背景:脑型疟疾(CM)是疟疾最致命的并发症,幸存者通常会忍受神经系统后遗症。值得注意的是,浸润性疟原虫激活的CD8+T细胞对脑微血管内皮细胞的细胞毒性作用是具有血脑屏障破坏的实验性CM(ECM)模型的突出特征.然而,CD8+T细胞浸润脑实质对神经元的损伤作用尚不清楚.基于PD-1/PD-L1通路对T细胞的免疫抑制作用,我们之前的研究表明,全身上调PD-L1抑制CD8+T细胞功能可以有效缓解ECM小鼠的症状。然而,神经元是否可以通过PD-1/PD-L1阴性免疫调节途径抑制CD8+T细胞的致病作用尚未见报道。作为CM的重要炎症因子,干扰素可以根据神经免疫微环境通过不同的分子机制诱导PD-L1的表达。因此,本研究旨在探讨CD8+T细胞与神经元之间的直接相互作用,以及神经元通过上调IFN诱导的PD-L1减轻CD8+T细胞致病作用的机制。
方法:使用感染伯氏疟原虫ANKA(PbA)的C57BL/6J小鼠的ECM模型,通过电子显微镜和IF染色进行体内形态学观察。通过IF染色和延时摄影观察了ECMCD8T细胞(来自ECM小鼠脾脏的免疫磁珠分选)与体外原代培养的皮质神经元之间的相互作用。RNA-seq分析IFNβ或IFNγ诱导的神经元PD-L1上调的信号通路,并通过q-PCR验证,WB,IF染色,和使用IFNAR或IFNGR基因敲除小鼠的体外和体内流式细胞术。腺病毒介导的PD-L1IgGFc融合蛋白表达的保护作用在体内脑立体定向注射的ECM小鼠和体外通过病毒感染的原代培养神经元中得到验证。
结果:体内,ECM小鼠脑实质显示活化的CD8+T细胞浸润和神经元损伤。体外,ECMCD8+T细胞与神经元直接接触并诱导轴突损伤,作为一种积极的行为。在ECM小鼠神经元和IFNβ诱导的原代培养神经元中PD-L1蛋白水平升高,IFNγ,或体外ECMCD8+T细胞。此外,IFNβ或IFNγ诱导的PD-L1神经元表达是通过IFN受体增加STAT1/IRF1途径介导的。在删除IFNAR或IFNGR后,PbA感染期间神经元中PD-L1表达的增加减弱。腺病毒增加的PD-L1表达在体外和体内部分保护神经元免受CD8+T细胞介导的损伤。
结论:我们的研究表明,I型和II型IFN均可通过IFN受体介导的STAT1/IRF1途径诱导神经元上调PD-L1,以防止活化的CD8+T细胞介导的损伤,提供了一个靶向途径来减轻ECM过程中的神经炎症。
BACKGROUND: Cerebral malaria (CM) is the most lethal complication of malaria, and survivors usually endure neurological sequelae. Notably, the cytotoxic effect of infiltrating Plasmodium-activated CD8+ T cells on cerebral microvasculature endothelial cells is a prominent feature of the experimental CM (ECM) model with blood-brain barrier disruption. However, the damage effect of CD8+ T cells infiltrating the brain parenchyma on neurons remains unclear. Based on the immunosuppressive effect of the PD-1/PD-L1 pathway on T cells, our previous study demonstrated that the systemic upregulation of PD-L1 to inhibit CD8+ T cell function could effectively alleviate the symptoms of ECM mice. However, it has not been reported whether neurons can suppress the pathogenic effect of CD8+ T cells through the PD-1/PD-L1 negative immunomodulatory pathway. As the important inflammatory factor of CM, interferons can induce the expression of PD-L1 via different molecular mechanisms according to the neuro-immune microenvironment. Therefore, this study aimed to investigate the direct interaction between CD8+ T cells and neurons, as well as the mechanism of neurons to alleviate the pathogenic effect of CD8+ T cells through up-regulating PD-L1 induced by IFNs.
METHODS: Using the ECM model of C57BL/6J mice infected with Plasmodium berghei ANKA (PbA), morphological observations were conducted in vivo by electron microscope and IF staining. The interaction between the ECM CD8+ T cells (immune magnetic bead sorting from spleen of ECM mice) and primary cultured cortical neurons in vitro was observed by IF staining and time-lapse photography. RNA-seq was performed to analyze the signaling pathway of PD-L1 upregulation in neurons induced by IFNβ or IFNγ, and verified through q-PCR, WB, IF staining, and flow cytometry both in vitro and in vivo using IFNAR or IFNGR gene knockout mice. The protective effect of adenovirus-mediated PD-L1 IgGFc fusion protein expression was verified in ECM mice with brain stereotaxic injection in vivo and in primary cultured neurons via viral infection in vitro.
RESULTS: In vivo, ECM mice showed infiltration of activated CD8+ T cells and neuronal injury in the brain parenchyma. In vitro, ECM CD8+ T cells were in direct contact with neurons and induced axonal damage, as an active behavior. The PD-L1 protein level was elevated in neurons of ECM mice and in primary cultured neurons induced by IFNβ, IFNγ, or ECM CD8+ T cells in vitro. Furthermore, the IFNβ or IFNγ induced neuronal expression of PD-L1 was mediated by increasing STAT1/IRF1 pathway via IFN receptors. The increase of PD-L1 expression in neurons during PbA infection was weakened after deleting the IFNAR or IFNGR. Increased PD-L1 expression by adenovirus partially protected neurons from CD8+ T cell-mediated damage both in vitro and in vivo.
CONCLUSIONS: Our study demonstrates that both type I and type II IFNs can induce neurons to upregulate PD-L1 via the STAT1/IRF1 pathway mediated by IFN receptors to protect against activated CD8+ T cell-mediated damage, providing a targeted pathway to alleviate neuroinflammation during ECM.