Fig. 5: Altering fatty acid metabolism by orlistat affects MGC formation.
From: Monocyte progenitors give rise to multinucleated giant cells
a cMoP from Cx3cr1gfp/+ mice were stimulated with BCG (MOI 20), heat-fixed BCG (106/ml), heat-fixed M.tb (50 µg/ml) and treated with orlistat (100 µmol, beige) or vehicle control (ethanol, blue) for 6 days. MGC were quantified by Hemacolor staining (scale bar: 100 µm). Gamma-correction was adjusted to 0.45 in both images. Bars depict mean ± SEM of n = 3 (BCG, BCG hf) and n = 4 (control, M.tb) biologically independent samples. ns = not significant (p = 0.1532), **p = 0.0022, ****p < 0.0001 (two-way ANOVA, Sidak’s MCT). b Hoechst staining of cMoP from Cx3cr1gfp/+ mice stimulated with BCG (MOI 20) and treated with orlistat (100 µmol) or ethanol (control) for 6 days. MGC were quantified as polyploid cells (N > 4) from histogram peaks. Bars show mean ± SEM of n = 3 biologically independent samples. **p < 0.01 (one-way ANOVA, Tukey’s MCT). c Cell density determined by FACS counting particles of cMoP from Cx3cr1gfp/+ mice treated as indicated in Fig. 5b. Bars show mean ± SEM of n = 5 biologically independent samples. d TNFα production of cMoP from Cx3cr1gfp/+ mice treated as indicated in Fig. 5b. LPS (100 ng/ml) was added 5 h prior to analysis. Bars show mean ± SEM of n = 3 biologically independent samples. ****p < 0.0001 (one-way ANOVA, Sidak’s MCT). e Sterol analysis of cMoP (blue) and MC (green) from Cx3cr1gfp/+ mice stimulated with heat-fixed BCG (106/ml, squares) for 6 days compared to M-CSF (50 ng/ml, circles) control (-). Shown are cholesterol, desmosterol, and 7-dehydrocholesterol (7-DHC) content normalized to cell number. Depicted are mean ± SEM of n = 4 biologically independent samples (cMoP control with n = 3 as one sample could not be analyzed for technical reasons). *p = 0.0195, **p = 0.0019 (desmosterol), **p = 0.002 (7-DHC, BCG), **p = 0.0063 (7-DHC, control), ***p = 0.0001, ****p < 0.0001 (two-way ANOVA, Tukey’s MCT). f Sqle, Dhcr24, Hmgcr and Fasn mRNA expression levels of cMoP and MC stimulated with heat-fixed BCG (106/ml, diamonds, hatched bars) for 6 days relative to Gapdh compared to M-CSF (50 ng/ml) controls (circles, gray bars). Bars show mean ± SEM of n = 5 biologically independent samples in two experiments. *p = 0.0328 (Fasn), *p = 0.0155 (Hmgcr, cMoP-control — cMoP-BCG), *p = 0.0313 (Hmgcr, MC-control — cMoP-BCG), **p = 0.0057 (Sqle), **p = 0.0013 (Dhcr24), **p = 0.001 (Hmgcr), **p = 0.0016 (Fasn), ***p = 0.001, ****p < 0.0001 (two-way ANOVA, Tukey’s MCT).
