Divergent effects of exendin- 4 and interleukin- 6 on rat colonic secretory and contractile activity are associated with changes in regional vagal afferent signaling

Background: Thepro-inflammatorycytokine,interleukin(IL)-6iselevatedinindividu-alswiththefunctionalboweldisorder,irritablebowelsyndrome(IBS).IL-6canin- dependentlymodifyintestinalsecreto-motorfunction,therebycontributingtoIBS signals altered vagal afferent activity. Methods: Colonic secretory currents and circular muscle contractile activity was investigatedinSpragueDawleyratsusingUssingchamberandorganbathelectro-physiology. Regional afferent signaling was assessed using extracellular electrophysi ological recordings from colonic vagal afferents. Key Results: ApplicationoftheGLP-1receptoragonist,exendin-4(Ex-4)inthepres-ence of IL-6 potentiated colonic secretory currents and transepithelial resistance.

Indeed,wehaveobservedthatadditionofEx-4inthepresenceof IL-6 had divergent effects on the enteric plexi. Calcium responses evoked by Ex-4 in the presence of IL-6 in submucosal neurons were enhanced, whereas calcium responses in myenteric neurons were suppressed in comparison with Ex-4 alone. 9 The aims of this study were to determine whether the modulatory effects of Ex-4 on colonic secretory and contractile function were impacted by the presenceofIL-6.Moreover,wesoughttoinvestigatewhethervagal afferents acted as signaling conduits for relaying such information to thecentralnervoussystem(CNS).

| Ethical approval
All experiments were in full accordance with the European Community Council Directive (86/609/EEC) and local guidelines fromUniversityCollegeCorkanimalethicalcommittee(#2011/015).

Key points
• IL-6 and GLP-1 analogues can independently modify colonic secreto-motor function. Both factors are modified in IBS and may contribute to bowel dysfunction.
• Similar patterns of activation were detected in vagal afferents originating in the submucosal andmyentericplexi,respectively.
• Immune-endocrine interactions differentially modify colonic function, and regional activation of vagal afferents mimics these patterns. removed to expose the submucosal plexus. To expose myenteric neurons,thecircularmusclelayerswerepeeledawayusingforcepsleaving a longitudinal muscle myenteric plexus preparation. Sections of colon stripped of the mucosa and orientated to measure circular muscle contractileactivitywereusedforgutbathstudies,whereasmucosasubmucosa tissue preparations were used for experiments in the Ussingchambers.Thedissectiontechniqueusedforthecolon-vagal nerve tissue preparation is described in detail below.
Reagents were added to the basolateral chamber. For studies examining afferents originating in the submucosal or myentericplexi,thecolonwasopenedlongitudinally,offcenterto the mesentery border to orientate the intact nerve fibers to the edge of the opened tissue. The mucosa was carefully peeled away using forceps to expose the submucosal neurons. To expose myenteric neurons, the submucosal layer and circular muscle fibers were removed. Reagents were added to the colonic bath in random order topreventpotentialconfoundingresultsduetodesensitization.

| Colonic-Afferent nerve electrophysiological recordings
Multi-unit neural activity was recorded using platinum bipolar recording electrodes (World Precision Instruments) attached to a Powerlabdevice(ADInstruments).Reagentsweredirectlyapplied to the colonic bath. Nerve activity was viewed and analyzed with Chart7(ADInstruments).Changesinmulti-unitneuralactivityinthe vagalnervearepresentedasfrequencyfromrawtraces.Bothraw and rectified traces are presented.

| Statistical analyses
DatawereanalyzedusingGraphPadPrismforwindows(version6, Graphpad Software). The data are represented as data plots with mean±thestandarddeviation.One-wayANOVAwithTukeymultiple comparison post hoc tests or paired t tests, as appropriate were used to compare data. p ≤ 0.05 was considered statistically significant.

| Activity in vagal afferents originating in the submucosal plexus is enhanced by ex-4 and il-6
Anexvivopreparationofratcolonwithintactvagalafferentswas usedtodeterminetheeffectsofEx-4andIL-6onregionalvagalafferentactivity.Inthiscolonicpreparation,thesubmucosalneurons and the extrinsic afferent nerve endings innervating this gut layer were exposed to the receptor agonists. Consistent with previous reports, 33

| Application of ex-4 in the presence of il-6 had a suppressive effect on colonic contractile activity
WepreviouslyfoundthatapplicationEx-4inthepresenceofIL-6suppressedthecalciumresponseinmyentericneurons,whencompared toresponsesevokedbyeitherEx-4orIL-6alone. 9 To assess whether this observation at the cellular level had functional consequences, the receptor agonists were applied to a colonic smooth muscle tissue preparation laterally orientated to assess circular muscle contractile activity. Baseline colonic circular smooth muscle contractions were

| DISCUSS ION
Circulating factors can be more than simple biomarkers of a par- The findings from this study build upon our recent report of divergenteffectsofIL-6andtheGLP-1Ragonist,Ex-4,insubmucosal and myenteric neurons. 29 Consistent with those findings in thesubmucosalplexus,theneuronalregulatorsofabsorptiveand secretoryactivityinthegut,wherecalciumresponsesevokedby Ex-4werepotentiatedwhenIL-6wasco-applied, 29 we observed that application of Ex-4 in the presence of IL-6 stimulated a secretory current from the colonic epithelium that was larger than Communication between the immune system and the CNS is criticaltoinitiatingtheinflammatoryreflex,aphysiologicalresponse wherevagalafferentsarestimulatedbypro-inflammatorycytokines resulting in activation of vagal efferents which regulate the release ofpro-inflammatorymediators. 41 Vagal afferents express receptors forpro-inflammatorycytokinessuchasTNFαandIL-1β, 49,50 and in our studies, we found that IL-6 also stimulated vagal afferent activity, although the response was less than that evoked by Ex-4.
When Ex-4 was applied in the presence of IL-6, there was an increase in frequency but it was not significantly greater than Ex-4 alone,whichissimilartowhatweobservedwithveratridine-evoked neurally regulated secretory currents. However, a caveat to these compound extracellular recordings is that signals are incorporated from multiple afferent fibers. Thus, it is not possible to determine whetherthesamefibersarebeingstimulatedbybothEx-4andIL-  This study provides further evidence that endocrine and immune factors can modulate gut function and supports the concept of interoceptive signaling via colonic vagal afferents. Kelliherforherinputtothesestudies.

CO N FLI C T O F I NTE R E S T S
The authors have no competing interests.

D ECL A R ATI O N
RO'B and MMB performed the research and analyzed the data.
DO'M designed the research study, wrote the paper, and secured funding.

DATA AVA I L A B I L I T Y S TAT E M E N T
Alldatasetsareavailableuponrequesttothecorrespondingauthor.