Molecular and cellular dissection of the pathogenesis of herpes simplex encephalitis with iPSC-derived CNS and PNS cells

项目摘要 单纯疱疹病毒1型脑炎(HSV-1)是西方国家最常见的散发性病毒性脑炎。健康儿童的前脑HSE(通过嗅觉神经元发育)可能是由于TLR3途径的先天错误(TLR3、UNK93B1、TRIF、TRAF3、TBK1和IRF3的突变)，而脑干HSE(通过三叉神经(TG)神经元)可能是由于RNA套叠代谢(DBR1)的先天错误。具有NEMO产生受损或对所有IFN反应受损(STAT1)的更广泛缺陷的儿童容易发生HSE和其他感染。我们通过从诱导的多能干细胞(IPSC)(NIH R01NS072381)中分离出外周和中枢神经系统(PNS)细胞来分析HSE的细胞学基础。与星形胶质细胞和神经干细胞不同，TLR3缺陷的前脑皮质神经元和少突胶质细胞前体细胞损害了抗HSV-1细胞的固有免疫；小胶质细胞未被检测。此外，IPSC来源的TG神经元并不依赖TLR3来控制HSV-1；没有对嗅觉神经元进行测试。最后，TLR3控制皮质神经元的基础干扰素水平和抗HSV-1免疫的早期步骤。因此，儿童HSE是由非造血性、中枢神经系统特异性的细胞固有免疫的先天错误造成的，尤其影响皮质神经元和少突胶质细胞。自那以后，我们已经发现了与TLR3-干扰素回路(MEX3B，IFNAR1)、TLR3-坏死性下垂途径(RIPK3)以及未知功能(SNORA31，TMEFF1)相关的新的前脑HSE致病基因。在这一新的应用中，我们假设前脑HSE的遗传病因损害了皮质神经元、少突胶质细胞和可能的小胶质细胞的固有免疫，而DBR1缺陷损害了脑干和/或TG神经元的固有免疫，可能还损害了小胶质细胞的固有免疫。首先，我们将评估IPSC来源的嗅觉和皮质神经元以及其他中枢神经系统细胞对TLR3、干扰素-α/β或干扰素-λ以及单纯疱疹病毒1型刺激的反应。第二，我们将研究核糖核酸的积累，对单纯疱疹病毒1型感染的免疫力，以及对干扰素-α/β或干扰素-λ的反应，在对照组和DBR1缺乏的IPSC分化的TG和脑干神经元中。第三，通过研究TLR3激动剂干扰素-α/β、干扰素-λ和单纯疱疹病毒1型在分离培养和神经元共培养中的反应，评估小胶质细胞在前脑和脑干高血压病中的作用。将使用通过基因编辑纠正或引入突变的患者和等基因iPSC株。将对HSV-1的细胞内源性免疫及其分子基础进行分析。已经获得了令人兴奋的初步数据，包括(1)HSE的新的遗传病因，(2)TLR3控制皮质神经元中HSV-1的新机制，以及(3)分化脑干神经元和小胶质细胞的新方案。这项基于人类IPSC的HSE研究的扩展将使我们能够更广泛和深入地剖析其在患有中枢神经系统固有免疫缺陷的儿童中对HSV-1的分子和细胞基础。

Project Summary Herpes simplex virus 1 (HSV-1) encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries. Forebrain HSE (which develops via olfactory neurons) in otherwise healthy children can result from inborn errors of the TLR3 pathway (mutations in TLR3, UNC93B1, TRIF, TRAF3, TBK1, and IRF3), whereas brainstem HSE (via trigeminal (TG) neurons) can result from inborn errors of RNA lariat metabolism (DBR1). Children with a broader defect of impaired production of (NEMO) or response to all IFNs (STAT1) are prone to HSE and other infections. We analyzed the cellular basis of HSE by deriving peripheral and central nervous system (PNS and CNS) cells from induced pluripotent stem cells (iPSC) (NIH R01NS072381). TLR3-deficient forebrain cortical neurons and oligodendrocyte precursors have impaired anti-HSV-1 cell-intrinsic immunity, unlike astrocytes and neural stem cells; microglial cells were not tested. Moreover, iPSC-derived TG neurons do not rely on TLR3 to control HSV-1; olfactory neurons were not tested. Finally, TLR3 controls both basal IFN levels and early steps of anti-HSV-1 immunity in cortical neurons. Childhood HSE results thus from inborn errors of non-hematopoietic, CNS-specific, cell-intrinsic immunity, affecting cortical neurons and oligodendrocytes in particular. We have since identified new forebrain HSE-causing genes that are connected to the TLR3-IFN circuit (MEX3B, IFNAR1), the TLR3-necroptosis pathway (RIPK3), and of unknown function (SNORA31, TMEFF1). In this renewal application, we hypothesize that genetic etiologies of forebrain HSE impair intrinsic immunity in cortical but not olfactory neurons, and in oligodendrocytes and probably microglial cells, and DBR1 deficiency impairs intrinsic immunity in brainstem and/or TG neurons and probably microglial cells. First, we will assess the responses of iPSC-derived olfactory and cortical neurons, and other CNS cells, from controls and patients mutated in new forebrain HSE-causing genes, to TLR3, IFN-α/β or IFN-λ, and HSV-1 stimulation. Second, we will study RNA lariat accumulation, immunity to HSV-1 infection, and responses to IFN-α/β or IFN- λ, in control and DBR1-deficient iPSC-differentiated TG and brainstem neurons. Third, the role of microglial cells in forebrain vs brainstem HSE will be assessed by studying the responses to TLR3 agonists, IFN-α/β, IFN-λ and HSV-1, in isolation and in neuronal co-culture. Patient and isogenic iPSC lines in which the mutation is corrected or introduced by gene editing will be used. Cell-intrinsic immunity to HSV-1 and its molecular basis will be analyzed. Exciting preliminary data have been obtained, including (1) novel genetic etiologies of HSE, (2) novel mechanisms by which TLR3 controls HSV-1 in cortical neurons, and (3) novel protocols to differentiate brainstem neurons and microglial cells. The expand of this human iPSC-based study of HSE will enable us to dissect in greater breadth and depth its molecular and cellular basis in children with inborn errors of CNS-intrinsic immunity to HSV-1.