IL-34 and CSF-1 display an equivalent macrophage differentiation ability but a different polarization potential

Abstract

CSF-1 and IL-34 share the CSF-1 receptor and no differences have been reported in the signaling pathways triggered by both ligands in human monocytes. IL-34 promotes the differentiation and survival of monocytes, macrophages and osteoclasts, as CSF-1 does. However, IL-34 binds other receptors, suggesting that differences exist in the effect of both cytokines. In the present study, we compared the differentiation and polarization abilities of human primary monocytes in response to CSF-1 or IL-34. CSF-1R engagement by one or the other ligands leads to AKT and caspase activation and autophagy induction through expression and activation of AMPK and ULK1. As no differences were detected on monocyte differentiation, we investigated the effect of CSF-1 and IL-34 on macrophage polarization into the M1 or M2 phenotype. We highlighted a striking increase in IL-10 and CCL17 secretion in M1 and M2 macrophages derived from IL-34 stimulated monocytes, respectively, compared to CSF-1 stimulated monocytes. Variations in the secretome induced by CSF-1 or IL-34 may account for their different ability to polarize naïve T cells into Th1 cells. In conclusion, our findings indicate that CSF-1 and IL-34 exhibit the same ability to induce human monocyte differentiation but may have a different ability to polarize macrophages.

Figure 1

IL-34 and CSF-1 induce equivalent macrophagic differentiation of primary monocytes. Human peripheral blood monocytes from healthy donors were exposed to 100 ng/mL CSF-1 or 100 ng/mL IL-34 for the indicated times. (a,b) Macrophage differentiation was examined morphologically (fibroblastic shape) and by 2-color flow cytometric analysis. The percentage indicates cells that express both CD71 and CD163. (c) Macrophagic differentiation of monocytes from 3 different healthy donors was followed by 2-color flow cytometric analysis. The percentage indicates cells that express both CD71 and CD163. *P < 0.05 according to a paired student t test.

Figure 2

Caspases and autophagy are activated upon IL-34 or CSF-1 treatment. Human peripheral blood monocytes from healthy donors were exposed to 100 ng/mL CSF-1 or 100 ng/mL IL-34 for the indicated times. (a) Immunoblot analysis of indicated proteins in monocytes following CSF-1 or IL-34 stimulation. P indicate phosphorylated proteins. Each panel is representative of at least 3 independent experiments. (b) Immunoblot analysis of indicated proteins in monocytes following CSF-1 or IL-34 stimulation. The ratio of the LC3-II protein level to that of LC3-I protein level was determined using ImageJ software. Actin was detected as the loading control. Asterisks indicate cleavage fragments. Each panel is representative of at least 3 independent experiments. (c) Caspase activity was quantified by flow cytometry analysis using DEVD-FITC. The results are expressed as the fold induction compared with untreated cells and represent the mean ± SD of 3 independent experiments performed in duplicate. n.s. denotes non-significant according to a paired student t test.

Figure 3

Caspases and autophagy are required for CSF-1 or IL-34 induced macrophagic differentiation. (a) Human monocytes were exposed for 2 days to 100 ng/mL CSF-1 or 100 ng/mL IL-34 alone or in combination with either 50 µM qVD or 3 mM 3MA (3-MethylAdenine), which were added 30 min before CSF-1 or IL-34 treatment. The expression of cleaved NPM (NPM*), LC3B and cleaved cathepsin B (CTSB*) was analyzed by immunoblotting. (b) Human monocytes were treated as in Fig. 3a. (c,d) Monocytes were transfected with siRNA targeting LUCIFERASE (LUC), CASPASE-8 (CASP8) or BECLIN (BEC) and exposed for 2 days to 100 ng/mL CSF-1 or 100 ng/mL IL-34. (c) Expression of Caspase-8, Beclin and LC3B was analyzed by immunoblotting. Actin is used as a loading control. Asterisk indicates a cleavage fragment. (d) Differentiation was examined as previously described.

Figure 4

IL-34 macrophages have a different polarization potential as compared to CSF-1-macrophages. (a) Human monocytes were differentiated during 7 days with 100 ng/mL CSF-1 or 100 ng/mL IL-34 and then polarized into M1-macrophages (LPS + IFNγ) or M2-macrophages (IL-4) for 2 days. Macrophage polarization was evaluated by 2-color flow cytometric analysis. (b) Functional assay of monocytes exposed for 2 or 5 days to 100 ng/mL CSF-1 or 100 ng/mL IL-34. The results are expressed as MFI and represent the mean ± SD of 3 independent experiments performed in duplicate. n.s. denotes not statistically significant according to a paired student t test. (c) Functional assay of monocytes exposed for 7 days with 100 ng/mL CSF-1 or 100 ng/mL IL-34 and then polarized into M2-macrophages (IL-4) for 2 days. The results are expressed as the fold induction compared to CSF-1 macrophages and represent the mean of 3 independent experiments performed in duplicate. *P < 0.05 according to a paired student t test (versus CSF-1-macrophages). (d,e) Human monocytes were differentiated during 7 days with 100 ng/mL CSF-1 or 100 ng/mL IL-34 and then polarized into M1-macrophages (LPS + IFNγ) or M2-macrophages (IL-4) for 24 hours. The expression of the indicated mRNA  was analyzed by qPCR (mean ± SEM of 5 independent experiments). *P < 0.05, ***P < 0.001 according to a paired student t test (versus CSF-1 macrophages). The production of IL-10 and CCL17 was analyzed using Multi-Analyte ELISArray kit as described in Material and Methods section. The results are expressed as ng/mL per million of cells and represent the mean ± SD of 2 independent experiments performed in duplicate.