Phenotype and function of myeloid-derived cells in different strains of mice.

<p>A, Bone marrow and blood cells collected from different strains of mice were stained for different cell surface markers and analysed by flow cytometry. Mean ± SEM, N = 6. B, Cytokine gene expression in LPS stimulated BM-DMs of different strains of mice. Mean ± SEM, N = 3. *, P<0.05; **, P<0.01; compared to WT cells. Unpaired Student t test. Experiments were performed four times. C–D, Nitrotyrosine (green) and PI (red) staining in the eye of a 20-month old WT mouse (C) and a 20-month old CCL2 KO mouse (D). E, isotype control staining. Ch, choroid; RPE, retinal pigment epithelium. F, Endocytosis of BM-DMs using dextran method (see Materials & Methods). MFI, mean fluorescence intensity. G, Phagocytosis of E. coli by BM-DMs (see Materials & Methods). Mean ± SEM, N = 3. *, P<0.05, **, P<0.01 compared to WT cells of the same time point. Dunnett Multiple comparison test. Experiments were performed twice.</p

TEM images of RPE/photoreceptor of different strains of mice.

<p>A, an image from a 22-month old WT mouse showing RPE basal laminar deposits (asterisk). B, an image from 22-month old CCR2 KO mouse showing a photoreceptor outer segment (arrowhead) in parallel with RPE cells, multiple vacuoles (white arrows) in RPE cells and basal laminar deposits (asterisk). C, an image from a 22-month old CCL2 KO mouse showing multiple vacuoles in RPE cells (white arrows) and basal laminar deposits (asterisk). D, an image from a 24-month old CCL2 KO mouse showing multiple vacuoles and reduced melanin granules in degenerated RPE cells, basal laminar deposits (asterisk). E, an image from a 24-month old CCR2 KO mouse showing photoreceptor inner segment degeneration (black arrows), and a gap between RPE cells and photoreceptor outer segments. F, an image from a 20-month old CCL2 KO mouse showing photoreceptor inner segment degeneration (black arrows), reduced melanin granules in RPE cells and RPE basal laminar deposition (asterisk). G, an image from a 20-month old CCL2 KO mouse showing a patch of RPE atrophy, disorganised photoreceptor outer segments, a lack of choriocapillaris, and fibrotic tissues in the choroid (black asterisks). H and I, images from a 24-month old CCL2 KO mouse showing the loss of electron-dense materials in the cytoplasm, the loss of cytoplasm organelles and membrane, and a nucleus with reduced electron-dense materials in the RPE layer; choriocapillaris basal laminar deposition in the choroid; a layer of melanin granule-containing cells (block arrowheads) on top of the degenerated RPE cells with their process extending towards the photoreceptor outer segments. RPE, retinal pigment epithelia. (J), A confocal image of RPE/choroidal flatmount of a 24-month CCL2 KO mouse showing accumulation of macrophages/microglia at the site of RPE damage. RPE damages were highlighted with disorganised actins (phalloidin staining) and subretinal macrophages were labelled with Iba-1.</p

C3d and C5b-9 deposition at the retina/choroidal interface of different strains of mice.

<p>Cryosections of eyes from 22–24-month old mice were stained for C3d (red) or C5b-9 (green in F and G) and observed by confocal microscopy. A, an image from a 22-momth old WT mouse. B, an image from a 22-momth old CCR2 KO mouse. C, an image from a 22-momth old CCL2 KO mouse. D and E, images taken from a 24-momth old CCL2 KO mouse showing area of RPE cell death (asterisks) and extensive C3d deposition (red). F and G, images taken from a 24-momth old CCR2 KO mouse (F) and a 24-month old CCL2 KO mouse showing areas of RPE cell death (arrows) with C3d deposition (red), but no C5b-9 deposition (green). H, a retina from a mouse with EAU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022818#pone.0022818-Xu2" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022818#pone.0022818-Copland1" target="_blank">[35]</a> was used as positive control for C5b-9 staining (green). I, Isotype control antibody staining for C3d in a 24-month CCL2 KO mouse showing no background staining. ONL, outer nuclear layer. RPE, retinal pigment epithelia. Ch, choroid.</p

Histology of mouse eyes.

<p>Mouse eyes were taken from 18–24 months old mice and processed for H-E staining. A–C, representative images of a 22-month WT (A) a 22-month CCL2 KO (B) and a 22-month CCR2 KO (C) mouse showing photoreceptors. D & E, quantitative analysis of the number of photoreceptor nuclei. D, a schematic image showing the locations where the photoreceptor nuclei were counted. E, the numbers of photoreceptor in 20–24 months old mice WT, CCL2- or CCR2-deficient mice. *, P<0.05 compared to the WT mice at the same location . Mean ± SEM, n = 8–10 eyes, ANOVA (Kruskal-Wallis test) followed by Dunn's multiple comparison test. F–I, Representative images from aged CCL2 KO (F), CCR2 KO (G), and WT (H) mice showing RPE vacuolation (arrows). I, the number of RPE vacuoles in different strains of mice. Mean ± SEM, n = 12 eyes, *, P<0.05; **, P<0.01. ANOVA Dunn's multiple comparison test. J & K, retinal images from a 20-month (J) and a 24-month (K) CCL2 KO mouse showing areas of RPE cell damage (arrowheads) and inflammatory cell infiltration (insert in J). A layer of pigmented cells on top of degenerated (damaged) RPE cells was observed in K.</p

Subretinal microglia in different strains of mice.

<p>A–D, Autofluorescent (AF) images of a 3-month WT mouse (A), a 24-month WT mouse (B), a 24-month old CCR2 KO mouse, (C) and a 24-month CCL2 KO mouse (D). E–G, confocal images of RPE flatmounts stained for microglia with Iba-1 antibody (see Methods) from a 20-month old WT mouse (E), a 20-month old CCR2 KO mouse (F) and a 20-month old CCL2 KO mouse (G). (H) The number of subretinal Iba-1⁺ microglia in 20-month old of different strains of mice. Mean ± SD, N = 8∼12. *, P<0.05 compared to WT, ANOVA Dunn's multiple comparison test. I, a TEM image from a CCL2 KO mouse showing a subretinal microglial cell (MG) with lipofuscin (arrows) on the surface of RPE cells.</p

Primers used in real-time PCR.

<p>Primers used in real-time PCR.</p

Phenotypes and functions of BM-DCs of different strains of mice.

<p>A, The expression of CD11b and CD11c in bone marrow-derived dendritic cells (BMDCs) of different strains of mice. B, cytokine production by immature BM-DCs (iDC) and mature BM-DCs (mDC) of different strains of mice. C, endocytosis of FITC-dextran by iDC of different strains of mice. Mean ± SEM, n = 4. *, P<0.05, **, P<0.01 in comparison to WT cells. n = 4, unpaired Student t test.</p

Chemotaxis of bone marrow-derived dendritic cells (BMDCs).

<p>A, migration of BMDCs of WT mice in response to different stimuli. *, P<0.05, **, P<0.01 in comparison to RPMI. Mean ± SEM, n = 3, Unpaired Student t test. B, the migration of BMDCs of different strains of mice in response to RPMI, RPE supernatant, TNF-a treated RPE supernatant and chemokine CCL21. *, P<0.05, **, P<0.01 in comparison to WT cells. Mean ± SEM, n = 3, Unpaired Student t test. Experiments were repeated twice.</p

Phenotype of subretinal macrophage/microglia.

<p>RPE/choroidal flatmounts from aged (20–24 months) WT (A, C) and CCL2 KO (B, D) mice were dual stained for Iba-1/arginase-1 (A, B), or CD68/P2Y12 (C, D) and observed by confocal microscopy. Images presented are representatives from 6 mice in each group. E, Z-stack images of a retinal flatmount stained for Iba-1 (green) and Propidium iodide (PI) showing three Iba-1⁺ cells in the photoreceptor outer segment layer. Arrows: cell dendrites pointing toward the inner retina in z-sections.</p

Retinal degeneration in the Ins2^Akita mouse.

<p>(A) SD-OCT image from 9-month old control retina. Retinal thickness was measured at 300, 600 and 900 µm eccentricities in dorso-ventral and nasal-temporal retinal regions, from NFL-OS (total retinal thickness), NFL-INL (inner retinal thickness) and OPL-OS (outer retinal thickness). Isodensity maps (B) and quantitative analysis (C) of total retinal thickness from control and Ins2^Akita mice at different ages. (D) The cumulative reduction of outer, inner and total retinal thickness in different ages of control and Ins2^Akita mice. Fundus (E, H) and OCT images (F-G, I-J) taken from 9-month old control (A-C) and Ins2^Akita mice (D-F). (B-D) n = 6 mice per strain/age, *<i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 compared to control mice of the same age. Student's <i>t</i>-test. RPE, retinal pigment epithelium; IS/OS, photoreceptor inner/outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL/NFL, ganglion/nerve fibre layer.</p