Epithelial responses to rotavirus induced purinergic signaling

上皮对轮状病毒诱导的嘌呤信号的反应

• 批准号: 10665537
• 负责人: Kristen Engevik
• 金额: $ 5.19万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10665537
• 关键词: 5 year old; Agonist; Animal Model; Apical; Body Weight decreased; Calcium; Calcium Oscillations; Calcium Signaling; Cell Line; Cells; Cessation of life; Child; Childhood; Chloride Channels; Chlorides; Clustered Regularly Interspaced Short Palindromic Repeats; Cytoplasmic Granules; Data; Dehydration; Developing Countries; Development; Diarrhea; Distant; Dysentery; Enteral; Enterocytes; Epithelial Cells; Epithelium; Event; Fluids and Secretions; Foundations; Functional disorder; Gastrointestinal Physiology; Generations; Genetic; Goals; Goblet Cells; Histologic; Histopathology; Human; ITPR1 gene; Image; Immune response; In Vitro; Infection; Intestines; Knock-out; Knowledge; Life; Liquid substance; Location; Mediating; Molecular; Morbidity - disease rate; Morphology; Mucins; Mucous body substance; Mus; Oral Rehydration Therapy; Organoids; Paracrine Communication; Pathogenesis; Pathway interactions; Pharmacotherapy; Physiology; Play; Predisposition; Production; Purine Nucleotides; Reporter; Research; Resolution; Role; Rotavirus; Rotavirus Infections; Signal Pathway; Signal Transduction; Small Intestines; Supportive care; Symptoms; System; Therapeutic Agents; Vaccines; Villus; Virus; Virus Shedding; Vomiting; cell motility; cell type; cytokine; diarrheal disease; experimental study; extracellular; global health; improved; in vivo; inhibitor; insight; intercellular communication; intestinal epithelium; intestinal villi; mortality; mouse model; new therapeutic target; novel; novel therapeutics; paracrine; pharmacologic; purinoceptor P2Y1; response; sobriety; statistics; targeted treatment; vaccine access

PROJECT SUMMARY Pediatric diarrheal diseases are a major cause of morbidity in children under 5 years old. Rotavirus is an enteric virus that causes diarrhea and vomiting and can be fatal in children without supportive care. Rotavirus infection results in 128,500 deaths per year. Despite causing severe, potentially life-threatening diarrhea, rotavirus infection is highly localized to the enterocytes in the villus tips of the small intestine. Until recently, it was unclear how these cell-cell signals were propagated from infected cells to surrounding uninfected cells. Using high resolution live imaging, we found that rotavirus infection increases calcium (Ca2+) signaling both within infected enterocytes as well as surrounding uninfected cells, which manifests as intercellular Ca2+ waves. These Ca2+ waves originate from the infected cell and propagate through uninfected cells. We have identified that Ca2+ waves are mediated by extracellular purinergic signaling; primarily through the purine nucleotide ADP and the P2Y1 receptor. However, the functional consequence of these intercellular Ca2+ waves remains unknown. Our central hypothesis is that rotavirus-induced paracrine signaling, via P2Y1 purinergic signaling, functionally dysregulates neighboring uninfected epithelium response and is required for the hallmarks of rotavirus infection. Potential downstream targets of P2Y1 signaling include chloride (Cl-) and mucin secretion. Rotavirus infection is characterized by both Cl- driven secretory diarrhea and loss of mucin-filled goblet cells; hallmarks which are considered to promote infection. We are focusing on rotavirus-epithelial interactions as these events may be able to mediate the major consequence of infection. The objective of this research is to elucidate the role of purinergic signaling in host intestinal epithelial cells in response to rotavirus infection and the mechanistic consequences of rotavirus dysregulation of these pathways. Aim 1 will determine the role of purinergic signaling via P2Y1 during rotavirus infection in vivo. We anticipate deficiency of P2Y1, by genetic loss of P2Y1 (P2Y1-/- mice) or inhibition of P2Y1 with the pharmacological inhibitor MRS2500, will reduce rotavirus symptoms including decreased diarrhea, weight loss, histopathology and cytokine production, and viral shedding. In Aim 2 we will characterize the signaling pathways in vitro that drive epithelial functions (cell susceptibility, Cl- secretion, and mucus expulsion) during rotavirus infection in human intestinal enteroids (HIEs). We predict that pretreatment of HIEs with ADP or P2Y1 agonist increases susceptibility to infection and results in increased infected cells, apical Cl- secretion and fluid secretion. We anticipate ADP activation of P2Y1 stimulations Ca2+ activated chloride channels (CaCC) via PLC and IP3R, which also plays a role in mucin secretion from goblet cells. We will use fluorescent reporters, pharmacological inhibitors and P2Y1 and P2Y2 CRISPR knockout HIEs to identify these epithelial signaling cascades. This study will provide the first evidence for purinergic signaling in epithelial cells in the context of rotavirus infection. Collectively, these experiments will enhance our knowledge of rotavirus pathophysiology and may provide insight for novel therapeutic targets.

项目概要 小儿腹泻病是 5 岁以下儿童发病的主要原因。轮状病毒是一种肠道病毒 该病毒会导致腹泻和呕吐，如果没有支持性护理，可能会导致儿童死亡。轮状病毒感染 每年导致 128,500 人死亡。尽管轮状病毒会引起严重的、可能危及生命的腹泻， 感染高度集中于小肠绒毛尖端的肠细胞。直到最近还不清楚 这些细胞间信号如何从受感染的细胞传播到周围未受感染的细胞。使用高 分辨率实时成像，我们发现轮状病毒感染会增加感染体内的钙 (Ca2+) 信号传导 肠上皮细胞以及周围未感染的细胞，表现为细胞间 Ca2+ 波。这些Ca2+ 波源自受感染的细胞并通过未受感染的细胞传播。我们已经确定 Ca2+ 波 由细胞外嘌呤能信号介导；主要通过嘌呤核苷酸ADP和P2Y1 受体。然而，这些细胞间 Ca2+ 波的功能后果仍然未知。我们的中央 假设是轮状病毒诱导的旁分泌信号通过 P2Y1 嘌呤能信号在功能上发挥作用 失调邻近未感染的上皮反应，并且是轮状病毒特征所必需的 感染。 P2Y1 信号传导的潜在下游靶标包括氯 (Cl-) 和粘蛋白分泌。轮状病毒 感染的特征是氯驱动的分泌性腹泻和充满粘蛋白的杯状细胞的损失；标志 这被认为会促进感染。我们关注轮状病毒与上皮细胞的相互作用，因为这些事件 可能能够介导感染的主要后果。这项研究的目的是阐明 宿主肠上皮细胞嘌呤信号对轮状病毒感染的反应及其机制 轮状病毒对这些途径失调的后果。目标 1 将确定嘌呤能信号传导的作用 在体内轮状病毒感染期间通过P2Y1。我们预计 P2Y1 会因 P2Y1 的遗传缺失而缺乏 (P2Y1-/- 小鼠）或用药理学抑制剂 MRS2500 抑制 P2Y1，将减轻轮状病毒症状，包括 腹泻、体重减轻、组织病理学和细胞因子产生以及病毒脱落减少。在目标 2 中，我们将 表征驱动上皮功能的体外信号通路（细胞敏感性、Cl-分泌和 人肠肠类（HIE）轮状病毒感染期间的粘液排出）。我们预测预处理 具有 ADP 或 P2Y1 激动剂的 HIE 会增加感染的易感性，并导致感染细胞增多，顶端 Cl-分泌和液体分泌。我们预计 ADP 激活 P2Y1 刺激 Ca2+ 活化氯化物 通过 PLC 和 IP3R 的通道（CaCC），它也在杯状细胞的粘蛋白分泌中发挥作用。我们将使用 荧光报告基因、药理学抑制剂以及 P2Y1 和 P2Y2 CRISPR 敲除 HIE 来识别这些 上皮信号级联反应。这项研究将为上皮细胞中嘌呤能信号传导提供第一个证据 在轮状病毒感染的情况下。总的来说，这些实验将增强我们对轮状病毒的了解 病理生理学，并可能为新的治疗靶点提供见解。

项目成果

Bioinformatics reveal elevated levels of Myosin Vb in uterine corpus endometrial carcinoma patients which correlates to increased cell metabolism and poor prognosis.

• DOI: 10.1371/journal.pone.0280428
• 发表时间: 2023
• 期刊: PLOS ONE
• 影响因子: 3.7
• 作者: Engevik, Kristen A.;Engevik, Melinda A.;Engevik, Amy C.
• 通讯作者: Engevik, Amy C.

Myosin 5b is required for proper localization of the intermicrovillar adhesion complex in the intestinal brush border.

肌球蛋白 5b 是微绒毛间粘附复合物在肠刷状缘正确定位所必需的。

• DOI: 10.1152/ajpgi.00212.2022
• 发表时间: 2022
• 期刊: American journal of physiology. Gastrointestinal and liver physiology
• 作者: Dooley,SarahA;Engevik,KristenA;Digrazia,Jessica;Stubler,Rachel;Kaji,Izumi;Krystofiak,Evan;Engevik,AmyC
• 通讯作者: Engevik,AmyC