repeating the experiments with other TLR stimuli. HAGGs together with LPS or R848 induced a modest and variable effect on TNF and IL-6 production (data not shown), but a consistent and significant increase in IL-10 production by all macrophage subsets, with the exception of MWIL-4 (p,0.05 compared to TLR stimulation without HAGGs) (Figure 4). Thus, costimulation of in vitro polarized human macrophages with HAGGs and TLR ligands resulted in an increased IL-10 production by all polarized macrophage subsets, excepting MWIL-4. Effect of soluble ICs on the IL-10 production by MWM-CSF and MWGM-CSF Both phenotypic analyses and cytokine profiling indicated that GM-CSF polarized macrophages (MWGM-CSF) tend to resemble MWIFN-c, whereas M-CSF mimics the effects of alternative polarization stimuli, in particular IL-10 [8,32,33,36]. We therefore investigated whether stimulation with HAGGs also modulated the IL-10 production of MWGM-CSF and MWM-CSF. HAGGs alone did not have any effect on the IL-10 production. However, after co- stimulation with TLR ligands, HAGGs induced an increased IL- 10 production by MWM-CSF but not MWGM-CSF (p,0.05 for P3C, and p,0.01 for R848) (Figure 5). Concluding, MWM-CSF showed a higher TLR-induced IL-10 production after stimulation with HAGGs. Effect of Immune Complexes on Macrophage Subsets Increased LPS-induced IL-10 versus IL-23 and IL-12 expression after stimulation with soluble ICs In order to confirm the increase in TLR-induced IL-10 production by polarized macrophages after stimulation with HAGGs, we measured the mRNA expression of IL-10 by each macrophage subset after 7 hours of stimulation with LPS, in the presence or absence of HAGGs. As shown in figure 6, HAGGs increased the expression of IL-10 in all macrophage subsets (p,0.05 for MWIFN-c and MWIL-10). As IL-23 and IL-12 protein levels were undetectable in the supernatants of the previous experiments, we investigated the effect of HAGGs on these cytokines at mRNA level. In contrast with IL-10, we did not observe any significant effect of HAGGs on IL-23p19 and IL- 12p35 mRNA expression. Taken together, these data confirm the specific increase in IL-10 expression after costimulation with HAGGs and TLR ligands, with the strongest effects observed in MWIFN-c and MWIL-10. Immobilized IgG enhances the cytokine production of polarized macrophages without shifting the balance towards IL-10 As the consistent increase in IL-10 production by HAGGs in our experiments contrasted with previously published data on increased TNF production by human macrophages upon Figure 3. TNF, IL-6, and IL-10 production by MWIFN-c, MWIL-4, and MWIL-10 after stimulation with HAGGs and P3C. Healthy peripheral blood monocytes were cultured for 4 days in medium or in medium supplemented with IFN-c, IL-4, or IL-10. Polarized macrophages were not stimulated, or stimulated for 20 hours with P3C, in the presence or absence of HAGGs. Bars represent the mean 6 SEM of 9 independent experiments. *p,0.05, **p,0.01. doi:10.1371/journal.pone.0035994.g003 PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35994