Distinct Redox Signalling following Macrophage Activation Influences Profibrotic Activity

Figure 5

PDBu-stimulated hydrogen peroxide generation in activated human macrophages. PDBu-differentiated THP-1 macrophages were left untreated (MΦ) or activated with IFN-γ+LPS or IL-4 for 72 hours. (a) Average trace depicting the accumulation of PDBu (10 μM)-stimulated hydrogen peroxide in the culture media over 90 min detected via Amplex Red fluorescence. (b) Hydrogen peroxide concentration was calculated at 90 minutes and expressed as . (c) M(IFN-γ+LPS) and M(IL-4) hydrogen peroxide concentration expressed as a fold change relative to the untreated macrophage (MΦ) control, . (d) Representative histogram and gating strategy for DCF⁺ macrophages, depicting M1 versus M2 polarisation and the effect of catalase-polyethylene glycol (PEG-Cat; 1000 U/ml) on the M2 signal. (e) Mean DCF⁺ macrophages, normalised to the response in untreated macrophages (MΦ), . (f) SOD1, (g) SOD2, and (h) SOD3 mRNA expression in activated THP-1 macrophages at 24, 48, and 72 hours, determined by RT-qPCR and expressed relative to the average MΦ (untreated) value, . (i) SOD2 and (j) SOD3 protein expression in activated THP-1 macrophages at 72 hours, determined by western blotting, . Representative blots, depicting for , shown on RHS, with GAPDH used as a loading control. All results expressed as , , (1-way ANOVA followed by Dunnett’s post hoc test (g, h, i) or Student’s unpaired -test (c, e)).