Müller cells and macrophages/microglia are likely important for the development of diabetic retinopathy; however, the interplay between these cells in this disease is not well understood. An inflammatory process is linked to the onset of experimental diabetic retinopathy. CD40 deficiency impairs this process and prevents diabetic retinopathy. Using mice with CD40 expression restricted to Müller cells, we identified a mechanism by which Müller cells trigger proinflammatory cytokine expression in myeloid cells. During diabetes, mice with CD40 expressed in Müller cells upregulated retinal tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), intracellular adhesion molecule 1 (ICAM-1), and nitric oxide synthase (NOS2), developed leukostasis and capillary degeneration. However, CD40 did not cause TNF-α or IL-1β secretion in Müller cells. TNF-α was not detected in Müller cells from diabetic mice with CD40+ Müller cells. Rather, TNF-α was upregulated in macrophages/microglia. CD40 ligation in Müller cells triggered phospholipase C–dependent ATP release that caused P2X7-dependent production of TNF-α and IL-1β by macrophages. P2X7−/− mice and mice treated with a P2X7 inhibitor were protected from diabetes-induced TNF-α, IL-1β, ICAM-1, and NOS2 upregulation. Our studies indicate that CD40 in Müller cells is sufficient to upregulate retinal inflammatory markers and appears to promote experimental diabetic retinopathy and that Müller cells orchestrate inflammatory responses in myeloid cells through a CD40-ATP-P2X7 pathway.

Toxoplasma gondii resides in an intracellular compartment (parasitophorous vacuole) that excludes transmembrane molecules required for endosome - lysosome recruitment. Thus, the parasite survives by avoiding lysosomal degradation. However, autophagy can re-route the parasitophorous vacuole to the lysosomes and cause parasite killing. This raises the possibility that T. gondii may deploy a strategy to prevent autophagic targeting to maintain the non-fusogenic nature of the vacuole. We report that T. gondii activated EGFR in endothelial cells, retinal pigment epithelial cells and microglia. Blockade of EGFR or its downstream molecule, Akt, caused targeting of the parasite by LC3+ structures, vacuole-lysosomal fusion, lysosomal degradation and killing of the parasite that were dependent on the autophagy proteins Atg7 and Beclin 1. Disassembly of GPCR or inhibition of metalloproteinases did not prevent EGFR-Akt activation. T. gondii micronemal proteins (MICs) containing EGF domains (EGF-MICs; MIC3 and MIC6) appeared to promote EGFR activation. Parasites defective in EGF-MICs (MIC1 ko, deficient in MIC1 and secretion of MIC6; MIC3 ko, deficient in MIC3; and MIC1-3 ko, deficient in MIC1, MIC3 and secretion of MIC6) caused impaired EGFR-Akt activation and recombinant EGF-MICs (MIC3 and MIC6) caused EGFR-Akt activation. In cells treated with autophagy stimulators (CD154, rapamycin) EGFR signaling inhibited LC3 accumulation around the parasite. Moreover, increased LC3 accumulation and parasite killing were noted in CD154-activated cells infected with MIC1-3 ko parasites. Finally, recombinant MIC3 and MIC6 inhibited parasite killing triggered by CD154 particularly against MIC1-3 ko parasites. Thus, our findings identified EGFR activation as a strategy used by T. gondii to maintain the non-fusogenic nature of the parasitophorous vacuole and suggest that EGF-MICs have a novel role in affecting signaling in host cells to promote parasite survival.

Autophagy degrades pathogens in vitro. The autophagy gene Atg5 has been reported to be required for IFN-γ-dependent host protection in vivo. However, these protective effects occur independently of autophagosome formation. Thus, the in vivo role of classic autophagy in protection conferred by adaptive immunity and how adaptive immunity triggers autophagy are incompletely understood. Employing biochemical, genetic and morphological studies, we found that CD40 upregulates the autophagy molecule Beclin 1 in microglia and triggers killing of Toxoplasma gondii dependent on the autophagy machinery. Infected CD40−/− mice failed to upregulate Beclin 1 in microglia/macrophages in vivo. Autophagy-deficient Beclin 1+/− mice, mice with deficiency of the autophagy protein Atg7 targeted to microglia/macrophages as well as CD40−/− mice exhibited impaired killing of T. gondii and were susceptible to cerebral and ocular toxoplasmosis. Susceptibility to toxoplasmosis occurred despite upregulation of IFN-γ, TNF-α and NOS2, preservation of IFN-γ-induced microglia/macrophage anti-T. gondii activity and the generation of anti-T. gondii T cell immunity. CD40 upregulated Beclin 1 and triggered killing of T. gondii by decreasing protein levels of p21, a molecule that degrades Beclin 1. These studies identified CD40-p21-Beclin 1 as a pathway by which adaptive immunity stimulates autophagy. In addition, they support that autophagy is a mechanism through which CD40-dependent immunity mediates in vivo protection and that the CD40-autophagic machinery is needed for host resistance despite IFN-γ.

Aims/hypothesis Microangiopathy is a leading complication of diabetes that commonly affects the retina. Degenerate capillaries are a central feature of diabetic retinopathy. An inflammatory process has been linked to the development of diabetic retinopathy but its regulation is incompletely understood. Cluster of differentiation (CD) 40 is a member of the TNF receptor superfamily that promotes the development of certain inflammatory disorders. The role of CD40 in diabetic microangiopathy is unknown. Methods B6 and Cd40−/− mice were administered streptozotocin to induce diabetes. Leucostasis was assessed using fluorescein isothiocyanate-conjugated concanavalin A. Retinal Icam1 and Cd40 mRNA levels were examined using real-time PCR. Protein nitration was assessed by immunohistochemistry. Histopathology was examined in the retinal vasculature. CD40 expression was assessed by flow cytometry and immunohistochemistry. Intercellular adhesion molecule 1 (ICAM-1) and nitric oxide synthase 2 (NOS2) were examined by immunoblot and/or flow cytometry. Nitric oxide production was examined by immunoblot and Griess reaction. Results In mouse models of diabetes, Cd40−/− mice exhibited reduced retinal leucostasis and did not develop capillary degeneration in comparison with B6 mice. Diabetic Cd40−/− mice had diminished ICAM-1 upregulation and decreased protein nitration. Cd40 mRNA levels were increased in the retinas of diabetic B6 mice compared with non-diabetic controls. CD40 expression increased in retinal Müller cells, endothelial cells and microglia of diabetic animals. CD40 stimulation upregulated ICAM-1 in retinal endothelial cells and Müller cells. CD40 ligation upregulated NOS2 and nitric oxide production by Müller cells. Conclusions/interpretation CD40-deficient mice were protected from the development of diabetic retinopathy. These mice exhibited diminished inflammatory responses linked to diabetic retinopathy. CD40 stimulation of retinal cells triggered these pro-inflammatory responses.

Retinopathies are major causes of visual impairment. We used a model of ischemic retinopathy to examine the role of CD40 in the pathogenesis of retinal injury. Retinal inflammation, loss of ganglion cells, and capillary degeneration were markedly attenuated in ischemic retinas of CD40−/− mice. Up-regulation of NOS2 and COX2 after retinal ischemia were blunted in CD40−/− mice. NOS2-COX-2 up-regulation in ischemic retinas from wild-type mice was at least in part explained by recruitment of NOS2+COX-2+ leukocytes. Up-regulation of KC/CXCL1 and ICAM-1 also required CD40. Retinal endothelial and Muller cells expressed CD40. Stimulation of these cells through CD40 caused ICAM-1 up-regulation and KC/CXCL1 production. Bone marrow transplant experiments revealed that leukocyte infiltration, ganglion cell loss, and up-regulation of proinflammatory molecules after retinal ischemia were dependent on CD40 expression in the retina and not peripheral blood leukocytes. These studies identified CD40 as a regulator of retinal inflammation and neurovascular degeneration. They support a model in which CD40 stimulation of endothelial and Muller cells triggers adhesion molecule up-regulation and chemokine production, promoting the recruitment of leukocytes that express NOS2/COX-2, molecules linked to neurovascular degeneration.

Gamma interferon (IFN-␥

No abstract

Targeting of Toxoplasma gondii by autophagy is an effective mechanism by which host cells kill the protozoan. Thus, the parasite must avoid autophagic targeting to survive. Here we show that the mammalian cytoplasmic molecule Focal Adhesion Kinase (FAK) becomes activated during invasion of host cells. Activated FAK appears to accompany the formation of the moving junction (as assessed by expression the parasite protein RON4). FAK activation was inhibited by approaches that impaired β1 and β3 integrin signaling. FAK caused activation of Src that in turn mediated Epidermal Growth Factor Receptor (EGFR) phosphorylation at the unique Y845 residue. Expression of Src-resistant Y845F EGFR mutant markedly inhibited ROP16-independent activation of STAT3 in host cells. Activation of FAK, Y845 EGFR or STAT3 prevented activation of PKR and eIF2α, key stimulators of autophagy. Genetic or pharmacologic inhibition of FAK, Src, EGFR phosphorylation at Y845, or STAT3 caused accumulation of the autophagy protein LC3 and LAMP-1 around the parasite and parasite killing dependent on autophagy proteins (ULK1 and Beclin 1) and lysosomal enzymes. Parasite killing was inhibited by expression of dominant negative PKR. Thus, T. gondii activates a FAK→Src→Y845-EGFR→STAT3 signaling axis within mammalian cells, thereby enabling the parasite to survive by avoiding autophagic targeting through a mechanism likely dependent on preventing activation of PKR and eIF2α.