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-1β 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-1β (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. Graphical abstract

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Purpose CD40 is upregulated in the retinas of diabetic mice, drives pro-inflammatory molecule expression, and promotes diabetic retinopathy. The role of CD40 in diabetic retinopathy in humans is unknown. Upregulation of CD40 and its downstream signaling molecules TNF receptor associated factors (TRAFs) is a key feature of CD40-driven inflammatory disorders. We examined the expression of CD40, TRAF2, and TRAF6 as well as pro-inflammatory molecules in retinas from patients with diabetic retinopathy. Methods Posterior poles from patients with diabetic retinopathy and non-diabetic controls were stained with antibodies against von Willebrand factor (labels endothelial cells), cellular retinaldehyde-binding protein (CRALBP), or vimentin (both label Müller cells) plus antibodies against CD40, TRAF2, TRAF6, ICAM-1, CCL2, TNF-α, and/or phospho-Tyr783 phospholipase Cγ1 (PLCγ1). Sections were analyzed by confocal microscopy. Results CD40 expression was increased in endothelial and Müller cells from patients with diabetic retinopathy. CD40 was co-expressed with ICAM-1 in endothelial cells and with CCL2 in Müller cells. TNF-α was detected in retinal cells from these patients, but these cells lacked endothelial/Müller cell markers. CD40 in Müller cells from patients with diabetic retinopathy co-expressed activated phospholipase Cγ1, a molecule that induces TNF-α expression in myeloid cells in mice. CD40 upregulation in endothelial cells and Müller cells from patients with diabetic retinopathy was accompanied by TRAF2 and TRAF6 upregulation. Conclusions CD40, TRAF2, and TRAF6 are upregulated in patients with diabetic retinopathy. CD40 associates with expression of pro-inflammatory molecules. These findings suggest that CD40-TRAF signaling may promote pro-inflammatory responses in the retinas of patients with diabetic retinopathy.

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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-1β, and NOS2 mRNA levels. CD40 stimulation in retinal endothelial cells upregulated ICAM-1 but not TNF-α, IL-1β, 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 P2X 7 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.