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.

概括 预防病毒感染的发病率和死亡率以及活力病毒疫苗的并发症 免疫需要鉴定会导致病毒自然抗性的基因。胚胎病毒 （ECTV），摩西蛋白的药物，是小鼠的天然病原体。当ECTV接种到脚下时 在小鼠中，它通过传入淋巴机迅速扩散到排水淋巴结（DLN）。复制后 DLN，ECTV通过有效的淋巴管扩散到血液，通过血液，它达到了其主要 靶器官，肝脏和脾脏。在易感小鼠（例如BALB/C）的敏感性中，肝脏的复制 在感染后7天（DPI）时达到〜109个斑块形成单位（PFU）/g。这导致肝坏死 和死亡。然而，在C57BL/6（B6）和其他抗菌抗抗小鼠菌株中，ECTV也变得全身但 7 dpi处的肝脏中的病毒载荷仅达到〜104 pfu/g，ECTV通过强烈的免疫反应清除 没有主要疾病迹象。 我们一直在研究B6小鼠多年来B6小鼠抵抗Mousepox的机制。我们找到了 阻力的主要原因是DLN中高度编排的先天免疫反应，它延迟了病毒 散布和优先涉及炎症单核细胞（IMO），必须产生和感知I型 干扰素（IFN-I）。该赠款的具体目的是发现与IMO介导的受保护有关的新基因 抗病毒先天免疫通过小鼠的产生，其中imos特别缺乏干扰素刺激 基因（ISG）。在Subaim A中，我们将在5个ISG中生产带有floxed等位基因的小鼠，我们已经通过 RNA-seq。这些ISG在体内上调高水平，至少来自幼稚对照的三倍 以IFN-I的方式感染和/或未感染的IMO。他们不是众所周知的，但在 人类。为了产生这些小鼠，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核糖核蛋白传递给E0.7胚胎。我们已经成功 在我们的实验室中建立了伊戈纳德。这使我们能够以低成本快速生产突变小鼠。在Subaim B中： Floxed小鼠将用LYZ2-CRE小鼠繁殖，该小鼠在单核细胞/巨噬细胞线上删除了flox的基因。 我们将使用这些小鼠来确定单核细胞/巨噬细胞是否特别需要这些基因 开发和IMO介导的对摩托巨星的抗性。我们预测，其中至少有一些基因将 对于抗菌丝的抵抗至关重要。此外，我们推测这些基因对于抵抗至关重要 多种病原体，对于人类对病毒感染的抵抗和缺乏 活疫苗免疫后的并发症。将来可以对此进行测试。