Fig. 3: cMoP-derived MΦ keep their potential to form MGC and show a characteristic metabolic profile.

a Dot plot of cMoP analyzed immediately after isolation for initial surface marker expression. Surface marker expression of cMoP and MC from Cx3cr1^gfp/+ mice analyzed by FACS related to CD45⁺ cells after 4, 8, 12 or 48 hours in culture with M-CSF. Depicted are mean ± SEM of n = 3–4 (CD117), n = 5–9 (CD11b), n = 3–5 (F4/80) biologically independent samples. Detailed sample numbers (n) are presented in Supplementary Table 4. ns = not significant, *p = 0.0161, ***p = 0.0009 (CD117), ***p = 0.0005 (CD11b), ****p < 0.0001 (two-way ANOVA, Sidak’s MCT). b Representative FACS dot plots for F4/80 and CD11b expression of cMoP and MC after 48 h in medium supplemented with M-CSF. c cMoP or MC from Cx3cr1^gfp/+ mice were stimulated either directly with LM (4 μg/ml) for 4 days or after 2 days in culture with M-CSF (cond-cMoP, cond-MC). MGC formation was quantified based on Hemacolor stainings (scale bar: 100 µm). Bars show mean ± SEM of n = 4 (cMoP) and n = 3 (MC) independent biological samples. ns = not significant (cMoP — cond-cMoP: p = 0.8162; MC — cond-MC: p = 0.9942), *p = 0.0256 (cMoP — MC), *p = 0.0109 (cond-cMoP — cond-MC), (one-way ANOVA, Tukey’s MCT). d Heatmaps of gene expression in cMoP and MC from Cx3cr1^gfp/+ mice after LM stimulation (4 µg/ml, 48 hours) with M-CSF (50 ng/ml). M-CSF alone served as control (-). Depicted are standardized log2 differences of significantly up- (red) or downregulated (blue) genes in cMoP-LM compared to MC-LM or cMoP (-) compared to MC (-). The analysis was performed with two biologically independent samples per group (1 = sample 1; 2 = sample 2) by gene-ontology terms (GO-Terms), (GO:0006631 fatty acid metabolic process, GO:0008203 cholesterol metabolic process, GO:0001516 prostaglandin biosynthetic process, GO:0006694 steroid biosynthetic process, GO:0006869 lipid transport). If GO-Terms overlapped, respective genes were only assigned to one group. e Dhcr24 and Fasn mRNA expression levels relative to Gapdh in cMoP and MC, stimulated with LM (12 µg/ml, light gray, diamonds) for 48 h compared to M-CSF control (dark gray, triangles) determined by qRT-PCR. Bars show mean ± SEM of n = 3 biologically independent samples. **p = 0.0015 (Dhcr24), **p = 0.0091 (Fasn), ***p = 0.0005 (two-way ANOVA, Sidak’s MCT). f Panther pathway analysis of gene expression data described in Fig. 3d. Depicted are significantly up- (red) or downregulated (blue) pathways in cMoP-LM versus MC-LM.