Identification of a quorum sensing-dependent communication pathway mediating bacteria-gut-brain cross talk

Despite recently established contributions of the intestinal microbiome to human health and disease, our understanding of bacteria-host communication pathways with regard to the gut-brain axis remains limited. Here we provide evidence that intestinal neurons are able to “sense” bacteria independently of the host immune system. Using supernatants from cultures of the opportunistic pathogen Staphylococcus aureus (S. aureus) we demonstrate the release of mediators with neuromodulatory properties at high population density. These mediators induced a biphasic response in extrinsic sensory afferent nerves, increased membrane permeability in cultured sensory neurons, and altered intestinal motility and secretion. Genetic manipulation of S. aureus revealed two key quorum sensing-regulated classes of pore forming toxins that mediate excitation and inhibition of extrinsic sensory nerves, respectively. As such, bacterial mediators have the potential to directly modulate gut-brain communication to influence intestinal symptoms and reflex function in vivo, contributing to homeostatic, behavioral, and sensory consequences of infection

In vitro effects of resistin on epithelial to mesenchymal transition (EMT) in MCF-7 and MDA-MB-231 breast cancer cells - qRT-PCR and Westen blot analyses data.

Resistin is an adipokine produced by the white adipocytes and adipose-derived macrophages, which mediates inflammation and insulin resistance Huang et al., 1997 and Renehan et al., 2008 Feb. Here, we provide data on the effect of resistin on epithelial to mesenchymal transition (EMT) in breast cancer cells in vitro. As model systems, we used human MCF-7 (low-metastatic) and MDA-MB-231 (high-metastatic) breast cancer cell lines. To optimize experimental conditions, we treated the cells with various concentrations of resistin (12.5, 25 and 50 ng/ml) for different time intervals (6 and 24 hours), and measured SOCS3 mRNA expression by using qRT-PCR analysis. Further, we used qRT-PCR and Western blot analyses to measure the expression of various epithelial (E-cadherin, claudin-1) and mesenchymal (SNAIL, SLUG, ZEB1, TWIST1, fibronectin, and vimentin) markers after resistin treatment. This data article is part of a study Avtanski et al., 2019 May, where detailed interpretation and discussion can be found

Dietary cholesterol, female gender and n-3 fatty acid deficiency are more important factors in the development of non-alcoholic fatty liver disease than the saturation index of the fat

<p>Abstract</p> <p>Background</p> <p>The central feature of NAFLD is a disturbed fatty-acid metabolism with hepatic lipid accumulation. However, the factors that determine the severity of NAFLD, including the role of nutrition, gender, and plasma lipid levels, remain to be determined.</p> <p>Methods</p> <p>High-fat diets (42 en% fat), containing 0.2% cholesterol, were fed to male and female wild-type and hyperlipidemic <it>APOE2ki </it>C57BL/6J mice for three weeks. The fats were, in order of decreasing saturation, fractionated palm fat (fPF; ~95%), cocoa butter (CB; ~60%), olive oil (OO; ~15%), sunflower oil (SO; ~12%), and high-oleic-acid sunflower oil (hoSO; ~7%). Plasma and liver triglycerides (concentration and composition), liver inflammation (<it>Ccl2</it>, <it>Cd68</it>, <it>Tnf-α </it>mRNA), and infiltration of macrophages (Cd68, Cd11b immunohistochemistry) and neutrophils (Mpo) were quantified.</p> <p>Results</p> <p>Addition of cholesterol to a low-fat diet decreased plasma HDL and increased (V)LDL levels in APOE2ki mice. Plasma cholesterol levels in female, but not male APOE2ki mice correlated significantly with inflammation. Kupffer cells of inflamed livers were swollen. Wild-type mice refused the highly saturated fPF diet. The high-fat CB, OO, and SO diets induced hyperglycemia and a 2-fold increase in hepatic fat content in male, but not female wild-type mice (in females, hepatic fat content was similar to that in males fed a high-fat diet). All high-fat diets induced macrovesicular setatosis. APOE2ki mice were protected against high-fat diet-induced steatosis and hyperglycemia, except when fed a hoSO diet. This diet caused a 5-fold increase in liver triglyceride and mead-acid content, and an increased expression of lipogenic genes, suggesting a deficiency in poly-unsaturated fatty acids. Irrespective of the composition of the high-fat diet, oleic acid was the main triglyceride component of liver fat in wild-type and APOE2ki mouse livers. Liver inflammation was dependent on genotype (APOE2ki > wild type), gender (female > male), and cholesterol content (high > low) of the diet, but not on dietary fat composition.</p> <p>Conclusions</p> <p>Dietary cholesterol plays a determining, independent role in inflammation, especially in female mice. The fatty-acid saturation of the diet hardly affected hepatic steatosis or inflammation.</p

Serotonin exerts a direct modulatory role on bladder afferent firing in mice

Serotonin (5-HT) is an excitatory mediator, which in the gastrointestinal (GI) tract, plays a physiological role in gut-brain signaling and which is dysregulated in functional GI disorders such as irritable bowel syndrome (IBS). Patients suffering from IBS frequently suffer from urological symptoms characteristic of interstitial cystitis/painful bladder syndrome, which manifests due to cross-sensitization of shared innervation pathways between the bladder and colon. However, a direct modulatory role of 5-HT in bladder afferent signaling and its role in colon-bladder neuronal crosstalk remain elusive. The aim of this study was to investigate the action of 5-HT on bladder afferent signaling in normal mice and mice with chronic visceral hypersensitivity (CVH) following trinitrobenzenesulfonic acid (TNBS) induced colitis. Bladder afferent activity was recorded directly using ex vivo afferent nerve recordings. Expression of 14 5-HT receptor subtypes, the serotonin transporter (SERT) and 5-HT producing enzymes were determined in the urothelium using RT-PCR. Retrograde labelling of bladder projecting dorsal root ganglion (DRG) neurons was used to investigate expression of 5-HT3 receptors using single cell RT-PCR, while sensory neuronal and urothelial responses to 5-HT were determined by live cell calcium imaging. 5-HT elicited bladder afferent firing predominantly via 5-HT3 receptors expressed on afferent terminals. CVH animals showed a downregulation of SERT mRNA expression in urothelium, suggesting increased 5-HT bioavailability. Granisetron, a 5-HT3 antagonist, reversed bladder afferent hypersensitivity in CVH mice. These data suggest 5-HT exerts a direct effect on bladder afferents to enhance signaling. 5-HT3 antagonists could therefore be a potential therapeutic target to treat functional bladder and bowel disorders

Serotonin exerts a direct modulatory role on bladder afferent firing in mice

Key points Functional disorders (i.e., interstitial cystitis/painful bladder syndrome and irritable bowel syndrome) are associated with hyperexcitability of afferent nerves innervating the urinary tract and the bowel respectively. Various non‐5‐HT3 receptor mRNA transcripts are expressed in mouse urothelium and exert functional responses to 5‐HT. Whilst 5‐HT3 receptors were not detected in mouse urothelium, 5‐HT3 receptors expressed on bladder sensory neurons plays a role in bladder afferent excitability under both normal conditions and in a mouse model of chronic visceral hypersensitivity (CVH). These data suggest that the role 5‐HT3 receptors play in bladder afferent signaling warrants further study as a potential therapeutic target for functional bladder disorders. Abstract Serotonin (5‐HT) is an excitatory mediator, which in the gastrointestinal (GI) tract, plays a physiological role in gut‐brain signaling and which is dysregulated in functional GI disorders such as irritable bowel syndrome (IBS). Patients suffering from IBS frequently suffer from urological symptoms characteristic of interstitial cystitis/painful bladder syndrome, which manifests due to cross‐sensitization of shared innervation pathways between the bladder and colon. However, a direct modulatory role of 5‐HT in bladder afferent signaling and its role in colon‐bladder neuronal crosstalk remain elusive. The aim of this study was to investigate the action of 5‐HT on bladder afferent signaling in normal mice and mice with chronic visceral hypersensitivity (CVH) following trinitrobenzenesulfonic acid (TNBS) induced colitis. Bladder afferent activity was recorded directly using ex vivo afferent nerve recordings. Expression of 14 5‐HT receptor subtypes, the serotonin transporter (SERT) and 5‐HT producing enzymes were determined in the urothelium using RT‐PCR. Retrograde labelling of bladder projecting dorsal root ganglion (DRG) neurons was used to investigate expression of 5‐HT3 receptors using single cell RT‐PCR, while sensory neuronal and urothelial responses to 5‐HT were determined by live cell calcium imaging. 5‐HT elicited bladder afferent firing predominantly via 5‐HT3 receptors expressed on afferent terminals. CVH animals showed a downregulation of SERT mRNA expression in urothelium, suggesting increased 5‐HT bioavailability. Granisetron, a 5‐HT3 antagonist, reversed bladder afferent hypersensitivity in CVH mice. These data suggest 5‐HT exerts a direct effect on bladder afferents to enhance signaling.5‐HT3 antagonists could therefore be a potential therapeutic target to treat functional bladder and bowel disorders

TGR5 agonists induce peripheral and central hypersensitivity to bladder distension

The mechanisms underlying chronic bladder conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) and overactive bladder syndrome (OAB) are incompletely understood. However, targeting specific receptors mediating neuronal sensitivity to specific stimuli is an emerging treatment strategy. Recently, irritant-sensing receptors including the bile acid receptor TGR5, have been identified within the viscera and are thought to play a key role in neuronal hypersensitivity. Here, in mice, we identify mRNA expression of TGR5 (Gpbar1) in all layers of the bladder as well as in the lumbosacral dorsal root ganglia (DRG) and in isolated bladder-innervating DRG neurons. In bladderinnervating DRG neurons Gpbar1 mRNA was 100% co-expressed with Trpv1 and 30% co-expressed with Trpa1. In vitro live-cell calcium imaging of bladder-innervating DRG neurons showed direct activation of a sub-population of bladder-innervating DRG neurons with the synthetic TGR5 agonist CCDC, which was diminished in Trpv1−/− but not Trpa1−/− DRG neurons. CCDC also activated a small percentage of non-neuronal cells. Using an ex vivo mouse bladder afferent recording preparation we show intravesical application of endogenous (5α-pregnan-3β-ol-20-one sulphate, Pg5α) and synthetic (CCDC) TGR5 agonists enhanced afferent mechanosensitivity to bladder distension. Correspondingly, in vivo intravesical administration of CCDC increased the number of spinal dorsal horn neurons that were activated by bladder distension. The enhanced mechanosensitivity induced by CCDC ex vivo and in vivo was absent using Gpbar1−/− mice. Together, these results indicate a role for the TGR5 receptor in mediating bladder afferent hypersensitivity to distension and thus may be important to the symptoms associated with IC/BPS and OAB.Ashlee Caldwell, Luke Grundy, Andrea M. Harrington, Sonia Garcia, Caraballo, Joel Castro, Nigel W. Bunnett, Stuart M. Brierle

Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1

Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1-1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation.Marco C. Inserra, Mathilde R. Israel, Ashlee Caldwell, Joel Castro, Jennifer R. Deuis, Andrea M. Harrington, Angelo Keramidas, Sonia Garcia-Caraballo, Jessica Maddern, Andelain Erickson, Luke Grundy, Grigori Y. Rychkov, Katharina Zimmermann, Richard J. Lewis, Stuart M. Brierley and Irina Vette

Core-shell Fe@Fex_xOy_y nanoring system: A versatile platform for biomedical applications

Iron oxide (maghemite and magnetite) nanoparticles are the most commonly used magnetic materials in nanomedicine because of their high biocompatibility. However, their low saturation magnetization (60–90 emu/g) limits their applicability. Here, we report a new core–shell (Fe@Fe$_x$O$_y$) nanoring system, which combines the high magnetic saturation of a metallic iron core (220 emu/g) and the biocompatibility of an iron oxide shell. To produce these nanostructures, hematite (α-Fe$_2$O$_3$) nanorings were annealed in a H$_2$ gas atmosphere for different periods to optimize the amount of metallic iron percentage (δ) in the system. Thus, nanostructures with different magnetic saturation (97 to 178 emu/g) could be obtained; based on their metallic iron content, these particles are labeled as Vortex Iron oxide Particle δ (VIPδ). Micromagnetic simulations confirmed that the VIPδ nanorings exhibit a vortex configuration, guaranteeing low remanence and coercitivity. Moreover, the system shows good biocompatibility in various assays as determined through cell viability measurements performed using two different human cell lines, which were exposed to VIP78% for 24 h. Therefore, VIPδ nanorings combine a magnetic vortex state and biocompatibility with their high magnetic saturation and can thus serve as a platform that can be tuned during the synthesis based on desired biomedical application

Olorinab (APD371), a peripherally acting, highly selective, full agonist of the cannabinoid receptor 2, reduces colitis-induced acute and chronic visceral hypersensitivity in rodents

Published on 17 April 2021Abdominal pain is a key symptom of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), for which there are inadequate therapeutic options. We tested whether olorinab-a highly selective, full agonist of the cannabinoid receptor 2 (CB2)-reduced visceral hypersensitivity in models of colitis and chronic visceral hypersensitivity (CVH). In rodents, colitis was induced by intrarectal administration of nitrobenzene sulfonic acid derivatives. Control or colitis animals were administered vehicle or olorinab (3 or 30 mg/kg) twice daily by oral gavage for 5 days, starting 1 day before colitis induction. CVH mice were administered olorinab (1, 3, 10, or 30 mg/kg) twice daily by oral gavage for 5 days, starting 24 days after colitis induction. Visceral mechanosensitivity was assessed in vivo by quantifying visceromotor responses (VMR) to colorectal distension (CRD). Ex vivo afferent recordings determined colonic nociceptor firing evoked by mechanical stimuli. Colitis and CVH animals displayed significantly elevated VMRs to CRD and colonic nociceptor hypersensitivity. Olorinab treatment significantly reduced VMRs to control levels in colitis and CVH animals. Additionally, olorinab reduced nociceptor hypersensitivity in colitis and CVH states in a concentration- and CB2-dependent manner. In contrast, olorinab did not alter VMRs nor nociceptor responsiveness in control animals. CB2 mRNA was detected in colonic tissue, particularly within epithelial cells, and dorsal root ganglia, with no significant differences between healthy, colitis, and CVH states. These results demonstrate olorinab reduces visceral hypersensitivity via CB2 agonism in animal models, suggesting that olorinab may provide a novel therapy for IBD- and IBS-associated abdominal pain.Joel Castro, Sonia Garcia-Caraballo, Jessica Maddern, Gudrun Schober, Amanda Lumsden, Andrea Harrington, Shirdi Schmiel, Beatriz Lindstrom, John Adams, Stuart M. Brierle

Activation of MrgprA3 and MrgprC11 on bladder-innervating afferents induces peripheral and central hypersensitivity to bladder distension

Understanding the sensory mechanisms innervating the bladder is paramount to developing efficacious treatments for chronic bladder hypersensitivity conditions. The contribution of Mas-gene-related G protein-coupled receptors (Mrgpr) to bladder signaling is currently unknown. Using male and female mice, we show with single-cell RT-PCR that subpopulations of DRG neurons innervating the mouse bladder express MrgprA3 (14%) and MrgprC11 (38%), either individually or in combination, with high levels of coexpression with Trpv1 (81%-89%). Calcium imaging studies demonstrated MrgprA3 and MrgprC11 agonists (chloroquine, BAM8-22, and neuropeptide FF) activated subpopulations of bladder-innervating DRG neurons, showing functional evidence of coexpression between MrgprA3, MrgprC11, and TRPV1. In ex vivo bladder-nerve preparations, chloroquine, BAM8-22, and neuropeptide FF all evoked mechanical hypersensitivity in subpopulations (20%-41%) of bladder afferents. These effects were absent in recordings from Mrgpr-clusterD2/2 mice. In vitro whole-cell patch-clamp recordings showed that application of an MrgprA3/C11 agonist mixture induced neuronal hyperexcitability in 44% of bladder-innervating DRG neurons. Finally, in vivo instillation of an MrgprA3/C11 agonist mixture into the bladder of WT mice induced a significant activation of dorsal horn neurons within the lumbosacral spinal cord, as quantified by pERK immunoreactivity. This MrgprA3/C11 agonist-induced activation was particularly apparent within the superficial dorsal horn and the sacral parasympathetic nuclei of WT, but not Mrgpr-clusterD2/2 mice. This study demonstrates, for the first time, functional expression of MrgprA3 and MrgprC11 in bladder afferents. Activation of these receptors triggers hypersensitivity to distension, a critically valuable factor for therapeutic target development.Luke Grundy, Ashlee Caldwell, Sonia Garcia-Caraballo, David Grundy, Nick J. Spencer, Xinzhong Dong ... et al