Varicella-zoster virus hijacks the type I interferon response and antigen presentation pathways in matured hiPSC-derived neurospheroids

Varicella-zoster virus (VZV) encephalitis and meningitis are potential central nervous system (CNS) complications following primary VZV infection or reactivation. With innate immune signalling, and more specifically type I interferon (IFN) signalling, being an important first line cellular defence mechanism against VZV infection, we here investigated the triggering of innate immune responses in a human CNS-like environment. For this, we established and characterised a 5-month matured hiPSC-derived neurospheroid (NSPH) model containing TuJ1+ MAP2+ NeuN+ neurons and GFAP+ S100b+ AQP4+ CD49f+ astrocytes. Immune competence of these NSPHs was demonstrated by secretion of IL-6 and CXCL10 following stimulation with a cocktail of pro-inflammatory stimuli. Subsequently, NSPHs were infected with reporter strains of VZV (eGFP-ORF23 VZV) and Sendai virus (eGFP SeV). Live cell and immunocytochemical analyses demonstrated VZV infection within NSPHs, while SeV infection was limited to the outer NSPH border. Next, a transcript-level immune profiling was performed using NanoString technology to explore innate immune signatures of virus-infected NSPHs. While SeV-infected NSPHs displayed a clear Type I IFN response, in VZV-infected NSPHs no Type I IFN response was activated. Even more, in the latter a strong suppression of genes related to IFN signalling and antigen presentation was noted. Validating these opposite immune signatures in VZV- and SeV-infected NSPHs, cytokine profiling of NSPH supernatant revealed increased secretion of IL6 and CXCL10 by SeV-infected NSPHs, but not by VZV-infected NSPHs. Similarly, immunocytochemical analysis demonstrated upregulation of type I IFN activated anti-viral proteins Mx1, IFIT2 and ISG15 in SeV-infected NSPHs, but not in VZV-infected NSPHs. Furthermore, CD74, a key part of the MHC class II antigen presentation pathway was found to be suppressed in VZV-infected NSPHs. Finally, even though VZV-infection seems to be immunologically ignored in NSPHs, its presence does result in the formation of stress granules throughout the entire NSPH. Concluding, in this study we demonstrate that 5-month matured hiPSC-derived NSPHs display functional innate immune reactivity towards SeV infection, as well as their capability to recapitulate the strong immune evasive behaviour of VZV. Subsequently, this NSPH model has the potential to study viral neuro-immune responses and evasion strategies in a human CNS-like environment. At 7 days post stimulation/infection, NSPHs for RNA extraction were washed with ice cold PBS, snap frozen in liquid nitrogen and stored at -80°C. RNA extraction was performed on 2 pooled NSPHs of each condition (in triplicate) using the RNeasy Mini Kit (Qiagen, 74104) according to the manufacturer's protocol. The RNA samples were used for nCounter (NanoString) analysis on a nCounter® MAX Analysis System. Briefly, RNA extracts were hybridized to ± 600 unique capture/reporter pairs (50bp each) targeting 585 immune transcripts and 15 housekeeping genes, as defined in the Human Immunology nCounter® panel, as well as six positive and eight negative control probes (all from NanoString). Results were sequentially corrected for background (negative control probes), technical variation (positive control probes) and RNA content (housekeeping genes) using nSolver 4.0 (NanoString), followed by differential gene expression analysis and gene set enrichment analysis (based on GO-terms) using Omics Playground (BigOmics Analytics).

水痘带状疱疹病毒（Varicella-zoster virus, VZV）脑炎与脑膜炎是原发性VZV感染或病毒再激活后可能出现的中枢神经系统（central nervous system, CNS）并发症。先天免疫信号通路，尤其是I型干扰素（type I interferon, IFN）信号通路，是抵御VZV感染的重要第一道细胞防御机制，本研究旨在探究人类中枢神经系统模拟环境中先天免疫应答的触发机制。为此，我们构建并表征了一种经5个月成熟培养的人类诱导多能干细胞（human induced pluripotent stem cell, hiPSC）来源的神经球（neurospheroid, NSPH）模型，该模型包含TuJ1+ MAP2+ NeuN+神经元以及GFAP+ S100b+ AQP4+ CD49f+星形胶质细胞。通过促炎刺激物混合物刺激后，该神经球可分泌IL-6与CXCL10，证实了其免疫活性。随后，我们用VZV报告株（eGFP-ORF23 VZV）与仙台病毒（Sendai virus, SeV）感染神经球。活细胞成像与免疫细胞化学分析显示，VZV可在神经球内部建立感染，而SeV感染仅局限于神经球外层边界。接下来，我们采用NanoString技术开展转录组水平的免疫谱分析，以探究病毒感染神经球后的先天免疫特征。SeV感染的神经球呈现出显著的I型IFN应答，而VZV感染的神经球未激活I型IFN应答。更为显著的是，VZV感染组中与IFN信号通路及抗原呈递相关的基因受到强烈抑制。为验证VZV与SeV感染神经球后截然不同的免疫特征，我们对神经球上清液进行细胞因子谱分析，结果显示SeV感染组的IL-6与CXCL10分泌量显著升高，而VZV感染组未出现该变化。类似地，免疫细胞化学分析证实，SeV感染组的I型IFN激活的抗病毒蛋白Mx1、IFIT2与ISG15表达上调，而VZV感染组未观察到此现象。此外，我们发现MHC II类抗原呈递通路的关键组分CD74在VZV感染的神经球中受到抑制。最后，尽管VZV感染在神经球中似乎未被免疫系统触发应答，但其存在仍会导致整个神经球内形成应激颗粒。本研究最终证实，经5个月成熟培养的hiPSC来源神经球对SeV感染具有功能性先天免疫应答能力，同时可复现VZV强大的免疫逃逸行为。该神经球模型有望在人类中枢神经系统模拟环境中研究病毒神经免疫应答与逃逸策略。感染/刺激后7天，用于RNA提取的神经球经冰态PBS洗涤后，用液氮快速冷冻并保存于-80℃。我们将每个条件下的2个神经球混合（每组设置3次生物学重复），采用RNeasy Mini Kit（Qiagen, 74104）按照制造商说明书进行RNA提取。提取的RNA样本用于nCounter（NanoString）分析，检测平台为nCounter® MAX分析系统。简要而言，RNA提取物与约600对独特的捕获/报告探针（每对长度50bp）进行杂交，这些探针靶向585个免疫转录本与15个持家基因（对应Human Immunology nCounter®试剂盒），同时包含6个阳性对照探针与8个阴性对照探针（均购自NanoString）。我们使用nSolver 4.0（NanoString）对结果依次进行背景校正（基于阴性对照探针）、技术偏差校正（基于阳性对照探针）与RNA含量校正（基于持家基因）；随后采用Omics Playground（BigOmics Analytics）进行差异基因表达分析与基于基因本体论（Gene Ontology, GO）术语的基因集富集分析。