Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Joseph-Stelzmann-Str. 9, D-50931, Cologne, Germany.
Center for Molecular Medicine Cologne, Cologne, Germany.
J Neuroinflammation. 2020 Nov 3;17(1):327. doi: 10.1186/s12974-020-01999-8.
Retinal degenerative diseases significantly contribute to visual impairment and blindness. Microglia reactivity is a hallmark of neurodegenerative diseases including retinal cell death and immunomodulation emerges as a therapeutic option. Indole-3-carbinol (I3C) is a natural ligand of aryl hydrocarbon receptor (AhR), with potent immunomodulatory properties. Here, we hypothesized that I3C may inhibit microglia reactivity and exert neuroprotective effects in the light-damaged murine retina mimicking important immunological aspects of retinal degeneration.
BV-2 microglia were treated in vitro with I3C followed by lipopolysaccharide (LPS) stimulation to analyze pro-inflammatory and anti-oxidant responses by quantitative real-time PCR (qRT-PCR) and Western blots. Nitric oxide (NO) secretion, caspase 3/7 levels, phagocytosis rates, migration, and morphology were analyzed in control and AhR knockdown cells. I3C or vehicle was systemically applied to light-treated BALB/cJ mice as an experimental model of retinal degeneration. Pro-inflammatory and anti-oxidant responses in the retina were examined by qRT-PCR, ELISA, and Western blots. Immunohistochemical staining of retinal flat mounts and cryosections were performed. The retinal thickness and structure were evaluated by in vivo imaging using spectral domain-optical coherence tomography (SD-OCT).
The in vitro data showed that I3C potently diminished LPS-induced pro-inflammatory gene expression of I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 and induced anti-oxidants gene levels of NQO1, HMOX1, and CAT1 in BV-2 cells. I3C also reduced LPS-induced NO secretion, phagocytosis, and migration as important functional microglia parameters. siRNA-mediated knockdown of AhR partially prevented the previously observed gene regulatory events. The in vivo experiments revealed that I3C treatment diminished light-damage induced I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 transcripts and also reduced CCL2, I-NOS, IL-1ß, p-NFkBp65 protein levels in mice. Moreover, I3C increased anti-oxidant NQO1 and HMOX1 protein levels in light-exposed retinas. Finally, I3C therapy prevented the accumulation of amoeboid microglia in the subretinal space and protected from retinal degeneration.
The AhR ligand I3C potently counter-acts microgliosis and light-induced retinal damage, highlighting a potential treatment concept for retinal degeneration.
视网膜退行性疾病是导致视力损害和失明的主要原因。小胶质细胞的反应是神经退行性疾病的一个标志，包括视网膜细胞死亡和免疫调节，这已成为一种治疗选择。吲哚-3-甲醇（I3C）是芳香烃受体（AhR）的天然配体，具有很强的免疫调节特性。在这里，我们假设 I3C 可能抑制小胶质细胞的反应，并在模拟视网膜变性重要免疫学方面的光损伤小鼠视网膜中发挥神经保护作用。
用 I3C 处理体外培养的 BV-2 小胶质细胞，然后用脂多糖（LPS）刺激，通过定量实时 PCR（qRT-PCR）和 Western blot 分析促炎和抗氧化反应。在对照和 AhR 敲低细胞中分析一氧化氮（NO）分泌、半胱天冬酶 3/7 水平、吞噬率、迁移和形态。I3C 或载体系统地应用于光处理的 BALB/cJ 小鼠，作为视网膜变性的实验模型。通过 qRT-PCR、ELISA 和 Western blot 检测视网膜中的促炎和抗氧化反应。对视网膜扁平标本和冷冻切片进行免疫组织化学染色。使用光谱域光学相干断层扫描（SD-OCT）进行体内成像评估视网膜厚度和结构。
体外数据显示，I3C 可强力减弱 LPS 诱导的 BV-2 细胞中诱导型一氧化氮合酶（I-NOS）、白细胞介素-1β（IL-1β）、NLRP3、白细胞介素-6（IL-6）和 C 型趋化因子配体 2（CCL2）的促炎基因表达，并诱导抗氧化剂 NQO1、血红素加氧酶 1（HMOX1）和 CAT1 的基因水平。I3C 还降低了 LPS 诱导的 NO 分泌、吞噬和迁移等重要的小胶质细胞功能参数。AhR 的 siRNA 介导的敲低部分阻止了先前观察到的基因调控事件。体内实验表明，I3C 治疗可减弱光损伤诱导的 I-NOS、IL-1β、NLRP3、IL-6 和 CCL2 转录物，并降低光暴露视网膜中的 CCL2、I-NOS、IL-1β、p-NFkBp65 蛋白水平。此外，I3C 增加了抗氧化剂 NQO1 和 HMOX1 的蛋白水平。最后，I3C 治疗可防止阿米巴样小胶质细胞在视网膜下腔的积聚，并防止视网膜变性。
AhR 配体 I3C 可有效拮抗小胶质细胞增生和光诱导的视网膜损伤，为视网膜变性提供了一种潜在的治疗方法。
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