Fig 2: Peripheral blood-derived origin of myeloid cells contributing to homeostatic turnover of eye macrophages ALeft, 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 (cbMF, triangles, Mann–Whitney ns P > 0.05), and cornea (cMF, circles, Kruskal–Wallis **P = 0.0024) of parabiotic mice. Blood chimerism of CD45+CD11b+Ly6ChiGFP+ 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–DRepresentative 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.EFlow cytometric quantification of RFP+ cells in Ccr2-RFP mice among CD45+CD11b+CD115-Ly6Cint granulocytes (triangles, n = 4), CD45+CD11b+CD115+Ly6Clo monocytes (filled squares, n = 4 mice), CD45+CD11b+CD115+Ly6Chi monocytes (open squares, n = 4 mice), CD45loCD11b+ rMG (squares, n = 6 mice), CD45+CD11b+CD64+F4/80+ cbMF (triangles, n = 6 samples from 12 mice), and CD45+CD11b+CD64+F4/80+ cMF (filled circles, n = 12 mice, unpaired t-test ***P < 0.0001). Data were obtained from one (rMG) or two independent experiments (blood, cbMF, cMF). Data are presented as means ± s.e.m.F–HFlow cytometry of eye macrophages from healthy Ccr2 RFP/+ mice (left) and representative histograms (right, Ccr2 RFP/+ solid red line, Ccr2 +/+ controls dotted gray line).IFlow 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-Ly6Cint granulocytes (gate 1), CD45+CD11b+CD115+Ly6Chi inflammatory (gate 2), and CD45+CD11b+CD115+Ly6Clo resident monocytes (gate 3), respectively. Right: representative histograms are shown (Ccr2 RFP/+ solid red line, Ccr2 +/+ controls dotted gray line). Four mice were investigated.JTop 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.

Fig 3: Flow cytometric gating strategy for eye macrophages and monocytes Gating strategy of eye macrophages. CD11b+ or CD45+ cells underwent doublet exclusion by FSC-W, -A, and -H gating and subsequent dead cell exclusion (fixable viability dye, FVD). Retinal microglia were specified as CD45loCD11b+, macrophages in the ciliary body and cornea as CD45loCD11b+CD64+F4/80+.Gating strategy of blood monocytes. Leukocytes underwent doublet exclusion by FSC-W, -A, and -H gating and subsequent dead cell exclusion (fixable viability dye, FVD). CD45+CD11b+ myeloid blood cells were further subdivided in CD45+CD11b+CD115+Ly6Chi and CD45+CD11b+CD115+Ly6Clo monocytes.

Fig 4: Contribution of definitive hematopoiesis to eye macrophage subsets during steady-state Scheme of a fate mapping experiment using adult Cx3cr1CreERT2:Rosa26-YFP mice. Tamoxifen (TAM) injection was performed at postnatal day 42 (P42). Mice were evaluated at 2, 12, and 24 weeks post-injections. Administration of TAM leads to the excision of a stop sequence flanked by loxP sites (gray triangles) in Cx3cr1 expressing cells in the eye which causes stable YFP expression under the control of the Rosa26 promotor.Flow cytometric measurement of the persistence of YFP+ retinal microglia (rMG), cliliary body (cb) MF, and corneal (c) MF in adult Cx3cr1CreERT2:Rosa26-YFP mice. Doublets and dead cells were excluded by FSC-W and viability dye. Representative flow cytometry plots from two independent experiments with at least six mice are displayed.Kinetics of YFP labeling in macrophages of the healthy eye. Symbols represent means ± s.e.m. rMG are shown as squares (2 weeks: n = 10 mice, 12 weeks: n = 9 mice, 24 weeks: n = 12 mice, Kruskal–Wallis ns P > 0.05), cbMF are depicted as triangles (2 weeks: n = 4 samples from eight mice, 12 weeks: n = 3 samples from six mice, 24 weeks: n = 6 samples from twelve mice, Kruskal–Wallis ns P > 0.05) and cMF as circles (2 weeks: n = 6 mice, 12 weeks: n = 8 mice, 24 weeks: n = 12 mice, one-way ANOVA ***P < 0.0001). Data were obtained from two (cMF: 2 weeks, cbMF: 2 and 12 weeks), three (rMG: 2 weeks, cbMF: 24 weeks), or four (rMG: 12 and 24 weeks, coMF: 12 and 24 weeks) independent experiments.Sketch of the Flt3-dependent Cre-mediated recombination system with excision of the loxP-flanked stop-sequences leading to expression of YFP under the control of the Rosa26 promotor in Flt3Cre:Rosa26-YFP mice.Left: Representative flow cytometric characterization of rMG by CD45 and CD11b and cbMF and cMF by CD45, CD11b, CD64, and F4/80 in Flt3Cre:Rosa26-YFP mice. Doublets and dead cell were excluded. Right: representative flow cytometric images depicting YFP expression in eye tissue macrophages of 12- or 52-week-old Flt3Cre:Rosa26-YFP mice. Typical images were taken from two independent experiments with six to seven mice.Quantification of the percentage of YFP+ eye macrophages at 12 and 52 weeks of age. rMG are shown as squares (12 weeks: n = 7 mice, 52 weeks: n = 6 mice), cbMF as triangles (12 weeks: n = 3 samples from six mice, 52 weeks: n = 3 samples from six mice), and cMF as circles (12 weeks: n = 7 mice, 52 weeks: n = 6 mice). Data are presented as means ± s.e.m. and were acquired in two independent experiments.