encephalitis (EAE). The relative expression of CX3CR1-GFP and CCR2-RFP allowed us for the first time to distinguish monocyte/ macrophage subsets and discriminate between resident microglia and infiltrating monocyte-derived macrophages in the brain. Results Generation of CCR2RFP knock-in mouse We used an expression cassette with a cDNA that encoded monomeric RFP [24] to replace the first 279 base pairs of the amino terminus of the CCR2 open reading frame (Figure 1A). The CCR2 promoter in the targeting vector was unchanged up to the first methionine of the open reading frame, as determined by sequence analysis. The RFP gene construct included a poly-A termination signal to exclude the remainder of the CCR2 sequence from the mature transcript. Homologous recombinations in embryonic stem cells and mouse derivation were accomplished by standard methods [25], and the neomycin-resistance gene was deleted. Southern blot hybridization confirmed the correct genomic structure (Figure 1B). Flow cytometric analysis of Ccr2RFP/+ and Ccr2RFP/RFP peripheral blood cells stained for cell- surface CCR2 (Figure 1C) confirmed bi-allelic expression of CCR2 and RFP in heterozygous mice and deletion of CCR2 in Ccr2RFP/RFP homozygous mice. All CCR2-positive cells were RFP positive, but some RFP-positive cells failed to express CCR2 at the cell surface. RFP and CCR2 RNA expression in leukocytes CCR2 and RFP transcript levels were determined by quantitative RT-PCR of cell populations isolated by fluores- cence-activated cell sorting (FACS). In blood cells from Ccr2RFP/+ mice, CCR2 and RFP mRNA levels tracked closely, as expected for bi-allelic expression from the CCR2 promoter (Figure 2A). RFP transcripts were undetectable in wildtype (WT) cells, and CCR2 was absent in Ccr2RFP/RFP cells. Monocytes that stained positively for surface CCR2 invariably contained RFP protein and expressed three- to fourfold more CCR2 and RFP transcripts than monocytes not expressing cell-surface CCR2 (Figure 2A). Prelim- inary flow cytometry surveys of Ccr2RFP/+ mice indicated that some natural killer (NK) and T cells also were positive for RFP (85–95% and 5–10% respectively; data not shown). NK and T cells were sorted into RFP-positive and RFP-negative fractions. Despite lacking CCR2 immunoreactivity by flow cytometry, T cells and NK cells from WT and Ccr2RFP/+ mice contained CCR2 transcripts, and CCR2 and RFP mRNA levels were similar in both cell types (Figure 2B). These results indicate possible post- transcriptional control of CCR2 expression. RFP and CCR2 protein expression in monocyte subsets To evaluate RFP and CCR2 expression in blood monocyte subsets in an inflammatory setting, we crossed Ccr2RFP mice with apolipoprotein E–deficient (Apoe2/2) mice, fed them a high-fat diet for 8 weeks, and analyzed RFP and CCR2 surface expression in CD115+ monocytes by flow cytometry (Figure 2C). Both Ly6Chi and Ly6Clo monocytes were RFP-positive, but the Ly6Chi cells were significantly brighter (Figure 2D and E). Approximately 86% of Ly6Chi monocytes expressed cell-surface CCR2 (Figure 2F), as did 18% of Ly6Clo monocytes (Figure 2G). These results indicate that RFP expression can predict CCR2 expression levels in monocytes. Cytoplasmic CCR2 in lymphocytes and monocyte subsets Some CCR2 surface-negative leukocytes contained abundant levels of CCR2 mRNA (and RFP protein and mRNA) (Figure 2). Novel CCR2-RFP Mice in EAE To ask whether Ly6Clo monocytes contained cytoplasmic CCR2 that failed to traffic to the cell surface, we compared CCR2 staining on permeabilized and nonpermeabilized monocytes by FACS (Figure S1). Monocytes from CCR2 knockout mice served as negative controls. In Ly6Chi monocytes, permeabilization increased the CCR2 signal twofold, indicating that approximately 50% of total CCR2 was cytoplasmic, but had little effect on CCR2 staining in Ly6Clo monocytes. Similarly, intracellular CCR2 was undetectable in T cells or NK cells, despite the presence of CCR2 mRNA (data not shown). These results suggest that T cells, NK cells and some Ly6Clo monocytes may be capable of expressing CCR2 protein but do not do so constitutively. Analysis of monocytes from Ccr2RFPCx3cr1GFP mice To evaluate blood monocyte subsets for expression of CCR2 or CX3CR1 during chronic systemic inflammation, we crossed Ccr2RFP/RFP mice with Cx3cr1GFP/GFP mice [23] and examined Ccr2RFP/+Cx3cr1GFP/+Apoe2/2 mice fed a high-fat diet for 8 weeks. Ly6Chi and Ly6Clo monocytes were both GFP positive (Figure 2H). Ly6Clo cells were clearly brighter than Ly6Chi cells, which had a wider range of GFP intensities. Both Ly6Chi and Ly6Clo monocytes were RFP positive, but Ly6Chi cells were brighter (Figure 2I). There was a broad range of RFP intensities in the Ly6Clo monocytes (Figure 2I), though many were as bright as those in the Ly6Chi population. We next examined RFP expression and CCR2 surface staining on monocytes stratified by their level of Ly6C. As expected from the Ccr2RFP mice (Figure 2F,G), virtually all of the Ly6Chi monocytes were markedly positive for both RFP and CCR2 staining (Figure 2J). While most but not all Ly6Clo monocytes were negative for CCR2 staining, some Ly6Clo monocytes expressed CCR2 at levels similar to those in Ly6Chi monocytes. Ccr2RFP/RFP mice served as negative controls to set the FACS gates for CCR2 staining (Figure 2K). To investigate the relative contributions of CCR2 and CX3CR1 to monocyte/macrophage recruitment in vivo, we used a mouse model of thioglycollate-induced peritonitis. In naive mice, the vast majority of F4/80hi peritoneal macrophages were negative for GFP and RFP and were probably resident macrophages (Figure 3A). After instillation of thioglycollate for 24 h to induce peritonitis, Ccr2RFP/+Cx3cr1GFP/+ F4/80dim monocytes were ac- tively recruited to the peritoneum, and almost all expressed high levels of both RFP and GFP (Figure 3A). Recruited monocytes were Ly6Chi (not shown). In WT and Ccr2RFP/+Cx3cr1GFP/+ mice, equal numbers of F4/80+ monocyte/macrophages accumulated in the peritoneum (Figure 3B). Monocyte recruitment was little affected by loss of the second CX3CR1 allele (Ccr2RFP/+Cx3cr1GFP/GFP), but was markedly reduced by loss of the second CCR2 allele (Ccr2RFP/RFP Cx3cr1GFP/+) (Figure 3B). Recruitment was indistinguishable in double-knockout mice (Ccr2RFP/RFPCx3cr1GFP/GFP) and CCR2 knock- outs (Ccr2RFP/RFPCx3cr1GFP/+). Monocyte counts in the blood and bone marrow were also indistinguishable in the two genotypes. These results revealed that the vast majority of monocytes recruited to the peritoneum in response to sterile inflammation are CCR2/CX3CR1 double-positive, although their recruitment is almost exclusively driven by CCR2. These experiments also demonstrated the feasibility of using CCR2-RFP and CX3CR1-GFP to track monocyte subsets in vivo. Experimental autoimmune encephalomyelitis in CCR2- deficient mice To extend these results in an established model of organ-specific inflammation, we compared Ccr2RFP/+Cx3cr1GFP/+ and Ccr2RFP/RFP Cx3cr1GFP/+ mice with EAE by monitoring disease severity daily for 40 days after immunization. All mice were highly PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13693