Placental barrier culture to delineate the mechanism of hepatitis E virus infection at the maternal and fetal interface

胎盘屏障培养描绘母体和胎儿界面戊型肝炎病毒感染的机制

• 批准号: 10716971
• 负责人: Wen Li
• 金额: $ 8万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-14 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10716971
• 关键词: Acute; Acute Hepatitis; Acute Liver Failure; Animal Model; Animals; Antiviral resistance; Apical; Astrocytes; Blood; Blood - brain barrier anatomy; CD8-Positive T-Lymphocytes; Cell Culture Techniques; Cells; Central Nervous System; Cessation of life; Clinical; Coculture Techniques; Conditioned Culture Media; Disease; Eclampsia; Electrical Resistance; Endothelial Cells; Environment; Epidermal Growth Factor; Extracellular Matrix; Family suidae; Fetal Mortality Statistics; Fetus; Forskolin; Gene Expression; Gene Proteins; Genotype; Goals; Hemorrhage; HepG2; Hepatitis; Hepatitis E virus; Hepatobiliary; Hepatocyte; Human; IL18 gene; Immunoglobulin M; In Vitro; India; Infection; Inflammatory; Interferon Type II; Interferon alpha; Interferon-beta; Interferons; Invaded; Low Birth Weight Infant; Maternal Mortality; Maternal-Fetal Exchange; Measures; Membrane; Modeling; Mothers; Neonatal Mortality; Newborn Infant; Permeability; Persons; Physiological; Placenta Diseases; Predisposition; Pregnancy; Pregnant Women; Premature Birth; Prevention strategy; Production; Proteins; Reporting; Rupture; Side; Spontaneous abortion; System; T cell response; TNF gene; Tissues; Trophoblastic Cell; Umbilical vein; Vertical Transmission; Viral Load result; Viremia; Virion; Virus; Virus Diseases; Virus Replication; blood-brain barrier crossing; brain endothelial cell; cytokine; exosome; fetal; fetal blood; fetal infection; fetal loss; fluorescein isothiocyanate dextran; in vivo; migration; mortality; nanoparticle; neutralizing antibody; occludin; particle; protein biomarkers; small molecule; stillbirth; syncytin; transmission process; treatment group; trophoblast; viral RNA; viral resistance; viral transmission; Acute; Acute Hepatitis; Acute Liver Failure; Animal Model; Animals; Antiviral resistance; Apical; Astrocytes; Blood; Blood - brain barrier anatomy; CD8-Positive T-Lymphocytes; Cell Culture Techniques; Cells; Central Nervous System; Cessation of life; Clinical; Coculture Techniques; Conditioned Culture Media; Disease; Eclampsia; Electrical Resistance; Endothelial Cells; Environment; Epidermal Growth Factor; Extracellular Matrix; Family suidae; Fetal Mortality Statistics; Fetus; Forskolin; Gene Expression; Gene Proteins; Genotype; Goals; Hemorrhage; HepG2; Hepatitis; Hepatitis E virus; Hepatobiliary; Hepatocyte; Human; IL18 gene; Immunoglobulin M; In Vitro; India; Infection; Inflammatory; Interferon Type II; Interferon alpha; Interferon-beta; Interferons; Invaded; Low Birth Weight Infant; Maternal Mortality; Maternal-Fetal Exchange; Measures; Membrane; Modeling; Mothers; Neonatal Mortality; Newborn Infant; Permeability; Persons; Physiological; Placenta Diseases; Predisposition; Pregnancy; Pregnant Women; Premature Birth; Prevention strategy; Production; Proteins; Reporting; Rupture; Side; Spontaneous abortion; System; T cell response; TNF gene; Tissues; Trophoblastic Cell; Umbilical vein; Vertical Transmission; Viral Load result; Viremia; Virion; Virus; Virus Diseases; Virus Replication; blood-brain barrier crossing; brain endothelial cell; cytokine; exosome; fetal; fetal blood; fetal infection; fetal loss; fluorescein isothiocyanate dextran; in vivo; migration; mortality; nanoparticle; neutralizing antibody; occludin; particle; protein biomarkers; small molecule; stillbirth; syncytin; transmission process; treatment group; trophoblast; viral RNA; viral resistance; viral transmission

Project Summary: Hepatitis E virus (HEV) infects >20 million people worldwide annually leading to 3.3 million clinical cases of hepatitis and >44,000 deaths due to hepatobiliary diseases. As a non-enveloped virus, HEV is surprisingly present as quasi-enveloped exosome-like virions in circulating blood that resist neutralization. Genotype 1 HEV (HEV-1) infection is associated with fulminant hepatitis with high mortality (>25%) in pregnant women. HEV-1 replicates in placental tissues, and HEV-1 vertical transmission is associated with a high neonatal mortality. Due to the lack of an efficient cell culture or animal model for HEV-1, the mechanism of HEV-1-associated severe diseases during pregnancy is unknown. Significantly higher levels of TNF-α were found in HEV-infected pregnant women with fulminant hepatitis, and HEV-infected pigs with detectable HEV RNA in CNS tissues had significantly higher levels of proinflammatory cytokines (TNF-α and IL-18) than in pigs without detectable HEV RNA in CNS tissues. The long-term goal is to delineate the mechanisms contributing to HEV-associated high mortality during pregnancy. Unfortunately, we currently do not have a suitable system, in vitro or in vivo, to study HEV-1 infection at the maternal-fetal interface. In aim 1, we will establish an in vitro placental barrier in Transwell insert to study HEV-1 infection in the maternal-fetal interface. We hypothesize that HEV-1 in circulating blood during peak viremia crosses the placental barrier leading to fetal infection. We will develop a placental barrier culture mimicking the critical maternal blood- and fetal blood-facing layers that constitute the human placental barrier in vivo, by co-culturing BeWo placental trophoblastic cells and human umbilical vein endothelial cells on basolateral and apical sides of an extracellular matrix-coated Transwell insert. The integrity of the barrier will be confirmed by measuring TEER and barrier permeability to small molecules. We will determine whether HEV-1 can cross the barrier by infecting barrier cultures in the maternal chamber with HEV-1 and HEV-3, respectively, and measuring the amount of HEV in the fetal chamber of the barrier. In aim 2, we will determine the mechanisms of HEV-1 infection in the maternal-fetal interface leading to fetal infection. We hypothesize that quasi-enveloped exosome-like HEVs in circulating maternal blood during peak viremia more easily cross the placental barrier when the barrier is inflamed by pro-inflammatory cytokines such as TNF-α and IL- 18 that are consistently produced during HEV infection, and that HEV-1 infection in the barrier produces type III IFNs to limit viral infection. We will inflame the barrier cultures with TNF-α and IL-18, separately or in combination, and then infect them with non-enveloped HEV-1, HEV-3, quasi-enveloped HEV-1, HEV-3, respectively, to determine the amounts of HEV that have crossed the barrier. We will also determine the expression levels of IFN-α, IFN-β and IFN-λs in infected cells, and determine if the antiviral resistance environment induced at the barrier can be transferred to HEV-susceptible liver cells. We anticipate to establish a placental barrier mimicking the critical maternal blood- and fetal blood-facing layers in vivo, and show that quasi-enveloped HEV-1 will more easily cross the barrier especially when the barrier is inflamed with proinflammatory cytokines TNF-α and IL-18. We also expect that HEV-1 induces IFN-λ1/λ2/λ3 in the barrier to limit virus replication, and that the antiviral resistance environment induced at the barrier is transferable to HEV-susceptible liver cells.

项目摘要：乙型肝炎病毒（HEV）感染>全球范围内> 2000万人，导致330万个临床 由于肝疾病造成的肝炎病例和> 44,000例死亡。作为一种非发育的病毒，HEV令人惊讶地存在 作为抗神经化的循环血液中准发育的外泌体样病毒。基因型1 HEV（HEV-1）感染 与孕妇高死亡率（> 25％）的暴发性肝炎有关。 HEV-1在斑点组织中复制， HEV-1垂直传播与新生儿死亡率高有关。由于缺乏有效的细胞培养或 HEV-1动物模型，妊娠期间与HEV-1相关的严重疾病的机制尚不清楚。显著地 在感染肝炎的HEV感染孕妇中发现了较高水平的TNF-α，并感染了HEV的猪 CNS组织中可检测的HEV RNA的促炎细胞因子（TNF-α和IL-18）的水平明显高于 在中枢神经系统组织中没有可检测到的HEV RNA的猪中。长期目标是描述有助于 HEV相关在怀孕期间的高死亡率。不幸的是，我们目前没有合适的系统，体外或 体内，研究在母亲界面上的HEV-1感染。在AIM 1中，我们将建立一个体外的位置屏障 Transwell插入以研究母体界界面中的HEV-1感染。我们假设循环血液中的HEV-1 在峰值病毒血症期间，会导致胎儿感染的位置屏障。我们将开发一种壁炉式屏障文化 模仿构成体内人体屏障的临界基质血液和胎儿血液的层 在基础和顶部的圆锥形细胞和人脐静脉内皮细胞和人的脐静脉细胞中共同培养的培养 细胞外基质涂层的transwell插入物。屏障的完整性将通过测量TEER和 屏障对小分子的渗透性。我们将确定HEV-1是否可以通过感染屏障培养 分别在带有HEV-1和HEV-3的母体室内，并测量胎儿室内的HEV量 障碍。在AIM 2中，我们将确定母体界面中HEV-1感染的机制，导致胎儿 感染。我们假设在峰值病毒血症期间，准发达的外泌体样HEV在循环的遗传性血液中 当屏障被促炎性细胞因子（例如TNF-α和IL-）发炎时，更容易越过lopenal屏障 18在HEV感染期间始终产生的，屏障中的HEV-1感染会产生III型IFNS 限制病毒感染。我们将分别或组合用TNF-α和IL-18炎症，然后 分别用未开发的HEV-1，HEV-3感染了它们，分别为Quasi Gevaled HEV-1，HEV-3，以确定金额 越过障碍物的HEV。我们还将确定感染中IFN-α，IFN-β和IFN-λs的表达水平 细胞，并确定在屏障处诱导的抗病毒抗性环境是否可以转移到可触觉的肝脏中 细胞。我们预计将建立一个模仿关键的母子血液和胎儿血液层的隔离屏障 Vivo，并表明准发达的HEV-1将更容易越过障碍，尤其是在屏障发炎时 促炎细胞因子TNF-α和IL-18。我们还期望HEV-1在屏障中诱导IFN-λ1/λ2/λ3以限制病毒 复制，并将在屏障处诱导的抗病毒抗性环境转移到可启发性肝细胞中。