microscopy revealed unanticipated characteristics of myeloid cell populations in the CNS. First, by evaluating monocyte/macrophage/microglial chemo- kine receptor expression in EAE infiltrates, we found that CD45lo microglia invariably expressed CX3CR1 but not CCR2 in both healthy and diseased CNS tissues. Thus, CCR2-RFP and CX3CR1-GFP can differentiate resident microglia (GFP only) from infiltrated monocyte-derived cells (RFP+ with various degrees of GFP expression). Resident microglia and infiltrating monocyte- derived macrophages fulfill very different functions, as most pointedly indicated by analysis of their functions in Alzheimer’s disease models [38]. Therefore, the capacity to distinguish resident microglia from infiltrating monocytes will be valuable for functional characterizations. Second, neutrophils, not Ly6Clo monocytes, replaced Ly6Chi monocytes in the brains of Ccr2RFP/RFPCx3cr1GFP/+ mice with EAE, accounting for the equivalent number of total leukocytes. Thus, our findings reveal that Ly6Clo/CX3CR1+ monocytes are relatively excluded from the inflamed CNS of mice with EAE, regardless of CCR2 expression. Therefore, Ly6Chi and Ly6Clo monocytes do no appear to contribute sequentially to tissue inflammation or tissue remodeling/repair in the CNS as reported in cardiac ischemic injury (16). We propose that endogenous microglia perform the repair and remodeling functions carried out by Ly6Clo monocytes in peripheral organs. Thus, CCR2 antagonists [39] will likely affect CCR2+ infiltrating monocytes but not resident CX3CR1+ microglia. A third major finding, made possible by the use of RFP as a surrogate marker for CCR2, was that the absence of CCR2 prevents monocytes from leaving the vasculature and entering the perivascular space. There is considerable controversy regarding whether CCR2 mediates monocyte trafficking out of hematopoi- etic organs into the blood, trafficking from blood to inflamed organs, or both [12,13]. CCR2 ligands, such as MCP-1, are made by parenchymal cells of the CNS [40], consistent with the hypothesis that the CCL2/CCR2 axis is critical for monocyte recruitment to the inflamed CNS from the blood [41]. Finally, RFP and GFP expression in infiltrated cells in the CNS overlapped with the patterns observed in the blood. In particular, mononuclear cells in the perivascular region of the brain displayed a range of RFP/GFP ratios consistent with those in the blood (Figures 2 5, and 6). For example, in FACS analyses, Ly6Chi cells in the diseased CNS of Ccr2RFP/+Cx3cr1GFP/+ mice with EAE were RFPhi and GFPmed/lo, while the infrequent Ly6Clo cells were GFPhi/RFPlo (Figure 2I). We used 7/4 as a surrogate for Ly6C staining in brain sections and found a corresponding correlation between the presence of RFP and GFP depending on the 7/4 status of the monocyte (Figure 6). In Ccr2RFP/RFPCx3cr1GFP/+ mice, the onset of EAE occurred 5 days later than in Ccr2RFP/+Cx3cr1GFP/+ control mice, but reached similar peak severity with the same kinetics as in littermate controls. The delay in EAE development was presumably due to the absence of CCR2+ monocytes in the CNS. Their particular contribution is a subject of future work, but it has been suggested that they express inflammatory mediators and can convert to DCs [17,37]. In support of this hypothesis, CCR2-deficient mice had 50% fewer CD11c+ cells in the brain with EAE, as compared with CCR2+ mice at the same sate of disease (unpublished data). Spinal cord total inflammatory burden was not affected by CCR2 deficiency (Figure S2). This finding most likely accounts for the similar peak disease severity in the Ccr2+/RFP and Ccr2RFP/RFP mice, since the neurological signs of EAE arise from spinal-cord involvement [30]. These results generally corre- spond to those reported in CCL2 knockout mice [35]. However, Novel CCR2-RFP Mice in EAE in the current study, we analyzed fully-back-crossed knockout mice at 24 weeks of age, which may explain the differences in neurobehavioral results between our studies and some previous studies [34,36,42]. In double-receptor knockout mice (Ccr2RFP/RFP Cx3cr1GFP/GFP), neurological signs of EAE were indistinguishable from those of CCR2-deficient Ccr2RFP/RFPCx3cr1+/GFP mice, indicating that CCR2 plays a dominant role in EAE severity (data not shown). There are several limitations to the usefulness of this model for in vivo imaging. First, because some T cells and NK cells express CCR2 transcripts and are thus RFP-positive, it is not possible to conclude that all red cells in an image are monocytes. Depending on the specific setting, additional markers may have to be included to allow more precise identification of the cells, as illustrated in the EAE experiments in Figure 6, in which we found that the vast majority, but not all RFP+ cells were also 7/4+. In addition, although RFP intensity correlates well with Ly6C intensity, the overlap is not perfect, and some RFPbright cells may be Ly6lo. Nonetheless, the ability to simultaneously image CCR2 and CX3CR1 expression in monocytes and other leukocytes represents a powerful new tool for investigating complex pathophysiology. In summary, our findings confirm, refine, and extend the concept of monocyte heterogeneity. We created and characterized a transgenic mouse model that allows simultaneous tracking of two key chemokine receptors, CCR2 and CX3CR1 and enumeration of distinct monocyte and myeloid subsets based on CCR2-RFP/ CX3CR1-GFP expression. We showed that CCR2 expression is controlled at a post-transcriptional stage in some Ly6Clo monocytes and NK and T cells. We found that Ly6Chi monocytes fail to enter the CNS of CCR2deficient mice during EAE and are mainly replaced by neutrophils, not Ly6Clo monocytes. We hypothesize that CCR2+ Ly6Chi monocytes initiate and maintain neuroinflammatory responses, while tissue remodeling is mediated by CX3CR1+ microglia. The CCR2-RFP/CX3CR1-GFP report- er mice should be useful for studying the roles of CCR2 and CX3CR1 in myeloid cell recruitment in a wide range of inflammatory diseases. The expression of CCR2 transcripts in some NK and T cells is of interest and must be taken into account when using this model. Materials and Methods Ethics Statement All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the appropriate committees: IACUC University of California, San Francisco APPROVAL NUMBER: AN079763-02; IACUC Cleveland Clinic APPROVAL NUMBER: ARC 07541; IACUC University of Texas, San Antonio APPROVAL NUMBER: MU060-08/12A0. Mice CCR2RFP mice were created as described [25] (see Figure 1 and Text S1). Founder mice were crossed with Cre-deleter mice [43] to remove the neo gene and backcrossed onto the C57Bl/6 line nine times. To generate homozygous Ccr2RFP/RFPCx3cr1GFP/GFP mice, we crossed Ccr2RFP/RFP with Cx3cr1GFP/GFP C57Bl/6 mice (a gift of D.R. Littman), and the progeny were backcrossed onto C57Bl/6. Mice that had undergone chromosome recombination between the CCR2 and CX3CR1 loci were selected by being positive for both RFP and GFP by flow cytometry of tail vein blood. PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13693