Epithelial responses to rotavirus induced purinergic signaling

项目摘要 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 一种引起腹泻和呕吐的病毒，如果没有支持性治疗，对儿童来说可能是致命的。轮状病毒感染 每年造成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， 感染高度局限于小肠绒毛尖端的肠上皮细胞。直到最近， 这些细胞间的信号是如何从感染细胞传播到周围未感染细胞的。使用高 分辨率实时成像，我们发现轮状病毒感染增加钙（Ca 2+）信号，无论是在感染 肠上皮细胞以及周围未感染的细胞，其表现为细胞间Ca 2+波。Ca2+ 波起源于受感染的细胞并通过未受感染的细胞传播。我们已经发现Ca 2+波 由细胞外嘌呤能信号传导介导;主要通过嘌呤核苷酸ADP和P2 Y1 受体的然而，这些细胞间的Ca 2+波的功能后果仍然未知。我们的中央 假设轮状病毒诱导的旁分泌信号，通过P2 Y1嘌呤能信号， 调节邻近未感染上皮细胞的反应，是轮状病毒标志所必需的 感染P2 Y1信号传导的潜在下游靶标包括氯离子（Cl-）和粘蛋白分泌。轮状 感染的特征是Cl-驱动的分泌性腹泻和充满粘蛋白的杯状细胞的丧失; 这被认为是促进感染。我们关注轮状病毒与上皮细胞的相互作用， 可能是导致感染的主要原因。本研究的目的是阐明 嘌呤能信号在轮状病毒感染后宿主肠上皮细胞中的作用及其机制 这些途径的轮状病毒失调的后果。目的1将确定嘌呤能信号的作用 通过P2 Y1在体内感染轮状病毒。我们预计P2 Y1的缺陷是由于P2 Y1的遗传丢失（P2 Y1-/-） 小鼠）或用药理学抑制剂MRS 2500抑制P2 Y1，将减少轮状病毒症状，包括 减少腹泻、体重减轻、组织病理学和细胞因子产生以及病毒脱落。在目标2中， 表征体外驱动上皮功能的信号通路（细胞易感性、Cl-分泌和 粘液排出）。我们预测，预处理的 含ADP或P2 Y1激动剂的HIE增加了对感染的易感性，并导致感染细胞增加， Cl-分泌和液体分泌。我们预期ADP激活P2 Y1刺激Ca 2+激活氯 通过PLC和IP 3R调节CaCC通道（CaCC），其也在杯状细胞的粘蛋白分泌中起作用。我们将使用 荧光报告分子、药理学抑制剂和P2 Y1和P2 Y2 CRISPR敲除HIE，以鉴定这些化合物。 上皮信号级联。本研究将首次为上皮细胞嘌呤能信号转导提供证据 在轮状病毒感染的情况下。总的来说，这些实验将提高我们对轮状病毒的认识 病理生理学，并可能提供新的治疗靶点的见解。

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.