Soluble Immune Complexes Shift the TLR-Induced Cytokine Production of Distinct Polarized Human Macrophage Subsets towards IL-10 Carmen A. Ambarus1*, Kim C. M. Santegoets2, Lenny van Bon 2 , Mark H. Wenink2, Paul P. Tak1, Timothy R. D. J. Radstake2, Dominique L. P. Baeten1 1 Department of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, Amsterdam, The Netherlands, 2 Department of Rheumatology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Abstract Background: Costimulation of murine macrophages with immune complexes (ICs) and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages. Materials and Methods: Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN- c, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs). Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/ or RT-qPCR. Results: HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MWIL-4. In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2). The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6. Conclusion: HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10. Citation: Ambarus CA, Santegoets KCM, van Bon L, Wenink MH, Tak PP, et al. (2012) Soluble Immune Complexes Shift the TLR-Induced Cytokine Production of Distinct Polarized Human Macrophage Subsets towards IL-10. PLoS ONE 7(4): e35994. doi:10.1371/journal.pone.0035994 Editor: Guillermo H. Giambartolomei, National Council of Sciences (CONICET), Argentina Received December 16, 2011; Accepted March 26, 2012; Published April 26, 2012 Copyright: 2012 Ambarus et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research is supported by a VIDI grant from The Netherlands Organization for Scientific Research (NWO) and by a grant from the Dutch Arthritis Foundation (Reumafonds). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: c.a.ambarus@amc.nl Introduction Macrophages play an important role in a wide variety of physiological and pathological processes including host defence, acute and chronic inflammation, and tissue homeostasis and remodelling. These pleiotropic cells can scavenge debris, sense microbial dangers signals, process and present antigens, and produce an array of pro- and anti-inflammatory mediators. Macrophage function, including the production of key cytokines such as TNF and IL-10, is not only determined by their activation but also by previous exposure to cytokines, growth factors, and other mediators during their differentiation from monocyte to macrophage. This so-called polarization process was originally proposed to distinguish classically activated macrophages (M1), which drive pro-inflammatory responses, from alternatively activated macrophages (M2), which steer immunoregulation and/or tissue remodelling [1–4]. Subsequent studies with mice and, to a lesser extent, human myeloid cells have lead to several more complex polarization models [5–7]. Using here the nomenclature proposed by Mantovani et al [5], the best characterized subsets are M1, M2a, and M2c, which are induced by IFN-c, IL-4, or IL-10, respectively. Functional differences are accompanied by distinct phenotypic profiles, and we recently validated in vitro a number of specific phenotypic markers for each of these three macrophage subsets [8]. Of particular interest in the model proposed by Mantovani [5], are the so-called M2b macrophages, which result from polariza- tion with ICs in combination with TLR ligands, such as LPS. Original studies showed that stimulation of mouse macrophages with ICs resulted in enhanced production of IL-10 and prostaglandins, especially PGE2 [9], while IL-6, IL-1, and TNF PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35994