Investigating miR-375-mediated regulation of intestinal helminth infection

研究 miR-375 介导的肠道蠕虫感染调节

• 批准号: 10495270
• 负责人: Praveen Sethupathy
• 金额: $ 24.08万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10495270
• 关键词: 3' Untranslated Regions; Address; Age; Architecture; Binding; Binding Sites; Bioinformatics; Biological Assay; Bone Marrow; CRISPR/Cas technology; Cell Count; Cell Differentiation process; Cell Lineage; Cell Maturation; Cells; Data; Development; Epithelial; Epithelial Cells; Exhibits; Follow-Up Studies; Foundations; Future; Gene Expression; Genes; Genetic; Genetic study; Goals; Helminths; Hookworm Infections; Human; Immune; Immune response; Individual; Infection; Infection prevention; Interleukin-13; Intestines; Knock-out; Knockout Mice; Knowledge; LoxP-flanked allele; Luciferases; Measures; Mediating; MicroRNAs; Modeling; Molecular; Morbidity - disease rate; Mus; Nematospiroides dubius; Parasites; Pathway interactions; Persons; Public Health; Regulation; Resistance; Role; Secretory Cell; Signal Transduction; Small Intestines; Soil; Testing; Therapeutic; Tissues; Transgenic Mice; Villus; Wild Type Mouse; base; cell type; gastrointestinal epithelium; helminth infection; improved; in vivo; intestinal crypt; intestinal epithelium; novel; protective effect; response; sex; single-cell RNA sequencing; stem; stem cells; tissue repair; transcription factor; transcriptomics; villin

PROJECT SUMMARY: Soil-transmitted intestinal helminth parasites infect billions of people worldwide, causing substantial morbidity and posing a significant global public health problem. Recent hallmark studies have shown that following helminth infection, specialized cells in the gut epithelium called tuft cells expand and trigger a host- protective type 2 immune response that promotes parasite expulsion and tissue repair. Tuft cells are derived from stem cells at the base of the intestinal epithelial crypt, and transcription factors such as Pou2f3 that drive the maturation of tuft cells have been identified. However, despite these advances, the molecular mechanisms that control tuft cell abundance and anti-helminth function remain incompletely understood. Although it is established that microRNAs (miRNAs) as a class of regulatory molecules are critical for proper intestinal architecture and function, the roles of individual miRNAs in the intestinal epithelium are just now starting to emerge. We recently discovered that the whole-body genetic deletion of a single miRNA, miR-375, was associated with lower worm burdens after infection with Heligmosomoides polygyrus, a helminth parasite of mice used as a model for human hookworm infection. Moreover, the 375-/- mice exhibited increased abundance of tuft cells in the small intestine. Notably, we also determined through a bioinformatic screen that Pou2f3 is a predicted target of miR-375. This proposal is focused on bridging two critical knowledge gaps: (1) Is the effect on H. polygyrus worm burden mediated by loss of miR-375 function in the intestinal epithelial cell (IEC) lineage, immune cell lineage, or both? and (2) Does the effect of miR-375 loss depend on Pou2f3-mediated maturation of tuft cells? In the first Aim, we will define the impact of IEC-specific miR-375 loss on resistance to H. polygyrus. To accomplish this goal, we will apply two independent, complementary strategies. Specifically, we will: (i) generate bone marrow chimeric mice, in which miR-375 deficiency is restricted to non-immune cells, and also (ii) breed miR-375fl/fl;Vil1-Cre mice, in which miR-375 deficiency is specific to IECs, and compare H. polygyrus worm burden after infection with what we have observed previously in whole-body miR-375-/- mice. In the second Aim, we will establish whether tuft cells are required for the effects of miR-375 loss on H. polygyrus worm burden. We will first determine whether Pou2f3 is a direct target of miR-375 and also leverage single cell transcriptomics to identify additional candidate miR-375 target genes whose expression levels are significantly increased in intestinal epithelial stem and/or secretory progenitor cells of 375-/- mice during the response to helminth infection. We will then perform genetic studies to determine whether loss of Pou2f3 is sufficient to negate the positive effects of miR-375 loss on H. polygyrus worm burden. The completion of these studies will substantially advance the field by establishing that an IEC miRNA regulates tuft cell-mediated anti-helminth function during H. polygyrus infection. Improved understanding of the key epithelial regulators of the host response to helminth will be critical for developing new strategies to treat and prevent infection.

项目摘要：土壤传播的肠道蠕虫寄生虫感染了全世界数十亿人，导致 发病率很高，并构成重大的全球公共卫生问题。最近的标志性研究表明 蠕虫感染后，肠道上皮中称为簇细胞的特殊细胞会扩张并触发宿主- 保护性 2 型免疫反应，促进寄生虫排出和组织修复。簇细胞衍生 来自肠上皮隐窝底部的干细胞，以及驱动转录因子（例如 Pou2f3） 簇细胞的成熟已被确定。然而，尽管取得了这些进展，分子机制 控制簇细胞丰度和抗蠕虫功能的机制仍不完全清楚。虽然它是 证实 microRNA (miRNA) 作为一类调节分子对于肠道正常运转至关重要 结构和功能，单个 miRNA 在肠上皮细胞中的作用现在才刚刚开始 出现。我们最近发现单个 miRNA miR-375 的全身基因缺失 与感染 Heligmosomoides polygyrus（一种小鼠​​蠕虫寄生虫）后较低的蠕虫负担有关 用作人类钩虫感染的模型。此外，375-/-小鼠表现出丰富的 小肠中的簇细胞。值得注意的是，我们还通过生物信息学筛选确定 Pou2f3 是 miR-375的预测靶标。该提案的重点是弥合两个关键的知识差距：（1）效果是否 肠上皮细胞 (IEC) 谱系中 miR-375 功能丧失介导的多回线虫负担， 免疫细胞谱系，或两者兼而有之？ (2) miR-375 丢失的影响是否取决于 Pou2f3 介导的成熟 簇细胞？在第一个目标中，我们将定义 IEC 特异性 miR-375 丢失对 H. polygyrus 抗性的影响。 为了实现这一目标，我们将应用两种独立、互补的策略。具体来说，我们将： (i) 产生骨髓嵌合小鼠，其中 miR-375 缺陷仅限于非免疫细胞，并且 (ii) 培育 miR-375fl/fl;Vil1-Cre 小鼠，其中 miR-375 缺陷是 IEC 特有的，并比较 H. polygyrus 我们之前在全身 miR-375-/- 小鼠中观察到的感染后的蠕虫负担。在第二个 目标是，我们将确定 miR-375 丢失对多回线虫负担的影响是否需要簇细胞。 我们将首先确定 Pou2f3 是否是 miR-375 的直接靶标，并利用单细胞转录组学 鉴定其他候选 miR-375 靶基因，其表达水平在 375-/-小鼠对蠕虫反应期间的肠上皮干细胞和/或分泌祖细胞 感染。然后，我们将进行遗传学研究，以确定 Pou2f3 的丢失是否足以抵消 miR-375 缺失对 H. polygyrus 蠕虫负担产生积极影响。这些研究的完成将大大 通过建立 IEC miRNA 调节簇细胞介导的抗蠕虫功能，推进了该领域的发展 H. Polygyrus 感染。提高对宿主对蠕虫反应的关键上皮调节因子的了解 对于制定治疗和预防感染的新策略至关重要。