Investigating roles of Topoisomerase 3b (TOP3B) in immune responses to infection by coronavirus and bacteria

研究拓扑异构酶 3b (TOP3B) 在冠状病毒和细菌感染免疫反应中的作用

• 批准号: 10913087
• 负责人: Weidong Wang
• 金额: $ 37.57万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10913087
• 关键词: Affect; Area; Autoantibodies; B-Lymphocytes; Bacteria; Bacterial Infections; Blood; Blood Cells; Bone Marrow Cells; Cell Line; Cells; Chemotaxis; Clinical Trials; Coagulation Process; Complex; Control Groups; Coronavirus; Coronavirus Infections; DNA; Data; Diffusion; Disease; Drosophila genus; Economics; Exhibits; FMR1; Family; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Harvest; Heterochromatin; Human; Immune System Diseases; Immune response; Immune signaling; Immune system; Infection; Inflammation; Inflammatory Response; Injury; Knockout Mice; Life; Listeria; Listeria monocytogenes; Longevity; Lung; Lymphocyte; Macrophage; Melanins; Messenger RNA; Modeling; Murine hepatitis virus; Mus; Numerical Chromosomal Abnormality; Patients; Pharmaceutical Preparations; Phenotype; Pigmentation physiologic function; Pigments; RNA; Recovery; Role; SARS coronavirus; Sampling; Schizophrenia; Serum; Signal Pathway; Site; Small Interfering RNA; Splenocyte; Stimulus; Study models; T-Lymphocyte; TOP1 gene; Testing; Time; Topoisomerase; Topoisomerase Inhibitors; Transcriptional Activation; Update; Viral; Viral hepatitis; Virus; Work; autism spectrum disorder; behavioral impairment; biological adaptation to stress; chemokine; cytokine; cytokine release syndrome; cytotoxic; enzyme activity; established cell line; experimental study; extracellular; fly; immunoregulation; inhibitor; insight; mRNA Translation; male; melanocyte; mouse model; neural; neurodevelopment; neutrophil; pathogen; pathogenic bacteria; pathogenic virus; prevent; response; transcriptome sequencing; wound response

Our group has discovered that Topoisomerase 3b (TOP3B) is a dual activity enzyme that can change the topology of not only DNA, but also RNA (XU et al. 2013). TOP3B forms a complex with TDRD3; this complex interacts with Fragile X Mental Retardation Protein (FMRP). Our recent studies show that Top3b-TDRD3 regulates stimulus-induced transcription, mRNA translation and turnover, as well as siRNA-guided heterochromatin formation (LEE et al. 2018; SU et al. 2022). The deletion of Top3b in human is associated with neural diseases, such as schizophrenia and autism (STOLL et al. 2013; XU et al. 2013; AHMAD et al. 2017). Our studies also showed that Top3b-KO mice had neurodevelopment and behavioral impairments (JOO et al. 2020). Interestingly, TOP3B-KO mice have shortened lifespan and immune dysfunction, exemplified by increased serum autoantibodies and an increase in numerical aberrations of chromosomes in splenocytes and bone marrow cells (KWAN et al. 2007). However, the mechanism of how Top3B functions in immune system is unclear. This new proposal is to investigate how TOP3B regulates immune response to mouse coronavirus MHV (Mouse Hepatitis virus) infection in mouse, and to bacterial infection in Drosophila. Severe Acute Respiratory Syndrome Coronavirus2 (SARS-Cov2) has caused economic and life damages over the past few years. The host immune response to SARS-Cov2 is responsible for viral elimination and recovery (GARCIA 2020). On the other hand, the strong inflammation (termed cytokine storm) elicited by the virus can also cause severe sickness or even lethality to the host (GARCIA 2020). Anti-inflammation drugs are often used to suppress cytokine storms in SARS-Cov2 patient. Notably, inhibitors of Topoisomerase 1 (Top1) have been developed as one class of anti-inflammation drugs, and are now being examined in clinical trials to treat SARS-Cov2 patients (HO et al. 2021). TOP3B resembles TOP1 in that both can promote transcriptional activation in response to extra cellular stimulus. We propose to study the roles of TOP3B in immune responses to infection by coronavirus and bacteria. The data should provide insights on whether TOP3B inhibitors can work as drugs to control immune responses. Specific Aims: Aim1: Investigate the functions of TOP3B in T and B cell response to MHV infection in mouse. Aim2: Investigate the functions of TOP3B in cytokine response to MHV infection in mouse-models and cell lines. Aim3: Investigate whether and how Top3b-Tdrd3 contributes to regulation of immune signaling pathways using a Drosophila model. Progress update: 1. Top3b-KO mice display altered levels of Lymphocytes and Neutrophils responding to MHV. We started to test whether Top3b-KO mouse-models show altered levels of lymphocytes and neutrophils in response to MHV infection. Four groups of mice, two wild type and two Top3b-KO, were inoculated with 105 pfu MHV or DMEM as a negative control. Blood was collected at three time points: seven days prior to inoculation, two days post inoculation, and 5 days post inoculation. Blood cell analysis was performed to test which cells were most affected during MHV infection in wild types and Top3b-KO mouse-models. We found that MHV-infected Top3b-KO mice showed a significant increase in lymphocytes and a robust decrease in Neutrophils compared to MHV-infected WT mice by subtraction of day 2 from day 5 reads. Meanwhile, control groups did not show any significant change between wildtype and Top3b-KO. The data suggest that Top3b is required for normal immune response to MHV. 2. A family of chemokines are upregulated in the lungs of Top3b-KO mice infected by MHV. We preformed RNA-seq analysis of lungs harvested on day 5 post MHV inoculation. 2 male mice of each group (1. WT MHV, 2. Top3b-KO MHV) were used. We found inflammatory response, lymphocyte chemotaxis, and macrophage chemotaxis genes were upregulated in MHV infected Top3b-KO mice compared to WT mice. Among these genes, a family of chemokines, involved in immunoregulation, was found upregulated in MHV infected Top3b-KO and in WT. We checked the changes of mRNA levels Chemokines (ccl2, ccl7, and ccl12) and Top3b (as a control)by RT-qPCR in each groups (1. WT MHV, 2. Top3b-KO MHV, 3. WT DMEM, and 4. Top3b-KO DMEM). Their levels , were normalized with GAPDH. Chemokine mRNA levels were increased in MHV-infected WT and Top3b-KO samples when compared with DMEM-treated controls. Consistent with the RNA-seq data, chemokine mRNA levels in MHV infected Top3b-KO mice are higher than in MHV-infected WT mice. The data are consistent with blood cell findings that TOP3B is required for normal immune response to MHV infection. 3. Top3b-KO and TDRD3-KO flies have dysregulated immune responses to bacterial infection Our preliminary study utilizing the pathogenic bacteria Listeria monocytogenes infected Drosophila shows that in the absence of Top3b and Tdrd3, expression of many genes involved in immune signaling, pathogen recognition, and stress response are altered, based on RNA-seq. These gene expression changes may account for a noticeable melanization phenotype between WT, Top3bko, and Tdrd3ko flies. In Drosophila, melanization is a unique rapid wound response akin to clotting and scab formation in humans. Around the site of infection or injury, melanocytes produce increasing amounts of melanin pigment to create a physical barrier in preventing further spread of pathogens and cytotoxic materials (DE GREGORIO et al. 2002). Upon infection, Top3b-/- and Tdrd3-/- flies exhibit both increased area and intensity of pigmentation. Interestingly, we observed a diffusion-like spreading of melanin pigment radiating outward from the original site of infection, suggesting a possible dysregulation in the melanization response. Our RNA-sequencing data reveals specific patterns of gene expression unique to Top3bko, where an immunosuppressive cytokine, Diedel, is highly upregulated and edin, a key player in the humoral immune response, is downregulated (data not shown). Together with data from MHV-infected mice, these findings suggest that Top3b is important for maintaining normal immune response to pathogens.

我们的小组发现拓扑异构酶3b (TOP3B) 是一种双重活性酶，不仅可以改变DNA 的拓扑结构，还可以改变RNA 的拓扑结构(XU et al. 2013)。 TOP3B与TDRD3形成复合物；该复合物与脆性 X 智力迟钝蛋白 (FMRP) 相互作用。我们最近的研究表明，Top3b-TDRD3 调节刺激诱导的转录、mRNA 翻译和转换，以及 siRNA 引导的异染色质形成（LEE 等人，2018 年；SU 等人，2022 年）。人类中 Top3b 的缺失与神经疾病有关，例如精神分裂症和自闭症（STOLL 等人，2013 年；XU 等人，2013 年；AHMAD 等人，2017 年）。我们的研究还表明，Top3b-KO 小鼠存在神经发育和行为障碍（JOO et al. 2020）。有趣的是，TOP3B-KO 小鼠寿命缩短和免疫功能障碍，具体表现为血清自身抗体增加以及脾细胞和骨髓细胞染色体数值畸变增加（KWAN 等人，2007）。然而，Top3B 在免疫系统中发挥作用的机制尚不清楚。这项新提案旨在研究 TOP3B 如何调节小鼠对小鼠冠状病毒 MHV（小鼠肝炎病毒）感染以及果蝇细菌感染的免疫反应。 严重急性呼吸系统综合症冠状病毒2（SARS-Cov2）在过去几年中造成了经济和生命损失。宿主对 SARS-Cov2 的免疫反应负责病毒的消除和恢复 (GARCIA 2020)。另一方面，病毒引起的强烈炎症（称为细胞因子风暴）也会导致宿主严重疾病甚至致命（GARCIA 2020）。抗炎药物常用于抑制 SARS-Cov2 患者的细胞因子风暴。值得注意的是，拓扑异构酶 1 (Top1) 抑制剂已被开发为一类抗炎药物，目前正在临床试验中进行检查以治疗 SARS-Cov2 患者 (HO et al. 2021)。 TOP3B 与 TOP1 相似，两者都可以响应细胞外刺激而促进转录激活。我们建议研究 TOP3B 在冠状病毒和细菌感染的免疫反应中的作用。这些数据应该有助于了解 TOP3B 抑制剂是否可以作为控制免疫反应的药物。 具体目标： 目的1：研究TOP3B在小鼠T细胞和B细胞对MHV感染反应中的功能。 目标 2：研究 TOP3B 在小鼠模型和细胞系中对 MHV 感染的细胞因子反应中的功能。 目标 3：使用果蝇模型研究 Top3b-Tdrd3 是否以及如何有助于调节免疫信号通路。 进度更新： 1. Top3b-KO 小鼠表现出响应 MHV 的淋巴细胞和中性粒细胞水平发生改变。 我们开始测试 Top3b-KO 小鼠模型是否因 MHV 感染而表现出淋巴细胞和中性粒细胞水平的改变。四组小鼠，两组野生型和两组Top3b-KO，接种105 pfu MHV或DMEM作为阴性对照。在三个时间点采集血液：接种前7天、接种后两天和接种后5天。通过血细胞分析来测试野生型和 Top3b-KO 小鼠模型中哪些细胞在 MHV 感染期间受影响最大。我们发现，通过从第 5 天读数中减去第 2 天，与感染 MHV 的 WT 小鼠相比，感染 MHV 的 Top3b-KO 小鼠表现出淋巴细胞显着增加和中性粒细胞大幅减少。同时，对照组在野生型和Top3b-KO之间没有表现出任何显着变化。数据表明 Top3b 是针对 MHV 的正常免疫反应所必需的。 2. 感染 MHV 的 Top3b-KO 小鼠肺部趋化因子家族上调。 我们对 MHV 接种后第 5 天收获的肺部进行了 RNA 测序分析。每组使用2只雄性小鼠(1.WT MHV，2.Top3b-KO MHV)。我们发现与 WT 小鼠相比，MHV 感染的 Top3b-KO 小鼠的炎症反应、淋巴细胞趋化性和巨噬细胞趋化性基因均上调。在这些基因中，参与免疫调节的趋化因子家族在 MHV 感染的 Top3b-KO 和 WT 中被发现表达上调。我们通过 RT-qPCR 检查了各组趋化因子（ccl2、ccl7 和 ccl12）和 Top3b（作为对照） mRNA 水平的变化（1. WT MHV、2. Top3b-KO MHV、3. WT DMEM 和 4） .Top3b-KO DMEM）。他们的水平用 GAPDH 标准化。与 DMEM 处理的对照相比，MHV 感染的 WT 和 Top3b-KO 样品中趋化因子 mRNA 水平增加。与 RNA-seq 数据一致，MHV 感染的 Top3b-KO 小鼠中的趋化因子 mRNA 水平高于 MHV 感染的 WT 小鼠。这些数据与血细胞研究结果一致，即 TOP3B 是对 MHV 感染的正常免疫反应所必需的。 3. Top3b-KO和TDRD3-KO果蝇对细菌感染的免疫反应失调 我们利用感染单核细胞增生李斯特氏菌的病原菌感染果蝇的初步研究表明，根据RNA-seq，在Top3b和Tdrd3缺失的情况下，涉及免疫信号、病原体识别和应激反应的许多基因的表达发生了改变。这些基因表达变化可能解释了 WT、Top3bko 和 Tdrd3ko 果蝇之间明显的黑化表型。在果蝇中，黑化是一种独特的快速伤口反应，类似于人类的凝血和结痂形成。在感染或损伤部位周围，黑素细胞产生越来越多的黑色素，以形成物理屏障，防止病原体和细胞毒性物质进一步传播（DE GREGORIO et al. 2002）。感染后，Top3b-/- 和 Tdrd3-/- 果蝇表现出色素沉着面积和强度的增加。有趣的是，我们观察到黑色素从原始感染部位向外辐射状扩散，表明黑色化反应可能存在失调。 我们的 RNA 测序数据揭示了 Top3bko 独特的基因表达的特定模式，其中免疫抑制细胞因子 Diedel 高度上调，而体液免疫反应的关键参与者 edin 下调（数据未显示）。结合 MHV 感染小鼠的数据，这些发现表明 Top3b 对于维持对病原体的正常免疫反应很重要。