Nicole J. Isaacs

Sensory neuro-immune interactions differ between Irritable Bowel Syndrome subtypes

Objective The gut is a major site of contact between immune and sensory systems and evidence suggests that patients with irritable bowel syndrome (IBS) have immune dysfunction. Here we show how this dysfunction differs between major IBS subgroups and how immunocytes communicate with sensory nerves. Design Peripheral blood mononuclear cell supernatants from 20 diarrhoea predominant IBS (D-IBS) patients, 15 constipation predominant IBS (C-IBS) patients and 36 healthy subjects were applied to mouse colonic sensory nerves and effects on mechanosensitivity assessed. Cytokine/chemokine concentration in the supernatants was assessed by proteomic analysis and correlated with abdominal symptoms, and expression of cytokine receptors evaluated in colonic dorsal root ganglia neurons. We then determined the effects of specific cytokines on colonic afferents. Results D-IBS supernatants caused mechanical hypersensitivity of mouse colonic afferent endings, which was reduced by infliximab. C-IBS supernatants did not, but occasionally elevated basal discharge. Supernatants of healthy subjects inhibited afferent mechanosensitivity via an opioidergic mechanism. Several cytokines were elevated in IBS supernatants, and levels correlated with pain frequency and intensity in patients. Visceral afferents expressed receptors for four cytokines: IL-1β, IL-6, IL-10 and TNF-α. TNF-α most effectively caused mechanical hypersensitivity which was blocked by a transient receptor potential channel TRPA1 antagonist. IL-1β elevated basal firing, and this was lost after tetrodotoxin blockade of sodium channels. Conclusions Distinct patterns of immune dysfunction and interaction with sensory pathways occur in different patient groups and through different intracellular pathways. Our results indicate IBS patient subgroups would benefit from selective targeting of the immune system.

A novel role for TRPM8 in visceral afferent function

&NA; Transient receptor potential ion channel melastatin subtype 8 (TRPM8) is activated by cold temperatures and cooling agents, such as menthol and icilin. Compounds containing peppermint are reported to reduce symptoms of bowel hypersensitivity; however, the underlying mechanisms of action are unclear. Here we determined the role of TRPM8 in colonic sensory pathways. Laser capture microdissection, quantitative reverse transcription‐polymerase chain reaction (RT‐PCR), immunofluorescence, and retrograde tracing were used to localise TRPM8 to colonic primary afferent neurons. In vitro extracellular single‐fibre afferent recordings were used to determine the effect of TRPM8 channel activation on the chemosensory and mechanosensory function of colonic high‐threshold afferent fibres. TRPM8 mRNA was present in colonic DRG neurons, whereas TRPM8 protein was present on nerve fibres throughout the wall of the colon. A subpopulation (24%, n = 58) of splanchnic serosal and mesenteric afferents tested responded directly to icilin (5 μmol/L). Subsequently, icilin significantly desensitised afferents to mechanical stimulation (P < .0001; n = 37). Of the splanchnic afferents responding to icilin, 21 (33%) also responded directly to the TRPV1 agonist capsaicin (3 μmol/L), and icilin reduced the direct chemosensory response to capsaicin. Icilin also prevented mechanosensory desensitization and sensitization induced by capsaicin and the TRPA1 agonist AITC (40 μmol/L), respectively. TRPM8 is present on a select population of colonic high threshold sensory neurons, which may also co‐express TRPV1. TRPM8 couples to TRPV1 and TRPA1 to inhibit their downstream chemosensory and mechanosensory actions. TRPM8 was localised to high‐threshold visceral afferent neurons. On visceral afferent peripheral endings, TRPM8 activation affected TRPV1 and TRPA1 downstream chemosensory and mechanosensory actions.

Diet-induced adaptation of vagal afferent function

Non‐technical summary  Obesity is the result of a disruption in the maintenance of energy balance such that energy intake exceeds expenditure. Why this occurs is unknown. We show that after food deprivation or consumption of a high fat diet gastric vagal afferent responses to distension are reduced. In addition, the effect of the orexigenic peptide ghrelin is enhanced. Thus the satiety signal is reduced not only after food deprivation but also after a high fat diet. This reduction in satiety signalling may explain the increase in energy intake and disruption in maintenance of energy balance in obesity.

Disordered control of intestinal sweet taste receptor expression and glucose absorption in type 2 diabetes.

We previously established that the intestinal sweet taste receptors (STRs), T1R2 and T1R3, were expressed in distinct epithelial cells in the human proximal intestine and that their transcript levels varied with glycemic status in patients with type 2 diabetes. Here we determined whether STR expression was 1) acutely regulated by changes in luminal and systemic glucose levels, 2) disordered in type 2 diabetes, and 3) linked to glucose absorption. Fourteen healthy subjects and 13 patients with type 2 diabetes were studied twice, at euglycemia (5.2 ± 0.2 mmol/L) or hyperglycemia (12.3 ± 0.2 mmol/L). Endoscopic biopsy specimens were collected from the duodenum at baseline and after a 30-min intraduodenal glucose infusion of 30 g/150 mL water plus 3 g 3-O-methylglucose (3-OMG). STR transcripts were quantified by RT-PCR, and plasma was assayed for 3-OMG concentration. Intestinal STR transcript levels at baseline were unaffected by acute variations in glycemia in healthy subjects and in type 2 diabetic patients. T1R2 transcript levels increased after luminal glucose infusion in both groups during euglycemia (+5.8 × 104 and +5.8 × 104 copies, respectively) but decreased in healthy subjects during hyperglycemia (−1.4 × 104 copies). T1R2 levels increased significantly in type 2 diabetic patients under the same conditions (+6.9 × 105 copies). Plasma 3-OMG concentrations were significantly higher in type 2 diabetic patients than in healthy control subjects during acute hyperglycemia. Intestinal T1R2 expression is reciprocally regulated by luminal glucose in health according to glycemic status but is disordered in type 2 diabetes during acute hyperglycemia. This defect may enhance glucose absorption in type 2 diabetic patients and exacerbate postprandial hyperglycemia.

Gastric vagal afferent modulation by leptin is influenced by food intake status

•  Obesity occurs when energy intake exceeds expenditure, and the excess energy is stored as fat. •  We show that, after a 14 h food deprivation or 12 weeks consumption of a high‐fat diet, gastric vagal afferent responses to mechanical stimulation in the presence of the satiety peptide leptin are altered. •  Leptin has an excitatory effect on gastric mucosal vagal afferents, which is abolished after food restriction or prolonged excess. •  In contrast, leptin has an inhibitory effect on gastric tension‐sensitive afferents, but only after food restriction or energy excess conditions. •  These changes in the response to leptin in the stomach, after food restriction or prolonged high‐fat feeding, occur in such a manner as to facilitate an increase in food intake in both conditions.

Sprouting of colonic afferent central terminals and increased spinal mitogen-activated protein kinase expression in a mouse model of chronic visceral hypersensitivity.

Visceral pain following infection or inflammation is a major clinical problem. Although we have knowledge of how peripheral endings of colonic afferents change in disease, their central projections have been overlooked. With neuroanatomical tracing and colorectal distension (CRD), we sought to identify colonic afferent central terminals (CACTs), the dorsal horn (DH) neurons activated by colonic stimuli in the thoracolumbar (T10–L1) DH, and determine how they are altered by postinflammatory chronic colonic mechanical hypersensitivity. Retrograde tracing from the colon identified CACTs in the DH, whereas immunohistochemistry for phosphorylated MAP kinase ERK 1/2 (pERK) identified DH neurons activated by CRD (80 mmHg). In healthy mice, CACTs were located primarily in DH laminae I (LI) and V (LV) and projected down middle and lateral DH collateral pathways. CRD evoked pERK immunoreactivity in DH neurons, the majority of which were located in LI and LV, the same regions as CACTs. In postinflammatory mice, CACTs were significantly increased in T12–L1 compared with healthy mice. Although CACTs remained abundant in LI, they were more widespread and were now present in deeper laminae. After CRD, significantly more DH neurons were pERK‐IR postinflammation (T12–L1), with abundant expression in LI and deeper laminae. In both healthy and postinflammatory mice, many pERK neurons were in close apposition to CACTs, suggesting that colonic afferents can stimulate specific DH neurons in response to noxious CRD. Overall, we demonstrate that CACT density and the number of responsive DH neurons in the spinal cord increase postinflammation, which may facilitate aberrant central representation of colonic nociceptive signaling following chronic peripheral hypersensitivity. J. Comp. Neurol. 520:2241–2255, 2012.

Increased κ-opioid receptor expression and function during chronic visceral hypersensitivity

In a recent article in Gut , Hughes et al 1 identified distinct patterns of immune dysfunction in IBS patients compared with healthy subjects. In particular, they showed that peripheral blood mononuclear cell (PBMC) supernatants from healthy subjects inhibited colonic afferents in a μ-opioid receptor (MOR)-mediated manner. These findings correlated with β-endorphin from T lymphocytes providing an important MOR-mediated antinociceptive influence in the healthy gut.2 Intriguingly, these inhibitory effects were lost with PBMC supernatants from constipation-predominant IBS patients suggesting a loss of MOR-mediated inhibition, coupled with increased excitatory mediators (TNF-α, IL-1β, IL-6), contributes to abdominal pain.1 We evaluated if this alteration was MOR specific or whether it extended to other members of the opioid receptor family. As clinical studies have shown varying outcomes on visceral pain perception with κ-opioid receptor (KOR) agonists,3–5 we postulated KOR expression and function are altered during visceral hypersensitivity. Therefore, we determined if the peripherally restricted selective KOR agonist, asimadoline, was able to modify colonic nociceptor function in health and during inflammatory and postinflammatory chronic visceral mechanical hypersensitivity (CVH).6 We performed in vitro afferent recordings from mouse splanchnic high-threshold nociceptors, which respond to focal compression and noxious stretch/distension.1 ,6 …

Characterization of duodenal expression and localization of fatty acid-sensing receptors in humans: relationships with body mass index

Fatty acids (FAs) stimulate the secretion of gastrointestinal hormones, including cholecystokinin (CCK) and glucagon like peptide-1 (GLP-1), which suppress energy intake. In obesity, gastrointestinal responses to FAs are attenuated. Recent studies have identified a key role for the FA-sensing receptors cluster of differentiation (CD)36, G protein-coupled receptor (GPR)40, GPR120, and GPR119 in mediating gastrointestinal hormone secretion. This study aimed to determine the expression and localization of these receptors in the duodenum of humans and to examine relationships with obesity. Duodenal mucosal biopsies were collected from nine lean [body mass index (BMI): 22 ± 1 kg/m2], six overweight (BMI: 28 ± 1 kg/m2), and seven obese (BMI: 49 ± 5 kg/m2) participants. Absolute levels of receptor transcripts were quantified using RT-PCR, while immunohistochemistry was used for localization. Transcripts were expressed in the duodenum of lean, overweight, and obese individuals with abundance of CD36>>GPR40>GPR120>GPR119. Expression levels of GPR120 (r = 0.46, P = 0.03) and CD36 (r = 0.69, P = 0.0004) were directly correlated with BMI. There was an inverse correlation between expression of GPR119 with BMI (r2 = 0.26, P = 0.016). Immunolabeling studies localized CD36 to the brush border membrane of the duodenal mucosa and GPR40, GPR120, and GPR119 to enteroendocrine cells. The number of cells immunolabeled with CCK (r = -0.54, P = 0.03) and GLP-1 (r = -0.49, P = 0.045) was inversely correlated with BMI, such that duodenal CCK and GLP-1 cell density decreased with increasing BMI. In conclusion, CD36, GPR40, GPR120, and GPR119 are expressed in the human duodenum. Transcript levels of duodenal FA receptors and enteroendocrine cell density are altered with increasing BMI, suggesting that these changes may underlie decreased gastrointestinal hormone responses to fat and impaired energy intake regulation in obesity.

Modulation of murine gastric vagal afferent mechanosensitivity by neuropeptide W.

Neuropeptide W (NPW) is an endogenous ligand for the receptors GPR7 and GPR8 and is involved in central regulation of energy homeostasis. NPW in the periphery is found in gastric gastrin (G) cells. In the stomach, energy intake is influenced by vagal afferent signals, so we aimed to determine the effect of NPW on mechanosensitive gastric vagal afferents under different feeding conditions.

Identifying spinal sensory pathways activated by noxious esophageal acid

The transient receptor potential vanilloid 1 (TRPV1) channel is critical for spinal afferent signaling of burning pain throughout the body. Such pain frequently originates from the esophagus, following acid reflux. The contribution of TRPV1 to spinal nociceptor signaling from the esophagus remains unclear. We aimed to identify the spinal afferent pathways that convey nociceptive signaling from the esophagus, specifically those sensitive to acid, and the extent to which TRPV1 contributes.