Ly6C^high monocytes reduce Ly6C expression, exhibit crawling behavior, and upregulate markers of alternative activation after infection.

<p>(<b>A</b>) Contour plots display Ly6C expression on monocytes isolated from <i>CX₃CR1^GFP/+</i> mice and transferred into uninfected or infected C57BL/6 mice (as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004080#ppat-1004080-g006" target="_blank">Figure 6</a>). Lines serve as guides to show the level of Ly6C expression. (<b>B</b>) Intravital confocal microscopy of an infected <i>Cx₃cr1^gfp/+</i> mouse showing crawling GFP+ cells (green) that are either Ly6C⁺ (white arrows) or Ly6C⁻ (yellow arrows) after intravenous anti-GR-1 (Ly6C/G) Ab labeling (red). (<b>C</b>) Time-lapse images from an 8-week infected <i>Cx₃cr1^gfp/+</i> mouse injected with anti-Ly6C/Ly6G Ab showing the movement of GFP+ Ly6C+ cells (white arrows), GFP+ Ly6C− cells (yellow arrows) and GFP− GR-1+ neutrophils (red arrows). (<b>D</b>) Tracks of GFP+ Ly6C+ cells (white lines), GFP+ Ly6C− cells (yellow lines) shown in the time lapse. GFP is shown in green, anti-Ly6C (GR-1) in red, and nuclei in blue. Scale bars = 50 µm. Data is representative of experiments from 2 mice. (<b>E–F</b>) Representative histograms (<b>E</b>) and graph (<b>F</b>) display the percentage of PD-L2+GFP+Ly6Chigh and PD-L2+GFP+Ly6Clow cells from the blood (by <i>in vivo</i> CD45 staining) of 6-week infected mice (black) or uninfected mice (gray). Cells are gated on single, live, lineage negative, <i>in vivo</i> CD45+, GFP+ cells. Data is representative of two independent experiments. N = 4 mice/group.