Novel CCR2-RFP Mice in EAE genomic DNA. DNA was digested with PstI, and hybridized with the indicated probe. (C) Flow cytometry analysis of peripheral blood leukocytes (PBL) from WT, Ccr2RFP/+, and Ccr2RFP/RFP mice. Cells were stained with anti-CCR2 antibody MC21. The results are representative of four similar experiments. doi:10.1371/journal.pone.0013693.g001 backcrossed onto the C57Bl6 background, and littermates were compared in each experiment. In Ccr2RFP/RFPCx3cr1GFP/+ mice, the onset of EAE was delayed by a mean of 4.5 days (day 17.460.6 (SD) vs. day 12.960.1 in Ccr2RFP/+Cx3cr1GFP/+ mice) (Figure 4A). The maximum disease scores were similarly delayed (day 20.360.5 for Ccr2RFP/RFPCx3cr1GFP/+ mice vs. day 16.660.4 for Ccr2RFP/+ Cx3cr1GFP/+ mice), indicating equivalent kinetics of disease evolution in the two groups (Figure 4B). Peak severity scores were lower in the CCR2-null mice, but the difference did not reach statistical significance (p = 0.11). A similar delay in onset followed by equivalent peak severity was observed in Ccr2RFP/RFPCx3cr1GFP/GFP mice (data not shown). The latter finding indicates that the effect of CCR2 deficiency is dominant on a CX3CR1-null background, given the observation that Cx3cr1GFP/GFP mice exhibited earlier disease onset than WT mice [26]. Infiltration by CD115+ monocytes is reduced in CNS of CCR2-deficient mice We previously showed that inflamed EAE tissues contain high levels of CCR2 and CX3CR1 ligands [26,27], making this tissue ideal for comparing the trafficking properties of monocytes bearing either of the two cognate receptors. To characterize the cellular infiltrate in the CNS of mice with EAE, we used flow cytometry to analyze CNS leukocytes isolated at the peak of disease severity (Figure 4C and 4D). Ccr2RFP/+Cx3cr1GFP/+ mice allowed us to simultaneously track CX3CR1-and CCR2-positive cells, and Ccr2RFP/RFPCx3cr1GFP/+ mice allowed us to examine the effect of CCR2 deficiency on monocyte recruitment during EAE. Leukocytes were stained with antibodies against CD45 to distinguish CD45hi infiltrating leukocytes from CD45lo microglial cells. At peak disease severity, similar numbers of total and CD45hi cells were recovered from the brains of Figure 2. Expression of CCR2 and RFP in sorted cell populations. (A) CD115+ monocytes were sorted by flow cytometry into populations that did or did not express cell-surface CCR2. Total RNA was isolated, pooled from three mice per genotype, and analyzed for CCR2 mRNA and RFP mRNA by quantitative RT-PCR. Probe specificity was confirmed with WT and Ccr22/2 cells. Expression levels were normalized to beta-actin, and the results are expressed relative to the concentration of CCR2 mRNA in monocytes expressing cell-surface CCR2. (B) NK and T cells were sorted by flow cytometry into RFP+ and RFP– fractions. Expression of CCR2 and RFP mRNA was characterized as in (A) and normalized to the amount of CCR2 RNA in the RFP+ T-cell fraction from Ccr2+/RFP mice. (C to G) WT, Ccr2RFP/+, and Ccr2RFP/RFP mice were bred onto the Apoe2/2 background and fed a high-fat diet for 8 weeks. Peripheral blood monocytes were stained for CD115, CCR2, and Ly6C and analyzed by flow cytometry. (C) Gating on monocytes. (D) Ly6C and RFP expression of monocytes. Dashed line indicates the cutoff for a positive RFP signal. (E) Mean RFP fluorescence intensity in Ly6Chi and Ly6Clo subsets. Bars indicate SEM. n = 4 mice/group. P,0.008. (F) CCR2 and RFP expression of Ly6Chi monocytes. (G) CCR2 and RFP expression in Ly6Clo monocytes. CCR2-positive and CCR2-negative gates were based on CCR2-deficient Ccr2RFP/RFP mice. (H to K) Flow cytometric analysis of Ly6C, RFP, GFP and cell-surface CCR2 expression on monocytes from Ccr2RFP/+Cx3cr1GFP/+ mice. CD115+ monocytes were gated (H) by Ly6C/GFP expression and analyzed for RFP (I) and CCR2 (J) expression. Ccr2RFP/RFPCx3cr1GFP/+ monocytes (K) were used as a negative control for CCR2 surface-staining. Results are representative of four mice in two similar experiments. doi:10.1371/journal.pone.0013693.g002 PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13693