Figure 7

In vitro GM-CSF neutralization induces a switch towards M2 macrophage polarization profile after mycobacterial infection. Mannose receptor CD206 expression was determined by flow cytometry in macrophages treated with GM-CSF neutralizing MAB (Clone A7.39) or IgG2b isotype control (1 µg/mL) and either unstimulated (medium), or stimulated with M. bovis BCG (M.O.I.:2) or LPS (100 ng/mL) during 24 hours. CD11b⁺ cells were pre-gated and F4/80⁺CD206⁺ double positive cells were selected, as shown on representative dot blots (A), together with cell frequency (B). Histograms (C) and MFI (D) of BMDMs treated with anti GM-CSF MAB (blue lines) or IgG2b isotype control (red lines) and labeled with rat anti-mouse CD206-APC (clone MR5D3) antibodies are shown, as compared to isotype control (grey). Data are expressed as mean ± SD of n = 2 independent cultures. M1 and M2-related genes expression was measured in macrophages after 24 hours of stimulation. The mRNA levels of nitric oxide synthase 2 (Nos2, E), arginase 1 (Arg1, F), chitinase-like 3 (Ym1, G), CD206 mannose receptor, C type 1 (Mrc1, H) and suppressor of cytokine signaling 3 (Socs3, I) were analyzed by RT-qPCR. The fold change of mRNA levels, normalized to Gapdh level in medium, is shown. Data are from two independent experiments and are expressed as mean values ± SEM of n = 4 independent cultures. Statistical comparisons are presented as compared to isotype control treated cells. ****p < 0.0001, **p < 0.01, *p < 0.05.