Novel CCR2-RFP Mice in EAE Figure 5. Expression of CX3CR1 and CCR2 in brain lesions of mice with EAE. (A–C) Low-magnification epifluorescence images of naive Ccr2RFP/+Cx3cr1GFP/+ (A), diseased Ccr2RFP/+Cx3cr1GFP/+ (B), and Ccr2RFP/RFPCx3cr1GFP/+ (C) brains show the extent of inflammation and anatomical location of the lesions. Higher magnification views of the boxed areas are shown in panels D–F. Confocal image of healthy perivascular (D) tissue shows GFP+ microglia (asterisks), small round RFP+ cells (arrowhead), and double-positive cells (circles). (E) Parenchymal lesion in a Ccr2RFP/+Cx3cr1GFP/+ mouse and (F) a perivascular lesion from a Ccr2RFP/RFPCx3cr1GFP/+ mouse at peak EAE disease. Dashed line in (F) indicates the approximate perivascular boundary. Within EAE lesions, note RFP/GFP double-positive cells (circles), GFP+ activated microglia (E, F; asterisks), large elongated RFP+ cells (E, F; arrows), and small round RFP+ cells (E, F; arrowheads). Small RFP+ cells were CD3+ (not shown). The particularly elongated RFP+ cells in the Ccr2RFP/+ Cx3cr1GFP/+ lesion (E) were much less abundant in the Ccr2RFP/RFPCx3cr1GFP/+ lesion (F). doi:10.1371/journal.pone.0013693.g005 expresses both CCR2 and CX3CR1. Inspection of Figure 6B confirms that this cell ‘‘1’’ appears yellow (co-localized RFP-GFP), and the green signal is best visualized in Figure 6C. Notably, the intense GFP-green in the microglial cells makes it difficult to visualize GFP-green signal in monocytes, without over-exposing the microglial cells. Inspection of Figure 6D confirms that cell ‘‘1’’, as well as the vast majority of RFP+ cells, are 7/4+ and thus likely represents a classical CCR2+ inflammatory monocyte. Cell ‘‘2’’ in Figure 6A is 7/42, RFP+, and GFP+ and is likely a to be a CX3CR1hi, CCR2lo, Ly6Clo monocyte. Cell ‘‘3’’ is 7/42, RFP+, but GFP2 and is interpreted as likely to be a T cell. Cell ‘‘4’’ is 7/ 4 2 , GFPbright,and RFP2 and has the morphological appearance of a microglial cell. Discussion In this study we created CCR2-RFP knock-in reporter mice and bred them with CX3CR1-GFP mice. Our goals were to quantify CCR2 and CX3CR1 expression in defined monocyte subsets in the blood to visualize infiltrating and resident myeloid cells in naive and challenged mice and to determine how CCR2 contributes to the accumulation of Ly6Chi/CCR2+ and Ly6Clo/ CX3CR1+ monocytes during autoimmune tissue inflammation in the CNS. Circulating blood monocytes in the Ccr2RFP/+ mice were uniformly positive for RFP by FACS, and approximately 80% expressed cell- surface CCR2, whereas CCR2 surface-negative monocytes were predominantly Ly6Clo, as expected. Mean RFP intensity was approximately twofold higher in Ly6Chi than Ly6Clo monocytes, consistent with reports of relative CCR2 mRNA levels in these populations [3]. The presence of RFP in Ly6Clo monocytes that expressed little or no surface CCR2 protein (.80% of monocytes in this population) raised the possibility that CCR2 was present in the cytoplasm but failed to traffic to the cell surface. However, permeabilization experiments with Ly6Clo monocytes did not reveal substantial intracellular stores of CCR2. Thus, Ly6Clo monocytes transcribe CCR2 but fail to translate it into protein. Similar results were seen in NK cells and T cells (data not shown). Additional signals might initiate CCR2 translation in these monocytes and thus provide the potential to rapidly upregulate CCR2 expression. To investigate the functions of monocyte subsets, we crossed the CCR2-RFP mice with CX3CR1-GFP knock-in mice [23] to create Ccr2RFP/+Cx3cr1GFP/+ mice. These ‘‘two-color’’ mice were examined in three pathological settings: Apoe deficiency on a high- fat diet, thioglycollate induced-peritonitis (localized sterile acute inflammation), and EAE (localized organ-specific autoimmune inflammation). Overall, the findings support the notion that CCR2 and CX3CR1 define functionally distinct monocyte subsets and PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13693