IL-9 触发的长非编码 RNA Gm13568 通过与 CBP/P300 的相互作用调节星形胶质细胞中的 Notch1：有助于实验性自身免疫性脑脊髓炎的发病机制。

IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis.

机构信息

Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogen Biology and Immunology and Laboratory of Infection and Immunity, Xuzhou Medical University, 209 Tongshan Road, Xuzhou, Jiangsu, 221004, People's Republic of China.

Neurology Department, The Affiliated Xuzhou Center Hospital of Nanjing University of Chinese Medicine, Xuzhou, People's Republic of China.

出版信息

J Neuroinflammation. 2021 May 11;18(1):108. doi: 10.1186/s12974-021-02156-5.

DOI:10.1186/s12974-021-02156-5

PMID:33971906

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8112022/

Abstract

BACKGROUND

Interleukin 9 (IL-9), produced mainly by T helper 9 (Th9) cells, has been recognized as an important regulator in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Astrocytes respond to IL-9 and reactive astrocytes always associate with blood-brain barrier damage, immune cell infiltration, and spinal injury in MS and EAE. Several long non-coding RNAs (lncRNAs) with aberrant expression have been identified in the pathogenesis of MS. Here, we examined the effects of lncRNA Gm13568 (a co-upregulated lncRNA both in EAE mice and in mouse primary astrocytes activated by IL-9) on the activation of astrocytes and the process of EAE.

METHODS

In vitro, shRNA-recombinant lentivirus with glial fibrillary acidic protein (GFAP) promoter were performed to determine the relative gene expression and proinflammatory cytokines production in IL-9 treated-astrocytes using Western blot, real-time PCR, and Cytometric Bead Array, respectively. RIP and ChIP assays were analyzed for the mechanism of lncRNA Gm13568 regulating gene expression. Immunofluorescence assays was performed to measure the protein expression in astrocytes. In vivo, H&E staining and LFB staining were applied to detect the inflammatory cells infiltrations and the medullary sheath damage in spinal cords of EAE mice infected by the recombinant lentivirus. Results were analyzed by one-way ANOVA or Student's t test, as appropriate.

RESULTS

Knockdown of the endogenous lncRNA Gm13568 remarkably inhibits the Notch1 expression, astrocytosis, and the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) as well as the production of inflammatory cytokines and chemokines (IL-6, TNF-α, IP-10) in IL-9-activated astrocytes, in which Gm13568 associates with the transcriptional co-activators CBP/P300 which are enriched in the promoter of Notch1 genes. More importantly, inhibiting Gm13568 with lentiviral vector in astrocytes ameliorates significantly inflammation and demyelination in EAE mice, therefore delaying the EAE process.

CONCLUSIONS

These findings uncover that Gm13568 regulates the production of inflammatory cytokines in active astrocytes and affects the pathogenesis of EAE through the Notch1/STAT3 pathway. LncRNA Gm13568 may be a promising target for treating MS and demyelinating diseases.

摘要

背景

白细胞介素 9（IL-9）主要由辅助性 T 细胞 9（Th9）细胞产生，已被认为是多发性硬化症（MS）及其动物模型实验性自身免疫性脑脊髓炎（EAE）的重要调节剂。星形胶质细胞对 IL-9 有反应，反应性星形胶质细胞总是与 MS 和 EAE 中的血脑屏障损伤、免疫细胞浸润和脊髓损伤有关。已经在 MS 的发病机制中鉴定出几种表达异常的长链非编码 RNA（lncRNA）。在这里，我们研究了 lncRNA Gm13568（EAE 小鼠和 IL-9 激活的小鼠原代星形胶质细胞中共同上调的 lncRNA）对星形胶质细胞激活和 EAE 过程的影响。

方法

体外，用胶质纤维酸性蛋白（GFAP）启动子的 shRNA 重组慢病毒处理，通过 Western blot、实时 PCR 和流式细胞术分别检测 IL-9 处理的星形胶质细胞中的相对基因表达和促炎细胞因子的产生。RIP 和 ChIP 测定用于分析 lncRNA Gm13568 调节基因表达的机制。免疫荧光测定用于测量星形胶质细胞中的蛋白质表达。体内，通过重组慢病毒感染的 EAE 小鼠的 H&E 染色和 LFB 染色检测脊髓中的炎症细胞浸润和髓鞘损伤。结果采用单因素方差分析或学生 t 检验进行分析。

结果

内源性 lncRNA Gm13568 的敲低显着抑制 Notch1 表达、星形胶质细胞增生以及信号转导和转录激活因子 3（p-STAT3）的磷酸化以及促炎细胞因子和趋化因子（IL-6、TNF-α、IP-10）的产生在 IL-9 激活的星形胶质细胞中，其中 Gm13568 与转录共激活因子 CBP/P300 结合，CBP/P300 在 Notch1 基因的启动子中富集。更重要的是，用慢病毒载体抑制星形胶质细胞中的 Gm13568 显着改善了 EAE 小鼠的炎症和脱髓鞘，从而延缓了 EAE 过程。