Investigating miR-375-mediated regulation of intestinal helminth infection

项目摘要：土壤传播的肠道蠕虫寄生虫感染全球数十亿人，造成 严重的发病率，并构成一个重大的全球公共卫生问题。最近的标志性研究表明 在蠕虫感染后，肠道上皮中称为丛生细胞的特殊细胞扩张并触发宿主- 保护性的2型免疫反应，促进寄生虫的排出和组织修复。丛生细胞是由 来自肠道上皮腺底部的干细胞和转录因子，如Pou2f3 已鉴定出簇状细胞的成熟。然而，尽管取得了这些进展，但分子机制 控制簇状细胞丰度和抗蠕虫功能的机制仍不完全清楚。尽管它是 证实了microRNAs(MiRNAs)作为一类调节分子对正常的肠道至关重要 结构和功能，单个miRNAs在肠道上皮细胞中的作用才刚刚开始 浮出水面。我们最近发现，单个miRNA miR-375的全身遗传缺失是 与感染小鼠蠕虫寄生虫多回螺旋体后的低蠕虫负担有关 作为人类钩虫感染的模型。此外，375-/-小鼠表现出更多的 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Pou2f3是一种 MIR-375的预测目标。这项建议的重点是弥合两个关键的知识差距：(1)是效果 在肠上皮细胞(IEC)谱系中miR-375功能丧失所介导的多脑回线虫负担上， 免疫细胞谱系，还是两者兼而有之？(2)miR-375缺失的影响是否依赖于Pou2f3介导的成熟 绒毛细胞？在第一个目标中，我们将定义IEC特异性miR-375缺失对多脑回耐药的影响。 为了实现这一目标，我们将采用两个独立、互补的战略。具体来说，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，并且还 (Ii)培育miR-375fl/fl；Vil1-Cre小鼠，其中miR-375缺陷是IECS特有的，并与多回小鼠进行比较。 我们之前在全身miR-375-/-小鼠身上观察到的感染后的蠕虫负担。在第二个 目的：我们将确定miR-375缺失是否需要丛生细胞来影响多脑回线虫的负担。 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 寻找其他表达水平显著提高的候选miR-375靶基因 375-/-小鼠肠道上皮干细胞和/或分泌祖细胞对蠕虫的应答 感染。然后，我们将进行遗传学研究，以确定Pou2f3的丢失是否足以否定 MiR-375缺失对多脑回虫负担的积极影响。这些研究的完成将大大 通过建立IEC miRNA调节丛生细胞介导的抗蠕虫功能来促进该领域的发展 H.多回感染。提高对宿主对蠕虫反应的关键上皮调节因子的理解 将对制定治疗和预防感染的新战略至关重要。

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.