Figure 6: FNDC4 treatment affects key macrophage processes and downregulates proinflammatory gene expression.

(a) Phagocytosis of fluorescently labelled zymosan A particles. Bone marrow macrophages were treated for 24 h with 100 nM hFc-FNDC4 or hFc control followed by incubation with zymosan A particles for 30 minutes. Mean±s.e.m., N=4. *P<0.05 (T-test). This experiment was performed twice with similar results. (b) Survival of bone marrow-derived macrophages after 48 h in starvation media, treated with 100 nM hFc-FNDC4 or hFc control. Mean±s.e.m., N=4. *P<0.05 (T-test). This effect was validated in two individual repeat experiments. (c) Gene expression of a selection of cytokines and chemokines on hFc-FNDC4 treatment (or hFc as carrier-control) (100 nM). N=6–7 (d) Gene expression of a selection of cytokines and chemokines on hFc-FNDC4 treatment (24 h, 100 nM) in the presence of TNFα. N=6–7. (e–f) Gene expression of M1 (e) and M2 (f) markers. N=6–7. (g) Gene expression of Tnf, Il1b and Nos2 after LPS-mediated polarization. Cells were treated with 10 ng ml⁻¹ LPS for 3 days before 24 h treatment with 100 nM hFc-FNDC4 or hFc control. N=4. (h) Gene expression of M2 markers Clec10a and Mrc1 after IL-4-mediated polarization. Cells were treated with 10 ng ml⁻¹ IL-4 for 3 days before 24 h treatment with 100 nM hFc-FNDC4 or hFc control. N=4. (i) Gene expression of the general leukocyte marker Ptprc (Cd45) and the general macrophage markers Emr1 (F4/80) and Itgam (Cd11b). N=3. Mean±s.e.m. NT, non-treated. *P<0.05 and q<0.10 for the comparison hFc-FNDC4 versus the corresponding hFc control group; one-way analysis of variance with Tukey’s post hoc tests, followed by the Benjamini-Hochberg FDR correction. These genes were regulated to a similar extend in the gene expression array and similar effects on gene expression were seen in multiple (>3) independent validation experiments.