Disruption of retinal inflammation and the development of diabetic retinopathy in mice by a CD40-derived peptide or mutation of CD40 in Müller cells.

AIMS/HYPOTHESIS: CD40 expressed in Müller cells is a central driver of diabetic retinopathy. CD40 causes phospholipase Cγ1 (PLCγ1)-dependent ATP release in Müller cells followed by purinergic receptor (P2X7)-dependent production of proinflammatory cytokines in myeloid cells. In the diabetic retina, CD40 and P2X7 upregulate a broad range of inflammatory molecules that promote development of diabetic retinopathy. The molecular event downstream of CD40 that activates the PLCγ1-ATP-P2X7-proinflammatory cytokine cascade and promotes development of diabetic retinopathy is unknown. We hypothesise that disruption of the CD40-driven molecular events that trigger this cascade prevents/treats diabetic retinopathy in mice. METHODS: B6 and transgenic mice with Müller cell-restricted expression of wild-type (WT) CD40 or CD40 with mutations in TNF receptor-associated factor (TRAF) binding sites were made diabetic using streptozotocin. Leucostasis was assessed using FITC-conjugated concanavalin A. Histopathology was examined in the retinal vasculature. Expression of inflammatory molecules and phospho-Tyr783 PLCγ1 (p-PLCγ1) were assessed using real-time PCR, immunoblot and/or immunohistochemistry. Release of ATP and cytokines were measured by ATP bioluminescence and ELISA, respectively. RESULTS: Human Müller cells with CD40 ΔT2,3 (lacks TRAF2,3 binding sites) were unable to phosphorylate PLCγ1 and release ATP in response to CD40 ligation, and could not induce TNF-α/IL-1ß secretion in bystander myeloid cells. CD40-TRAF signalling acted via Src to induce PLCγ1 phosphorylation. Diabetic mice in which WT CD40 in Müller cells was replaced by CD40 ΔT2,3 failed to exhibit phosphorylation of PLCγ1 in these cells and upregulate P2X7 and TNF-α in microglia/macrophages. P2x7 (also known as P2rx7), Tnf-α (also known as Tnf), Il-1ß (also known as Il1b), Nos2, Icam-1 (also known as Icam1) and Ccl2 mRNA were not increased in these mice and the mice did not develop retinal leucostasis and capillary degeneration. Diabetic B6 mice treated intravitreally with a cell-permeable peptide that disrupts CD40-TRAF2,3 signalling did not exhibit either upregulation of P2X7 and inflammatory molecules in the retina or leucostasis. CONCLUSIONS/INTERPRETATION: CD40-TRAF2,3 signalling activated the CD40-PLCγ1-ATP-P2X7-proinflammatory cytokine pathway. Src functioned as a link between CD40-TRAF2,3 and PLCγ1. Replacing WT CD40 with CD40 ΔT2,3 impaired activation of PLCγ1 in Müller cells, upregulation of P2X7 in microglia/macrophages, upregulation of a broad range of inflammatory molecules in the diabetic retina and the development of diabetic retinopathy. Administration of a peptide that disrupts CD40-TRAF2,3 signalling reduced retinal expression of inflammatory molecules and reduced leucostasis in diabetic mice, supporting the therapeutic potential of pharmacological inhibition of CD40-TRAF2,3 in diabetic retinopathy.

A cell-penetrating CD40-TRAF2,3 blocking peptide diminishes inflammation and neuronal loss after ischemia/reperfusion.

While the administration of anti-CD154 mAbs in mice validated the CD40-CD154 pathway as a target against inflammatory disorders, this approach caused thromboembolism in humans (unrelated to CD40 inhibition) and is expected to predispose to opportunistic infections. There is a need for alternative approaches to inhibit CD40 that avoid these complications. CD40 signals through TRAF2,3 and TRAF6-binding sites. Given that CD40-TRAF6 is the pathway that stimulates responses key for cell-mediated immunity against opportunistic pathogens, we examined the effects of pharmacologic inhibition of CD40-TRAF2,3 signaling. We used a model of ischemia/reperfusion (I/R)-induced retinopathy, a CD40-driven inflammatory disorder. Intravitreal administration of a cell-penetrating CD40-TRAF2,3 blocking peptide impaired ICAM-1 upregulation in retinal endothelial cells and CXCL1 upregulation in endothelial and Müller cells. The peptide reduced leukocyte infiltration, upregulation of NOS2/COX-2/TNF-α/IL-1ß, and ameliorated neuronal loss, effects that mimic those observed after I/R in Cd40-/- mice. While a cell-penetrating CD40-TRAF6 blocking peptide also diminished I/R-induced inflammation, this peptide (but not the CD40-TRAF2,3 blocking peptide) impaired control of the opportunistic pathogen Toxoplasma gondii in the retina. Thus, inhibition of the CD40-TRAF2,3 pathway is a novel and potent approach to reduce CD40-induced inflammation, while likely diminishing the risk of opportunistic infections that would otherwise accompany CD40 inhibition.

Toxoplasma gondii induces prolonged host epidermal growth factor receptor signalling to prevent parasite elimination by autophagy: Perspectives for in vivo control of the parasite.

Toxoplasma gondii causes retinitis and encephalitis. Avoiding targeting by autophagosomes is key for its survival because T. gondii cannot withstand lysosomal degradation. During invasion of host cells, T. gondii triggers epidermal growth factor receptor (EGFR) signalling enabling the parasite to avoid initial autophagic targeting. However, autophagy is a constitutive process indicating that the parasite may also use a strategy operative beyond invasion to maintain blockade of autophagic targeting. Finding that such a strategy exists would be important because it could lead to inhibition of host cell signalling as a novel approach to kill the parasite in previously infected cells and treat toxoplasmosis. We report that T. gondii induced prolonged EGFR autophosphorylation. This effect was mediated by PKCα/PKCß âž” Src because T. gondii caused prolonged activation of these molecules and their knockdown or incubation with inhibitors of PKCα/PKCß or Src after host cell invasion impaired sustained EGFR autophosphorylation. Addition of EGFR tyrosine kinase inhibitor (TKI) to previously infected cells led to parasite entrapment by LC3 and LAMP-1 and pathogen killing dependent on the autophagy proteins ULK1 and Beclin 1 as well as lysosomal enzymes. Administration of gefitinib (EGFR TKI) to mice with ocular and cerebral toxoplasmosis resulted in disease control that was dependent on Beclin 1. Thus, T. gondii promotes its survival through sustained EGFR signalling driven by PKCα/ß âž” Src, and inhibition of EGFR controls pre-established toxoplasmosis.

CD40 in Endothelial Cells Restricts Neural Tissue Invasion by Toxoplasma gondii.

Little is known about whether pathogen invasion of neural tissue is affected by immune-based mechanisms in endothelial cells. We examined the effects of endothelial cell CD40 on Toxoplasma gondii invasion of the retina and brain, organs seeded hematogenously. T. gondii circulates in the bloodstream within infected leukocytes (including monocytes and dendritic cells) and as extracellular tachyzoites. After T. gondii infection, mice that expressed CD40 restricted to endothelial cells exhibited diminished parasite loads and histopathology in the retina and brain. These mice also had lower parasite loads in the retina and brain after intravenous (i.v.) injection of infected monocytes or dendritic cells. The protective effect of endothelial cell CD40 was not explained by changes in cellular or humoral immunity, reduced transmigration of leukocytes into neural tissue, or reduced invasion by extracellular parasites. Circulating T. gondii-infected leukocytes (dendritic cells used as a model) led to infection of neural endothelial cells. The number of foci of infection in these cells were reduced if endothelial cells expressed CD40. Infected dendritic cells and macrophages expressed membrane-associated inducible Hsp70. Infected leukocytes triggered Hsp70-dependent autophagy in CD40+ endothelial cells and anti-T. gondii activity dependent on ULK1 and beclin 1. Reduced parasite load in the retina and brain not only required CD40 expression in endothelial cells but was also dependent on beclin 1 and the expression of inducible Hsp70 in dendritic cells. These studies suggest that during endothelial cell-leukocyte interaction, CD40 restricts T. gondii invasion of neural tissue through a mechanism that appears mediated by endothelial cell anti-parasitic activity stimulated by Hsp70.

Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy.

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 ß1 and ß3 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.

Loss of CD40 attenuates experimental diabetes-induced retinal inflammation but does not protect mice from electroretinogram defects.

Chronic low grade inflammation is considered to contribute to the development of experimental diabetic retinopathy (DR). We recently demonstrated that lack of CD40 in mice ameliorates the upregulation of inflammatory molecules in the diabetic retina and prevented capillary degeneration, a hallmark of experimental diabetic retinopathy. Herein, we investigated the contribution of CD40 to diabetes-induced reductions in retinal function via the electroretinogram (ERG) to determine if inflammation plays a role in the development of ERG defects associated with diabetes. We demonstrate that diabetic CD40-/- mice are not protected from reduction to the ERG b-wave despite failing to upregulate inflammatory molecules in the retina. Our data therefore supports the hypothesis that retinal dysfunction found in diabetics occurs independent of the induction of inflammatory processes.

Identification of Signaling Pathways by Which CD40 Stimulates Autophagy and Antimicrobial Activity against Toxoplasma gondii in Macrophages.

CD40 is an important stimulator of autophagy and autophagic killing of Toxoplasma gondii in host cells. In contrast to autophagy induced by nutrient deprivation or pattern recognition receptors, less is known about the effects of cell-mediated immunity on Beclin 1 and ULK1, key regulators of autophagy. Here we studied the molecular mechanisms by which CD40 stimulates autophagy in macrophages. CD40 ligation caused biphasic Jun N-terminal protein kinase (JNK) phosphorylation. The second phase of JNK phosphorylation was dependent on autocrine production of tumor necrosis factor alpha (TNF-α). TNF-α and JNK signaling were required for the CD40-induced increase in autophagy. JNK signaling downstream of CD40 caused Ser-87 phosphorylation of Bcl-2 and dissociation between Bcl-2 and Beclin 1, an event known to stimulate the autophagic function of Beclin 1. However, TNF-α alone was unable to stimulate autophagy. CD40 also stimulated autophagy via a pathway that included calcium/calmodulin-dependent kinase kinase ß (CaMKKß), AMP-activated protein kinase (AMPK), and ULK1. CD40 caused AMPK phosphorylation at its activating site, Thr-172. This effect was mediated by CaMKKß and was not impaired by neutralization of TNF-α. CD40 triggered AMPK-dependent Ser-555 phosphorylation of ULK1. CaMKKß, AMPK, and ULK1 were required for CD40-induced increase in autophagy. CD40-mediated autophagic killing of Toxoplasma gondii is known to require TNF-α. Knockdown of JNK, CaMKKß, AMPK, or ULK1 prevented T. gondii killing in CD40-activated macrophages. The second phase of JNK phosphorylation-Bcl-2 phosphorylation-Bcl-2-Beclin 1 dissociation and AMPK phosphorylation-ULK1 phosphorylation occurred simultaneously at ∼4 h post-CD40 stimulation. Thus, CaMKKß and TNF-α are upstream molecules by which CD40 acts on ULK1 and Beclin 1 to stimulate autophagy and killing of T. gondii.

Blockade of CD40-TRAF2,3 or CD40-TRAF6 is sufficient to inhibit pro-inflammatory responses in non-haematopoietic cells.

Inhibition of the CD40-CD154 pathway controls inflammatory disorders. Unfortunately, administration of anti-CD154 monoclonal antibodies causes thromboembolism. Blockade of signalling downstream of CD40 may represent an approach to treat CD40-driven inflammatory disorders. Blocking tumour necrosis factor receptor-associated factor 6 (TRAF6) signalling downstream of CD40 in MHC II(+) cells diminishes inflammation. However, CD40-TRAF6 blockade may cause immunosuppression. We examined the role of CD40-TRAF2,3 and CD40-TRAF6 signalling in the development of pro-inflammatory responses in human non-haematopoietic and monocytic cells. Human aortic endothelial cells, aortic smooth muscle cells, renal proximal tubule epithelial cells, renal mesangial cells and monocytic cells were transduced with retroviral vectors that encode wild-type CD40, CD40 with a mutation that prevents TRAF2,3 recruitment (ΔT2,3), TRAF6 recruitment (ΔT6) or both TRAF2,3 plus TRAF6 recruitment (ΔT2,3,6). Non-haematopoietic cells that expressed CD40 ΔT2,3 exhibited marked inhibition in CD154-induced up-regulation of vascular cell adhesion molecule 1, intercellular adhesion molecule 1 (ICAM-1), monocyte chemotactic protein 1 (MCP-1), tissue factor and matrix metalloproteinase 9. Similar results were obtained with cells that expressed CD40 ΔT6. Although both mutations impaired ICAM-1 up-regulation in monocytic cells, only expression of CD40 ΔT6 reduced MCP-1 and tissue factor up-regulation in these cells. Treatment of endothelial and smooth muscle cells with cell-permeable peptides that block CD40-TRAF2,3 or CD40-TRAF6 signalling impaired pro-inflammatory responses. In contrast, while the CD40-TRAF2,3 blocking peptide did not reduce CD154-induced dendritic cell maturation, the CD40-TRAF6 blocking peptide impaired this response. Hence, preventing CD40-TRAF2,3 or CD40-TRAF6 interaction inhibits pro-inflammatory responses in human non-haematopoietic cells. In contrast to inhibition of CD40-TRAF6 signalling, inhibition of CD40-TRAF2,3 signalling did not impair dendritic cell maturation. Blocking CD40-TRAF2,3 signalling may control CD40-CD154-dependent inflammatory disorders.

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.

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/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 fromthe development of diabetic retinopathy. These mice exhibited diminished inflammatory responses linked to diabetic retinopathy. CD40 stimulation of retinal cells triggered these pro-inflammatory responses.

Advanced Glycation End Products Upregulate CD40 in Human Retinal Endothelial and Müller Cells: Relevance to Diabetic Retinopathy.

CD40 induces pro-inflammatory responses in endothelial and Müller cells and is required for the development of diabetic retinopathy (DR). CD40 is upregulated in these cells in patients with DR. CD40 upregulation is a central feature of CD40-driven inflammatory disorders. What drives CD40 upregulation in the diabetic retina remains unknown. We examined the role of advanced glycation end products (AGEs) in CD40 upregulation in endothelial cells and Müller cells. Human endothelial cells and Müller cells were incubated with unmodified or methylglyoxal (MGO)-modified fibronectin. CD40 expression was assessed by flow cytometry. The expression of ICAM-1 and CCL2 was examined by flow cytometry or ELISA after stimulation with CD154 (CD40 ligand). The expression of carboxymethyl lysine (CML), fibronectin, and laminin as well as CD40 in endothelial and Müller cells from patients with DR was examined by confocal microscopy. Fibronectin modified by MGO upregulated CD40 in endothelial and Müller cells. CD40 upregulation was functionally relevant. MGO-modified fibronectin enhanced CD154-driven upregulation of ICAM-1 and CCL2 in endothelial and Müller cells. Increased CD40 expression in endothelial and Müller cells from patients with DR was associated with increased CML expression in fibronectin and laminin. These findings identify AGEs as inducers of CD40 upregulation in endothelial and Müller cells and enhancers of CD40-dependent pro-inflammatory responses. CD40 upregulation in these cells is associated with higher CML expression in fibronectin and laminin in patients with DR. This study revealed that CD40 and AGEs, two important drivers of DR, are interconnected.

CD40 induces anti-Toxoplasma gondii activity in nonhematopoietic cells dependent on autophagy proteins.

Toxoplasma gondii infects both hematopoietic and nonhematopoietic cells and can cause cerebral and ocular toxoplasmosis, as a result of either congenital or postnatally acquired infections. Host protection likely acts at both cellular levels to control the parasite. CD40 is a key factor for protection against cerebral and ocular toxoplasmosis. We determined if CD40 induces anti-T. gondii activity at the level of nonhematopoietic cells. Engagement of CD40 on various endothelial cells including human microvascular brain endothelial cells, human umbilical vein endothelial cells, and a mouse endothelial cell line as well as human and mouse retinal pigment epithelial cells resulted in killing of T. gondii. CD40 stimulation increased expression of the autophagy proteins Beclin 1 and LC3 II, enhanced autophagy flux, and led to recruitment of LC3 around the parasite. The late endosomal/lysosomal marker LAMP-1 accumulated around the parasite in CD40-stimulated cells. This was accompanied by killing of T. gondii dependent on lysosomal enzymes. Accumulation of LAMP-1 and killing of T. gondii were dependent on the autophagy proteins Beclin 1 and Atg7. Together, these studies revealed that CD40 induces toxoplasmacidal activity in various nonhematopoietic cells dependent on proteins of the autophagy machinery.

CD40 and tumour necrosis factor-α co-operate to up-regulate inducuble nitric oxide synthase expression in macrophages.

Regulation of inducible nitric oxide synthase (NOS2) expression is important given the role of this enzyme in inflammation, control of infections and immune regulation. In contrast to tumour necrosis factor-α (TNF-α) alone or CD40 stimulation alone, simultaneous stimulation of mouse macrophages through CD40 ligation and TNF-α led to up-regulation of NOS2 and nitric oxide production. This response was of functional relevance because CD40/TNF-α-stimulated macrophages acquired nitric oxide-dependent anti-Leishmania major activity. CD40 plus TNF-α up-regulated NOS2 independently of interferon-γ, interferon-α/ß and interleukin-1. TNF receptor-associated factor 6 (TRAF6), an adapter protein downstream of CD40, appears to be required for NOS2 up-regulation because a CD40-TRAF6 blocking peptide inhibited up-regulation of NOS2 in CD40/TNF-α-stimulated macrophages. CCAAT/enhancer-binding protein-ß (C/EBPß), a transcription factor activated by TNF-α but not CD40, was required for NOS2 up-regulation because this enzyme was not up-regulated when C/EBPß(-/-) macrophages received CD40 plus TNF-α stimulation. These results indicate that CD40 and TNF-α co-operate to up-regulate NOS2, probably via the effect of TRAF6 and C/EBPß.

CD40 Expressed in Endothelial Cells Promotes Upregulation of ICAM-1 But Not Pro-Inflammatory Cytokines, NOS2 and P2X7 in the Diabetic Retina.

Purpose: CD40 is an upstream inducer of inflammation in the diabetic retina. CD40 is upregulated in retinal endothelial cells in diabetes. The purpose of this study was to determine whether expression of CD40 in endothelial cells is sufficient to promote inflammatory responses in the retina of diabetic mice. Methods: Transgenic mice with CD40 expression restricted to endothelial cells (Trg-CD40 EC), transgenic control mice (Trg-Ctr), B6, and CD40-/- mice were made diabetic using streptozotocin. Leukostasis was assessed using FITC-conjugated ConA. Pro-inflammatory molecule expression was examined by real-time PCR, immunohistochemistry, ELISA, or flow cytometry. Release of ATP was assessed by ATP bioluminescence. Results: Diabetic B6 and Trg-CD40 EC mice exhibited increased retinal mRNA levels of ICAM-1, higher ICAM-1 expression in endothelial cells, and increased leukostasis. These responses were not detected in diabetic mice that lacked CD40 (CD40-/- and Trg-Ctr). Diabetic B6 but not Trg-CD40 EC mice upregulated TNF-α, IL-1ß, and NOS2 mRNA levels. CD40 stimulation in retinal endothelial cells upregulated ICAM-1 but not TNF-α, IL-1ß, or NOS2. CD40 ligation did not trigger ATP release by retinal endothelial cells or pro-inflammatory cytokine production in bystander myeloid cells. In contrast to diabetic B6 mice, diabetic Trg-CD40 EC mice did not upregulate P2X7 mRNA levels in the retina. Conclusions: Endothelial cell CD40 promotes ICAM-1 upregulation and leukostasis. In contrast, endothelial cell CD40 does not lead to pro-inflammatory cytokine and NOS2 upregulation likely because it does not activate purinergic-mediated pro-inflammatory molecule expression by myeloid cells or induce expression of these pro-inflammatory molecules in endothelial cells.

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.

Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry.

PURPOSE: Advances in the understanding of the pathogenesis of retinal disorders can be facilitated by a methodology to measure expression of proinflammatory molecules in various subsets of retinal cells. METHODS: We examined whether a multiparameter flow cytometric assay can be used to identify various subsets of retinal cells and examine expression of molecules involved in inflammatory responses in the retina. Single-cell suspensions freshly obtained after enzymatic digestion of normal mouse retinas were stained with antibodies against cluster of differentiation 11b (CD11b), cluster of differentiation 31 (CD31), Glial fibrillary acidic protein (GFAP), rhodopsin, Thy-1, and vimentin. These markers were previously shown by immunohistochemistry to label retinal microglia/macrophages, endothelial cells, astrocytes, photoreceptors, ganglion neurons, and Müller cells respectively in normal mouse retinas. RESULTS: Costaining with antibodies against intercellular adhesion molecule-1 (ICAM-1) and CD40 revealed that ICAM-1 is normally expressed at various levels on all subsets of retinal cells examined. In contrast, CD40 was detected only on CD11b(+), CD31(+), Thy-1(+), and vimentin(+) cells. Ischemia-reperfusion of the retina resulted in upregulation of ICAM-1 on CD105(+) and vimentin(+) cells and upregulation of nitric oxide synthase 2 in CD11b(+) cells. DISCUSSION: These results indicate that flow cytometry can be used to readily quantitate expression of surface and intracellular molecules of relevance to retinopathies in freshly isolated retinal cells.

Epidermal growth factor receptor promotes cerebral and retinal invasion by Toxoplasma gondii.

Little is known about strategies used by pathogens to facilitate CNS invasion. Toxoplasma gondii reaches the CNS by circulating in blood within leukocytes or as extracellular tachyzoites. T. gondii induces EGFR signaling in vitro during invasion of mammalian cells. We examined the effects of endothelial cell EGFR on CNS invasion. Transgenic mice whose endothelial cells expressed a dominant negative (DN) EGFR (inhibits EGFR signaling) exhibited diminished parasite load and histopathology in the brain and retina after T. gondii infection. I.V. administration of infected leukocytes or extracellular tachyzoites led to reduced parasite loads in mice with DN EGFR. This was not explained by enhanced immunity or reduced leukocyte recruitment. Endothelial cell infection is key for CNS invasion. Parasite foci in brain endothelial cells were reduced by DN EGFR. DN EGFR in these cells led to recruitment of the autophagy protein LC3 around T. gondii and spontaneous parasite killing dependent on the autophagy protein ULK1 and lysosomal enzymes. The autophagy inhibitor 3-MA prevented DN EGFR mice from exhibiting reduced CNS invasion. Altogether, EGFR is a novel regulator of T. gondii invasion of neural tissue, enhancing invasion likely by promoting survival of the parasite within endothelial cells.

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 1ß (IL-1ß), 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-1ß 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-1ß by macrophages. P2X7-/- mice and mice treated with a P2X7 inhibitor were protected from diabetes-induced TNF-α, IL-1ß, 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.

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-1ß 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.