phenotypic effects of IFN-c, IL-4, or IL-10, with the exception of CD32 downregulation on MWIL-10 (p,0.01) (Figure 1). Taken together, these experiments did not show any polarizing effect of HAGGs on the phenotype of human macrophage subsets. Macrophage polarization determines the TLR-induced production of pro- and anti-inflammatory cytokines Numerous reports indicate that, besides phenotype, a main difference between distinct polarized macrophage subsets is the production of key cytokines such as TNF, IL-6, IL-10, IL-12 and IL-23 upon activation [5,7,13,15,31–33]. Before evaluating the effect of HAGGs on the cytokine production of polarized human macrophages, we first determined the cytokine production profile of MWIFN-c, MWIL-4, and MWIL-10 upon TLR stimulation. As depicted in Figure 2, the production of TNF, IL-6 and IL-10 was very low for all macrophage subsets in the absence of TLR stimulation. In these basal conditions, MWIL-10 produced signif- icantly more IL-10 than all other macrophage subsets (p,0.01). Following TLR stimulation, MWIFN-c produced more IL-6 than all other macrophage subsets (p,0.001 for the P3C and R848 stimulations, and p,0.01 for the LPS stimulation). There was a similar albeit less pronounced trend for TNF (p,0.05 versus MWIL-10 for the LPS stimulation), which was partially due to the fact that MWIL-4 also produced more TNF, especially after LPS and R848 stimulation. IL-10 levels were similar in all polarized Effect of Immune Complexes on Macrophage Subsets subsets, with the exception of a higher production by MWIL-4 versus MWIFN-c after LPS stimulation (p,0.05). Despite the fact that some comparisons did not reach statistical significance, these findings confirm previous reports of a pro-inflammatory profile of MWIFN-c versus a more neutral to anti-inflammatory profile for MWIL-4 and MWIL-10 upon TLR stimulation [5,7,13,15]. Soluble ICs enhance the TLR-induced IL-10 production by polarized macrophages Since ICs were described to induce an immunoregulatory function in mouse macrophages [10,18,29,34,35], we next investigated whether HAGGs alter the pro- versus anti-inflamma- tory cytokine production of differentially polarized human macrophages. In the absence of TLR stimulation, HAGGs did not alter the low basal production of TNF, IL-6, and IL-10 by any of the macrophage subsets (Figure 3). Using P3C as prototypical TLR stimulation, costimulation with HAGGs had variable effects on the production of pro-inflammatory cytokines, with a decrease in TNF production by unpolarized macrophages and MWIL-10, and an increased IL-6 production by MWIFN-c (p,0.05 compared to P3C alone for all 3 comparisons) (Figure 3). In contrast, HAGGs consistently enhanced IL-10 production by all macro- phage subsets, with the exception of MWIL-4 (p,0.01 for all comparisons) (Figure 3). In particular, HAGGs almost doubled the IL-10 secretion by MWIFN-c. These data were confirmed by Figure 2. TLR-induced production of TNF, IL-6, and IL-10 by MWIFN-c, MWIL-4, and MWIL-10. 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, LPS, or R848. Bars represent the mean 6 SEM of 6 independent experiments. *p,0.05, **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0035994.g002 PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35994