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: 10313481
• 负责人: Kristen Engevik
• 金额: $ 6.64万
• 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/10313481
• 关键词: 5 year old; Address; Agonist; Animal Model; Apical; Body Weight decreased; Calcium; 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; Epithelial Cells; 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; Pharmacology; Pharmacotherapy; Physiology; Play; Predisposition; Production; Purine Nucleotides; Reporter; Research; Resolution; Role; Rotavirus; Rotavirus Infections; Signal Pathway; Signal Transduction; Small Intestines; Supportive care; Symptoms; Therapeutic Agents; Vaccines; Villus; Virus; Virus Shedding; Vomiting; cell motility; cell type; cytokine; diarrheal disease; experimental study; extracellular; global health; improved; in vivo; inhibitor/antagonist; insight; intercellular communication; intestinal epithelium; intestinal villi; mortality; mouse model; new therapeutic target; novel; novel therapeutics; paracrine; purinoceptor P2Y1; response; 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.

项目总结 儿科腹泻病是5岁以下儿童发病率的主要原因。轮状病毒是一种肠道病毒 导致腹泻和呕吐的病毒，在没有支持性护理的儿童中可能是致命的。轮状病毒感染 每年导致128,500人死亡。尽管会导致严重的、可能危及生命的腹泻，轮状病毒 感染高度局限于小肠绒毛尖端的肠细胞。直到最近，人们还不清楚 这些细胞-细胞信号是如何从感染细胞传播到周围未感染细胞的。使用高 分辨肝脏成像，我们发现轮状病毒感染增加了感染体内的钙(Ca~(2+))信号 肠道细胞以及周围未感染的细胞，表现为细胞间的钙波。这些钙离子 电波源自受感染的细胞，并通过未受感染的细胞传播。我们已经确认了钙离子波 由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，这些细胞间钙波的功能结果仍不清楚。我们的中央 假设轮状病毒通过P2Y1嘌呤能信号功能诱导旁分泌信号 调节邻近未受感染的上皮细胞的反应，是轮状病毒特征所必需的 感染。P2Y1信号的潜在下游靶标包括氯(Cl-)和粘蛋白的分泌。轮形病毒 感染的特征是由氯引起的分泌性腹泻和粘蛋白填充的杯状细胞丢失；特征 它们被认为会促进感染。我们将重点放在轮状病毒与上皮细胞的相互作用上，因为这些事件 可能能够调解感染的主要后果。这项研究的目的是阐明 宿主肠道上皮细胞嘌呤能信号在轮状病毒感染应答中的作用及其机制 轮状病毒对这些通路的失调的后果。目标1将确定嘌呤能信号的作用 在轮状病毒体内感染过程中通过P2Y1。我们预测，由于P2Y1(P2Y1-/-)基因缺失，导致了P2Y1的缺失 或用药物抑制剂MRS2500抑制P2Y1，将减少轮状病毒的症状，包括 减少腹泻、体重减轻、组织病理学和细胞因子的产生，以及病毒的脱落。在《目标2》中我们将 描述驱动上皮细胞功能的体外信号通路(细胞敏感性、氯离子分泌和 在人类肠道类(HIE)中轮状病毒感染期间的粘液排出)。我们预测，前处理 含ADP或P2Y1激动剂的HIE增加感染易感性，并导致感染细胞增加 CL-分泌和液体分泌。我们预计P2Y1刺激的钙激活氯对ADP的激活 通过PLC和IP3R的通道(CACC)，这也在杯状细胞的粘蛋白分泌中发挥作用。我们将使用 荧光记者、药理抑制剂和P2Y1和P2Y2 CRISPR基因敲除HIE以识别这些 上皮性信号级联。这项研究将为上皮细胞中的嘌呤能信号提供第一个证据 在轮状病毒感染的背景下。总的来说，这些实验将加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洞察。