Three-dimensional human epithelial cultures as a model for evaluation of flavivirus-host interactions driving infection in the skin

三维人类上皮培养物作为评估驱动皮肤感染的黄病毒-宿主相互作用的模型

• 批准号: 10303730
• 负责人: Susan R Weiss
• 金额: $ 20.31万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 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/10303730
• 关键词: 3-Dimensional; Address; Adherent Culture; Affect; Animal Model; Anti-Inflammatory Agents; Antiviral Agents; Arboviruses; Automobile Driving; Biology; CRISPR/Cas technology; Cell Line; Cell model; Cells; Cellular Tropism; Clinical; Culicidae; DNA Viruses; Dendritic Cells; Dengue; Dengue Virus; Dermatology; Dermis; Development; Disease; Disease Outcome; Environment; Epithelial; Evaluation; Event; Exhibits; Extracellular Matrix; Fibroblasts; Flavivirus; Flavivirus Infections; Funding; Future; Generations; Genes; Genetic; Human; Human Cell Line; IFNAR1 gene; Imaging Techniques; Immune; Immune response; Immunocompetent; Immunology; Impairment; Individual; Infection; Infection prevention; Integration Host Factors; Investigation; Knock-out; Knowledge; Langerhans cell; Measures; Modeling; Molecular; Outcome; Pathogenesis; Plaque Assay; Play; Primary Infection; Production; Proteins; Public Health; RNA Viruses; Reagent; Research; Ribonucleases; Role; Saliva; Salivary; Site; Skin; Skin Tissue; Study models; System; Testing; Tissue Model; Tissues; Tropism; Viral; Viral Drug Resistance; Virus; Virus Replication; West Nile viral infection; West Nile virus; Work; ZIKA; ZIKV infection; Zika Virus; burden of illness; cell immortalization; cell motility; cell type; human disease; human model; human pathogen; immunoregulation; keratinocyte; macrophage; mosquito-borne; spatiotemporal; therapeutic target; tissue tropism; topical antiviral; transmission process; virology; virus host interaction

PROJECT SUMMARY Zika virus (ZIKV), dengue virus (DENV), and West Nile virus (WNV) all cause severe human disease as a result of their widespread mosquito-borne transmission. While these flaviviruses share a common primary site of infection in the dermis, they disseminate to varying secondary sites of infection with highly divergent clinical outcomes. We propose that discrepancies in early virus-host interactions among ZIKV, DENV, and WNV are responsible for these differences in disease; however, there is a gap in our knowledge of the molecular mechanisms responsible. Elucidation of distinct virus-host interactions occurring at the initial infection site is thus key to understanding how these viruses establish productive infections in the skin and elsewhere. The proposed research will establish organotypic epithelial cultures as a genetically tractable and immunocompetent 3D human skin model of flavivirus infection, to be used for virus-host interaction studies. We will expand on the current model commonly used to study DNA viruses, typically comprised solely of human fibroblasts and keratinocytes, with the addition of human skin-resident dendritic cells and macrophages, to accurately recreate natural flavivirus infection in the skin. Additionally, we will incorporate mosquito saliva, which includes immunomodulatory and anti-inflammatory proteins, into the virus inoculum to further recapitulate the molecular events occurring at the primary site of flavivirus infection. We will determine individual infection conditions for ZIKV, DENV, and WNV, and evaluate cell tropism and host responses with each virus. Once established, we will employ this 3D human skin model to evaluate the role of the host ribonuclease L (RNase L) protein during ZIKV infection in the skin, which will validate this system for flavivirus-host interaction studies. While antiviral RNase L activity is well described, we have recently discovered proviral RNase L activity during ZIKV infection. In contrast, we observed canonical antiviral RNase L activity during DENV and WNV infections. These studies were performed in 2D monolayer culture systems with immortalized cell lines, therefore we will use our 3D skin model containing primary cells to test the hypothesis that RNase L plays a role in ZIKV cell tropism and spread in the skin in a more relevant system. We will use CRISPR-Cas9 gene editing to delete RNase L from the different skin cells comprising epithelial cultures, and subsequently generate RNase L-deficient skin cultures for infections with ZIKV, DENV, or WNV. We will assess effects of RNase L deletion on infection and spread of the different flaviviruses in the skin, as well as how host responses are impacted. The proposed studies will characterize the role of RNase L as an important host factor repurposed by ZIKV during infection in the skin. Furthermore, this project will establish organotypic epithelial cultures as an exciting new human model amenable to gene editing which will facilitate inquiries of arbovirus-host interactions and host responses at the common primary infection site of the skin.

项目摘要 寨卡病毒（ZIKV）、登革病毒（DENV）和西尼罗河病毒（WNV）都可导致严重的人类疾病 蚊子传播的广泛传播。虽然这些黄病毒共享一个共同的主要位点， 在真皮中的感染，它们传播到不同的继发感染部位，具有高度不同的临床 结果。我们提出ZIKV、DENV和WNV之间早期病毒-宿主相互作用的差异是 负责这些疾病的差异;然而，在我们的分子知识中存在差距， 机制负责。因此，阐明了在初始感染部位发生的不同病毒-宿主相互作用， 了解这些病毒如何在皮肤和其他地方建立生产性感染的关键。拟议 研究将建立器官型上皮细胞培养作为一种遗传上易处理和免疫活性的 黄病毒感染的3D人体皮肤模型，用于病毒-宿主相互作用研究。我们将扩大 目前通常用于研究DNA病毒的模型，通常仅由人成纤维细胞组成， 角质形成细胞，加上人类皮肤驻留树突状细胞和巨噬细胞，以准确地重建 皮肤自然黄病毒感染。此外，我们将纳入蚊子唾液，其中包括 免疫调节和抗炎蛋白，以进一步概括病毒接种物的分子， 在黄病毒感染的原发部位发生的事件。我们将确定个人感染情况， ZIKV、DENV和WNV，并评估每种病毒的细胞嗜性和宿主应答。一旦建立，我们 将采用这种3D人类皮肤模型来评估宿主核糖核酸酶L（RNase L）蛋白在 皮肤中的ZIKV感染，这将验证该系统用于黄病毒-宿主相互作用研究。虽然抗病毒 RNA酶L活性被充分描述，我们最近发现ZIKV感染期间的前病毒RNA酶L活性。 相反，我们观察到典型的抗病毒RNase L活性在DENV和WNV感染。这些研究 在具有永生化细胞系的2D单层培养系统中进行，因此我们将使用3D皮肤 包含原代细胞的模型，以检验RNA酶L在ZIKV细胞向性中起作用的假设， 在皮肤中传播的更相关的系统。我们将使用CRISPR-Cas9基因编辑来删除RNase L， 所述不同的皮肤细胞包括上皮培养物，并且随后产生RNase L缺陷的皮肤培养物 ZIKV、DENV或WNV感染。我们将评估RNase L缺失对感染和传播的影响。 皮肤中不同的黄病毒，以及宿主反应如何受到影响。拟议的研究将 表征RNase L作为在皮肤中感染期间由ZIKV再利用的重要宿主因子的作用。 此外，该项目将建立器官型上皮细胞培养物，作为一种令人兴奋的新的人类模型， 基因编辑将有助于研究虫媒病毒与宿主的相互作用和宿主的反应， 皮肤的主要感染部位。