFs grown on ARdH hydrogel and indirectly interacted with granulocyte subsets, determined by qPCR assay—normalized to β-actin and represented as fold change compared with control Cf (non-interacted); (B) Concentration of soluble TGF-β1 quantified in conditioned media following indirect interaction as evaluated by ELISA assay; n = 3, * p < 0.05, ** p < 0.01. Data is represented as mean ± SEM.

Figure 4. Effect of N1/N2 secretome (SN1/SN2) in modulating the inflammatory phase—day 1 post-MI. (A,B) Immunofluorescence of Ly6G (red) and MPO (red) in cardiac tissue from MI mice with or without treatment with SN1/SN2, 1-day post-MI. Nuclei are counterstained with DAPI (cyan). Scale bar always represents 100 μm. (C) Neutrophil elastase activity detected directly in explanted mouse hearts at 1-day post-MI; (D–F) Levels of IL-6, MCP-1, and TGF-β1, respectively, in plasma from MI mice treated or not with SN1/SN2, as evaluated by ELISA. n ≥ 3. Data is represented as mean ± SD.

Figure 5. Gene expression of pro-inflammatory, matrix remodeling and fibrotic mediators at day 1 post-MI in left ventricle heart tissue. (A) Pro-inflammation markers IL-1β, IL-6 and MCP-1; (B) Metalloproteases MMP-1, -9, and -13, (C) Fibrotic molecules CCN2, CCN5, and α-SMA, quantified in LV tissue of SHAM, MI mice with or without SN1/SN2, as well as in the corresponding remote area from each MI mouse. Gene expression data were generated by qPCR, normalized to RPL41 housekeeping gene, and expressed relative to control—untreated mice (dotted line). n = 5/6 mice per group, * MI vs. MI with SN1/SN2, # MI tissue vs. corresponding remote tissue from each MI mouse. #, * p < 0.05, ##, ** p < 0.01, ###, *** p < 0.001. Data is represented as mean ± SD.

Figure 6. Gene expression of pro-inflammatory, matrix-degrading, fibrotic and ECM remodeling mediators at day 7 post-MI in LV heart tissue. (A) Pro-inflammatory molecules IL-1β, IL-6 and MCP-1; (B) Fibrotic molecules α-SMA, CCN2, CCN5, and FAP; (C) Metalloproteases MMP-1, -9, and -13; and (D) Extracellular matrix molecules proteins COL I, COL III, Fibronectin and Periostin, quantified in LV tissue of SHAM, mice with MI with or without SN1/SN2, as well as in the corresponding remote area from each MI mouse. Gene expression data were generated by qPCR and normalized to healthy control mice. n = 5/6 mice per group, * MI vs. MI with SN1/SN2, # MI tissue vs. corresponding remote tissue from each MI mouse. #, * p < 0.05, ##, ** p < 0.01, ###, *** p < 0.001. Data is represented as mean ± SD.

Figure 7. Effect of N1/N2 secretome (SN1/SN2) in the reparative phase—7 days post-MI. (A) Immunofluorescence staining of α-SMA (red) and MPO (red) in mouse cardiac tissue from MI mice with or without treatment with SN1/SN2. Nuclei are counterstained with DAPI (cyan). Scale bar always represents 100 μm; Signal intensity was quantified within the infarcted zone, selected from slide scans acquired at 20× magnification, and expressed as mean fluorescence intensity (MFI) per infarct area (a1,a2). (B–D) Levels of IL-6, MCP-1 and TGF-β1, respectively, in plasma from mice with MI, treated or not with SN1/SN2, as evaluated by ELISA assay. n ≥ 3, * p < 0.05, ** p < 0.01. Data is represented as mean ± SD.