RESEARCH ARTICLE In Acute Experimental Autoimmune Encephalomyelitis, Infiltrating Macrophages Are Immune Activated, Whereas Microglia Remain Immune Suppressed I. D. Vainchtein, 1 J. Vinet, 2 N. Brouwer, 1 S. Brendecke, 3 G. Biagini, 2 K. Biber, 4 H. W. G. M. Boddeke, 1 and B. J. L. Eggen 1 Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) characterized by loss of myelin accompanied by infiltration of T-lymphocytes and monocytes. Although it has been shown that these infiltrates are important for the progression of MS, the role of microglia, the resident macrophages of the CNS, remains ambiguous. There- fore, we have compared the phenotypes of microglia and macrophages in a mouse model for MS, experimental autoimmune encephalomyelitis (EAE). In order to properly discriminate between these two cell types, microglia were defined as CD11b pos CD45 int Ly-6C neg , and infiltrated macrophages as CD11b pos CD45 high Ly-6C pos . During clinical EAE, microglia displayed a weakly immune-activated phenotype, based on the expression of MHCII, co-stimulatory molecules (CD80, CD86, and CD40) and proinflammatory genes [interleukin-1b (IL-1b) and tumour necrosis factor-a (TNF-a)]. In contrast, CD11b pos CD45 high Ly- 6C pos infiltrated macrophages were strongly activated and could be divided into two populations Ly-6C int and Ly-6C high , respectively. Ly-6C high macrophages contained less myelin than Ly-6C int macrophages and expression levels of the proinflam- matory cytokines IL-1b and TNF-a were higher in Ly-6C int macrophages. Together, our data show that during clinical EAE, microglia are only weakly activated whereas infiltrated macrophages are highly immune reactive. GLIA 2014;00:000–000 Key words: microglia, macrophage, Ly-6C, EAE, multiple sclerosis Introduction M ultiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by demyelin- ation and axonal damage (Lassmann et al., 2007). Throughout the relapsing/remitting (RR) MS phase, patients develop inflammatory and demyelinated lesions in the white and gray matter (Lassmann et al., 2007). The first white matter MS lesions appear around small veins and consist of inflammatory infiltrates mainly composed of T-lymphocytes and infiltrating macrophages (Lassmann and van Horssen, 2011). This has led to the hypothesis that myelin-specific auto-reactive T-lymphocytes, triggered by unknown factors in the periphery, migrate to the CNS and induce demyelination resulting in neurological damage. The interplay between the innate immune system and T-lymphocytes at target locations is essen- tial, as infiltrating T-lymphocytes require antigen presentation in order to be re-stimulated; thereby the innate immune system controls the effector functions of T-lymphocytes (Becher et al., 2006; Gandhi et al., 2010; Weiner, 2008). The local innate immune cells of the CNS are the microglia, specialized tissue macrophages that monitor the neuronal environment for changes and tissue injury (Kreutzberg, 1996; Nimmerjahn et al., 2005). Chronic inflammation of the CNS or neurodege- nerative conditions has been shown to result in microglia acti- vation, which is associated with progressive neurodegeneration (Cunningham, 2013). View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22711 Published online Month 00, 2014 in Wiley Online Library (wileyonlinelibrary.com). Received Jan 30, 2014, Accepted for publication June 12, 2014. Address correspondence to: B. J. L. Eggen, A. Deusinglaan 1, 9713 AV Groningen, the Netherlands. E-mail: b.j.l.eggen@umcg.nl From the 1 Department of Neuroscience, Section Medical Physiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; 2 Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; 3 Department of Neuropathology, University Medical Center Freiburg, Freiburg, Germany; 4 Department of Psychiatry and Psychotherapy, University Medical Center Freiburg, Freiburg, Germany. Additional Supporting Information may be found in the online version of this article. V C 2014 Wiley Periodicals, Inc. 1