Riding the calcium waves Rotavirus causes severe diarrhea and vomiting in children worldwide, yet how infection causes these diseases remains poorly understood. A leading theory is that virus-infected cells secrete paracrine signaling molecules that dysregulate epithelial cells. Chang-Graham et al. found that rotavirus-infected cells trigger cell-to-cell signaling that manifests as intercellular calcium waves (see the Perspective by Stanifer and Boulant). This signal results from the repeated release of adenosine 5′-diphosphate by rotavirus-infected cells. This release activates receptors on nearby uninfected cells, resulting in a calcium signal. The intercellular calcium waves activate chloride and serotonin secretion, which contributes to diarrhea and vomiting. Blocking this paracrine signal could represent a target for antidiarrheal pharmacotherapy. Science, this issue p. eabc3621; see also p. 909 Rotavirus, responsible for severe diarrhea in children, triggers extracellular release of ADP from infected cells, which dysregulates nearby uninfected cells. INTRODUCTION Rotavirus (RV) causes severe diarrheal disease in children worldwide by broadly dysregulating intestinal homeostasis. The mechanisms of RV diarrhea are multifactorial and still not completely understood. RV infects epithelial enterocytes and enteroendocrine cells at the tip and middle of villi in the small intestine, but not all of the cells susceptible to RV are infected during disease. A hallmark of RV infection is that RV dysregulates host cell calcium signaling pathways to increase cytosolic calcium, which is required for RV replication. RATIONALE A long-held concept in how RV infection causes life-threatening diarrhea—despite a small percentage of infected cells—is that RV-infected cells release potent signaling molecules that can dysregulate neighboring, uninfected cells. Previous studies have found increased levels of signaling molecules during RV infection, such as RV enterotoxin nonstructural protein 4 (NSP4), nitric oxide (NO), and prostaglandins (PGE2). However, whether signaling molecule(s) spread from infected to uninfected cells is not clear. Examination of the intercellular signaling between infected and uninfected cells during RV infection may provide insights into the pathophysiology of RV and other viral diarrheas. RESULTS In this study, we used long-term live fluorescence calcium imaging throughout RV infection to show that RV-infected cells produce paracrine signals that manifest as intercellular calcium waves (ICWs), an intercellular communication pathway in which increases in cytosolic calcium occur in an expanding circular pattern from a central initiating cell. We observed RV-induced ICWs in both cell lines and human intestinal enteroids (HIEs). Blocking previously known RV-induced paracrine signaling pathways (enterotoxin NSP4, NO, and PGE2) did not inhibit the ICWs, but the addition of apyrase, an enzyme that degrades extracellular adenosine 5′-triphosphate (ATP) and adenosine 5′-diphosphate (ADP), greatly reduced the ICWs. The RV-induced ICWs were mediated by extracellular ADP, which activates P2Y1 purinergic receptors on neighboring cells. ICWs were blocked by P2Y1 antagonists or CRISPR-Cas9 knockout of the P2Y1 receptor. Inhibiting the paracrine ADP signal reduced RV replication and inhibited the RV-induced increases in COX2 and iNOS expression that mediate PGE2 and NO production, respectively. Blocking ADP signaling also decreased RV-induced serotonin release from HIEs and fluid secretion in an HIE swelling assay. Furthermore, BPTU and MRS2500, small molecule inhibitors of the P2Y1 receptor, reduced RV diarrhea severity in neonatal mice. CONCLUSION The current concept of RV pathogenesis proposes that paracrine signaling from RV-infected cells dysregulates surrounding uninfected cells and contributes to life-threatening diarrhea. Here, characterization of ICWs originating from RV-infected cells has provided direct experimental proof for the role of virus-induced paracrine signaling in gastrointestinal pathophysiology. Furthermore, ADP signaling provides both a potent paracrine signal and the most dominant calcium signal observed in RV infection. These results point to purinergic signaling as a therapeutic target for developing antidiarrheal drugs for RV and potentially other viral diarrheas. Our studies provide direct evidence that viruses can exploit purinergic signaling and intercellular calcium waves to potentially amplify pathophysiological signaling that is important for disease. ADP released from rotavirus-infected cells drives pathogenesis. (Top) Rotavirus-infected intestinal epithelial cells release ADP, which diffuses to neighboring, uninfected cells. (Middle) ADP activates the purinergic P2Y1 receptor, which increases intracellular calcium, observed as an intercellular calcium wave (see filmstrip, right). (Bottom) ADP and P2Y1 activation triggers the release of inflammatory mediators, fluid secretion contributing to diarrhea, and the release of serotonin from enteroendocrine cells to activate the enteric nervous system (ENS). Rotavirus causes severe diarrheal disease in children by broadly dysregulating intestinal homeostasis. However, the underlying mechanism(s) of rotavirus-induced dysregulation remains unclear. We found that rotavirus-infected cells produce paracrine signals that manifested as intercellular calcium waves (ICWs), observed in cell lines and human intestinal enteroids. Rotavirus ICWs were caused by the release of extracellular adenosine 5′-diphosphate (ADP) that activated P2Y1 purinergic receptors on neighboring cells. ICWs were blocked by P2Y1 antagonists or CRISPR-Cas9 knockout of the P2Y1 receptor. Blocking the ADP signal reduced rotavirus replication, inhibited rotavirus-induced serotonin release and fluid secretion, and reduced diarrhea severity in neonatal mice. Thus, rotavirus exploited paracrine purinergic signaling to generate ICWs that amplified the dysregulation of host cells and altered gastrointestinal physiology to cause diarrhea.

中文翻译：

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轮状病毒通过ADP信号诱导细胞间钙波

驾驭钙波 轮状病毒在全世界引起严重的儿童腹泻和呕吐，但人们对感染如何引起这些疾病仍知之甚少。一个主要的理论是，病毒感染的细胞分泌旁分泌信号分子，使上皮细胞失调。Chang-Graham 等人。发现轮状病毒感染的细胞触发细胞间信号传导，表现为细胞间钙波（参见 Stanifer 和 Boulant 的观点）。该信号是由轮状病毒感染的细胞重复释放腺苷 5'-二磷酸引起的。这种释放会激活附近未感染细胞上的受体，从而产生钙信号。细胞间钙波激活氯化物和血清素分泌，从而导致腹泻和呕吐。阻断这种旁分泌信号可能代表止泻药物治疗的目标。科学，这个问题 p。eabc3621；另见 p. 导致儿童严重腹泻的 909 轮状病毒会触发受感染细胞的细胞外 ADP 释放，从而使附近未受感染的细胞失调。简介 轮状病毒 (RV) 通过广泛失调肠道稳态导致全世界儿童严重腹泻。RV 腹泻的机制是多因素的，但仍未完全了解。RV感染小肠绒毛顶端和中部的上皮肠细胞和肠内分泌细胞，但并非所有对RV敏感的细胞在疾病期间都被感染。RV 感染的一个标志是 RV 失调宿主细胞钙信号通路以增加细胞溶质钙，这是 RV 复制所必需的。基本原理 关于 RV 感染如何导致危及生命的腹泻（尽管感染细胞的比例很小）的一个长期存在的概念是，RV 感染的细胞释放有效的信号分子，这些信号分子可以使邻近的未感染细胞失调。先前的研究发现 RV 感染期间信号分子水平增加，例如 RV 肠毒素非结构蛋白 4 (NSP4)、一氧化氮 (NO) 和前列腺素 (PGE2)。然而，信号分子是否从感染细胞传播到未感染细胞尚不清楚。检查 RV 感染期间感染细胞和未感染细胞之间的细胞间信号传导可能有助于深入了解 RV 和其他病毒性腹泻的病理生理学。结果 在这项研究中，我们在整个 RV 感染过程中使用长期活体荧光钙成像来显示 RV 感染的细胞产生旁分泌信号，表现为细胞间钙波 (ICW)，这是一种细胞间通讯途径，其中细胞溶质钙的增加以扩展的圆形模式从中央起始细胞。我们在细胞系和人肠小肠 (HIE) 中观察到 RV 诱导的 ICW。阻断先前已知的 RV 诱导的旁分泌信号通路（肠毒素 NSP4、NO 和 PGE2）不会抑制 ICW，但添加腺苷三磷酸双磷酸酶是一种降解细胞外腺苷 5'-三磷酸 (ATP) 和腺苷 5'-二磷酸的酶。 ADP)，大大减少了 ICW。RV 诱导的 ICW 由细胞外 ADP 介导，它激活邻近细胞上的 P2Y1 嘌呤能受体。ICW 被 P2Y1 拮抗剂或 P2Y1 受体的 CRISPR-Cas9 敲除阻断。抑制旁分泌 ADP 信号可减少 RV 复制并抑制 RV 诱导的 COX2 和 iNOS 表达增加，这分别介导 PGE2 和 NO 的产生。阻断 ADP 信号也减少了 HIE 中 RV 诱导的血清素释放和 HIE 肿胀试验中的液体分泌。此外，BPTU 和 MRS2500（P2Y1 受体的小分子抑制剂）可降低新生小鼠 RV 腹泻的严重程度。结论 目前 RV 发病机制的概念提出，来自 RV 感染细胞的旁分泌信号调节周围未感染细胞的失调并导致危及生命的腹泻。这里，对源自 RV 感染细胞的 ICW 的表征为病毒诱导的旁分泌信号转导在胃肠道病理生理学中的作用提供了直接的实验证据。此外，ADP 信号提供了一种有效的旁分泌信号和在 RV 感染中观察到的最主要的钙信号。这些结果表明嘌呤能信号可作为开发 RV 和其他潜在病毒性腹泻止泻药的治疗靶点。我们的研究提供了直接证据，证明病毒可以利用嘌呤能信号和细胞间钙波来潜在地放大对疾病很重要的病理生理信号。轮状病毒感染细胞释放的 ADP 驱动发病机制。（上）轮状病毒感染的肠上皮细胞释放 ADP，ADP 扩散到邻近的未感染细胞。（中）ADP 激活嘌呤能 P2Y1 受体，增加细胞内钙，观察为细胞间钙波（见幻灯片，右）。（下）ADP 和 P2Y1 激活触发炎症介质的释放、导致腹泻的液体分泌，以及肠内分泌细胞释放血清素以激活肠神经系统 (ENS)。轮状病毒通过广泛失调肠道稳态导致儿童严重腹泻病。然而，轮状病毒引起的失调的潜在机制仍不清楚。我们发现轮状病毒感染的细胞会产生旁分泌信号，表现为细胞间钙波 (ICW)，在细胞系和人肠小肠中观察到。轮状病毒 ICW 是由细胞外腺苷 5'-二磷酸 (ADP) 的释放引起的，ADP 激活邻近细胞上的 P2Y1 嘌呤能受体。ICW 被 P2Y1 拮抗剂或 P2Y1 受体的 CRISPR-Cas9 敲除阻断。阻断 ADP 信号可减少轮状病毒复制，抑制轮状病毒诱导的血清素释放和液体分泌，并降低新生小鼠的腹泻严重程度。因此，轮状病毒利用旁分泌嘌呤能信号产生 ICW，从而放大宿主细胞的失调并改变胃肠道生理以引起腹泻。并降低新生小鼠的腹泻严重程度。因此，轮状病毒利用旁分泌嘌呤能信号产生 ICW，从而放大宿主细胞的失调并改变胃肠道生理以引起腹泻。并降低新生小鼠的腹泻严重程度。因此，轮状病毒利用旁分泌嘌呤能信号产生 ICW，从而放大宿主细胞的失调并改变胃肠道生理以引起腹泻。