Jose Andres C Portillo

The CD40-Autophagy Pathway Is Needed for Host Protection Despite IFN-Γ-Dependent Immunity and CD40 Induces Autophagy via Control of P21 Levels

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.

Toxoplasma gondii-Induced Activation of EGFR Prevents Autophagy Protein-Mediated Killing of the Parasite

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.

CD40 Mediates Retinal Inflammation and Neurovascular Degeneration

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.

Photoreceptor cells constitutively express functional TLR4

Toll-like receptor 4 (TLR4) is expressed on a number of cells including neurons in the brain. However, it has yet to be determined if TLR4 is expressed on photoreceptor cells in the retina. In this report, we examined primary photoreceptor cells and an established photoreceptor cell line (661W). We found that functional TLR4 is constitutively expressed on photoreceptor cells, and can be activated by LPS. We conclude that TLR4 on photoreceptor cells could directly contribute to retinal inflammatory diseases and photoreceptor cell survival.

CD40 in Retinal Müller Cells Induces P2X7-Dependent Cytokine Expression in Macrophages/Microglia in Diabetic Mice and Development of Early Experimental Diabetic Retinopathy.

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.

The protein kinase double-stranded RNA-dependent (PKR) enhances protection against disease cause by a non-viral pathogen.

PKR is well characterized for its function in antiviral immunity. Using Toxoplasma gondii, we examined if PKR promotes resistance to disease caused by a non-viral pathogen. PKR−/− mice infected with T. gondii exhibited higher parasite load and worsened histopathology in the eye and brain compared to wild-type controls. Susceptibility to toxoplasmosis was not due to defective expression of IFN-γ, TNF-α, NOS2 or IL-6 in the retina and brain, differences in IL-10 expression in these organs or to impaired induction of T. gondii-reactive T cells. While macrophages/microglia with defective PKR signaling exhibited unimpaired anti-T. gondii activity in response to IFN-γ/TNF-α, these cells were unable to kill the parasite in response to CD40 stimulation. The TRAF6 binding site of CD40, but not the TRAF2,3 binding sites, was required for PKR phosphorylation in response to CD40 ligation in macrophages. TRAF6 co-immunoprecipitated with PKR upon CD40 ligation. TRAF6-PKR interaction appeared to be indirect, since TRAF6 co-immunoprecipitated with TRAF2 and TRAF2 co-immunoprecipitated with PKR, and deficiency of TRAF2 inhibited TRAF6-PKR co-immunoprecipitation as well as PKR phosphorylation induced by CD40 ligation. PKR was required for stimulation of autophagy, accumulation the autophagy molecule LC3 around the parasite, vacuole-lysosomal fusion and killing of T. gondii in CD40-activated macrophages and microglia. Thus, our findings identified PKR as a mediator of anti-microbial activity and promoter of protection against disease caused by a non-viral pathogen, revealed that PKR is activated by CD40 via TRAF6 and TRAF2, and positioned PKR as a link between CD40-TRAF signaling and stimulation of the autophagy pathway.

CD40 promotes the development of early diabetic retinopathy in mice

Aims/hypothesisMicroangiopathy 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.MethodsB6 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.ResultsIn 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/interpretationCD40-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.

Modulation of Retinal Müller Cells by Complement Receptor C5aR

PURPOSE Müller cells, a major type of glial cell found in the eye, are postulated to play an important role in many retinal diseases, including diabetic retinopathy (DR). Complement is an integral part of innate immunity, and the activation of complement has been associated with retinal diseases. However, the role of complement in the regulation of Müller cell function remains unclear. We were trying to address these issues in this study. METHODS Using primary human Müller cells and a spontaneously immortalized human Müller cell line, we examined the expression of complement receptor C5aR both at mRNA and protein levels. Regulation of C5aR expression on Müller cells by prostaglandin E2 and by hyperglycemia, both of which are integrally involved in DR, were studied. Significance of C5aR on Müller cells was also investigated by examining relevant cytokine productions and their impacts on retinal endothelial cell proliferation/permeability after ligating the receptor using its ligand, C5a. RESULTS C5aR is constitutively expressed in human Müller cells. Prostaglandin E2 and hyperglycemia individually and synergistically upregulate C5aR expression in Müller cells. Signaling through C5aR on Müller cells upregulates production of IL-6 and VEGF, which promotes the proliferation of human retinal endothelial cells and increases their permeability. CONCLUSIONS These results indicate that complement can regulate Müller cells through C5aR, which may contribute to the pathogenesis of retinal diseases, including DR.

Proinflammatory Responses Induced by CD40 in Retinal Endothelial and Müller Cells are Inhibited by Blocking CD40-Traf2,3 or CD40-Traf6 Signaling

PURPOSE The cell surface receptor CD40 is required for the development of retinopathies induced by diabetes and ischemia/reperfusion. The purpose of this study was to identify signaling pathways by which CD40 triggers proinflammatory responses in retinal cells, since this may lead to pharmacologic targeting of these pathways as novel therapy against retinopathies. METHODS Retinal endothelial and Müller cells were transduced with vectors that encode wild-type CD40 or CD40 with mutations in sites that recruit TNF receptor associated factors (TRAF): TRAF2,3 (ΔT2,3), TRAF6 (ΔT6), or TRAF2,3 plus TRAF6 (ΔT2,3,6). Cells also were incubated with CD40-TRAF2,3 or CD40-TRAF6 blocking peptides. We assessed intercellular adhesion molecule-1 (ICAM-1), CD40, monocyte chemoattractant protein-1 (MCP-1), VEGF, and prostaglandin E₂ (PGE₂) by fluorescence-activated cell sorting (FACS), ELISA, or mass spectrometry. Mice (B6 and CD40(-/-)) were made diabetic using streptozotocin. The MCP-1 mRNA was assessed by real-time PCR. RESULTS The CD40-mediated ICAM-1 upregulation in endothelial and Müller cells was markedly inhibited by expression of CD40 ΔT2,3 or CD40 ΔT6. The CD40 was required for MCP-1 mRNA upregulation in the retina of diabetic mice. The CD40 stimulation of endothelial and Müller cells enhanced MCP-1 production that was markedly diminished by CD40 ΔT2,3 or CD40 ΔT6. Similar results were obtained in cells incubated with CD40-TRAF2,3 or CD40-TRAF6 blocking peptides. The CD40 ligation upregulated PGE₂ and VEGF production by Müller cells, that was inhibited by CD40 ΔT2,3 or CD40 ΔT6. All cellular responses tested were obliterated by expression of CD40 ΔT2,3,6. CONCLUSIONS Blockade of a single CD40-TRAF pathway was sufficient to impair ICAM-1, MCP-1, PGE₂, and VEGF upregulation in retinal endothelial and/or Müller cells. Blockade of CD40-TRAF signaling may control retinopathies.

Ligation of CD40 in Human Müller Cells Induces P2X7 Receptor-Dependent Death of Retinal Endothelial Cells.

Purpose Cluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the retinal endothelial cells (REC) death. However, CD40 activates prosurvival signals in endothelial cells. The purpose of this study was to identify a mechanism by which CD40 triggers programmed cell death (PCD) of RECs and address this paradox. Methods Human RECs and Müller cells were incubated with CD154 and L-N6-(1-Iminoethyl)lysine (L-Nil, nitric oxide synthase 2 inhibitor), α-lipoic acid (inhibitor of oxidative stress), anti-Fas ligand antibody, or A-438079 (P2X7 adenosine triphosphate [ATP] receptor inhibitor). Programmed cell death was analyzed by fluorescence-activated cell sorting (FACS) or Hoechst/propidium iodide staining. Release of ATP was measured using a luciferase-based assay. Mice were made diabetic with streptozotocin. Expression of P2X7 was assessed by FACS, quantitative PCR, or immunohistochemistry. Results Ligation of CD40 in primary RECs did not induce PCD. In contrast, in the presence of primary CD40+ Müller cells, CD40 stimulation caused PCD of RECs that was not impaired by L-Nil, α-lipoic acid, or anti-Fas ligand antibody. We found CD40 did not trigger TNF-α or IL-1β secretion. Primary Müller cells released extracellular ATP in response to CD40 ligation. Inhibition of P2X7 (A-438079) impaired PCD of RECs; CD40 upregulated P2X7 in RECs, making them susceptible to ATP/P2X7–mediated PCD. Diabetic mice upregulated P2X7 in the retina and RECs in a CD40-dependent manner. Conclusions Cluster of differentiation 40 induces PCD of RECs through a dual mechanism: ATP release by Müller cells and P2X7 upregulation in RECs. These findings are likely of in vivo relevance since CD40 upregulates P2X7 in RECs in diabetic mice and CD40 is known to be required for retinal capillary degeneration.