Fig 2: Activation of the CSF1R signaling pathway by IL-34 and CSF-1 and inhibition of IL-34-mediated cell growth using IL-34 neutralizing antibodies. (A) N13 microglia cell line was stimulated with IL-34 (50 or 100 ng/mL), CSF-1 (50 or 100 ng/mL) or LPS (1 µg/mL) for 5 or 10 min. Cell lysates were subjected to Western blotting which showed increased phosphorylation of CSF1R and downstream ERK1/2 and AKT after IL-34 and CSF-1 stimulation. (B) IL-34 neutralizing antibodies used in this study (mouse monoclonal IgG2A [v1.1, (24)), rat monoclonal IgG2A (MAB5195, R&D Systems) and sheep polyclonal IgG (AF5195, R&D Systems)] prevented IL-34-dependent cell growth of M-NFS-60 in a similar nanomolar range. n = 3, data shown represent mean ± SEM, two-way ANOVA followed by Tukey's multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, comparisons are stimulations vs. unstimulated control (C) for each time point.

Fig 3: Effect of CSF1R- and IL-34 antibody treatment on blood immune cell compartment. (A) Macgreen mice were treated with anti-CSF1R or anti-IL-34 (both rat monoclonal IgG2A) by intraperitoneal injections of 250 µg antibody 3x a week for 3 weeks. (B–D) Levels of rat IgG2a, CSF-1 and IL-34 were measured in blood plasma after the treatment by ELISA, which showed increased IL-34 and CSF-1 levels after CSF1R- but not IL-34 blockade. (E) Flow cytometric analysis of CSF1R+ cells in the blood of anti-CSF1R- and anti-IL-34- treated mice demonstrated a significant reduction after CSF1R- but not IL-34 antibody treatment. Graphs indicate respective cell numbers per 5 × 104 CD45+ cells. (F) Flow cytometry of eGFP+ Ly6Chi and eGFP+ Ly6Clo subpopulations of CSF1R-expressing cells in the blood shows a reduction in Ly6Clo cells after anti-CSF1R treatment, while Ly6Chi cells were not affected. Graphs indicate respective cell numbers per 5 × 104 CD45+ cells. PBS n = 8, isotype n = 8, anti-CSF1R n = 8, anti-IL-34 n = 7, data shown represent mean ± SEM, two-way ANOVA followed by Tukey's multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Effect of CSF1R- and IL-34 antibody treatment on tissue macrophages. (A) Histological analysis of CSF1R-positive cells in liver and kidney shows a reduction after anti-CSF1R treatment, but not after anti-IL-34 treatment. Skin Langerhans cells were significantly reduced after both CSF1R – and IL-34 blockade. (B) Microglia in cortex, hippocampal CA1 and dentate gyrus were not majorly affected by CSF1R or IL-34 antibody administration. Scale bar 100 µm. PBS n = 8, isotype n = 8, anti-CSF1R n = 8, anti-IL-34 n = 7, data shown represent mean ± SEM, two-way ANOVA followed by Tukey's multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001.