Monocytes and neutrophils promote cardiac fibroblast pro-fibrotic phenotypes through IL-6 and MIF

Figures

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  FIGURE 1. Acute exposure to both THP-1 and HL-60 cells induces increases HCF collagen I and fibronectin deposition in cell-derived matrices. (A) Example single channel images of collagen I, fibronectin and α-SMA (all shown in white), and merged nuclei (blue; DAPI), α-SMA (green; Alexa-488) and F-actin (magenta; Alexa-568) of HCF in CDM after 12 days culture. (B) Representative merged images of nuclei (blue; DAPI), α-SMA (green; Alexa-488) and F-actin (magenta; Alexa-568) of HCF in CDM after 12 days, cultured for first 2 days with noted immune cells. α-SMA channel is shown below each in white. Scale bars represent 50 μm. (C) Quantification of mean intensity from images as in (B). Data shown is from one experiment, representative of three independent experiments (5 fields of view per coverslip, per condition). (D) Representative images of collagen I and fibronectin in CDM with or without immune cell (THP-1 or HL-60) co-culture for the first 2 days; quantification of local mean intensity for (E) collagen I and (F) fibronectin. Data shown is from one experiment, representative of three independent experiments. n = 160 regions divided from 10 fields of view from one coverslip analyzed per…

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  FIGURE 2. Direct and indirect co-culture with monocytes or neutrophils enhances HCF collagen gel contraction. Representative images of collagen gels over 2 days containing HCF alone or (A) co-cultured with immune cell lines THP-1 and HL-60, (C) co-cultured with human primary monocytes and neutrophils and (E) cultured with conditioned media (CM) from HCF-THP-1 or HCF-HL-60 cell co-cultures. (B, D, F) Quantification of collagen gel surface area over time for (A, C, E) respectively. Data shown is from one experiment (n = 3 gels per condition), representative of three independent experiments; statistical analysis performed by two-way ANOVA, followed by Bonferroni’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 3. Co-culture with immune cells induces HCF IL-6 and MIF release. (A) Quantification of cytokines detected from cytokine array. Statistical analysis performed by one-way ANOVA, followed by Dunnett’s multiple comparisons test. n = 4 experiments shown. *p < 0.05, **p < 0.01, ***p < 0.001. (B–D) Quantification of ELISAs for MIF, IL-6 and TGF-β1 respectively in conditioned media from HCF with or without immune cell co-cultures for 48 h. Statistical analysis performed by one-way ANOVA, followed by Dunnett’s multiple comparisons test. n = 3 independent experiments shown for B-D. *p < 0.05, ***p < 0.001.

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  FIGURE 4. IL-6 and MIF release promote HCF-induced collagen gel contraction. (A) Representative images of collagen gels containing HCF treated with recombinant IL-6 (100 ng/mL) or MIF (20 ng/mL) over 2 days (B) Quantification of collagen gel surface area from images as in (A). (C,E) Representative images of collagen gels containing HCF co-cultured with immune cells treated with IgG control antibody or (C) anti-IL6 or (E) anti-MIF at day 2. (D,F) Quantification of collagen gel surface area from images as in (C,E) respectively. Data shown in B, D and F is from one experiment (4 samples per condition), representative of three independent experiments. Statistical analysis performed by (B) one-way ANOVA, followed by Dunnett’s multiple comparisons test and (D,F), two-way ANOVA, followed by Bonferroni’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 5. IL-6 promotes THP-1-induced HCF migration in 3D collagen gels. (A) Representative images of stills from confocal live cell time lapse movies of HCF alone (green, Alexa-488) or co-cultured with THP-1 (magenta, cyan arrows) or HL-60 (magenta, yellow arrows) cells embedded in 3D collagen gels. Cultures were treated with IgG or IL-6/MIF neutralizing antibodies (0.5 μg/mL) from start of experiment. Scale bar represents 50 μm. (B,C) Quantification of velocity over imaging period with or without IL-6 or MIF neutralizing antibody (B) HCF alone and in presence of immune cells and of (C) HL-60 alone and in presence of HCF. Data shown from one experiment (n = 7–13 movies tracked per condition with >3 cells per field of view), representative of three independent experiments; statistical analysis performed by two-way ANOVA, followed by Tukey’s multiple comparisons test. *p < 0.05, ***p < 0.001.