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Intracellularly expressed rotavirus NSP4 stimulates release of serotonin (5-HT) from human enterochromaffin cells
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Rotavirus (RV) is associated with diarrhoea and vomiting, but the mechanisms behind these symptoms remain unresolved. While RV have been shown to infect and stimulate secretion of serotonin (5-hydroxytryptamine; 5-HT) from human enterochromaffin (EC) cells and to infect EC cells in the small intestine of mice, it remains to identify which intracellularly expressed viral protein (VP) being responsible for this novel property.

To address this issue, human EC cells were transfected with small interfering RNA (siRNA) targeting the structural (VP4, VP6 and VP7) and the non-structural protein 4 (NSP4) followed by infection with Rhesus rotavirus (RRV). siRNA specific to NSP4 (siRNANSP4) significantly attenuated secretion of 5-HT compared to siRNAVP4, siRNAVP6 , siRNAVP7 and non-targeting (Nt) siRNAnt. Intracellular calcium clamping with BABTA/AM showed that intracellularly expressed NSP4-stimulated secretion of 5-HT from EC cells was calcium-dependent. Furthermore RV down-regulated the 5-HT transporter (SERT) mRNA in ileum but not tryptophan hydroxylase 1 (TPH1) mRNA the rate-limiting enzyme for 5-HT synthesis. The unaffected expression of TPH1 mRNA in the intestinal segments suggests that release of 5- HT primarily originates from pre-made 5-HT rather than from newly synthesised 5-HT mRNA. Moreover, down-regulation of SERT mRNA in ileum presumably resulted in reduced re- uptake of 5-HT by SERT to EC cells and thus increased extracellular 5-HT in the small intestine. Moreover, 7/7 infant mice responded following intraperitoneal administration of 5-HT with rapid (<30 min) diarrhoea in dose-dependent manner. In the light of these results and the fact that both 5-HT and NSP4 can induce diarrhoea in mice, a disease mechanism to RV diarrhoea is proposed.

National Category
Microbiology Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-117893OAI: oai:DiVA.org:liu-117893DiVA: diva2:811727
Available from: 2015-05-13 Created: 2015-05-13 Last updated: 2015-05-13Bibliographically approved
In thesis
1. Rotavirus Disease Mechanisms Diarrhea, Vomiting and Inflammation: How and Why
Open this publication in new window or tab >>Rotavirus Disease Mechanisms Diarrhea, Vomiting and Inflammation: How and Why
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Rotavirus infections cause diarrhea and vomiting that can lead to severe dehydration. Despite extensive tissue damage and cell death, the inflammatory response is very limited. The focus of this thesis was to study pathophysiological mechanisms behind diarrhea and vomiting during rotavirus infection and also to investigate the mechanism behind the limited inflammatory response.

An important discovery in this thesis was that rotavirus infection and the rotavirus toxin NSP4 stimulate release of the neurotransmitter serotonin from intestinal sensory enterochromaffin cells, in vitro and ex vivo. Interestingly, serotonin is known to be a mediator of both diarrhea and vomiting. Moreover, mice pups infected with rotavirus responded with central nervous system (CNS) activation in brain structures associated with vomiting, thus indicating a cross-talk between the gut and brain in rotavirus disease.

Our finding that rotavirus infection activates the CNS led us to address the hypothesis that rotavirus infection not only activates the vagus nerve to stimulate vomiting, but also suppresses the inflammatory response via the cholinergic anti-inflammatory pathway, both of which are mediated by activated vagal afferent nerve signals into the brain stem. We found that mice lacking an intact vagus nerve, and mice lacking the α7 nicotine acetylcholine receptor (nAChR), being involved in cytokine suppression from macrophages, responded with a higher inflammatory response.

Moreover, stimulated cytokine release from macrophages, by the rotavirus toxin NSP4, could be attenuated by nicotine, an agonist of the α7 nAChR. Thus, it seems most reasonable that the cholinergic anti-inflammatory pathway contributes to the limited inflammatory response during rotavirus infection. Moreover, rotavirus-infected mice displayed increased intestinal motility at the onset of diarrhea, which was not associated with increased intestinal permeability. The increased motility and diarrhea in infant mice could be attenuated by drugs acting on the enteric nervous system, indicating the importance and contribution of nerves in the rotavirus mediated disease.

In conclusion, this thesis provides further insight into the pathophysiology of diarrhea and describe for the first time how rotavirus and host cross-talk to induce the vomiting reflex and limit inflammation. Results from these studies strongly support our hypothesis that serotonin and activation of the enteric nervous system and CNS contributes to diarrhea, vomiting and suppression of the inflammatory response in rotavirus disease.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. 56 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1463
National Category
Cell and Molecular Biology Microbiology in the medical area
Identifiers
urn:nbn:se:liu:diva-117895 (URN)10.3384/diss.diva-117895 (DOI)978-91-7519-052-5 (print) (ISBN)
Public defence
2015-06-05, Berzeliussalen, Campus US, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2015-05-13 Created: 2015-05-13 Last updated: 2015-05-13Bibliographically approved

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