Elucidating the Role of Purinergic and Calcium Signaling During Rotavirus Infection

Abstract

Calcium (Ca2+) is a ubiquitous messenger that influences numerous cellular processes, and therefore Ca2+ signaling is tightly regulated by cells. Ca2+ signaling dysregulation results in severe and potentially life‐threatening diseases, which is exemplified by rotavirus (RV) infection. RV is an enteric virus that causes life‐threatening diarrhea in children, resulting in ~198,000 deaths each year. While pathophysiological consequences of RV infection are widely studied, host Ca2+ signaling pathways and the mechanisms by which RV exploits them to cause diarrhea remain incompletely characterized. We have previously found that RV infection increases Ca2+ signaling both within infected enterocytes and in surrounding uninfected cells through paracrine signaling. This manifests as intercellular Ca2+ waves that originate from the infected cell and propagates to surrounding uninfected cells mainly through ADP activation of the P2Y1 receptor, which is expressed in many cell types in the intestine. We hypothesize that RV‐induced paracrine purinergic signaling functionally dysregulates neighboring uninfected enterocytes and modulates innate immune responses from both epithelial and intestinal immune cells. We generated cell lines and human intestinal enteroids (HIEs) stably expressing cytosolic genetically‐encoded Ca2+ indicators to characterize Ca2+ signaling throughout RV infection by time‐lapse imaging. We found that P2Y1‐mediated signaling was critical for activation of secretory epithelial cells, including induction of serotonin secretion of enterochromaffin cells and mucus secretion from goblet cells in human intestinal enteroids (HIEs) and mucin‐producing intestinal cell lines. Further, signaling from RV‐infected MA104 monkey kidney cells chemoattracted mouse bone‐marrow derived macrophages, which was blocked by pharmacological inhibitors of the P2Y1 receptor. Consistent with our in vitro findings, we observed that murine RV infection promoted secretion of serotonin, mucin and accumulation of macrophages. These effects of minimized in the presence of P2Y1 inhibitors and in P2Y1 knock out mice. Furthermore, we found that the RV‐induced increase in NFKb and IL‐1α are significantly attenuated by P2Y1 receptor blockers. Collectively these findings indicate that intercellular Ca2+ waves caused by paracrine purinergic signaling have a broad influence on intestinal pathophysiology during RV infection. Understanding the cellular consequences of P2Y1 activation during RV will generate new mechanistic insights into the homeostatic function of purinergic signaling in the GI tract.Support or Funding InformationR01 DK115507 (Hyser)T32 DK007664 (Engevik)