Browsing by Author "Sharkey, Keith A."
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Item Open Access Endogenous cannabinoid signalling and energy balance(2008) Chambers, Adam; Sharkey, Keith A.Item Open Access Enteric glial cells play a role in neuronal signalling and in inflammation in the gastrointestinal tract(2007) Nasser, Yasmin; Sharkey, Keith A.The functions of glial cells in the enteric nervous system remain poorly understood. We hypothesized that enteric glia play a role in enteric neurotransmission and in intestinal inflammation. Enteric glia may participate in neurotransmission as we observed that these cells expressed receptors for neurotransmitters, such as the metabotropic glutamate receptors (mGluR) 2/3 and 5, the a2a-adrenergic receptor and the somatostatin 2A (SSTR2A) receptor. Glial receptor expression was altered during colitis. We observed a redistribution in mGluR immunoreactivity in both trinitrobenzene sulphonic acid (TNBS) colitis in guinea pigs and dextran sodium sulphate (OSS) colitis in mice, while mGluR expression was decreased in the inflamed colons of interleukin-10 gene-deficient (I L-10-1-) mice. SSTR2A immunoreactivity was redistributed in mice given DSS but unchanged in IL-10-1- mice. No changes to enteric ganglia or glial fibrillary acidic protein expression were observed in DSS and IL-10_,_ mice. Stimulation of glial mGluR5 receptors increased the expression of the early immediate gene product, Fos, as well as the phosphorylated form of the extracellular signal-regulated kinase 1/2 (pERK1/2), but did not alter intracellular calcium, demonstrating that glial receptors were functionally activated. Studies in cultured enteric glia proved inconclusive, as their phenotype no longer resembled that in situ. The gliotoxin fluorocitrate specifically stimulated pERK1/2 expression in enteric glia and reduced ilea! motility in vitro and gastrointestinal transit in vivo. No changes in colonic transit or ion transport were observed, nor were there any signs of inflammation, suggesting that glia modulate the enteric neural circuits underlying gastrointestinal motility. Enteric glia of the mouse colon constitutively expressed the inducible nitric oxide synthase, likely due to "physiological" inflammation. The nitric oxide precursor, L-arginine was localized to neurons and glia in the mouse, suggesting that glial cells were not the sole source of L-arginine. No changes in gut transit or in the distribution of glial and neuronal markers were observed in mice deficient for the inducible L-arginine transporter. Taken together, these data demonstrate that enteric glia play a role in enteric neurotransmission, particularly in gastrointestinal motility and that changes in glial receptor expression are a feature of colitis.Item Open Access Enteroendocrine cells and mucosal serotonin availability in experimental models of intestinal inflammation(2007) O'Hara, Jennifer R.; Sharkey, Keith A.Item Open Access Helminth Parasites and the Modulation of Joint Inflammation(2011-04-18) Matisz, Chelsea E.; McDougall, Jason J.; Sharkey, Keith A.; McKay, Derek M.There is an urgent need to develop better therapeutics for autoimmune and autoinflammatory diseases, of which musculoskeletal disorders such as rheumatoid arthritis are particularly prevalent and debilitating. Helminth parasites are accomplished masters at modifying their hosts' immune activity, and so attention has focused on rodent-helminth model systems to uncover the workings of the mammalian immune response to metazoan parasites, with the hope of revealing molecules and/or mechanisms that can be translated into better treatments for human autoimmune and idiopathic disorders. Substantial proof-of-principal data supporting the concept that infection with helminth parasites can reduce the severity of concomitant disease has been amassed from models of mucosal inflammation. Indeed, infection with helminth parasites has been tried as a therapy in inflammatory bowel disease, and there are case reports relating to other conditions (e.g., autism); however, the impact of infection with parasitic helminths on musculoskeletal diseases has not been extensively studied. Here, we present the view that such a strategy should be applied to the amelioration of joint inflammation and review the literature that supports this contention.Item Open Access Helminth Regulation of the Colonic Microbiome: Implications for the Control of Colitis(2020-11-18) Shute, Adam; McKay, Derek Mark; Buret, André Gerald; Sharkey, Keith A.; Strous, MarcThe mammalian gut is a dynamic and complex organ composed of host cells, bacteria, fungi and viruses; while parasitic protozoa and helminths can be transient residents in the intestine. Through the advancements in high throughput sequencing, an increasing number of studies have catalogued taxonomic compositional changes in the gut microbiota following infection with parasitic helminths. However, the functional implications of such helminth-microbiota interactions on the host are not well understood, especially in the context of controlling inflammation. The McKay laboratory continuously demonstrates that mice infected with the rat tapeworm, Hymenolepis diminuta, are protected from dinitrobenzene sulfonic acid (DNBS)-induced colitis. My thesis seeks to determine if H. diminuta is dependent on the intestinal microbiota to protect from colonic inflammation and to determine the underlying mechanisms that are responsible for this anti-inflammatory event. Although H. diminuta does not require the microbiota to infect or be recognized/expelled by the murine host’s immune system, distinct compositional changes to the colonic microbiota occur during infection with H. diminuta. Removing microbes by using germ-free mice or disrupting the intestinal microbiota through broad-spectrum antibiotic use inhibits the anti-inflammatory mechanism of H. diminuta towards DNBS-colitis. In addition to characterizing the compositional shift in the intestinal microbiota of mice infected with H. diminuta, we demonstrated a significant increase in fecal short chain fatty acids (SCFA), in particular acetate and butyrate. Infecting free fatty receptor 2 (ffar2)-knockout mice with H. diminuta inhibited the beneficial effect towards DNBS, and in parallel to this, treating mice with neutralizing IL-10 antibodies also blocked the protective effect of H. diminuta when challenged with DNBS. Immunostaining for IL-10Rα demonstrated increased reactivity in colonic tissues of mice that were either treated with butyrate enemas or infected with H. diminuta, as compared to tissue from naive mice. The data in this thesis provides proof-of-principle evidence that H. diminuta modulates both the gut microbiota and the immune system of its host to protect from chemically induced colitis. Where the removal of these constituents, inhibits H. diminuta’s ability to protect the host from colitis.Item Open Access Intestinal microbiota shapes gut physiology and regulates enteric neurons and glia(2021-10-26) Vicentini, Fernando A.; Keenan, Catherine M.; Wallace, Laurie E.; Woods, Crystal; Cavin, Jean-Baptiste; Flockton, Amanda R.; Macklin, Wendy B.; Belkind-Gerson, Jaime; Hirota, Simon A.; Sharkey, Keith A.Abstract Background The intestinal microbiota plays an important role in regulating gastrointestinal (GI) physiology in part through interactions with the enteric nervous system (ENS). Alterations in the gut microbiome frequently occur together with disturbances in enteric neural control in pathophysiological conditions. However, the mechanisms by which the microbiota regulates GI function and the structure of the ENS are incompletely understood. Using a mouse model of antibiotic (Abx)-induced bacterial depletion, we sought to determine the molecular mechanisms of microbial regulation of intestinal function and the integrity of the ENS. Spontaneous reconstitution of the Abx-depleted microbiota was used to assess the plasticity of structure and function of the GI tract and ENS. Microbiota-dependent molecular mechanisms of ENS neuronal survival and neurogenesis were also assessed. Results Adult male and female Abx-treated mice exhibited alterations in GI structure and function, including a longer small intestine, slower transit time, increased carbachol-stimulated ion secretion, and increased intestinal permeability. These alterations were accompanied by the loss of enteric neurons in the ileum and proximal colon in both submucosal and myenteric plexuses. A reduction in the number of enteric glia was only observed in the ileal myenteric plexus. Recovery of the microbiota restored intestinal function and stimulated enteric neurogenesis leading to increases in the number of enteric glia and neurons. Lipopolysaccharide (LPS) supplementation enhanced neuronal survival alongside bacterial depletion, but had no effect on neuronal recovery once the Abx-induced neuronal loss was established. In contrast, short-chain fatty acids (SCFA) were able to restore neuronal numbers after Abx-induced neuronal loss, demonstrating that SCFA stimulate enteric neurogenesis in vivo. Conclusions Our results demonstrate a role for the gut microbiota in regulating the structure and function of the GI tract in a sex-independent manner. Moreover, the microbiota is essential for the maintenance of ENS integrity, by regulating enteric neuronal survival and promoting neurogenesis. Molecular determinants of the microbiota, LPS and SCFA, regulate enteric neuronal survival, while SCFA also stimulates neurogenesis. Our data reveal new insights into the role of the gut microbiota that could lead to therapeutic developments for the treatment of enteric neuropathies. Video abstractItem Open Access Investigating the Role of Central Endocannabinoids and Inflammation in Comorbid Anxiety-Like Behaviour and Colitis(2020-01-08) Vecchiarelli, Haley Alleson; Hill, Matthew Nicholas; Sharkey, Keith A.; Trang, TuanThere is a well-established connection between inflammatory disorders and stress-associated neuropsychiatric disorders. For example, in patients with chronic inflammatory diseases, there are increased indices of anxiety and depression. However, despite this clinically significant relationship, there is not a comprehensive insight of the mechanisms linking inflammatory diseases with co-morbid mood and anxiety disorders. The endocannabinoid system, which is the system in the body that the psychoactive constituents of cannabis act on, regulates both anxiety and inflammation—indicating that it has the potential to underly these comorbidities. We hypothesize that in a rodent model of peripheral (gastrointestinal) inflammation, there would be alterations in endocannabinoid signaling that drive anxiety-like behaviours; that by boosting endocannabinoid signaling, these behaviours can be reversed; that neuroinflammation contributes to changes in central endocannabinoids.; and that endocannabinoids have the potential to regulate inflammatory processes. We find, using a rat model of colitis (intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS)), that there are reductions of an endocannabinoid, anandamide, in brain regions that regulate anxiety, which contributes to the generation of anxiety-like behaviour. We further show that these reductions are driven through corticotropin releasing factor receptor 1 (CRF-R1) and neuroinflammation mechanisms. Using a transgenic mouse model which presents elevated anandamide levels, we find a role for anandamide in regulating peripheral and central inflammatory changes induced by colitis. These studies link endocannabinoids and neuroinflammation to anxiety-like behaviour and show a protective role for endocannabinoid signaling in colitis-induced anxiety in rats and in peripheral and central inflammation in mice. This work contributes to our understanding of the mechanisms underlying inflammation induced anxiety.Item Open Access Microbial Dynamics and Metabolism in Gnotobiotic Mouse Models of Autism Spectrum Disorder(2021-09-17) Yee, Jenine R.; McCoy, Kathleen D.; Reimer, Raylene A.; Sharkey, Keith A.Autism Spectrum Disorder (ASD) is a highly heterogeneous disorder with diagnoses based on core behaviours centering on limited social interest, anxiety, and obsessive and repetitive movements. Recent studies have revealed a potential link between composition of the gut microbiota and ASD. The bacterial species Clostridium innocuum was found to be highly abundant in children with ASD and significantly reduced following fecal microbiota transplantation. The maternal immune activation (MIA) model was chosen as an experimental model to investigate whether C. innocuum would alter susceptibility to ASD. As the MIA model has been shown to require segmented filamentous bacterium (SFB) induction of Interleukin 17 (IL-17) during pregnancy, a co-colonization system was required. In this thesis, we investigated the dynamics between SFB and two different strains of C. innocuum, one isolated from an ASD participant and the other isolated from a healthy donor. In this mouse model, SFB co-colonized similarly with either strain of C. innocuum. To further investigate the effect presence of the two C. innocuum strains, serum metabolite profiles were analyzed using liquid chromatography-mass spectrometry for semi-untargeted metabolomics. Although serum samples from mice co-colonized with SFB and either of the two C. innocuum strains had similar metabolite profiles, significantly different metabolomic profiles were found following induction of maternal immune activation. Aspartate, hippurate and glycerol-3-phosphate were found to be significantly upregulated in the mice co-colonized with the C. innocuum isolated from an ASD participant when MIA was induced. These metabolites have been found in altered levels in children with ASD and ASD mouse models. Finally, in vitro cultures of other bacterial species found post-FMT produced metabolites involved with cellular repair, energy production and antioxidant activity such as N-acetyl-L-cysteine, glutathione, inosine and glutamine compared to media controls. Metabolites produced by the C. innocuum from the ASD participant may potentiate ASD behaviours and unravel targets for future research.Item Open Access The Mind-Gut Connection: A virtual reality education program on the relationship between the digestive system, nervous system, and microbiome.(2019-11-26) Lee, Ryan M.; Jacob, Christian; Sharkey, Keith A.The Mind-Gut Connection is a virtual reality education application on the relationship between the digestive system, nervous system, and microbiome. Together, these systems form the gut-brain axis and communicate with one another to carry out physiological processes associated with digestion. By illustrating this complex medical topic in a virtual reality environment, we have addressed the lack of accurate or comprehensive depictions of the gut-brain axis. Additionally, the use of virtual reality in education may allow for a broader audience to be exposed to this information. Learning about digestion in relation to the gut-brain axis is beneficial for everyone because of the impact our diets and lifestyles have on our physical and mental health. The use of this virtual reality program has the potential to better engage and inform the general public so that they are more aware of how our different body systems are interconnected. Not only is this program novel in addressing such a unique but important topic, it also exhibits innovation upon current virtual reality practices surrounding movement and motion sickness. The use of full-body virtual reality and a natural form of locomotion using arm swinging builds upon existing methods to improve the level of immersion and believability.Item Open Access Myc, fos and jun proteins in enteric and sympathetic neurons that supply the intestines(1997) Parr, Edward J.; Sharkey, Keith A.Item Open Access Neuropeptide y regulation of mouse colon epithelial ion transport in health and colitis(2009) Klompus, Matthew; McKay, Derek M.; Sharkey, Keith A.Item Open Access Plasticity of the enteric synapse in basal conditions and following gastrointestinal inflammation(2011) Hons, Ian; Sharkey, Keith A.Item Open Access Recruitment of α4β7 monocytes and neutrophils to the brain in experimental colitis is associated with elevated cytokines and anxiety-like behavior(2022-04-04) Cluny, Nina L.; Nyuyki, Kewir D.; Almishri, Wagdi; Griffin, Lateece; Lee, Benjamin H.; Hirota, Simon A.; Pittman, Quentin J.; Swain, Mark G.; Sharkey, Keith A.Abstract Background Behavioral comorbidities, such as anxiety and depression, are a prominent feature of IBD. The signals from the inflamed gut that cause changes in the brain leading to these behavioral comorbidities remain to be fully elucidated. We tested the hypothesis that enhanced leukocyte–cerebral endothelial cell interactions occur in the brain in experimental colitis, mediated by α4β7 integrin, to initiate neuroimmune activation and anxiety-like behavior. Methods Female mice treated with dextran sodium sulfate were studied at the peak of acute colitis. Circulating leukocyte populations were determined using flow cytometry. Leukocyte–cerebral endothelial cell interactions were examined using intravital microscopy in mice treated with anti-integrin antibodies. Brain cytokine and chemokines were assessed using a multiplex assay in animals treated with anti-α4β7 integrin. Anxiety-like behavior was assessed using an elevated plus maze in animals after treatment with an intracerebroventricular injection of interleukin 1 receptor antagonist. Results The proportion of classical monocytes expressing α4β7 integrin was increased in peripheral blood of mice with colitis. An increase in the number of rolling and adherent leukocytes on cerebral endothelial cells was observed, the majority of which were neutrophils. Treatment with anti-α4β7 integrin significantly reduced the number of rolling leukocytes. After anti-Ly6C treatment to deplete monocytes, the number of rolling and adhering neutrophils was significantly reduced in mice with colitis. Interleukin-1β and CCL2 levels were elevated in the brain and treatment with anti-α4β7 significantly reduced them. Enhanced anxiety-like behavior in mice with colitis was reversed by treatment with interleukin 1 receptor antagonist. Conclusions In experimental colitis, α4β7 integrin-expressing monocytes direct the recruitment of neutrophils to the cerebral vasculature, leading to elevated cytokine levels. Increased interleukin-1β mediates anxiety-like behavior.Item Open Access The role of serotonin in the immunoregulation by the helminth parasite Hymenolepis diminuta(2019-12-19) Wang, Susan Joanne; McKay, Derek Mark; Sharkey, Keith A.; Nasser, Yasmin; Geuking, Markus B.While infection with parasitic helminths remains a major concern for a quarter of the worlds’ population in non-westernized societies, the incidence of infection has significantly reduced in westernized societies. Simultaneously, the incidence of autoimmune diseases, such as inflammatory bowel disease, is on the rise in the industrialized-west. Studies of infection with various helminths, including the rat tapeworm, Hymenolepis diminuta, in murine models of inflammatory disease reveal the potential of helminth-based therapeutics. Serotonin (5-hydroxytryptamine, 5-HT) has previously been shown to be responsive to infection with a helminth. It has a variety of immunomodulatory actions in the gastrointestinal tract, yet its potential role in helminth expulsion and helminth therapy is largely unknown. Thus, the research presented in this thesis sought to test the hypothesis that infection with H. diminuta evokes increases in mucosal serotonin levels, important for fine-tuning type 2 (Th2) immunity and are involved in H. diminuta-mediated inhibition of dinitrobenzene sulphonic acid (DNBS)-induced colitis. Three specific aims were: (1) assess changes in murine intestinal serotonin following infection with H. diminuta, (2) determine if serotonin was important in the expulsion of H. diminuta from its non-permissive mouse host; and, (3) evaluate if manipulation of the enteric serotonergic system affected H. diminuta-evoked suppression of murine colitis caused by DNBS. This study did not find evidence of an increase in serotonin in the small intestine of H. diminuta-infected male BALB/c mice, and the extrinsic manipulation of the serotoninergic system via systemic delivery of a tryptophan hydroxylase inhibitor (para-chlorophenylalanine), 5-HT7 receptor antagonist (SB269970), or selective serotonin reuptake transporter inhibitor (fluoxetine) had negligible effects on worm expulsion or systemic Th2 events post-infection. Thus, the data do not support the hypothesis that enteric serotonin is a major influence of the immune response to infection with H. diminuta. Preliminary findings, however, suggest that the stress response to consecutive daily handling and i.p. injections may abrogate the helminth-evoked suppression of DNBS-induced colitis in mice, highlighting the neuro-immune axis and stress component of experimental design when studying inflammatory disorders.Item Open Access Role of the endocannabinoid system in the regulation of intestinal permeability: Effects of diet(2020-09-24) Cuddihey, Hailey; Sharkey, Keith A.; MacNaughton, Wallace K.; Hill, Matthew NicholasThe endocannabinoid system in the gastrointestinal tract plays a role in the peripheral regulation of energy balance and has been implicated in the control of intestinal barrier function. Previous studies have shown that high-fat diet (HFD) increases endocannabinoid tone and changes in endocannabinoid tone have been associated with a disruption in gut barrier function. Whether the cannabinoid 1 (CB1) receptor is expressed on the intestinal epithelium and is involved in the acute regulation of intestinal barrier function has not been determined. Therefore, the research presented in this thesis sought to test the hypothesis that CB1 receptor activation acutely regulates intestinal barrier function. The expression and function of the CB1 receptor was examined in the small intestine of mice fed standard chow or HFD (45% kcal) for 2, 6 and 12 weeks. We used immunohistochemistry and quantitative polymerase chain reaction (qPCR) to assess the expression and localization of the CB1 receptor on the gastrointestinal epithelium and Ussing chambers to assess intestinal barrier function using 4kDa fluorescein isothiocyanate-dextran (FD4) as a marker of paracellular permeability. Two structurally distinct CB1 agonists (AM841 [1μM] and CP55,940 [100nm]), an inverse agonist/antagonist (AM251 [1μM]) and a neutral antagonist (AM6545 [1μM]) were used. We found that the CB1 receptor is expressed on the small intestinal epithelium and its protein expression is upregulated in response to 2 weeks of HFD feeding. Acute application of CB1 agonists and antagonists following 2 weeks of HFD feeding reduces intestinal permeability, although these drugs have no effect in tissues from animals fed a chow diet or on tissues from animals fed HFD for a longer duration. The CB1 antagonists block the effect of the agonists and the effects of CB1 agonists and antagonists are absent in CB1 knock-out (KO) mice fed a 2-week HFD. Taken together, this study demonstrates that the CB1 receptor is involved in the acute regulation of small intestinal permeability in conditions where baseline intestinal permeability is perturbed such as in initial exposure to HFD. Further studies are required to elucidate the mechanism by which agonists and antagonists of the CB1 receptor give rise to similar responses in the small intestine.Item Open Access The effects of inflammation on the proportions of neuronal subtypes in the guinea pig ileum(1996) Amundson, Krista R.; Sharkey, Keith A.Item Open Access The response of the central nervous system to experimental colitis(1998) Ran, Israeli Shlomo; Sharkey, Keith A.Item Open Access The role of calcitonin gene-related peptide (CGRP) in colonic inflammation and secretion in the rat distal colon(1999) Esfandyari, Tuba; Sharkey, Keith A.Item Open Access The role of the endogenous cannabinoid system in the regulation of emesis(2006) Van Sickle, Marja; Sharkey, Keith A.Cannabinoids inhibit nausea and vomiting (emesis), but their mechanism is unknown. The aim of the following studies was to determine if cannabinoids are part of an endogenous cannabinoid system involved in inhibiting emesis in the brainstem. An anatomical substrate for the anti-emetic effect was demonstrated by the presence of the cannabinoid receptor 1 (CB1r) in the nucleus of the solitary tract and the dorsal motor nucleus of the vagus in the brainstem by immunohistochemistry and Western blotting. CB1r was also found in the myenteric plexus of the stomach and duodenum. A role for the CB1r in the modulation of emesis was revealed by observing a reduction in the number of episodes of retching and vomiting in ferrets that had been administered an emetic stimulus and a CB1r agonist and showing that the action of cannabinoids was reversed by a selective CB1r antagonist. A low dose of the cannabinoid delta-9- tetrahydrocannabinol inhibited emesis when applied directly to the brainstem demonstrating a central site of action. Fos immunohistochemistry was used to show that cannabinoids reduce emesis-induced neuronal activation in the area postrema, nucleus of the solitary tract and dorsal motor nucleus of the vagus nerve. The presence of endocannabinoids was demonstrated by evaluating the anti-emetic potential of endocannabinoids and determining their levels in the brainstem during an emetic response. The enzymes that degrade endogenous cannabinoids were also found in the brainstem by immunohistochemistry. A relationship between endocannabinoids and other neurotransmitter systems involved in emesis was suggested by the comparison of the immunoreactivity of CB1 r with substance P, the NK1 receptor and serotonin. Another cannabinoid receptor, CB2r, was shown to be present and functional and involved in the antiemetic effect of cannabinoids. The involvement of the endocannabinoid system in emesis and the establishment of the site of action in the dorsal vagal complex of the brainstem provides new avenues for further study of the targeting of pharmaceuticals and future research into the cellular mechanism of cannabinoids in the brain.