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Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...

Baylor College of Medicine

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.

Rotavirus induces intercellular calcium waves through ADP signaling.

Nucleotides are potent extracellular signaling molecules during homeostasis, infection, and injury due to their ability to activate purinergic receptors. The nucleotide ATP activates P2X receptors (P2RXs), whereas the nucleotides ADP, ATP, UTP, and UDP-glucose selectively activate different P2Y receptors (P2RYs). Several studies have established crucial roles for P2 receptors during intestinal inflammatory and infectious diseases, yet the most extensive characterization of purinergic signaling has focused on immune cells and the central and enteric nervous systems. As epithelial cells serve as the first barrier against irritants and infection, we hypothesized that the gut epithelium may express multiple purinergic receptors that respond to extracellular nucleotide signals. Using the Human Protein Atlas and Gut Cell Survey, we queried single-cell RNA sequencing (RNAseq) data for the P2 purinergic receptors in the small and large intestines. In silico analysis reveals robust mRNA expression of P2RY1, P2RY2, P2RY11, and P2RX4 throughout the gastrointestinal tract. Human intestinal organoids exhibited a similar expression pattern with a prominent expression of P2RY1, P2RY2, and P2RX4, but this purinergic receptor repertoire was not conserved in T84, Caco2, and HT29 intestinal epithelial cell lines. Finally, P2YR1 and P2YR2 agonists elicited robust calcium responses in human intestinal organoids, but calcium responses were weaker or absent in the cell lines. These findings suggest that the gastrointestinal epithelia respond to extracellular purinergic signaling via P2RY1, P2RY2, P2RY11, and P2RX4 receptors and highlight the benefit of using intestinal organoids as a model of intestinal purinergic signaling. Several studies have revealed crucial roles for P2 receptors during inflammatory and infectious diseases, however, these have largely been demonstrated in immune cells and the enteric nervous system. Although epithelial cells serve as the first barrier against infection and inflammation, the role of purinergic signaling within the gastrointestinal tract remains largely unknown. This work expands our knowledge of purinergic receptor distribution and relative expression along the intestine.

Distribution of P2Y and P2X purinergic receptor expression within the intestine.

Many viruses exploit host Ca signaling to facilitate their replication; however, little is known about how Ca signals from different host and viral channels contribute to the overall dysregulation of Ca signaling or promote virus replication. Using cells lacking IPR, a host ER Ca channel, we delineated intracellular Ca signals within virus-infected cells and intercellular Ca waves (ICWs), which increased Ca signaling in neighboring, uninfected cells. In infected cells, IPR was dispensable for rotavirus-induced Ca signaling and replication, suggesting the rotavirus NSP4 viroporin supplies these signals. However, IPR-mediated ICWs increase rotavirus replication kinetics and spread, indicating that the Ca signals from the ICWs may prime nearby uninfected cells to better support virus replication upon eventual infection. This "pre-emptive priming" of uninfected cells by exploiting host intercellular pathways in the vicinity of virus-infected cells represents a novel mechanism for viral reprogramming of the host to gain a replication advantage.

Host IPR channels are dispensable for rotavirus Ca signaling but critical for intercellular Ca waves that prime uninfected cells for rapid virus spread.

Francesca J. Scribano

Alkek Center for Metagenomics and Microbiome Research,
Department of Molecular Virology and Microbiology,
Alkek Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology

Baylor College of Medicine

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Francesca J. Scribano

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Alkek Center for Metagenomics and Microbiome Research,Department of Molecular Virology and Microbiology,Alkek Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology

Baylor College of Medicine

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Alkek Center for Metagenomics and Microbiome Research,
Department of Molecular Virology and Microbiology,
Alkek Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology