Figure 5. Peripheral blood‐derived origin of myeloid cells contributing to homeostatic turnover of eye macrophages.

• A
  Left, Experimental setup of surgically connected parabiotic mice. Acta1 ^GFP/+ and Acta1^+/+ mice underwent parabiosis for 2, 12, and 20 weeks before analysis. Right, quantification of GFP⁺Iba1⁺ microglia in the retina (rMG, squares, n.d. = not detectable), ciliary body (cbMΦ, triangles, Mann–Whitney ns P > 0.05), and cornea (cMΦ, circles, Kruskal–Wallis **P = 0.0024) of parabiotic mice. Blood chimerism of CD45⁺CD11b⁺Ly6C^hiGFP⁺ cells in the analyzed wild‐type mice was 37.7 ± 3.2% (2 weeks), 27.5 ± 2.7% (12 weeks), and 34.7 ± 3.7% (20 weeks). Symbols represent mean ± s.e.m. of three (2 weeks), four (12 weeks) or five (20 weeks) individual mice. Scale bars represent 50 µm.
• B–D
  Representative immunofluorescence images from the retina (20 weeks), ciliary body (12 weeks), and cornea (20 weeks) from flat mounts of Acta1 ^+/+ parabiotic mice. GFP⁺Iba1⁺ double‐positive cells are marked by arrows, GFP⁻Iba1⁺ single‐positive cells are labeled by asterisks and GFP⁺Iba1⁻ leukocytes are indicated by arrow heads. Pictures are representative of three animals.
• E
  Flow cytometric quantification of RFP⁺ cells in Ccr2‐RFP mice among CD45⁺CD11b⁺CD115⁻Ly6C^int granulocytes (triangles, n = 4), CD45⁺CD11b⁺CD115⁺Ly6C^lo monocytes (filled squares, n = 4 mice), CD45⁺CD11b⁺CD115⁺Ly6C^hi monocytes (open squares, n = 4 mice), CD45^loCD11b⁺ rMG (squares, n = 6 mice), CD45⁺CD11b⁺CD64⁺F4/80⁺ cbMΦ (triangles, n = 6 samples from 12 mice), and CD45⁺CD11b⁺CD64⁺F4/80⁺ cMΦ (filled circles, n = 12 mice, unpaired t‐test ***P < 0.0001). Data were obtained from one (rMG) or two independent experiments (blood, cbMΦ, cMΦ). Data are presented as means ± s.e.m.
• F–H
  Flow cytometry of eye macrophages from healthy Ccr2 ^RFP/+ mice (left) and representative histograms (right, Ccr2 ^RFP/+ solid red line, Ccr2 ^+/+ controls dotted gray line).
• I
  Flow cytometry of myeloid blood cells from Ccr2 ^RFP/+ mice. Left: CD45⁺CD11b⁺ cells were further subdivided according to the expression of Ly6C and CD115 into CD45⁺CD11b⁺CD115⁻Ly6C^int granulocytes (gate 1), CD45⁺CD11b⁺CD115⁺Ly6C^hi inflammatory (gate 2), and CD45⁺CD11b⁺CD115⁺Ly6C^lo resident monocytes (gate 3), respectively. Right: representative histograms are shown (Ccr2 ^RFP/+ solid red line, Ccr2 ^+/+ controls dotted gray line). Four mice were investigated.
• J
  Top left, sketch of Ccr2‐RFP construct. Top right, typical confocal picture for CCR2 (red), Iba1 (green), and collagen IV (Coll IV, white) revealing Ccr2‐RFP expression in a blood vessel (arrow, left image, scale bar represents 20 µm) and no RFP signal in retinal microglia (right images, scale bar represents 100 µm). Bottom, representative pictures of the ciliary body and cornea immunolabeled with F4/80 (green), CCR2 (red), and DAPI (blue). Arrows indicate RFP⁺F4/80⁺ cells. Asterisks point to RFP⁻F4/80⁺ cells. Representative images from two independent experiments with three mice are displayed. Scale bars represents 20 µm.