(2008). Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy: Vol. 4, No. 2, pp. 151-175.

Many intracellular pathogens, including Toxoplasma gondii, survive within macrophages by residing in vacuoles that avoid fusion with lysosomes. It is important to determine whether cell-mediated immunity can trigger macrophage antimicrobial activity by rerouting these vacuoles to lysosomes. We report that CD40 stimulation of human and mouse macrophages infected with T. gondii resulted in fusion of parasitophorous vacuoles and late endosomes/lysosomes. Vacuole/lysosome fusion took place even when CD40 was ligated after the formation of parasitophorous vacuoles. Genetic and pharmacological approaches that impaired phosphoinositide-3-class 3 (PIK3C3), Rab7, vacuolar ATPase, and lysosomal enzymes revealed that vacuole/lysosome fusion mediated antimicrobial activity induced by CD40. Ligation of CD40 caused colocalization of parasitophorous vacuoles and LC3, a marker of autophagy, which is a process that controls lysosomal degradation. Vacuole/lysosome fusion and antimicrobial activity were shown to be dependent on autophagy. Thus, cell-mediated immunity through CD40 stimulation can reroute an intracellular pathogen to the lysosomal compartment, resulting in macrophage antimicrobial activity.

Autophagy is a conserved feature of lysosome-mediated intracellular degradation. Dysregulated autophagy is implicated as a contributor in neurodegenerative diseases; however, the role of autophagy in retinal degeneration remains largely unknown. Here, we report that the photo-activated visual chromophore, all-trans-retinal, modulated autophagosome formation in ARPE19 retinal cells. Increased formation of autophagosomes in these cells was observed when incubated with 2.5 μm all-trans-retinal, a condition that did not cause cell death after 24 h in culture. However, autophagosome formation was decreased at concentrations, which caused cell death. Increased expression of activating transcription factor 4 (Atf4), which indicates the activation of oxidative stress, was recorded in response to light illumination in retinas of Abca4^−/−Rdh8^−/− mice, which showed delayed clearance of all-trans-retinal after light exposure. Expression of autophagosome marker LC3B-II and mitochondria-specific autophagy, mitophagy, regulator Park2, were significantly increased in the retinas of Abca4^−/−Rdh8^−/− mice after light exposure, suggesting involvement of autophagy and mitophagy in the pathogenesis of light-induced retinal degeneration. Deletion of essential genes required for autophagy, including Beclin1 systemically or Atg7 in only rod photoreceptors resulted in increased susceptibility to light-induced retinal damage. Increased photoreceptor cell death was observed when retinas lacking the rod photoreceptor-specific Atg7 gene were coincubated with 20 μm all-trans-retinal. Park2^−/− mice also displayed light-induced retinal degeneration. Ultra-structural analyses showed mitochondrial and endoplasmic reticulum impairment in retinas of these model animals after light exposure. Taken together, these observations provide novel evidence implicating an important role of autophagy and mitophagy in protecting the retina from all-trans-retinal- and light-induced degeneration.

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. Toxoplasma gondii resides in a parasitophorous vacuole that excludes transmembrane proteins required for recruitment of endosomes and lysosomes and thus, does not follow the path of classical lysosomal degradation. However, the non-fusogenic nature of the vacuole can be reverted when autophagy, a pathway to lysosomal degradation, is upregulated through the immune system or pharmacologically. Maintenance of the non-fusogenic nature of the vacuole is central to parasite survival. Thus, in addition to preventing degradation through a classical lysosomal pathway, T. gondii may also deploy strategies to prevent constitutive levels of autophagy from targeting the pathogen and causing its lysosomal degradation. We report that T. gondii accomplishes this task by causing EGFR activation in host cells. In cells that were not subjected to immune or pharmacologic upregulation of autophagy, blockade of EGFR resulted in parasite encasing by structures that expressed the autophagy protein LC3, vacuole-lysosomal fusion and autophagy protein-dependent killing of the parasite. Moreover, EGFR signaling also impaired targeting of the parasite by LC3⁺ structures in cells treated with stimulators of autophagy. Studies with T. gondii deficient in EGF domain containing-micronemal proteins (EGF-MICs) and recombinant EGF-MICs support the concept that these parasite adhesins contribute to EGFR activation.

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.

Autophagy is a homeostatic mechanism of lysosomal degradation. Defective autophagy has been linked to various disorders such as impaired control of pathogens and neurodegeneration. Autophagy is regulated by a complex array of signaling pathways that act upstream of autophagy proteins. Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy. In particular, it is not known if pathogens inhibit autophagy by modulation of upstream regulatory pathways. Cells infected with HIV-1 blocked rapamycin-induced autophagy and CD40-induced autophagic killing of Toxoplasma gondii in bystander (non-HIV-1 infected) macrophage/monocytic cells. Blockade of autophagy was dependent on Src-Akt and STAT3 triggered by HIV-1 Tat and IL-10. Neutralization of the upstream receptors VEGFR, β-integrin or CXCR4, as well as of HIV-1 Tat or IL-10 restored autophagy in macrophage/monocytic cells exposed to HIV-1-infected cells. Defective autophagic killing of T. gondii was detected in monocyte-derived macrophages from a subset of HIV-1⁺ patients. This defect was also reverted by neutralization of Tat or IL-10. These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways. The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection.