Macrophage phenotype and function in CCL2^−/−CX3CR1^GFP/GFP DKO mice.

<p>Bone marrow-derived macrophages (BM-DMs) were cultured from WT and CCL2^−/−CX3CR1^GFP/GFP mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061381#s2" target="_blank">Materials & Methods</a>). Cells were then used for gene expression and phagocytosis assay. In some experiments, BM-DMs were further polarized into M1 (with LPS) or M2 (with IL-4) macrophages. (A) Flow cytometry analysis showing F4/80 and CD11b expression in BM-DMs. (B) Real-time RT-PCR analysis of immune related genes in undifferentiated M0 macrophages from WT and DKO mice under normoxia culture condition. Gene expression level was normalised by housekeeping gene (18s). (C) Real-time RT-PCR analysis of immune related genes in different subsets of BM-DMs (M0, M1, and M2) from WT and DKO mice under hypoxia conditions (1% oxygen). Data shown are mRNA fold changes versus WT M0 BM-DMs under normoxia conditions. *, P<0.05; **<0.01, compared WT BM-DMs, n = 3. (D) Phagocytosis of M0 BM-DMs from WT and DKO mice determined by the pHrodo™ E. coli Bioparticles® Phagocytosis assay. *, P<0.05; **<0.01, compared to DKO BM-DMs at the same time point. N = 6.</p

Transmission electron microscopic (TEM) images of mouse eyes.

<p>(A) Image from an 18-month WT mouse. (B) Image from an 18-month CCL2^−/−/CX3CR1^GFP/GFP mouse. (C) The average thickness of Bruch membrane in WT and CCL2^−/−/CX3CR1^GFP/GFP mice. N = 6 (eyes). (D) Vacuolated changes in RPE mitochondria from CCL2^−/−/CX3CR1^GFP/GFP mice. (E) Vacuolated changes (asterisks) in RPE cells in CCL2^−/−CX3CR1^GFP/GFP mice. (F) Photoreceptor inner segment damage (arrow) in CCL2^−/−CX3CR1^GFP/GFP mice. (G) Photoreceptor inner segment damage (arrows) and outer segment disorientation (arrowheads) in an area without vacuolated RPE damage. BM, Bruch membrane; CP, choroidal capillaries; RPE, retinal pigment epithelium.</p

Primer sequences for real time RT-PCR.

<p>Primer sequences for real time RT-PCR.</p

Retinal GFAP expression in WT and CCL2^−/−CX3CR1^GFP/GFP mice.

<p>Cryosections of mouse eyes were stained for GFAP (red) and DAPI (blue) and observed by confocal microscopy. (A) 3-month WT mice. (B) 3-month CCL2^−/−CX3CR1^GFP/GFP mice. (C) 18-month WT mice, (D) 18-month CCL2^−/−CX3CR1^GFP/GFP mice. GL, ganglion layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar = 20 µm.</p

Retinal lesion in mice that were exposed to 800lux light.

<p>Retinal lesion in mice that were exposed to 800lux light.</p

Immune response in WT and CCL2^−/−CX3CR1^GFP/GFP mice.

<p>(A–C) flow cytometry analysis of blood cells (gated on CD45⁺ cells) from 3-month (A) and 18-month (B) old WT and CCL2^−/−CX3CR1^GFP/GFP mice. (C) The expression levels of MHC-II (geo-mean) in CD45⁺ blood cells in different groups of mice. (D) Real-time RT-PCR analysis of retinal gene expression. *, P<0.05; **<0.01 compared to WT mice, n = 6, unpaired Student t test.</p

Choroidal neovascularisation in CCL2^−/−CX3CR1^GFP/GFP DKO mice.

<p>(A) CNV was induced in adult (8∼12-week) WT and CCL2^−/−CX3CR1^GFP/GFP mice. RPE/choroidal flatmounts were collected at day 10 and stained for isolectin B4 (green) and collagen IV (red) and examined by confocal microscopy. (B) CNV size in WT and CCL2^−/−CX3CR1^GFP/GFP mice at day 10. N = 6. (C, D) VEGF-A mRNA expression in RPE cells (C) and BM-DMs (D) from WT and CCL2^−/−CX3CR1^GFP/GFP mice. N = 5.</p

Retinal lesion in different experiments under normal housing conditions.

*<p>, Light intensity was the average of measurements taken from 5 different areas inside the cage: front, middle, rear, under the food, under the water box.</p

Laser-induced CNV lesions are attenuated in RAGE−/− mice.

<p>Representative images demonstrating laser-burned spots immediately (A) and 7 days after photocoagulation (B) in RAGE−/− mice and WT controls. A) As assessed by cSLO, the retina from WT and RAGE−/− mice shows laser burn sites immediately after photocoagulation. The left image in each pair is fluorescein angiography and the right image is infrared reflectance. There is no obvious difference between the two animal groups with comparable leakage at the lesion (black arrow). B) 7 days after photocoagulation the CNV lesions are apparent in angiograms and infrared reflectance fundus images from WT mice although these are smaller in the retina of RAGE−/− mice (white arrows). C) Comparison of CNV lesion size between the WT and RAGE−/− mice. Retinal flat mounts were evaluated for the presence and size of clearly demarcated isolectin positive CNV lesions one week post-laser injury. RAGE −/− mice exhibited significantly less CNV than age matched controls (n = 12 animals/group, *p<0.05) (Scale bar = 100 µm).</p

Retinal lesion in CCL2^−/−CX3CR1^GFP/GFP mice.

<p>(A–F) Fundus image from 6-month (A), 12-month (B, C), 18-month (D), 10-month+light (E) CCL2^−/−CX3CR1^GFP/GFP mice, and 18-month WT mice (F). Arrows indicate retinal lesions. (G, H) Indocyanine green angiography showing retinal (G) and choroidal (H) circulation of an 18-month old CCL2^−/−CX3CR1^GFP/GFP mouse. CNV was not observed in CCL2^−/−CX3CR1^GFP/GFP mice.</p