Statistics Statistical analysis was performed using Prism software (GraphPad, La Jolla, CA). Data were expressed as mean 6 SEM. ANOVA followed by Bonferroni post test were used for comparisons between more than 2 groups. For comparisons between 2 groups (stimulation with and without HAGGs) we used the Wilcoxon matched pair test. A P value of less than 0.05 was considered to be statistically significant. Results Soluble ICs do not alter phenotypic polarization of human macrophages Several reports indicate that stimulation of murine macrophages with ICs in combination with TLR ligands induces alternative macrophage polarization [7,10,12–14,29]. Therefore, our first aim was to investigate whether HAGGs had a similar polarizing effect on human macrophages. We tested whether the expression of Effect of Immune Complexes on Macrophage Subsets phenotypic markers for specific polarized macrophage subsets was altered by HAGGs alone, in combination with LPS, or in combination with IFN-c, IL-4, or IL-10 as major polarizing cytokines [5,7]. IFN-c polarization resulted in upregulation of CD80 (p,0.05 compared to medium, IL-4 and IL-10) and CD64 (p,0.05 compared to IL-10, and p,0.01 compared to medium, IL-4 and LPS), IL-4 upregulated CD200R (p,0.01 for all comparisons) and downregulated CD14 (p,0.05 compared to IFN-c and IL-10, and p,0.01 compared to LPS), while IL-10 induced a higher expression of CD163 (p,0.05 for all compar- isons), CD16 (p,0.05 for all comparisons), and CD32 (p,0.05 compared to IFN-c, and p,0.01 compared to medium, IL-4 and LPS), respectively [8] (Figure 1). LPS used as polarizing stimulus strongly increased the expression of CD14 (p,0.01), as described previously [30]. Stimulation with HAGGs alone or in combination with LPS did not lead to upregulation of specific phenotypic markers, in particular those associated with IL-4 and IL-10 polarization (Figure 1). Additionally, HAGGs did not influence the Figure 1. Expression of phenotypic markers on in vitro polarized human macrophages. Healthy peripheral blood monocytes were cultured for 4 days in medium or in medium supplemented with IFN-c, IL-4, IL-10, or LPS in the absence (white bars) or presence (black bars) of HAGGs. Expression of MWIFN-c markers CD80 and CD64, MWIL-4 markers CD200R and CD14, and MWIL-10 markers CD163, CD16 and CD32 was measured by flow cytometry. Bars represent the mean 6 SEM of 4 independent experiments. *p,0.05, **p,0.01, #p,0.01. doi:10.1371/journal.pone.0035994.g001 PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35994