Interleukin-10 Overexpression Promotes Fas-Ligand-Dependent Chronic Macrophage-Mediated Demyelinating Polyneuropathy

BACKGROUND:Demyelinating polyneuropathy is a debilitating, poorly understood disease that can exist in acute (Guillain-Barré syndrome) or chronic forms. Interleukin-10 (IL-10), although traditionally considered an anti-inflammatory cytokine, has also been implicated in promoting abnormal angiogenesis in the eye and in the pathobiology of autoimmune diseases such as lupus and encephalomyelitis. PRINCIPAL FINDINGS:Overexpression of IL-10 in a transgenic mouse model leads to macrophage-mediated demyelinating polyneuropathy. IL-10 upregulates ICAM-1 within neural tissues, promoting massive macrophage influx, inflammation-induced demyelination, and subsequent loss of neural tissue resulting in muscle weakness and paralysis. The primary insult is to perineural myelin followed by secondary axonal loss. Infiltrating macrophages within the peripheral nerves demonstrate a highly pro-inflammatory signature. Macrophages are central players in the pathophysiology, as in vivo depletion of macrophages using clodronate liposomes reverses the phenotype, including progressive nerve loss and paralysis. Macrophage-mediate demyelination is dependent on Fas-ligand (FasL)-mediated Schwann cell death. SIGNIFICANCE:These findings mimic the human disease chronic idiopathic demyelinating polyneuropathy (CIDP) and may also promote further understanding of the pathobiology of related conditions such as acute idiopathic demyelinating polyneuropathy (AIDP) or Guillain-Barré syndrome

Interleukin-10 Promotes Pathological Angiogenesis by Regulating Macrophage Response to Hypoxia during Development

Aberrant angiogenesis in the eye is the most common cause of blindness. The current study examined the role of interleukin-10 (IL-10) in ischemia-induced pathological angiogenesis called neovascularization during postnatal development. IL-10 deficiency resulted in significantly reduced pathological retinal angiogenesis. In contrast to the choroicapillaris where IL-10 interferes with macrophage influx, IL-10 did not prevent anti-angiogenic macrophages from migrating to the retina in response to hypoxia. Instead, IL-10 promoted retinal angiogenesis by altering macrophage angiogenic function, as macrophages from wild-type mice demonstrated increased vascular endothelial growth factor (VEGF) and nitric oxide (NO) compared to IL-10 deficient macrophages. IL-10 appears to directly affect macrophage responsiveness to hypoxia, as macrophages responded to hypoxia with increased levels of IL-10 and STAT3 phosphorylation as opposed to IL-10 deficient macrophages. Also, IL-10 deficient macrophages inhibited the proliferation of vascular endothelial cells in response to hypoxia while wild-type macrophages failed to do so. These findings suggest that hypoxia guides macrophage behavior to a pro-angiogenic phenotype via IL-10 activated pathways

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

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

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