Investigating miR-375-mediated regulation of intestinal helminth infection

土壤传播的肠道蠕虫寄生虫感染全球数十亿人， 发病率很高，并构成严重的全球公共卫生问题。最近的研究表明， 在蠕虫感染后，肠道上皮中称为簇细胞的特化细胞扩张并触发宿主- 促进寄生虫排出和组织修复的保护性2型免疫反应。毛丛细胞来源于 来自肠上皮隐窝底部的干细胞，以及转录因子，如Pou 2f 3， 已经鉴定了簇细胞的成熟。然而，尽管有这些进展， 控制簇状细胞丰度和抗蠕虫功能的基因仍不完全清楚。虽然 建立了microRNAs（miRNAs）作为一类调节分子，对于肠道正常的 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，一个单一的miRNA，miR-375的全身遗传缺失， 与感染多回Heligmosomoides polygyrus（一种小鼠蠕虫寄生虫）后较低的蠕虫负荷相关 用作人类钩虫感染的模型。此外，375-/-小鼠表现出增加的 小肠中的簇状细胞。值得注意的是，我们还通过生物信息学筛选确定Pou 2f 3是一种 miR-375的预测靶点。本建议的重点是弥合两个关键的知识差距：（1）是影响 在H.肠上皮细胞（IEC）谱系中miR-375功能丧失介导的多脑回蠕虫负荷， 免疫细胞谱系还是两者都有？以及（2）miR-375缺失的影响是否依赖于Pou 2f 3介导的成熟 簇状细胞的在第一个目标中，我们将确定IEC特异性miR-375缺失对H.多脑回 为了实现这一目标，我们将采用两种独立的、互补的策略。具体而言，我们会：（i） 产生骨髓嵌合小鼠，其中miR-375缺陷仅限于非免疫细胞，以及 (ii)繁殖miR-375 fl/fl; Vil 1-Cre小鼠，其中miR-375缺陷是IEC特异性的，并比较H.多脑回 感染后的蠕虫负荷与我们先前在全身miR-375-/-小鼠中观察到的一样。在第二 目的是确定miR-375缺失对H.多脑回蠕虫负担。 我们将首先确定Pou 2f 3是否是miR-375的直接靶点，并利用单细胞转录组学 为了鉴定额外的候选miR-375靶基因，其表达水平显著增加， 375-/-小鼠肠道上皮干细胞和/或分泌祖细胞对蠕虫的反应 感染然后，我们将进行遗传研究，以确定Pou 2f 3的缺失是否足以否定 miR-375缺失对H.多脑回蠕虫负担。这些研究的完成将大大 通过建立IEC miRNA调节簇状细胞介导的抗蠕虫功能， H.多脑回感染提高对宿主对蠕虫反应的关键上皮调节因子的认识 将是制定新的战略来治疗和预防感染的关键。

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.