Macrophage responses to hypoxia.

<p>Splenic F4/80⁺ macrophages from IL-10^−/− mice (A) significantly inhibit (*P = 0.0163) the proliferation of HMVECs following exposure to hypoxia compared to hypoxia-treated wild-type macrophages. Splenic F4/80⁺ macrophages from wild-type mice do not significantly (P>0.05) inhibit HMVEC proliferation compared to untreated HMVECs. This could be due to (B) increased expression of VEGF in wild-type macrophages compared to IL-10^−/− macrophages. The IL-10 signaling pathway appears to be activated following exposure to hypoxia. RAW 264.7 murine macrophages (C) do not exhibit increased levels of phosphorylation of STAT3 under normoxic conditions (open histogram) compared to IgG stained cells (shaded histogram). However, (E) exposure to hypoxia (open histogram) results in a significant increase in phosphorylation of STAT3 compared to normoxia-treated cells (shaded histogram). This increase in phosphorylation of STAT3 is at levels similar to (D) RAW 264.7 cells exposed to recombinant IL-10 protein for 10 min (open histogram) versus normoxic cells (shaded histogram). Inset numbers indicate percentage of positive cells above normoxic controls. Phosphorylation of STAT3 protein following hypoxia in RAW 264.7 cells was also confirmed with (F) western blot analysis. Phosphorylation of STAT3 occurred following exposure to both recombinant IL-10 protein for 10 min and a 24 hour exposure to hypoxia. Probing of total STAT3 protein was used as a loading control. These findings in RAW macrophages were also observed in primary macrophages (G). Wild-type macrophages treated with recombinant IL-10 protein for 10 minutes or hypoxia for 24 hours demonstrated increased phosphorylation of STAT3 compared to baseline normoxic levels. IL-10^−/− macrophages, however, demonstrated decreased levels of pSTAT3 at baseline normoxic levels compared to wild-type normoxic macrophages, and did not upregulate pSTAT3 in response to hypoxia. STAT3 signaling is still intact in IL-10^−/− macrophages, as phosphorylation of STAT3 in increased in IL-10^−/− macrophages following stimulation with recombinant IL-10 protein. This increase in STAT3 phosphorylation may be due to the (H) significantly increased (*P = 0.0123) production of IL-10 protein by RAW 264.7 macrophages following exposure to hypoxia compared to normoxia-treated RAW macrophages.</p

IL-10^−/− mice demonstrate significantly reduced retinal neovascularization in response to ischemia.

<p>C57BL/6 and IL-10^−/− were exposed to 75%±2% O₂ from day P7 to P12. Mice were returned to normoxic conditions for 5 days, and on P17 animals were perfused with FITC-dextran, eyes harvested, and retina flatmounts made. Fluorescent microscopy of perfused retinas of (A) normoxic-treated wild-type mice (n = 5), (B) oxygen-treated wild-type mice (n = 5), and (C) oxygen-treated IL-10^−/− mice (n = 4) reveal decreased angiogenesis and increased areas of non-perfusion in IL-10^−/− mice. This experiment was repeated 2 additional times with similar results (D–G) H&E staining of ocular tissue sections from C57BL/6 oxygen-treated mice exhibit extensive preretinal neovascular loops (arrows), whereas (H–K) ocular tissue sections from IL-10^−/− oxygen-treated mice demonstrate a significant reduction in preretinal neovascular loops. Images were acquired at both 40× (D,F,H,J) and 100× (E,G,I,K). (L) The total area of retinal vascularization was quantified using Metamorph™ software as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003381#s4" target="_blank">Materials and Methods</a>, and plotted as a bar graph. IL-10^−/− mice exposed to oxygen had significantly reduced (**P = 0.0006) retinal vascularization compared to wild-type mice exposed to oxygen. (M) A bar graph represents the mean±SD number of neovascular loops of 10 separate sections, with IL-10^−/− mice demonstrating reduced neovascular loops (**P = 0.0071) compared to wild-type mice following OIR.</p

F4/80⁺ macrophages infiltrate ischemic retinas of both C57BL/6 and IL-10^−/− mice.

<p>Retinal flatmounts of P17, FITC-dextran perfused C57BL/6 normoxia-treated, C57BL/6 oxygen-treated, and IL-10^−/− oxygen-treated retinas were stained with Allophycocyanin (APC) anti-mouse F4/80 antibody. In contrast to the paucity of cells in the (A) normoxic wild-type retina, a significant number of F4/80⁺ macrophages are observed in the retinas of both (B) C57BL/6 oxygen-treated and (C) IL-10^−/− oxygen-treated mice. (D, E, F) Examining both macrophage infiltrate and blood vessels reveals that APC-labeled macrophages reside primarily along FITC-dextran perfused blood vessels. Since IL-10 did not prevent macrophage infiltration into the retina, we examined the genetic profile of retinal macrophages. Using splenic wild-type macrophages as a baseline, retinal macrophages from wild-type mice exhibit significantly (p<0.05) increased expression of the pro-angiogenic gene (G) nitric oxide compared to IL-10^−/− retinal macrophages following OIR, suggesting that IL-10 promotes angiogenesis by polarizing macrophages towards a pro-angiogenic phenotype.</p

GLUT1 is up-regulated in SIRT6-KO retina.

<p>a) GLUTl immunoreactivity in cross-section of WT and SIRT6-KO mice retina. Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner nuclear Layer (INL) Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Retinal Pigment Epithelium (RPE). GLUT1 protein (b) and mRNA levels (c) were determined by Western blot and RT-PCR respectively. β-actin was used as loading control. Data are mean ± SE (n = 6 eyes/group) **p<0.01</p

SIRT6 is active in the mouse retina.

<p>a) H3K56 acetylation is shown by immunofluoescence. b) Representative Western blot showing protein levels of SIRT6 and the acetylation levels of H3K56 and H3K9 in chromatin preparations from WT and KO mice retinas. Total H3 was used for normalization. c) Quantification of the intensity of bands was determined by using the ImageJ and is represented as arbitrary units. Data are mean ± SE (n = 6 eyes/group). **p<0.01, ***p<0.001</p

Grm6 is down-regulated in SIRT6-KO retinas.

<p>Whole retina mRNA from WT and KO mice was used to profile the expression of several key genes of glutamate receptors involved in the synaptic transmission in an Affymetrix Mouse Gene 2.1 ST DNA microarray. a) Heatmap representing the hierarchical cluster analysis shows the differential expressed mRNAs between WT and SIRT6 KO retinas. The graphic depicts the expression levels of ionotropic AMPA glutamate receptors (Gria1–4), Glutamate receptor, ionotropic kainate (Grik1-2-4-5), Glutamate [NMDA] receptors (Grin1-2a-c) and metabotropic glutamate receptors (Grm1–8). The expression data for the hierarchical clustering image has been row normalized to a range of zero to one with blue representing the row minimum and red representing the row maximum. b) RNA was purified from SIRT6 WT and KO retinas, and Grm6 levels analyzed by RT-PCR. c) immunofluorescence was performed in SIRT6 WT and KO retinas with the indicated antibodies. PKC-alpha was used as a marker for ON bipolar cells. Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner nuclear Layer (INL), Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Retinal Pigment Epithelium (RPE). Data are mean ± SE (n = 4) **p<0.01 d) Representative fluorescent images of TUNEL analysis performed in WT and SIRT6 KO retinal sections. Apoptotic nuclei (bright green dots) labeled with fluorescein-dUTP were visualized by fluorescence microscopy. Data are mean ± SE (n  = 3) **p<0.01</p

Retinal functional evaluation.

<p>Representative scotopic (A) and photopic (C) electroretinograms from WT and SIRT6-KO mice at different light intensities (dBs). Plots B and D depict average amplitudes of <i>a</i>-wave and <i>b</i>-wave. Note that the fold decrease of the scotopic <i>a</i>-wave amplitude (8) is greater than the fold decrease of the photopic <i>a</i>-wave amplitude (2,5). Data are mean ± SE (n =  4). **p<0.01, ***p<0.001.</p

Impaired autophagy in macrophages promotes inflammatory eye disease

<p>Autophagy is critical for maintaining cellular homeostasis. Organs such as the eye and brain are immunologically privileged. Here, we demonstrate that autophagy is essential for maintaining ocular immune privilege. Deletion of multiple autophagy genes in macrophages leads to an inflammation-mediated eye disease called uveitis that can cause blindness. Loss of autophagy activates inflammasome-mediated IL1B secretion that increases disease severity. Inhibition of caspase activity by gene deletion or pharmacological means completely reverses the disease phenotype. Of interest, experimental uveitis was also increased in a model of Crohn disease, a systemic autoimmune disease in which patients often develop uveitis, offering a potential mechanistic link between macrophage autophagy and systemic disease. These findings directly implicate the homeostatic process of autophagy in blinding eye disease and identify novel pathways for therapeutic intervention in uveitis.</p