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Irritable bowel syndrome (IBS) is characterized by chronic abdominal pain or discomfort and altered gastrointestinal function of unknown etiology. Studies of colonic mucosal biopsies from patients with IBS have suggested altered immune system function as a potential mechanism in the pathophysiology of IBS, but efforts to identify the mucosal mediators responsible for the manifestation of symptoms that define the disorder have been limited. In this issue of Neurogastroenterology and Motility, Buhner et al. and Balestra et al. report findings from studies linking increased excitability of the enteric nervous system with mucosal mediators released from biopsies of patients with IBS. These studies provide evidence for the concept that mediators present in the colonic mucosa may contribute to the manifestation of clinical symptoms present in IBS.
The intestinal mucosa is a complex network of cells that is essential to proper functioning of the gastrointestinal (GI) tract. The mucosal epithelium is positioned adjacent to the lumen where it serves as our body’s largest interface with its external environment. The epithelial cells, immune cells, regulatory cells, secretory cells, and associated microbiota that comprise this epithelial lining are in close proximity to resident blood vessels, nerves, and lymphoid tissue in the lamina propria, where integrated responses and reflex activity are initiated. Thus, the intestinal mucosa serves as a dynamic site of regulatory neural, humoral, and immune responses for both the intestine and the entire body.
Irritable bowel syndrome (IBS) is a functional GI disorder characterized by chronic abdominal pain or discomfort and altered GI function in the absence of alarm signs or overt inflammation.1 These patients also typically exhibit a reduced threshold for pain, or visceral hypersensitivity. Due to the absence of known structural abnormalities or clear pathological markers, the diagnosis of IBS is made on symptom-based criteria and further classified by the nature of the predominant stool pattern; IBS-C (constipation), IBS-D (diarrhea), or IBS-M (mixed or alternating).1 Substantial research has therefore focused on the study of the symptoms associated with IBS, but the innate heterogeneity of the disorder and lack of appropriate animal models have limited consensus on mechanisms of action and potential treatment options for IBS patients.
Previous acute GI inflammation has been established as a major risk factor for the development of postinfectious IBS (PI-IBS),2 and a significant body of evidence has emerged in support of microscopic inflammation as a primary factor in the pathophysiology of IBS without a clinical history of infection. Increased numbers of activated immune cells in the mucosa, including mast cells, have been reported in several studies of colonic mucosal biopsies from IBS patients.3–6 However, other groups have failed to detect these differences7–9 or have reported decreases10,11 in mucosal mast cell numbers in IBS patients compared with healthy controls. Furthermore, evaluation of pro-inflammatory cytokine mRNA expression profiles has not revealed differences in colonic mucosal biopsies from IBS patients compared with controls.8,12,13 Expression levels of other targets in mucosal biopsies, including key elements of serotonin signaling and various proteases, have also yielded conflicting results.
In this issue of Neurogastroenterology and Motility, articles by the research groups of Michael Schemann and Giovanni Barbara report the results of two independent studies examining the physiological effects of colonic mucosal biopsy supernatants from patients with IBS on enteric neurons. Furthermore, through pharmacological manipulations of their respective measures of neuronal activity, they reveal information about candidate mediators that may underlie the manifestation of IBS symptoms. These studies represent an important complement to molecular and histological approaches aiming to define the contribution of the colonic mucosa to the pathophysiology of IBS.
In early 2007, the first publications using colonic biopsy supernatants from patients with IBS were published by the research groups of Barbara and Vergnolle in Gastroenterology and The Journal of Clinical Investigation, respectively. Barbara et al. reported that application of colonic mucosal supernatants from IBS patients with enhanced levels of histamine, tryptase and prostaglandin E2 increased visceral afferent nerve activity.10 Vergnolle and colleagues published findings showing that the proteases tryptase and trypsin were upregulated in colonic mucosal biopsies from IBS patients, and these supernatants increased activation of mouse dorsal root ganglia neurons in a PAR-2-dependent manner.9 These papers illustrated, for the first time, that mediators released from mast cells in the mucosa of patients with IBS are capable of activating visceral afferent neuronal pathways and may play a mechanistic role in the manifestation of visceral hypersensitivity in IBS.
Since those initial studies, investigation of the actions of biopsy extracts has been adopted by others, including the group of Buhner, Schemann and colleagues. In 2009, they reported that colonic mucosal biopsy supernatants from patients with IBS, but not control patients, evoked action potentials from human submucosal neurons as measured with voltage-sensitive dye neuroimaging.14 This was the first demonstration of human mucosal biopsy supernatants from patients with IBS on human enteric neurons, or on submucosal neurons. They also demonstrated through use of pharmacological inhibitors that this increase in neuronal excitability is mediated by serine proteases, serotonin, and histamine. The present investigation made use of a similar experimental paradigm to study the effects on myenteric neurons; however, guinea pig distal colon longitudinal muscle-myenteric plexus (LMMP) and submucosal plexus preparations were used to compare the effects on the two plexuses. Submucosal neurons were found to be more sensitive to colonic mucosal biopsy supernatants than myenteric neurons. Interestingly, no differences were observed between IBS-C and IBS-D samples, and this study confirmed previous findings in human submucosal neurons that serine proteases, serotonin (5-HT3 receptors), and histamine (H1/H2 receptors) mediate the increased action potential generation seen following application of mucosal biopsy supernatants from IBS patients.15
The paper by Balestra et al. also sought to determine whether mediators present in supernatants from mucosal biopsies could influence neuronal signaling in guinea pig LMMP preparations. They reported increased cholinergic motor activity (measured as an increase in amplitude of cholinergic twitch contractions) in response to IBS supernatant application. The magnitude of this change was correlated with the number of mast cells in the lamina propria of patients with IBS (no difference between IBS-C and IBS-D); however, no correlation between mast cell number and cholinergic motor activity was observed in healthy controls. The increased amplitude of cholinergic twitch contractions was attenuated by the administration of prostaglandin D2, P2X receptor, and TRPV1 channel antagonists. Interestingly, based on pharmacological approaches, no changes in cholinergic motor function were found to involve serotonin or histamine receptors, or serine proteases, suggesting different profiles of mucosal mediators may underlie altered motor activity and secretion in IBS.16
While the findings from these two studies are novel, it remains unclear how relevant they and other similar studies assessing the effects of mucosal biopsy supernatants are to the pathophysiology of IBS in the intact gut. Future studies should address the role of potential mediators following mucosal administration in in vivo and ex vivo models of visceral hypersensitivity, motility, and secretion. It will also be important to assess release from the mucosal epithelium in more intact preparations, possibly with the aid of in vivo electrochemical monitoring and/or biosensors. Conversely, future studies are also needed to identify the presence and precise localization of receptors on nerve fibers in the lamina propria mediating these reflex behaviors, as well as differences in expression in IBS patients.
In conclusion, the studies of Buhner et al. and Balestra et al. further support the concept that immune cell activation may contribute to IBS symptomatology. In addition to previous data on the potential involvement for colonic mucosal mediators in visceral hypersensitivity, these studies provide new information regarding target transmitters for the secretory and motor disturbances observed in IBS patients. Furthermore, they underscore the importance of enteric nervous system and immune interactions that warrant further investigation.