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: 10416067
• 负责人: Luis J Sigal
• 金额: $ 19.5万
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10416067
• 关键词: Acute; Affect; Animal Model; 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; Persons; 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.

总结 预防病毒感染的发病率和死亡率以及活病毒疫苗的并发症 免疫需要鉴定赋予对病毒的天然抗性的基因。小鼠脱脚病病毒 鼠痘病毒（ECTV）是小鼠的一种自然病原体。当ECTV接种到脚垫中时 在小鼠中，它通过传入淋巴管迅速扩散到引流淋巴结（dLN）。复制后， dLN，ECTV通过传出神经系统传播到血流，并通过血液到达其主要的 靶器官肝脏和脾脏在敏感品系小鼠如BALB/c中， 是巨大的，在感染后（DPI）7天达到~109空斑形成单位（pfu）/g。这会导致肝坏死 与死然而，在C57 BL/6（B6）和其他鼠痘抗性小鼠品系中，ECTV也成为全身性的，但 感染后7天，肝脏中的病毒载量仅达到~104 pfu/g，ECTV被强烈的免疫应答清除 没有明显的疾病迹象。 多年来，我们一直在研究B6小鼠抵抗鼠痘的机制。我们发现 耐药的主要原因是dLN中高度设计的先天免疫应答， 扩散并主要涉及炎性单核细胞（iMO），其必须产生并感测I型 干扰素（IFN-I）。该资助的具体目标是发现参与imo介导的保护作用的新基因 通过产生其中iMO特异性缺乏干扰素刺激的小鼠的抗病毒先天免疫 基因（ISG）。在Subaim A中，我们将产生在5个ISG中具有floxed等位基因的小鼠，我们已经通过 RNA测序这些ISG在体内上调至高水平，并且是原始对照的至少三倍。 感染的和/或未感染的iMO。它们并不为人所知，但保存在 人类为了生产这些小鼠，我们将使用“输卵管核酸递送（i-GONAD）”方法， 通过原位电穿孔将CRISPR核糖核蛋白递送到E0.7胚胎。我们已经成功 在我们的实验室里建立了iGONAD。这使我们能够以低成本快速生产突变小鼠。在Subaim B： Floxed小鼠将与删除单核细胞/巨噬细胞谱系中floxed基因的Lyz 2-Cre小鼠一起繁殖。 我们将使用这些小鼠来确定这些基因是否是单核细胞/巨噬细胞特异性所需的。 发展和免疫组织化学介导的抗鼠痘。我们预测，这些基因中至少有一部分 对抵抗鼠痘至关重要。此外，我们推测，这些基因将是关键的抵抗， 多种病原体，并且对于人类抵抗病毒感染和缺乏抗病毒药物也很重要。 活疫苗免疫后的并发症。这可以在未来进行测试。