Beneficial effects of IL-4 and IL-6 on rat neonatal target cardiac cells

Figures

• 

  Figure 1. Total RNA sequencing of resected neonatal hearts. (A) Experimental design scheme for total RNA sequencing. Cardiac apex resection or false surgery was performed in 1-day-old and 7-day-old rats, and samples were collected one (D1) or five (D5) days after the procedure. We created this figure in the Mind the Graph platform (www.mindthegraph.com). (B) Only the apex portion (1/3 of the heart) was considered for the experiment; in the PCA diagram, samples were clustered considering three main parameters: age at time of procedure, procedure and time post-procedure. The more prevalent responses were age at time of procedure (x axis, corresponds to 42% of the variance) followed by time post-procedure (y axis, corresponds to 19% of the variance). (C) Biological processes and pathways related to the inflammatory response and cellular movement in comparison to their respective sham-operated groups at different time points (R1D1/S1D1; R1D5/S1D5; R7D1/S7D1 and R7D5/S7D5) through ingenuity enrichment analysis. Red and blue bars show activation and inactivation of pathways, respectively, showing that the overall inflammatory responses are contrasting between neonatal development times.

• 

  Figure 2. Macrophage infiltration after apex resection. (A) Macrophage infiltration evaluated (mean ± SD) by CD68-positive cell staining in the apex areas of R1D1 (n = 7), R1D5 (n = 7), R7D1 (n = 7) and R7D5 (n = 6). (B) Bars show total CD68-positive cells normalized by heart tissue area, considering that non-resected rats do not display macrophage infiltration in the apex area and that CD68 infiltration increases with time. Values are relative to P1D1. ns = non-significant; *p-value ≤ 0.05 and ***p-value ≤ 0.001. (C) Adult bone marrow-derived macrophages cultivated for 7 days were stained for CD68 and then polarized towards M1 and M2 profiles. (D) The gene expression pattern of polarized macrophages was analysed by real-time PCR. M1 polarized macrophages were confirmed for IL-1b, TNF-a and NOS2 gene expression (black bars), and M2 polarized macrophages were confirmed for Arginase 1, CCL2 and TGF-b (grey bars). Scale bars = 50 µm e 100 µm.

• 

  Figure 3. M2-conditioned media increase cardiomyocyte proliferation and reduce α-SMA gene and protein expression in TGF-β-stimulated fibroblasts. (A) Representative images of HCS of cardiomyocytes under normoxia treated with M1 and M2 compared to control culture medium. Staining for tropomyosin (green), Ki67 (red) and nuclei with DAPI (blue). Phenotypic parameters were retrieved for each single-cell identified and only cardiomyocytes were included in the analysis. Scale bars = 100 µm. (B) Percentage of Ki67-positive cardiomyocytes by HCS (A) (N = 8–9). *** p-value ≤ 0.001 ****p-value ≤ 0.0001. (C) Percentage of Ki67-positive cardiomyocyte-negative cells by HCS. Only cardiomyocytes-negative cells were included in the analysis (A). (D) Representative images of HCS of cardiomyocytes under hypoxia treated with M1 and M2 compared to control culture medium. Scale bars = 100 µm. (E) Percentage of Ki67-positive cardiomyocytes by HCS (D) (N = 29). **p-value ≤ 0.01. (F) Representative images of HCS of TGF-β1-stimulated fibroblasts treated with M1- and M2-conditioned media after 24 h of treatment. Staining for α-SMA (green) and DAPI (blue). Scale bars = 100 µm. (G) Mean expression of α- SMA per…

• 

  Figure 4. Protein and gene expression of M2 macrophages. (A) Cytokine array panel randomization of M0-, M1- and M2-conditioned media expressed cytokines. (B) Cytokines expressed from both M2 and M1, different from M0 and exclusively expressed M2 candidate cytokines, such as IL-4, IL-6, IL-1β and fractalkine. (C) Real-time PCR data analysis of the IL-4 gene confirmed that M2 macrophages express IL-4 after 48 h of polarization. **p-value ≤ 0.01. The membranes were generated and compared as pairs: (D) macrophage control means vs M2 and (E) M1 vs M2. The M1- and M2-conditioned media were obtained from the combination of independent polarization experiments (n = 7).

• 

  Figure 5. Candidate cytokines IL-4 and IL-6 recapitulate part of the pro-repair/regeneration responses. (A) Representative images of HCS of cardiomyocytes treated with M2, IL-4, IL-6 and IL-4/IL-6 compared to control culture medium. Scale bars = 100 µm. (B) Percentage of Ki67-positive cardiomyocytes by HCS (A). **p-value ≤ 0.01 and ****p-value ≤ 0.0001. (N = 3). (C) DNA content was measured by flow cytometry by tracing the fluorescence intensity of HUVECs treated with M2, IL-4, IL-6 and IL-4/IL-6. (D) The graphical representation of (C). **p-value ≤ 0.01. (E) Images show the tube formation of HUVECs treated with Crtl culture medium, M2, IL-4, IL-6 and IL-4/IL-6. (F) Quantification of the tubes formed of HUVECs treated (E). **p-value ≤ 0.01. (G) Real-time PCR data analysis of a-SMA, collagen I and collagen III in TGF-β1-stimulated fibroblasts treated with M2, IL-4, IL-6 and IL-4/IL-6 compared to control culture medium. (N = 3–6). **p-value ≤ 0.01. (H) Representative images of high-content screening of TGF-β1-stimulated fibroblasts treated with M2, IL-4, IL-6 and IL-4/IL-6 compared to control culture medium. Scale bars = 100 µm. (I) Quantification of α-SMA fluorescence intensity in the…