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

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：戊型肝炎病毒（HEV）每年感染全球超过2000万人，导致330万临床病例 肝炎病例和超过44 000例肝胆疾病死亡。作为一种无包膜病毒，HEV令人惊讶地存在于 作为循环血液中抵抗中和的类包膜外泌体样病毒体。基因1型戊型肝炎病毒（HEV-1）感染 与孕妇爆发性肝炎相关，死亡率高（>25%）。HEV-1在胎盘组织中复制， HEV-1垂直传播与高新生儿死亡率相关。由于缺乏有效的细胞培养或 HEV-1的动物模型，HEV-1相关的妊娠期严重疾病的机制尚不清楚。显著 在患有暴发性肝炎的HEV感染孕妇中发现了较高水平的TNF-α， CNS组织中可检测到的HEV RNA的促炎细胞因子（TNF-α和IL-18）水平显著高于 在CNS组织中检测不到HEV RNA的猪中。长期目标是阐明有助于 妊娠期间HEV相关的高死亡率。不幸的是，我们目前还没有合适的系统，无论是体外还是体内。 体内，以研究母胎界面的HEV-1感染。在目标1中，我们将在体外建立胎盘屏障， Transwell插入研究母胎界面中的HEV-1感染。我们假设循环血液中的HEV-1 在病毒血症高峰期穿过胎盘屏障导致胎儿感染。我们将培养胎盘屏障细胞 模拟体内构成人胎盘屏障的关键的母体血液和胎儿血液面对层， 在基底侧和顶侧共培养BeWo胎盘滋养层细胞和人脐静脉内皮细胞 细胞外基质包被的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。我们将通过感染屏障培养物来确定HEV-1是否可以穿过屏障 分别用HEV-1和HEV-3在母体腔室中，并测量胎儿腔室中HEV的量， 屏障。在目的2中，我们将确定母亲-胎儿界面中HEV-1感染导致胎儿死亡的机制。 感染我们假设，在病毒血症高峰期，母血循环中的类包膜外泌体样HEV 当屏障被促炎细胞因子（如TNF-α和IL-）炎症时，更容易穿过胎盘屏障 18，在HEV感染期间持续产生，并且屏障中的HEV-1感染产生III型IFN， 限制病毒感染。我们将单独或联合使用TNF-α和IL-18刺激屏障培养物，然后 分别用无包膜HEV-1、HEV-3、准包膜HEV-1、HEV-3感染，测定其含量 已经越过屏障的混合动力汽车。我们还将测定感染的大肠杆菌中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耐药环境是否可以转移到HEV敏感的肝脏 细胞我们期望建立一个胎盘屏障，模仿关键的母体血液和胎儿血液面对层， 体内，并显示准包膜的HEV-1将更容易穿过屏障，特别是当屏障被感染时， 促炎细胞因子TNF-α和IL-18。我们还预期HEV-1在屏障中诱导IFN-λ1/λ2/λ3以限制病毒 复制，并且屏障处诱导的抗病毒耐药性环境可以转移到对戊型肝炎病毒敏感的肝细胞。