Novel CCR2-RFP Mice in EAE Figure 6. Analysis of monocyte subsets in brain lesions of EAE Ccr2RFP/+Cx3cr1GFP/+ mice at peak EAE. Confocal images of the periventricular lesions were obtained after staining with the 7/4 antibody. Images represent the same lesion (A) Merged images of red/CCR2, green/ CX3CR1, and blue/7/4. (B) Red/CCR2 and green/CX3CR1. (C) Blue/7/4 and green/CX3CR1. (D) Red/CCR2 and blue/7/4. The majority of CCR2+ cells are 7/4+ and CX3CR1lo or negative, suggesting that they are classical infiltrating monocytes (see individual cell ‘‘1’’). Ly6Clo monocytes (CX3CR1hi and CCR2) are also present (cell ‘‘2’’), as well as T cells (‘‘3’’) and activated microglial cells (‘‘4’’). doi:10.1371/journal.pone.0013693.g006 for the first time we provide a model to visualize monocytes subsets in situ within brain infiltrates. Under the steady-state, low-level inflammatory conditions of a high-fat diet, Ly6Chi monocytes had higher RFP intensity and lower GFP intensity than Ly6Clo cells, allowing the subsets to be clearly distinguished by their RFP/GFP profiles. To investigate the trafficking of monocyte subsets in an acute inflammatory model, we used thioglycollate to induce sterile peritonitis. At 24 h, the vast majority of monocyte/macrophages recruited to the peritoneum in Ccr2+/RFPCx3cr1+/GFP mice expressed RFP and GFP. Because CCR2 is rapidly downregulated when monocytes differentiate into macrophages [33], these results suggest that the RFPhi cells were newly arrived monocyte/macrophages and show that the RFP and GFP markers are useful for tracking migrating monocytes. Interestingly, the absence of CX3CR1 in the Ccr2RFP/+ Cx3cr1GFP/GFP mice had little or no effect on the RFP/GFP profile or the number of recruited cells. By contrast, deletion of CCR2 in Ccr2RFP/RFPCx3cr1+/GFP mice drastically reduced peritoneal mac- rophage recruitment. CCR2 and CX3CR1 have been implicated as disease modifiers in EAE, a model for the inflammatory aspects of multiple sclerosis. EAE severity is restricted in CCR2-deficient mice on a mixed C57Bl/6 and 129/J background and in CCL2-deficient (MCP-1/ JE) mice [34–36]. It has been proposed that CCR2+ monocytes that enter the CNS during EAE differentiate into macrophages and DCs, thereby promoting EAE pathogenesis [17,37]. In contrast, CX3CR1 deficiency exacerbates EAE because regulatory NK cells are not recruited to the inflamed CNS [26]. Our EAE experiments revealed physiologically relevant and mechanistically intriguing functions of CCR2 expression and regulation of CNS myeloid inflammation. We predicted that CCR2 deficiency would preferentially reduce Ly6Chi monocyte infiltration into the CNS of mice with EAE. To test this hypothesis, we compared the pathogenesis of EAE in Ccr2RFP/+Cx3cr1GFP/+ and CCR2-deficient Ccr2RFP/RFPCx3cr1GFP/+ mice. Mice deficient in both CCR2 and CX3CR1 (Ccr2RFP/RFPCx3cr1GFP/GFP mice) were included to probe the interactions between these receptors in EAE. As predicted, Ly6Chi monocytes were virtually absent from the CNS of the Ccr2RFP/RFPCx3cr1GFP/+ mice with EAE. Unexpected- ly, the total numbers of leukocytes infiltrating the CNS were indistinguishable in control and CCR2-deficient mice. Detailed analysis of CNS infiltrates by flow cytometry and fluorescence PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13693