Background Reactive oxygen species (ROS) play a role in maintaining intestinal epithelial homeostasis and are normally kept at low levels via antioxidant compounds. Dysregulation of ROS can lead to intestinal inflammation and contribute to inflammatory bowel disease (IBD). Select gut microbes possess the enzymatic machinery to produce antioxidants whereas others can dysregulate levels of ROS. Our model microbe, Lactobacillus reuteri (ATCC PTA 6475), has been demonstrated to reduce intestinal inflammation in mice models. It contains the genes encoding two distinct GshA-like glutamylcysteine ligases. We hypothesize that L. reuteri can secrete γ-glutamylcysteine to suppress ROS, minimize NFκB activation and regulate secretion of e pithelial cytokines. Methods & Results Conditioned media from L. reuteri was analyzed via mass spectrometry to confirm the presence of γ-glutamylcysteine. All cysteine containing products including γ-glutamylcysteine were fluorescently tagged in the conditioned media and then incubated with HT29 cell monolayers as well as human jejunal enteroid (HJE) monolayers. γ-glutamylcysteine was demonstrated to enter intestinal epithelial cells based on microscopy. Next, a Thioltracker assay was used to show increased intracellular glutathione levels by L. reuteri secreted γ-glutamylcysteine. HT29 cells and HJEs were then treated with IL-1β or hydrogen peroxide, and L. reuteri metabolites as well as γ-glutamylcysteine significantly suppressed pro-inflammatory cytokine driven ROS and IL-8 production. L. reuteri secreted products also reduced activity of NFκB as determined by a luciferase reporter assay. γ-glutamylcysteine deficient mutants were generated by targeted mutagenesis of GshA genes, and these mutant L. reuteri strains had a diminished ability to suppress IL-8 production and ROS. To further test the role of L. reuteri secreted γ-glutamylcysteine in vivo, a 2,4,6-Trinitrobenzenesulfonic acid (TNBS)- induced mouse colitis model was used. Adolescent mice were orogavaged with PBS, L. reuteri, L. reuteri GshA2 mutant, or γ-glutamylcysteine for a week after which TNBS was rectally administered to induce colitis. We demonstrate that L. reuteri and γ-glutamylcysteine can suppress histologic inflammation compared to PBS control and L. reuteri GshA2 mutant groups. Conclusions Together these data indicate that L. reuteri secretes γ-glutamylcysteine which can enter the intestinal epithelial cells and modulate epithelial cytokine production. It acts via suppression of ROS and NFκB which then decreases IL-8 production. We are able to demonstrate this in vitro in both HT 29 cells and HJEs. We now also demonstrate this in vivo in a mouse colitis model. These experiments highlight a prominent role for ROS intermediates in microbiome-mammalian cell signaling processes involved in immune responses and intestinal inflammation.
26Rotavirus causes severe diarrheal disease in children worldwide. A hallmark of rotavirus 27 infection is an increase in cytosolic calcium in infected small intestine epithelial cells. However, 28 the underlying mechanism(s) of rotavirus-cell signaling remains incompletely characterized. Here 29 we show that rotavirus-infected cells produce paracrine signals that manifest as intercellular 30 calcium waves (ICWs); which are observed in both cell lines and human intestinal enteroids 31 (HIEs). Rotavirus ICWs are caused by the release of extracellular adenosine diphosphate (ADP) 32 that activates P2Y1 purinergic receptors on neighboring cells and are blocked by P2Y1 33 antagonists or CRISPR/Cas9 knockout of P2Y1. This paracrine purinergic signal is critical for 34 rotavirus replication and diarrhea. Blocking the ICW signal reduces rotavirus replication; inhibits 35 rotavirus-induced serotonin release and fluid secretion; and reduces diarrhea severity in neonatal 36 mice. This is the first evidence that viruses exploit intercellular calcium waves to amplify diarrheal 37 signaling; a finding which have broad implications for gastrointestinal physiology. signaling molecules during RV infection, including enterotoxin NSP4, prostaglandins (PGE2), and 57 nitric oxide (NO) 7,15-17 . In this model, enterotoxin NSP4 can bind to neighboring, uninfected 58 enterocytes to activate Ca 2+ -activated chloride channels and cause secretory diarrhea 18,19 , and 59 PGE2 and NO may further activate fluid secretion processes 20,21 . Dysregulation of neighboring 60 enteroendocrine cells triggers the Ca 2+ -dependent release of serotonin, which stimulates the 61 enteric nervous system both to activate vomiting centers in the central nervous system and to 62 activate secretory reflex pathways in the gastrointestinal (GI) tract 5,22 . Thus, this model of RV-63 induced diarrhea addresses how limited infection at the middle-to-upper villi may cause 64 widespread dysregulation of host physiology and life-threatening disease. 65Herein we demonstrate that RV-infected cells signal to uninfected cells via an extracellular 66 purinergic signaling pathway. This newly identified pathway is a dominant driver of observed RV 67 disease processes, including replication, upregulation of PGE2-and NO-producing enzymes, 68 serotonin secretion, fluid secretion, and diarrhea in a neonatal mouse model. Our findings provide 69 new insights into the mechanism(s) of viral diarrhea and gastrointestinal physiology. 70 Results 71 Low multiplicity infection reveals intercellular calcium waves 72Previous studies have shown that RV significantly increases cytosolic Ca 2+ during infection 73 and disrupts host Ca 2+ -dependent processes to cause disease [23][24][25] . We used African Green 74 monkey kidney MA104 cells stably expressing the genetically-encoded calcium indicator (GECI) 75GCaMP5G or GCaMP6s to observe changes in cytosolic Ca 2+ during RV infection using live-cell 76 time-lapse epifluorescence imaging. We did not observe differences in response using either...
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 Information R01 DK115507 (Hyser)T32 DK007664 (Engevik)
Despite causing severe, potentially life‐threatening diarrhea, most enteric viruses only infect a small percent of intestinal epithelial cells. Thus, the induction of paracrine signaling pathways to dysregulate both infected and neighboring uninfected cell remains a major premise of enteric virus pathogenesis. However, virus‐induced paracrine signaling has never been directly observed and therefore the mechanisms have yet to be defined. Rotavirus (RV) remains a major cause of severe diarrhea in children worldwide. A hallmark of RV infection is the activation of aberrant calcium (Ca2+) signaling, which is necessary for replication and activation of secretory pathways in GI epithelium. We sought to characterize RV‐induced calcium signaling dynamics and identify paracrine signaling pathways responsible for infected‐to‐uninfected cell signaling. We conducted live‐cell Ca2+ imaging throughout the entire infection using cell lines and human intestinal enteroids (HIEs) engineered to stably express the genetically‐encoded Ca2+ indicator GCaMP. We found that rotavirus significantly increases both steady‐state and transient Ca2+ signaling mediated by RV nonstructural protein 4 (NSP4) and mutation of the NSP4 ion channel domain altered the RV‐induced Ca2+ signaling pattern observed, particularly low amplitude Ca2+ puffs observed early in infected cells. Further, isolated rotavirus‐infected cells generated multiple intercellular calcium waves (ICWs), which was the most prominent paracrine signal observed in both MA104 cells and HIEs. RV infection induces several signaling molecules, such as enterotoxin NSP4, prostaglandins and nitric oxide, and in many cell types extracellular purinergic signaling by ATP/ADP are responsible for ICW propagation. We found that RV‐induced ICWs were abolished by blocking extracellular ATP/ADP signaling with apyrase or purinergic receptor inhibitors or by CRISPR/Cas9‐mediated deletion of the P2Y1 receptor, but not by blocking extracellular enterotoxin form of NSP4, nitric oxide, or prostaglandin signaling. Rotavirus‐induced paracrine signaling was critical for multiple aspects of rotavirus pathogenesis, including fluid secretion and serotonin release, as well as regulating host responses, such as upregulation of IL‐1α and mucin secretion. Finally, we investigated whether activation of aberrant Ca2+ signaling and paracrine purinergic signaling were features of enteric caliciviruses using Tulane virus (TV), a rhesus monkey calicivirus. Like RV, we found that TV encodes for a viral ion channel in the endoplasmic reticulum and induces aberrant Ca2+ signaling during infection. TV infected cells also trigger ICWs that are blocked by purinergic signaling inhibitors. Thus, enteric viruses employ mechanisms to not only disrupt signaling in the virus‐infected cell but also exploit paracrine purinergic signaling to generate ICWs that represent a potent mechanism to amplify the pathophysiological signals underlying viral diarrhea.
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