Rotavirus induces intercellular calcium waves through ADP signaling

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.