Novel CCR2-RFP Mice in EAE Figure 4. CCR2-deficient mice exhibit delayed onset EAE and decreased monocyte recruitment to the CNS. (A) Ccr2RFP/+Cx3cr1GFP/+ and Ccr2RFP/RFPCx3cr1GFP/+ mice were immunized with MOG peptide 33–55 and scored daily for neurological signs. (B) EAE symptom onset and peak disease. (C) Total number of brain mononuclear cells and CD45hi infiltrating cells at peak disease. (D) CD45hi infiltrating cells were analyzed to quantify total neutrophils (SSChi, CD1152, Ly6Clo), monocytes (CD115+), and monocyte subsets (CD115+/Ly6Chi/lo), (E) CX3CR1 GFP and CCR2-RFP fluorescence intensities in monocyte subsets and microglia. Data points represent individual mice. Values are mean 6 SEM. Results are representative of two similar experiments. doi:10.1371/journal.pone.0013693.g004 CD115 expression and CD115+ cells for Ly6C expression. Among inflammatory CD45hi cells, there were strikingly fewer CD115+ monocytes/macrophages in the absence of CCR2 (Figure 4D). In Ccr2RFP/+Cx3cr1GFP/+ mice, 88% of monocyte-lineage (CD115+) cells were Ly6Chi, indicating an overwhelming advantage for this subset in accessing the CNS during EAE (Figure 4D). In Ccr2RFP/RFP Cx3cr1GFP/+ mice, however, Ly6Chi cells were drastically reduced, both in number and as a percentage of CD45hi infiltrating cells, demonstrating the importance of CCR2 for recruitment of this subset into the CNS. In contrast, accumulation of the much less numerous Ly6Clocells was unaffected by CCR2 ablation (Fig 4D). CCR2 deficiency led to an increase in the number of neutrophils, from 8% to 16% of the total infiltrating cells (Figure 4D). Comparison of CX3CR1-GFP and CCR2-RFP fluorescence intensities in the various CD115+ populations from Ccr2RFP/+ Cx3cr1GFP/+ mice showed that Ly6Chi cells expressed higher levels of CCR2-RFP (P = 0.016) than Ly6Clo cells and lower levels of CX3CR1-GFP (P = 0.008) (Figure 4E). CD45lo microglial cells were CCR2-RFP negative at EAE peak severity and had the highest level of CX3CR1 expression. Thus, the diversity of the mononuclear myeloid cell infiltrate in EAE is mirrored by a population-specific pattern of CCR2 and CX3CR1 expression, suggesting that the Ly6Chi/CCR2+ and Ly6Clo/CX3CR1+ subsets retain functional specialization in the inflamed CNS. Differential distribution of CX3CR1 and CCR2 in the naive and inflamed brain To visualize CX3CR1-and CCR2-expressing cells in the brains of Ccr2RFP/+Cx3cr1GFP/+ and Ccr2RFP/RFPCx3cr1GFP/+ mice, we used fluorescence microscopy. At low magnification, no RFP signal was detected in the brains of healthy Ccr2RFP/+Cx3cr1GFP/+ mice (Figure 5A), although a diffuse GFP signal was visible, corresponding to that previously attributed to resident microglia [23]. At high magnification, RFP expression was found in occasional perivascular cells in healthy Ccr2RFP/+Cx3cr1GFP/+ brains (Figures 5A and D); flow cytometry, these RFP+ cells accounted for ,2% of the CD45+ marrow-derived cells in naive brain (data not shown). Otherwise RFP expression was absent from the parenchyma of healthy brains (Figure 5D and not shown). RFP+ and RFP/GFP double-positive perivascular cells were often in close proximity (Figure 5D) and co-localized in the perivascular zone. As previously shown by co-localization with the microglial marker iba-1 [28], CX3CR1-GFP was present in microglial cells throughout the brain in both healthy and diseased tissue (Figure 5A–F). In contrast, microglia did not express CCR2 under naive conditions or EAE-associated inflammation. These findings were confirmed by flow cytometry (Figure 4G). At peak EAE severity, CCR2-RFP+ cells were present in the perivascular and parenchymal regions of the brains of Ccr2RFP/+ Cx3cr1GFP/+ mice (Figure 5B), but were greatly reduced and only found perivascularly in Ccr2RFP/RFPCx3cr1GFP/+ mice (Figure 5C). RFP+ cells accumulated in localized lesions, whereas GFP+ cells were both widely distributed and present in clusters within the lesions. Detailed analysis of RFP/GFP expression and cellular morphology in the lesion cores provided further insight into the functions of CCR2 and Ly6Chi monocytes in EAE. The inflammatory lesions contained CX3CR1 single-positive cells with morphological features of activated microglia (larger cell bodies and thicker processes, designated by an ‘‘*’’, compare Figure 5D and 5E). In Ccr2RFP/+Cx3cr1GFP/+ mice, the lesion core contained strikingly elongated CCR2-RFP+ cells (arrow, Figure 5E) that were notably less abundant in lesions of CCR2-deficient mice (Figure 5F), strongly suggesting that these cells are derived from Ly6Chi monocytes. A number of CCR2-RFP+ cells were also GFP-positive, and thus appear faintly yellow (circled cells, Figure 5E), indicating that both CCR2+ and CCR2/CX3CR1 double-positive monocytes invade the brain parenchyma in response to EAE. We noted that the green fluorescence in the monocytes was much less intense than in the microglia, consistent with the flow analysis in Figure 4G. A population of small, round RFP+ GFP2 cells in EAE lesions of CCR2+ and CCR2-deficient mice (arrowheads, Figures 5E–F) was shown in double-labeling experiments to be predominantly CD3+ T cells, levels of which were not altered by absence of CCR2 (data not shown). RFP+ cells in the brains of Ccr2RFP/RFPCx3cr1GFP/+ mice were primarily located in the meninges, and RFP+ cells were clearly absent from parenchymal tissue (Figure 5F). Microglial cells near Ccr2RFP/RFP Cx3cr1GFP/+ lesions displayed a less activated morphology than microglia in Ccr2RFP/+Cx3cr1GFP/+ lesions (Figure 5E and 5F), suggesting that monocytic cells in particular contribute to activation of resident microglia. Lack of CCR2RFP expression by neuroepithelial cells The findings of studies using tissue immunohistochemistry and receptor autoradiography have suggested constitutive or inducible expression of CCR2 by neuroepithelial cells (e.g., neurons, astrocytes, neural stem cells) (reviewed in [31]). Aside from sparse perivascular macrophages, we detected no CCR2-RFP+ cells in the CNS of healthy mice and no RFP+ cells with neuroepithelial morphology in the CNS of Ccr2RFP/+ mice with EAE (Figure 5). Furthermore, CCR2-RFP expression did not overlap with markers of astrocytes, neurons, or oligodendrocytes (data not shown). In analyses of CNS tissues of CCR2-RFP mice, the location and morphology of CCR2-RFP cells provided no evidence of CCR2 expression by CNS neurons. These findings are consistent with recent reports that peripheral nervous system but not CNS neurons inducibly express CCR2 in pain models [32]. Three color imaging with a myeloid marker To further analyze the identity of recruited cells, we stained brain sections from Ccr2RFP/+Cx3cr1GFP/+ mice at peak EAE for the myeloid marker 7/4. The 7/4 antigen identifies Ly6Chi monocytes and neutrophils, but not NK or T cells. Figure 6A shows a periventricular lesion visualized for CCR2 (RFP, red), CX3CR1 (GFP, green), and 7/4 (Cy5, blue). Figure 6B shows the overlapping CX3CR1-GFP and CCR2-RFP channels only; 6C shows CX3CR1-GFP and 7/4-blue, and 6D shows 7/4-blue and CCR2-RFP, to allow more convenient visualization and compar- ison of the fluorescent signals. For example, cell ‘‘1’’ in Fig. 6A is 7/4+, RFP+ and GFPdim, suggesting it is a Ly6Chi monocyte that PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13693