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Rotavirus Disease Mechanisms Diarrhea, Vomiting and Inflammation: How and Why
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-117895DOI: 10.3384/diss.diva-117895ISBN: 978-91-7519-052-5 (print)OAI: oai:DiVA.org:liu-117895DiVA: diva2:811743
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
List of papers
1. Rotavirus Stimulates Release of Serotonin (5-HT) from Human Enterochromaffin Cells and Activates Brain Structures Involved in Nausea and Vomiting
Open this publication in new window or tab >>Rotavirus Stimulates Release of Serotonin (5-HT) from Human Enterochromaffin Cells and Activates Brain Structures Involved in Nausea and Vomiting
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2011 (English)In: PLOS PATHOGENS, ISSN 1553-7366, Vol. 7, no 7Article in journal (Refereed) Published
Abstract [en]

otavirus (RV) is the major cause of severe gastroenteritis in young children. A virus-encoded enterotoxin, NSP4 is proposed to play a major role in causing RV diarrhoea but how RV can induce emesis, a hallmark of the illness, remains unresolved. In this study we have addressed the hypothesis that RV-induced secretion of serotonin (5-hydroxytryptamine, 5-HT) by enterochromaffin (EC) cells plays a key role in the emetic reflex during RV infection resulting in activation of vagal afferent nerves connected to nucleus of the solitary tract (NTS) and area postrema in the brain stem, structures associated with nausea and vomiting. Our experiments revealed that RV can infect and replicate in human EC tumor cells ex vivo and in vitro and are localized to both EC cells and infected enterocytes in the close vicinity of EC cells in the jejunum of infected mice. Purified NSP4, but not purified virus particles, evoked release of 5-HT within 60 minutes and increased the intracellular Ca(2+) concentration in a human midgut carcinoid EC cell line (GOT1) and ex vivo in human primary carcinoid EC cells concomitant with the release of 5-HT. Furthermore, NSP4 stimulated a modest production of inositol 1,4,5-triphosphate (IP(3)), but not of cAMP. RV infection in mice induced Fos expression in the NTS, as seen in animals which vomit after administration of chemotherapeutic drugs. The demonstration that RV can stimulate EC cells leads us to propose that RV disease includes participation of 5-HT, EC cells, the enteric nervous system and activation of vagal afferent nerves to brain structures associated with nausea and vomiting. This hypothesis is supported by treating vomiting in children with acute gastroenteritis with 5-HT(3) receptor antagonists.

Place, publisher, year, edition, pages
Public Library of Science (PLoS), 2011
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-69989 (URN)10.1371/journal.ppat.1002115 (DOI)000293339300012 ()
Note
Original Publication: Marie Hagbom, Claudia Istrate, David Engblom, Thommie Karlsson, Jesus Rodriguez-Diaz, Javier Buesa, John A Taylor, Vesa Loitto, Karl-Eric Magnusson, Hakan Ahlman, Ove Lundgren and Lennart Svensson, Rotavirus Stimulates Release of Serotonin (5-HT) from Human Enterochromaffin Cells and Activates Brain Structures Involved in Nausea and Vomiting, 2011, PLOS PATHOGENS, (7), 7, . http://dx.doi.org/10.1371/journal.ppat.1002115 Licensee: Public Library of Science (PLoS) http://www.plos.org/Available from: 2011-08-12 Created: 2011-08-12 Last updated: 2015-05-13
2. Rotavirus Infection Increases Intestinal Motility but Not Permeability at the Onset of Diarrhea
Open this publication in new window or tab >>Rotavirus Infection Increases Intestinal Motility but Not Permeability at the Onset of Diarrhea
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2014 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 88, no 6, 3161-3169 p.Article in journal (Refereed) Published
Abstract [en]

The disease mechanisms associated with onset and secondary effects of rotavirus (RV) diarrhea remain to be determined and may not be identical. In this study, we investigated whether onset of RV diarrhea is associated with increased intestinal permeability and/or motility. To study the transit time, fluorescent fluorescein isothiocyanate (FITC)-dextran was given to RV-infected adult and infant mice. Intestinal motility was also studied with an opioid receptor agonist (loperamide) and a muscarinic receptor antagonist (atropine). To investigate whether RV increases permeability at the onset of diarrhea, fluorescent 4- and 10-kDa dextran doses were given to infected and noninfected mice, and fluorescence intensity was measured subsequently in serum. RV increased transit time in infant mice. Increased motility was detected at 24 h postinfection (h p.i.) and persisted up to 72 h p.i in pups. Both loperamide and atropine decreased intestinal motility and attenuated diarrhea. Analysis of passage of fluorescent dextran from the intestine into serum indicated unaffected intestinal permeability at the onset of diarrhea (24 to 48 h p.i.). We show that RV-induced diarrhea is associated with increased intestinal motility via an activation of the myenteric nerve plexus, which in turn stimulates muscarinic receptors on intestinal smooth muscles.

Place, publisher, year, edition, pages
American Society for Microbiology, 2014
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-105750 (URN)10.1128/JVI.02927-13 (DOI)000332126000010 ()
Available from: 2014-04-07 Created: 2014-04-04 Last updated: 2017-12-05
3. The Cholinergic Anti-Inflammatory Pathway Contributes to the Limited Inflammatory Response following Rotavirus Infection
Open this publication in new window or tab >>The Cholinergic Anti-Inflammatory Pathway Contributes to the Limited Inflammatory Response following Rotavirus Infection
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Rotavirus causes acute gastroenteritis in young children and is characterized by severe diarrhoea and vomiting. Surprisingly, although rotavirus infection results in significant intestinal pathology, the inflammatory response is limited. We tested the novel hypothesis that rotavirus infection stimulates the cholinergic anti-inflammatory pathway to suppress gut inflammation. The role of the vagus nerve and the α7 nicotinic acetylcholine receptor (α7 nAChR) in rotavirus infection were explored in α7 nAChR gene-deficient mice, vagotomized mice and wild-type mice treated with the α7 nAChR antagonist mecamylamine. TNF-α, IL-1β and IL-6 were measured in serum, spleen, duodenum, jejunum and ileum at 48 hours post infection. To determine if modulation of the inflammatory response affects virus shedding, α7 nAChRs was blocked and virus quantified in faeces. To investigate if stimulation of α7 nAChRs could attenuate rotavirus toxin NSP4-induced cytokine release, mouse peritoneal- and human blood-macrophages were treated with nicotine before NSP4 stimulation.

Our results shows that stimulation of the vagus nerve and α7 nAChRs attenuated the pro- inflammatory response during rotavirus infection and blockade of the α7 nAChR reduced virus shedding from infected mice. IL-6 was increased in duodenum (p<0.05) and serum (p<0.05) of vagotomized mice and in jejunum (p<0.05) and spleen (p<0.05) of α7 nAChR gene-deficient mice. Furthermore, IL-6 mRNA (p<0.01) and TNF-α mRNA (p<0.05) were increased in duodenum of vagotomized animals. Similarly, nicotine attenuated the release of TNF-α (p<0.05) and IL-6 (p<0.05) from macrophages stimulated by NSP4 in vitro, all suggesting that the cholinergic anti- inflammatory pathway contributes to attenuate inflammation during rotavirus infection.

National Category
Microbiology Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-117892 (URN)
Available from: 2015-05-13 Created: 2015-05-13 Last updated: 2015-05-13Bibliographically approved
4. Intracellularly expressed rotavirus NSP4 stimulates release of serotonin (5-HT) from human enterochromaffin cells
Open this publication in new window or tab >>Intracellularly expressed rotavirus NSP4 stimulates release of serotonin (5-HT) from human enterochromaffin cells
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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:nbn:se:liu:diva-117893 (URN)
Available from: 2015-05-13 Created: 2015-05-13 Last updated: 2015-05-13Bibliographically approved

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