LPS pretreatment suppressed CNV formation.

<p>Peritoneal injection of low-dose LPS (20 µg) was performed at 4, 3, 2 or 1 days (respectively, Day -4, -3, -2 and -1) before laser irradiation (Day 0), or at 2 days after laser irradiation (Day +2), and the CNV size was evaluated at 10 days after laser treatment (Day 10) (A). In all groups of LPS-pretreated mice, the size of CNV was significantly smaller than that in control mice (B, C). The smallest CNV was shown in the mice given LPS pretreatment 2 days before laser treatment. The bars show means ± SEM. <i>n</i> = 6 mice/group, *<i>P</i> = 0.002 compared with control.</p

Adoptive transfer of LPS-treated peritoneal macrophages suppressed CNV formation as well as did LPS pretreatment.

<p>The donor mice were injected with LPS (20 µg/PBS 200 µl) or PBS (200 µl) at Day -2. At Day 0, laser treatment was performed on the recipient mice. After that, peritoneal macrophages were harvested from the donor mice and 2×10⁶ or 1×10⁶ macrophages were transferred into the peritoneal cavity of the recipient mice (A). For comparison, laser treatment was performed in PBS-pretreated mice and LPS-pretreated mice without adoptive transfer. In the LPS-pretreated mice and the recipient mice with 2×10⁶ macrophages from LPS-treated donor mice, CNV was significantly smaller than that in the control mice and the recipient mice with macrophages from PBS-pretreated donor mice (B). The bars show means ± SEM. <i>n</i> = 6 mice/group, *<i>P</i> = 0.003 compared with control.</p

Anti-IL-10 antibody inhibited the CNV inhibitory effect of LPS pretreatment.

<p>Peritoneal injection of anti-IL-10 neutralizing antibody (IL-10Ab) inhibited the CNV inhibitory effect of LPS pretreatment in LPS-treated mice. The bars show means ± SEM. <i>n</i> = 6 mice/group, *<i>P</i> = 0.01.</p

LPS treatment increased serum IL-10 concentration and IL-10 expression in peritoneal macrophages and in the eye.

<p>After peritoneal injection of low-dose LPS, IL-10 expression in the peritoneal macrophages (A) and in the posterior part of the eye (the retina, RPE and choroid) (B) increased, approximately 8-fold and 4-fold, respectively, two days after LPS injection. The bars show means ± SEM. <i>n</i> = 6 mice/group, *<i>P</i><0.001 compared with control. Serum IL-10 concentration increased (C). <i>n</i> = 6, *<i>P</i><0.001 compared with baseline. It reached a peak on day 1 and gradually decreased. A significant increase was shown for at least 4 days.</p

Macrophages detected by immunohistochemistry of choroid-RPE flat mounts and flow cytometry.

<p>(<b>A</b>) Immunohistochemistry of macrophages in choroid-RPE flat mounts on Day 3. After photocoagulation, a large number of macrophages accumulated at the laser injury sites. INCB3344 suppressed this increase. Scale bar = 100 µm. (<b>B</b>) Left: Overlay histogram of flow cytometric results. Right: Flow cytometric analysis data with F4/80 staining of the macrophages in choroid-RPE on Day 3 after laser photocoagulation (Macrophage numbers per choroid-RPE complex). After photocoagulation, the number of macrophages significantly increased compared with no laser photocoagulation controls (relative to normal control, ^*<i>P</i><0.001 <i>n</i> = 5, ^**<i>P</i><0.001 <i>n</i> = 5). INCB3344 treatment significantly reduced the number of macrophages compared to the vehicle-treated group (^***<i>P</i><0.001, <i>n</i> = 5).</p

VEGF expression.

<p>(<b>A</b>) Immunohistochemistry of macrophages (green) and VEGF (red) in cryosections on Day 3. Significantly higher levels of VEGF were expressed in macrophages at the photocoagulated sites. VEGF localized mainly in infiltrating macrophages at the laser injury sites. INCB3344 apparently decreased VEGF immunoreactivity compared to vehicle treatment. The negative control omitting the primary antibody (second antibody only) had detectable auto-fluorescence in RPE. Scale bar = 100 µm. (<b>B</b>) VEGF protein levels in the choroid-RPE were quantitatively measured by ELISA. VEGF levels on Day 3 were significantly suppressed by INCB3344 treatment. (<i>n</i> = 8, *<i>P</i> = 0.012). (<b>C</b>) The expression of VEGF mRNA derived from macrophages isolated from choroid-RPE complexes was detected by real-time PCR on Day 3 after photocoagulation. The increased VEGF mRNA expression in infiltrating macrophages was significantly suppressed by INCB3344 treatment (**,***<i>P</i><0.001, <i>n</i> = 3).</p

Effect of INCB3344 on CNV formation.

<p>(<b>A</b>) Haematoxylin–eosin-stained light micrograph of CNV lesions on Day 14 after laser photocoagulation. Each photograph shows the central area of CNV lesions in vehicle-treated or INCB3344-treated mice. Scale bar = 100 µm. (ILM: internal limiting membrane; NFL: nerve fiber layer; GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS: inner segment; OS: outer segment; RPE: retinal pigment epithelium; C: choroid; S: sclera). (<b>B</b>) Representative micrographs of CNV lesions in the choroid-RPE flat mounts from laser-induced CNV in mice treated with vehicle or INCB3344. CNV areas were perfused with fluorescein isothiocyanate-dextran in flat-mount choroid-RPE complex. Scale bar = 100 µm. (<b>C</b>) Quantitative analysis of CNV size. Values are mean ± SE, vehicle, <i>n</i> = 78 spots, INCB3344, <i>n</i> = 81 spots. *<i>P</i><0.001.</p

Phosphorylated extracellular signal-regulated kinase (p-ERK1/2) expression in Western blot.

<p>(<b>A</b>) A representative blot. p-ERK expression in the choroid-RPE complex from vehicle-treated mice and INCB3344-treated mice on Day 3 after laser photocoagulation, and normal mice (without photocoagulation). Western blot analysis revealed that p-ERK expression increased after laser photocoagulation and was suppressed by INCB3344 treatment. (<b>B</b>) Semi-quantitative analysis of the band intensity showed an increase in relative p-ERK expression (values normalized to total ERK expression) in the eyes of photocoagulated mice compared with untreated mice(<i>n</i> = 8, *<i>P</i><0.001; <i>n</i> = 8, **<i>P</i><0.001), and INCB3344 suppressed this increase (<i>n</i> = 8, ***<i>P</i><0.001).</p