Discovering new genes involved in monocyte-mediated protective anti-viral innate immunity through the generation of mice with targeted mutations

通过产生具有靶向突变的小鼠，发现参与单核细胞介导的保护性抗病毒先天免疫的新基因

• 批准号: 10303725
• 负责人: Luis J Sigal
• 金额: $ 23.4万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-02 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303725
• 关键词: Acute; Affect; Animal Model; Antiviral Agents; Antiviral resistance; Attenuated Vaccines; Blood; Blood Circulation; CXCL9 gene; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Dendritic Cells; Development; Disease; Electroporation; Embryo; Future; Generations; Genes; Genetic; Genetic Transcription; Goals; Grant; Health; Hematopoietic; Human; IFNAR1 gene; Immune response; Immunization; In Situ; In Vitro; Inbred BALB C Mice; Individual; Infection; Infectious Ectromelia; Infectious Skin Diseases; Inflammatory; Inflammatory Response; Innate Immune Response; Interferon Type I; Interferon Type II; Interferons; Knowledge; Liver; LoxP-flanked allele; Lymphatic; Mammalian Oviducts; Mediating; Methods; Modeling; Morbidity - disease rate; Mouse Pox Virus; Mouse Strains; Mus; Mutant Strains Mice; Mutation; Natural Immunity; Natural Killer Cells; Natural Resistance; Organ; Outcome; Play; Prevention; Proteins; Resistance; Ribonucleoproteins; Role; Signal Transduction; Skin; Smallpox; Smallpox Vaccine; Spleen; Tamoxifen; Testing; Time; Vaccines; Variola major virus; Viral; Viral Load result; Viral Physiology; Virus; Virus Diseases; Work; adaptive immune response; chemokine; conditional mutant; cost; draining lymph node; expectation; experience; experimental study; gene discovery; genetic manipulation; hepatic necrosis; in vivo; interferon alpha receptor; mRNA sequencing; macrophage; monocyte; mortality; nucleic acid delivery; pathogen; prevent; recruit; response; tool; transcriptome sequencing

Summary Prevention of morbidity and mortality from viral infections and of complications from live-virus vaccine immunizations requires the identification of genes that confer natural resistance to viruses. Ectromelia virus (ECTV), the agent of mousepox, is a natural pathogen of the mouse. When ECTV is inoculated into the footpad of mice, it rapidly spreads to the draining lymph node (dLN) through afferent lymphatics. After replicating in the dLN, ECTV spreads to the bloodstream through efferent lymphatics, and through the blood, it reaches its main target organs, the liver and the spleen. In susceptible strain of mice such as BALB/c, the replication in the liver is massive, reaching ~109 plaque forming units (pfu)/g at 7 days post infection (dpi). This results in liver necrosis and death. Yet, in C57BL/6 (B6) and other mousepox resistant mouse strains, ECTV also becomes systemic but the viral load in the liver at 7 dpi reaches only ~104 pfu/g and ECTV is cleared by a strong immune response without major signs of disease. We have been studying the mechanisms whereby B6 mice resist mousepox for many years. We have found that a main cause of resistance is a highly choreographed innate immune response in the dLN which delays viral spread and preponderantly involves inflammatory monocytes (iMOs) which must produce and sense Type I interferon (IFN-I). The Specific Aim of this grant is to discover new genes involved in iMO-mediated protective anti-viral innate immunity through the generation of mice in which iMOs specifically lack interferon stimulated genes (ISGs). In Subaim A, we will produce mice with floxed alleles in 5 ISGs that we have identified through RNA-Seq. These ISGs where upregulated in vivo to high levels and at least three-fold from naïve controls in infected and/or uninfected iMOs in an IFN-I dependent manner. They are not well-known but are conserved in humans. To produce these mice, we will use the method “Oviductal Nucleic Acids Delivery (i-GONAD)” that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. We have successfully established iGONAD in our lab. This allows us to produce mutant mice rapidly and at a low cost. In Subaim B: Floxed mice will be bred with Lyz2-Cre mice which deletes floxed genes in the monocytes/macrophage linage. We will use these mice to determine whether the genes are specifically required for monocytes/macrophage development and for iMO-mediated resistance to mousepox. We predict that at least some of these genes will be critical for resistance to mousepox. Moreover, we speculate that these genes will be critical for resistance to a variety of pathogens and also important for the resistance of humans to viral infections and for lack of complications after live-vaccine immunization. This could be tested in the future.

摘要 预防病毒感染和活病毒疫苗并发症的发病率和死亡率 免疫需要识别对病毒具有天然抵抗力的基因。蜕皮病病毒 鼠痘病毒(ECTV)是鼠痘的天然病原体。当ECTV接种到脚垫上时 在小鼠中，它通过传入淋巴管迅速传播到引流淋巴(DLN)。在中复制后 DLN，ECTV通过传出淋巴管传播到血流，并通过血液到达主要 靶器官，肝脏和脾。在BALB/c等敏感品系小鼠中，肝脏中的复制 是巨大的，在感染后7天(Dpi)达到~109个空斑形成单位(Pfu)/克。这会导致肝脏坏死。 和死亡。然而，在C57BL/6(B6)和其他鼠痘抗性小鼠株系中，ECTV也成为全身性的，但 在7dpi时，肝脏中的病毒载量仅为~104pfu/g，ECTV通过强大的免疫反应被清除 没有重大的疾病征兆。 多年来，我们一直在研究B6小鼠抵抗鼠痘的机制。我们发现了 耐药性的一个主要原因是DLN中高度编排的先天免疫反应，它延迟了病毒的传播 广泛传播，主要涉及炎性单核细胞(IMO)，它必须产生和感觉I型 干扰素(IFN-I)。这项拨款的具体目的是发现与IMO介导的保护有关的新基因。 通过产生IMOS特异性缺乏干扰素刺激的小鼠来抗病毒先天免疫 基因(ISG)。在苏巴伊姆A，我们将在5个ISG中培育出带有FLOXED等位基因的小鼠，这些ISG是通过 RNA-Seq.这些ISGs在体内上调到高水平，至少是幼稚对照的三倍 感染和/或未感染的IMOS以干扰素-I依赖的方式。它们不是很出名，但很保守 人类。为了生产这些小鼠，我们将使用“输卵管核酸输送(I-性腺)”的方法 通过原位电穿孔将CRISPR核糖核蛋白运送到E0.7胚胎。我们已经成功地 在我们的实验室建立了iGONAD。这使我们能够以低成本快速培育出突变小鼠。在苏巴伊姆B： 克隆的小鼠将与Lyz2-Cre小鼠繁殖，这种小鼠删除了单核/巨噬细胞系中的克隆基因。 我们将利用这些小鼠来确定这些基因是否是单核/巨噬细胞所特有的 开发和海事组织介导的对鼠痘的抗药性。我们预测，这些基因中至少有一部分会 对鼠痘的抵抗力至关重要。此外，我们推测这些基因将是抗病的关键。 各种病原体，对人类对病毒感染的抵抗力和缺乏 活疫苗免疫后的并发症。这可能会在未来得到测试。