Diabetologia ( IF 8.4 ) Pub Date : 2022-08-03 , DOI: 10.1007/s00125-022-05775-6 Jose-Andres C Portillo 1 , Jin-Sang Yu 1 , Sarah Vos 1 , Reena Bapputty 2 , Yalitza Lopez Corcino 1 , Alyssa Hubal 1, 3 , Jad Daw 1 , Sahil Arora 1 , Wenyu Sun 4 , Zheng-Rong Lu 4 , Carlos S Subauste 1, 3
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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
中文翻译：
通过 CD40 衍生肽或 Müller 细胞中 CD40 突变破坏小鼠视网膜炎症和糖尿病视网膜病变的发展
目标/假设
Müller 细胞中表达的 CD40 是糖尿病视网膜病变的主要驱动因素。 CD40 导致 Müller 细胞中磷脂酶 Cγ1 (PLCγ1) 依赖性 ATP 释放，随后在骨髓细胞中嘌呤能受体 (P2X 7 ) 依赖性促炎细胞因子的产生。在糖尿病视网膜中，CD40 和 P2X 7上调多种炎症分子，促进糖尿病视网膜病变的发展。 CD40 下游激活 PLCγ1–ATP–P2X 7 -促炎细胞因子级联并促进糖尿病视网膜病变发展的分子事件尚不清楚。我们假设，破坏触发该级联的 CD40 驱动的分子事件可以预防/治疗小鼠糖尿病视网膜病变。
方法
使用链脲佐菌素使 B6 和具有 Müller 细胞限制性表达野生型 (WT) CD40 或具有 TNF 受体相关因子 (TRAF) 结合位点突变的 CD40 的转基因小鼠患上糖尿病。使用 FITC 缀合的刀豆球蛋白 A 评估白细胞沉积。在视网膜脉管系统中检查组织病理学。使用实时 PCR、免疫印迹和/或免疫组织化学评估炎症分子和磷酸-Tyr783 PLCγ1 (p-PLCγ1) 的表达。分别通过 ATP 生物发光和 ELISA 测量 ATP 和细胞因子的释放。
结果
具有 CD40 ΔT2,3（缺乏 TRAF2,3 结合位点）的人 Müller 细胞无法磷酸化 PLCγ1 并响应 CD40 连接释放 ATP，并且不能诱导旁观者骨髓细胞分泌 TNF-α/IL-1β。 CD40-TRAF 信号通过 Src 诱导 PLCγ1 磷酸化。 Müller 细胞中的 WT CD40 被 CD40 ΔT2,3 取代的糖尿病小鼠未能表现出这些细胞中 PLCγ1 的磷酸化，也未能上调小胶质细胞/巨噬细胞中的 P2X 7和 TNF-α。 P2x 7 （也称为P2rx7 ）、 Tnf-α （也称为Tnf ）、 Il-1β （也称为Il1b ）、 Nos2 、 Icam-1 （也称为Icam1 ）和Ccl2 mRNA 在这些小鼠中没有增加，并且小鼠没有出现视网膜白细胞停滞和毛细血管变性。用破坏 CD40-TRAF2,3 信号传导的细胞渗透性肽进行玻璃体内治疗的糖尿病 B6 小鼠没有表现出视网膜中 P2X 7和炎症分子的上调或白细胞停滞。
结论/解释
CD40–TRAF2,3 信号传导激活 CD40–PLCγ1–ATP–P2X 7 –促炎细胞因子途径。 Src 充当 CD40–TRAF2,3 和 PLCγ1 之间的链接。用 CD40 ΔT2,3 替换 WT CD40 会损害 Müller 细胞中 PLCγ1 的激活、小胶质细胞/巨噬细胞中 P2X 7的上调、糖尿病视网膜中多种炎症分子的上调以及糖尿病视网膜病变的发展。给予破坏 CD40-TRAF2,3 信号传导的肽可减少糖尿病小鼠视网膜炎症分子的表达并减少白细胞淤积，支持药物抑制 CD40-TRAF2,3 在糖尿病视网膜病变中的治疗潜力。
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