ARTICLE

Effects of tumor necrosis factor � on leptin-sensitive intestinal vagalmechanoreceptors in the catNathalie Quinson, Véronique Vitton, Michel Bouvier, Jean-Charles Grimaud, and Anne Abysique

Abstract: The involvement of tumour necrosis factor � (TNF-�) in inflammatory bowel disease (IBD) has been established, andanti-TNF-� has been suggested as a therapeutic approach for the treatment of these pathologies. We studied the effects of TNF-�on leptin-sensitive intestinal vagal units to determine whether TNF-� exerts its effects through the intestinal vagal mechanore-ceptors and to investigate its interactions with substances regulating food intake. The activity of intestinal vagal mechanore-ceptors was recorded via microelectrodes implanted into the nodose ganglion in anesthetized cats. TNF-� (1 �g, i.a.) increased thedischarge frequency of leptin-activated units (type 1 units; P < 0.05) and had no effect on the discharge frequency of leptin-inhibited units (type 2 units). When TNF-� was administered 20 min after sulfated cholecystokinin-8 (CCK), its excitatory effectson type 1 units were significantly enhanced (P < 0.0001) and type 2 units were significantly (P < 0.05) activated. Pre-treatment withIl-1ra (250 �g, i.a.) blocked the excitatory effects of TNF-� on type 1 units whereas the excitatory effects of TNF-� administrationafter CCK treatment on type 2 units were not modified. The activation of leptin-sensitive units by TNF-� may explain, at least inpart, the weight loss observed in IBD.

Key words: TNF-�, leptin, CCK, intestinal mechanoreceptors, vagus nerve, nodose ganglion.

Résumé : L'implication du TNF-� dans les maladies inflammatoires chroniques intestinales (MICI) est bien établie, ainsil'utilisation d'anti-TNF-� constitue une nouvelle approche thérapeutique. Pour déterminer l'implication des mécanorécepteursvagaux dans les effets du TNF-� et préciser ses interactions avec des substances modulant la prise alimentaire, nous avons étudiéses effets sur les unités vagales intestinales sensibles a la leptine. L'activité de ces unités est enregistrée, chez le chat anesthesié,dans le ganglion plexiforme. Le TNF-� augmente la fréquence de décharge des unités activées par la leptine (unités « type 1 ») etn'a pas d'effet sur celle des unités inhibées par la leptine (unités « type 2 »). Administré 20 min après CCK, les effets excitateursdu TNF-� sur les unités de type 1 sont potentialisés et les unités de type 2 sont activées. Un prétraitement par l'Il-1ra bloque leseffets excitateurs du TNF-� sur les unités de type 1 alors que, pour les unités de type 2, les effets excitateurs du TNF-� en présencede CCK ne sont pas modifiés. Les effets du TNF-� sur les mécanorécepteurs vagaux pourraient expliquer la perte de poidsobservée dans les MICI.

Mots-clés : TNF-�, leptine, CCK, mécanorécepteurs intestinaux, nerf vague, ganglion plexiforme.

IntroductionTumor necrosis factor alpha (TNF-�) is a pro-inflammatory cyto-

kine produced by various cell types including macrophages, lym-phoid cells, mast cells, and adipose tissue (Hotamisligil et al. 1993;Oshima and Oshima 2012). The involvement of TNF-� in inflam-matory bowel disease is well established (Podolsky 2002), and theuse of anti-TNF-� (antibody against TNF-�) is a new therapeuticapproach for the treatment of these pathologies. Weight loss isfrequently observed during the course of inflammatory boweldisease (Jahnsen et al. 2003; Filippi et al. 2006), and TNF-�, whichproduces cachexia characterized by anorexia and body weightloss (Plata-Salamán 2000), may play a role in these abnormalities.Indeed, interactions exist between proinflammatory cytokinessuch as TNF-�, and hormones that influence food intake, includ-ing leptin, which is an anorexigenic hormone (Loffreda et al. 1998;Faggioni et al. 2001; Sung et al. 2009). In addition, Franchimontet al. (2005) and Karmiris et al. (2007) have demonstrated thatpatients treated with anti-TNF-� exhibit a decrease in their leptinlevels. Many studies have reported weight gain in psoriatic and

spondyloarthropathic patients after treatment with anti-TNF-�(Briot et al. 2005; Gisondi et al. 2008; Saraceno et al. 2008).

Previous studies (Gaige et al. 2002) have indicated that in addi-tion to the direct effects observed on brain targets via hormonalpathways, leptin can act on the intestinal vagal afferent fibers,sending rapid signals to the central nervous system via neuronalpathways. Moreover, these studies established that leptin differ-entially affects 2 kinds of intestinal vagal afferent fibers: it acti-vates the type 1 fibers, and inhibits the type 2 fibers (Gaige et al.2002, 2003). In addition, interleukin-1� (Il-1�) activates both typesof fiber units (Gaige et al. 2004). Interactions between sulfatedcholecystokinin-8 (CCK), leptin, and Il-1� have been observed inthe intestine, which suggests that these afferent fibers play animportant role in the control of food intake (Gaige et al. 2002,2004; Peters et al. 2005, 2006a, 2006b). It has also been shown thatCCK and leptin interact to activate cultured vagal afferent neu-rons in the rat (Peters et al. 2004).

The excitatory effects of leptin on type 1 intestinal units involveIl-1�, since these effects can be blocked by interleukin-1 receptorantagonist (Il-1ra), which indicates that type 1 units potentially

Received 21 January 2013. Accepted 3 July 2013.

N. Quinson and A. Abysique. Aix Marseille Université, Physiologie et Physiopathologie du Système Nerveux Somatomoteur et Neurovégétatif (PPSN, EA4674), Avenue Escadrille NormandieNiemen, 13397 Marseille Cedex 20, France.V. Vitton and J.-C. Grimaud. AP HM, Hôpital Nord, Department of Gastroenterology, Chemin des Bourrely, 13915 Marseille Cedex 20, France; Aix Marseille Université, CNRS, Centre deRecherche en Neurobiologie et Neurophysiologie de Marseille (CRN2M, UMR7286), Boulevard Pierre Dramard, 13344 Marseille Cedex 15, France.M. Bouvier. Aix Marseille Université, CNRS, Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille (CRN2M, UMR7286), Boulevard Pierre Dramard, 13344 Marseille Cedex15, France.

Corresponding author: Anne Abysique (e-mail: anne.abysique@univ-amu.fr).

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Can. J. Physiol. Pharmacol. 91: 941–950 (2013) dx.doi.org/10.1139/cjpp-2013-0025 Published at www.nrcresearchpress.com/cjpp on 16 July 2013.

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regulate food intake under pathological conditions (Gaige et al.2002). This finding is consistent with data obtained from rats in astudy conducted by Luheshi et al. (1999), showing that the effectsof leptin on food intake and body temperature involve Il-1�. Nu-merous studies have demonstrated the existence of a correlationbetween Il-1� release and leptin levels in plasma (Grunfeld et al.1996; Barbier et al. 1998; Faggioni et al. 1998; Arnalich et al. 1999;Francis et al. 1999; La Cava and Matarese 2004; Biesiada et al. 2012).Other studies have suggested that leptin and Il-1� may coopera-tively mediate anorexia during inflammatory processes (Sarrafet al. 1997; Barbier et al. 1998; Francis et al. 1999; Luheshi et al.1999). Similar to leptin, Il-1� transmits information to the centralnervous system via at least 2 different pathways (Schwartz et al.1991; Konsman et al. 2002; Gaige et al. 2004): there is not only ahormonal pathway, but also a neural pathway, which consists ofthe vagus nerves. Therefore, it is important to investigate whetherTNF-� exerts its effects, in part, through the intestinal vagal affer-ents, and also to determine the interactions between leptin, Il-1�,TNF-�, and CCK. Thus, in this study, we investigated the effects ofTNF-� on leptin-sensitive intestinal vagal units.

Materials and methodsThe experimental procedures were performed in accordance

with the European guidelines for the care and use of laboratoryanimals (Council Directive 86/6009/EEC) and with CCAC Guide tothe Care and Use of Experimental Animals (CCAC 1993). These proce-dures received approval from the local ethics committee (ComitéRégional d'Ethique pour l'Expérimentation Animale, Provence,France). These experimental methods were similar to those de-scribed by Abysique et al. (1999) and Gaige et al. (2004). Twenty-one adult male and female European cats weighing 3.1–4.7 kg(mean: 3.6 kg) were used in these acute experiments. The animalswere fasted (water ad libitum) for 24 h prior to surgery.

AnesthesiaAnesthesia was induced by administering a gaseous mixture

(98% air; 2% isoflurane; Baxter SA, Belgium). The trachea was can-nulated, and a catheter was inserted into a radial vein, throughwhich the main anesthetic (chloralose, 80 mg·kg−1, intravenousinjection (i.v.); Sigma, Saint Quentin-Fallavier, France) was admin-istered. The anesthetic depth was monitored by checking for theabsence of the palpebral reflex and the constriction of the pupils.In addition, the blood pressure and heart rate were systematicallymonitored throughout the experiment to detect any changes inthe levels of anesthesia. The appropriate depth of anesthesiawas characterized by constricted pupils, stable blood pressure(113.5 ± 1 mm Hg, n = 21; 1 mm Hg = 133.322 Pa), heart rate (116.5 ±0.5 beats·min−1, n = 21), and by the absence of the palpebral reflex.Changes in any of these parameters indicating weakening of theanesthesia resulted in the administration of chloralose (8 mg·kg−1,i.v.). To prevent the occurrence of any movement artefacts due tothe electrical stimulation of the nerves, the animals were para-lyzed with 4 mg·kg−1 Flaxedil, i.v. (gallamine triethiodide; Sigma)and were ventilated with a respiratory pump. The cats were fixedin a Horsley–Clarke apparatus in the supine position, and theirbody temperature was maintained at 37 ± 1 °C with a heatingblanket. In each experiment, the surgical procedure began whenthe values of the parameters indicating the appropriate depth ofanesthesia were reached (after approximately 40–60 min). A sta-bilization time of 2 h allowed the time elapse between the end ofthe surgery and the start of the tests with the compounds. At theend of each experiment, the cats were euthanized with an over-dose (70 mg·kg−1, i.v.) of barbiturates (sodium thiopentone; Sanofi,Libourne, France).

Electrophysiological recording techniquesThe activity of the vagal interoceptors was routinely recorded at

the level of the right nodose ganglion (Mei 1978; Gaige et al. 2002,

2004), which was reached via a short lateral incision in the neck.The ganglion was then placed on a special metallic plate designedto prevent any artefacts from occurring as the result of the respi-ratory and carotid movements. The activity of the vagal afferentunits was recorded using extracellular glass microelectrodes filledwith 3 mol·L−1 KCl solution, as previously described (Mei 1970).Recordings obtained using glass electrodes filled with KCl(3 mol·L−1) are reportedly similar to those obtained from glasselectrodes filled with NaCl (2 mol·L−1) (Mei 1978) or with metalelectrodes (M.Bouvier, unpublished observations). This clearly in-dicates that using KCl (3 mol·L−1) would not affect the dischargepattern of vagal afferent neurons under our experimental condi-tions. The electrodes were fixed to a micromanipulator (Mei 1983)using a surgical microscope so that they could be implanted ex-actly into the gastro-intestinal region of the nodose ganglion,which is located caudally (Mei 1970; Zhuo et al. 1997). The elec-trodes were made using a vertical glass-stretching apparatus toobtain tips ranging between 1 and 3 �m in diameter with a resis-tance of 2 to 5 M�. The discharge patterns of the vagal afferentunits were displayed on an oscilloscope (type R564B; Tektronix,Beaverton, Oregon, USA). The discharge frequency of the vagalafferent fibers was calculated by data transfer using a computerconnected to a Powerlab system (Analog Digital System, France).With the amplitude discrimination (model 121; WPI, New Haven,Connecticut, USA) and shape recognition (Mac Laboratory, NewHaven, Conn.) procedures used, it was possible to select the elec-trical activity of a single afferent fiber and thus to work underunitary recording conditions. The data were presented in the formof frequency histograms showing the number of action potentialsper second (AP·s−1). To ensure that the full effects were displayed,the recordings began 2 min prior to administration of the injec-tions and lasted for 17 min.

Stimulation techniques

Electrical stimulation of the vagus nerveThe right cervical vagus nerve was dissected 1 cm along the

neck, 4–5 cm below the nodose ganglion, and was placed on stim-ulating electrodes consisting of 2 platinum wires placed in a Plexi-glass gutter. The rectangular stimulation shocks (1 ms in durationand 15–30 V in amplitude) were delivered via a Grass stimulatorconnected in series to an isolation unit. This electrical stimulationmade it possible to monitor all the responses via the recordingelectrode and to check whether the location of the electrode tipwas accurate. This set-up was also used to record the conductionvelocity (approximately 1 m·s−1) of the unmyelinated vagal fibers.

Mechanical stimulation of the mechanoreceptorsA segment of the small intestine including the duodenum and

the proximal portion of the jejunum was isolated between the2 cannulas. The anterior cannula was connected to a verticallyadjustable reservoir containing physiological saline at 37 °C, andinserted into the proximal duodenum so that various perfusionpressures could be obtained. The posterior cannula, used to emptythe intestinal loop, was placed 20 cm lower and was connected toa pressure transducer (Telco, Gentilly, France) to measure intralu-minal pressure. The isolated intestinal loop was distended bymoving the reservoir vertically. Using this experimental protocol,pressure adjustments could be made that ranged between 0 and75 mm Hg. We selectively activated the intestinal mechanorecep-tors with distensions of 15 mm Hg. Moderate digital pressure wasalso applied to locate these endings more exactly.

Chemical stimulation of the mechanoreceptorsWhen an interoceptor was mechanically identified, the intra-

arterial chemical stimuli were applied. Drugs were administeredby performing close intra-arterial injections through a catheterintroduced into the right femoral artery so that its tip was locatedat the coeliac artery, which irrigates the upper part of the small

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intestine. A post-mortem injection in the celiac artery of 1% meth-ylene blue (Sigma) was performed to confirm that the injectedsubstances were only distributed in the upper part of the smallintestine. To prevent the occurrence of any tachyphylaxic events,the time elapsed between the successive injections was set at60 min. When testing whether CCK modified the effects of TNF-�,and to ensure that the discharge frequency had returned to itsbaseline value, we chose an interval of 20 min. In our experimentsthe activating effects of CCK on the discharge frequency of theintestinal afferents lasted about 11 min, similar to what was pre-viously reported (12 min) by Schwartz et al. (1991). In addition, wehave previously reported that the excitatory effects of Il-1� onintestinal motility, which are most likely mediated via activationof the vagal mechanoreceptors, were present 20 min after pre-treatment by CCK (Gaige et al. 2003). Likewise, when testingwhether Il-1ra modified the effects of TNF-�, this interval wasreduced to 5 min, because this is when the antagonist blocks theinterleukin-1 receptors, as demonstrated for the effects of leptinand Il-1� (Gaige et al. 2002, 2003, 2004).

DrugsThe following drugs were used: TNF-� from mouse (Sigma),

monoclonal anti-TNF-� (Remicade; Schering-Plough, Kenilworth,USA), leptin from mouse (Sigma), recombinant human interleukin-1� receptor antagonist (Il-1ra; Amgen, Thousand Oaks, California,USA), CCK (Sigma), and atropine sulfate salt (Sigma). All of thesesubstances were diluted in 1 mL of physiological saline and admin-istered within 5 s. Each injection was followed by a flush of 1 mL ofsaline solution. The TNF-� and anti-TNF-� were injected at 1 �gand 250 �g, respectively. The CCK and leptin proteins were in-jected at a 10 �g dose, and all of these doses were similar to thosepreviously used by Gaige et al. (2002) to activate vagal afferentfibers. In addition, the Il-1ra and atropine were administered at a250 �g dose (Gaige et al. 2003, 2004; Bouvier and Gonella 1981).The weight of the animals was not taken into account, since thedrugs were injected by performing proximal intra-arterial injec-tions into the intestine.

Statistical analysisWhen the activity of several mechanoreceptors was recorded in

the same animal, only the first unit recorded was included in thestatistical analysis. For each injection, the mean discharge fre-quency (AP·s−1) of the afferent neurons was calculated during the40 s time period before (controls) and after the time of drug ad-ministration (effects). At each injection, we calculated the per-centage difference between the mean discharge frequencies. Theresults are expressed as the mean percentages ± SEM. Moreover,the values shown in parentheses are the mean discharge fre-quency before the injection ± SEM compared with the mean dis-charge frequency after the injection ± SEM. Statistical analysis wasperformed using an analysis of variance (ANOVA) followed by aFisher's protected least significant difference post-hoc test, whichwere also performed to compare the effects of multiple drug in-jections. The significance level was determined as P < 0.05.

ResultsTwenty-one vagal neurons of the small intestine responding to

mechanical stimulation were studied in 21 cats (one neuron percat). All of these neurons exhibited unmyelinated fibers with con-duction velocities ranging between 0.8 and 1.2 m·s−1. Their basaldischarge frequencies ranged from 0.05 to 4.65 AP·s−1 (mean value =1.57 ± 0.31 AP·s−1).

Effects of leptin on the activity of intestinalmechanoreceptors

Leptin was injected intra-arterially (i.a.) at a dose of 10 �g for21 mechanoreceptors. In 13 mechanoreceptors, leptin signifi-cantly increased (P < 0.05) the mean discharge frequency by 62 ±18% with a latency of 9.9 ± 2 s and a duration of 108 ± 24 s (Table 1;Fig. 1). In 8 mechanoreceptors, leptin significantly decreased(P < 0.05) the mean discharge frequency by 43 ± 8% with a latencyof 3.1 ± 0.9 s and a duration of 48 ± 10 s (Table 2; Fig. 1). Themechanoreceptors are referenced as type 1 units in the case ofthe 13 neurons that were activated, and as type 2 units in that ofthe 8 neurons that were inhibited.

Table 1. Effects of leptin, CCK, TNF-� (before and after CCK), TNF-� (before and after Il-1ra) and TNF-� (before and after anti-TNF-�) on thedischarge frequency of type 1 intestinal vagal mechanoreceptors.

Drugs AP·s−1 before injection AP·s−1 after injection P n Latency (s) Duration (s)

Leptin (10 �g) 1.43±0.39 1.87±0.52 <0.05 13 9.9±2 108±24CCK (10 �g) 1.48±0.46 4.34±1.37 <0.05 13 13±5 585±97TNF-� (1 �g) before CCK (10 �g) 1.27±0.39 1.88±0.49 <0.05 13 7±1 76±19TNF-� (1 �g) after CCK (10 �g) 1.10±0.36 2.36±0.71 <0.0001 13 7±1.7 116±21TNF-� (1 �g) before Il-1ra (250 �g) 0.47±0.13 0.74±0.31 <0.05 6 7.3±2 65±27TNF-� (1 �g) after Il-1ra (250 �g) 0.32±0.04 0.38±0.11 ns 6 — —TNF-� (1 �g) before anti-TNF-� (250 �g) 1.34±0.38 1.89±0.44 <0.05 13 6.9±1.1 71±13TNF-� (1 �g) after anti-TNF-� (250 �g) 1.49±0.34 1.50±0.34 ns 13 — —

Note: Values for discharge frequency (AP·s−1) are the mean ± SEM. The number of units (n) and the significance are indicated; ns, not significant; CCK, sulfatedcholecystokinin-8; TNF-�, tumour necrosis factor �; Il-1ra, interleukin-1 receptor antagonist; anti-TNF-�, antibody against TNF-�.

Fig. 1. Effects of leptin (10 �g, intra-arterial (i.a.)) on the dischargefrequency of intestinal vagal mechanoreceptors. Column C showsthe mean value of the control responses (100%). Values for theeffects of leptin are the mean compared with the control value.Error bars represent the SEM. *, P < 0.05; n (the number of units) isindicated in parentheses under the histograms.

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The activity of several mechanoreceptors was recorded in 6 cats.In 3 of these cats, either type 1 or 2 units were identified; and inthe others, both types of units were detected (not illustrated).

Effects of TNF-� on the activity of the 2 types of intestinalmechanoreceptors defined in terms of their response toleptin

TNF-� was injected, i.a., at a dose of 1 �g. The effects of TNF-� onthe 2 types of units defined in terms of their response to leptinwere analyzed. The effects of TNF-� administered to the 21 mecha-noreceptors were as follows: the 13 type-1 units were stronglyactivated (59 ± 23%; P < 0.05) (Fig. 2) with a mean latency of 7 ± 1 s

and a mean duration of 76 ± 19 s (Table 1, Fig. 4a); the discharge ofthe 8 type 2 units was not modified (Table 2; Figs. 3 and 4b).

The effects of the vehicle were investigated in the case of eachunit, and no significant changes in the spontaneous activity wererecorded after the intra-arterial administration.

In the 13 type-1 units, the excitatory responses of TNF-� (1 �g)were blocked by pre-treatment with anti-TNF-� (250 �g, i.a.)(Table 1).

In 3 type 1 units, pre-treatment of atropine (250 �g, i.a.), a mus-carinic receptor-blocking agent, administered prior to TNF-�(1 �g), did not affect the excitatory responses (Fig. 5). Sectioning of

Table 2. Effects of leptin, CCK, TNF-� (before and after CCK), and TNF-� (before and after anti-TNF-�) on the discharge frequency of type 2intestinal vagal mechanoreceptors.

Drugs AP·s−1 before injection AP·s−1 after injection P n Latency (s) Duration (s)

Leptin (10 �g) 1.81±0.51 1.21±0.41 <0.05 8 3.1±0.9 48±10CCK (10 �g) 0.79±0.28 1.74±0.56 <0.05 8 5.5±2 474±132TNF-� (1 �g) before CCK (10 �g) 1.18±0.28 1.05±0.16 ns 8 — —TNF-� (1 �g) after CCK (10 �g) 0.75±0.21 0.98±0.24 <0.05 8 6±3 84±25TNF-� (1 �g) before anti-TNF-� (250 �g) 0.93±0.60 1.29±0.55 <0.05 8 7.1±2.1 82±16TNF-� (1 �g) after anti-TNF-� (250 �g) 1.11±0.41 1.14±0.35 ns 8 — —

Note: Values for discharge frequency (AP·s−1) are the mean ± SEM. The number of units (n) and the significance are indicated; ns, not significant; CCK, sulfatedcholecystokinin-8; TNF-�, tumour necrosis factor �; anti-TNF-�, antibody against TNF-�.

Fig. 2. Effects of tumour necrosis factor � (TNF-�) (1 �g, intra-arterial (i.a.)) on type 1 intestinal vagal mechanoreceptors before and afteradministration of sulfated cholecystokinin-8 (CCK; 10 �g, i.a.). (A) Control; (B) TNF-� administered before CCK (arrow) induced activation of thereceptor; (C) administering CCK (arrow) also induced activation of the receptor; (D) administering TNF-� (arrow) 20 min after CCK inducedstrong activation of the receptor, indicating that the effects of TNF-� were enhanced by a previous injection of CCK. *, Indicates an example ofan analyzed vagal afferent unit.

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the vagus nerve caudally to the nodose ganglion abolished thespontaneous activity of the 2 types of units (type 1, n = 3; type 2, n =3), and no effects of TNF-� on the chemosensitive mechanorecep-tors were observed (n = 6) (not illustrated). The injection of TNF-�did not affect the intraluminal intestinal pressure, since no intes-tinal contraction was observed (31 ± 2 compared with 30 ±2 mm Hg, n = 21). Additionally, TNF-� did not affect the arterialpressure (108 ± 1 compared with 109 ± 1 mm Hg, n = 21) or thecardiac frequency (93 ± 1 compared with 94 ± 1 beats min−1, n = 21).

Effects of CCK on the activity of intestinalmechanoreceptors

CCK (10 �g, i.a.) increased the mean discharge frequency in all ofthe units tested (n = 21), including type 1 (n = 13) and type 2 (n = 8)units. In the type 1 units, a significant increase (P < 0.05) in themean discharge frequency (Table 1, Fig. 2) was observed, amount-ing to 223.5 ± 80%, with a latency of 13 ± 5 s and a duration of 585 ±97 s. The significant increase (P < 0.05) in the mean dischargefrequency of 136.6 ± 48% was found in the type 2 units after CCKadministration (Table 2, Fig. 3) and had a latency of 5.5 ± 2 s and aduration of 474 ± 132 s.

Effects of TNF-� on the activity of intestinalmechanoreceptors before and after the administration ofCCK

In the 13 type-1 units, TNF-� was administered in doses of 1 �g,20 min after CCK injection (10 �g, i.a.). After pre-treatment with

CCK, a significant increase in the mean discharge frequency(P < 0.0001) was observed, amounting to 115 ± 31%, with a latencyof 7 ± 1.7 s and a duration of 116 ± 21 s. The excitatory effects ofTNF-� administered after CCK injection were significantly greater(P < 0.05) than the excitatory effects of TNF-� administered beforeCCK pre-treatment (Table 1; Figs. 2 and 4a). The latencies were notsignificantly different from those obtained with TNF-� prior toCCK administration. The latencies of the effects of TNF-� did notdiffer significantly from those for the effects of CCK. However, theduration of the excitatory effects of TNF-� after CCK administra-tion was significantly greater (P < 0.05) than that of TNF-� injectedprior to CCK pre-treatment.

TNF-� (1 �g) administered 20 min after CCK injection signifi-cantly (P < 0.05) activated the 8 type-2 units (Table 2; Fig. 4b). Anexample of the effects of TNF-� on a type 2 unit after CCK pre-treatment is illustrated in Fig. 3. In addition, these excitatoryeffects of TNF-� (1 �g) were blocked by anti-TNF-� (250 �g, i.a.)pre-treatment (Table 2).

Interestingly, the injection of vehicle 20 min after CCK affectedneither type 1 (n = 3) nor type 2 units (n = 3). Two injections of 1 �gTNF-� administered within a 20 min interval induced a similarincrease in the discharge frequencies of the 2 types of units (type1 units (n = 3); type 2 units (n = 3)) (not illustrated). In 4 type-1 unitsand 3 type-2 units, the responses of TNF-� (1 �g) administered20 min after CCK, were not affected by atropine (250 �g, i.a.)pre-treatment (Figs. 5 and 6 respectively).

Fig. 3. Effects of tumour necrosis factor � (TNF-�; 1 �g, intra-arterial (i.a.)) on type 2 intestinal vagal mechanoreceptors before and afteradministration of sulfated cholecystokinin-8 (CCK; 10 �g, i.a.). (A) Control; (B) administering TNF-� before CCK (arrow) did not affect dischargeof the receptor; (C) CCK administration (arrow) induced strong activation of the receptor; (D) administering TNF-� (arrow), 20 min after CCK,induced receptor activation. *, Indicates an example of an analyzed vagal afferent unit.

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Effects of Il-1ra on the responses towards TNF-�Il-1ra, the endogenous antagonist of the Il-1� receptor, had no

significant effects on the discharge frequency of the type 1 units(n = 6) or type 2 units (n = 4).

In the 6 type 1 units tested with Il-1ra, the 1 �g TNF-� injection(alone) significantly (P < 0.05) increased the mean discharge fre-quency by 39 ± 18%, with a latency of 7.3 ± 2 s and a duration of 65 ±27 s (Table 1; Fig. 7). The same dose of TNF-� injected 5 min afterIl-1ra pre-treatment (250 �g, i.a.) had no further significant effects(Table 1; Fig. 7). In the 4 type-2 units examined, the excitatoryeffects of TNF-� injection after CCK were not modified by Il-1ra(250 �g, i.a.) pre-treatment.

DiscussionIn previous studies (Gaige et al. 2002, 2004) we showed that

leptin affected the spontaneous discharge patterns of the vagalchemosensitive mechanoreceptors in the intestine by increasingthe discharge frequency of type 1 units, and decreasing the dis-charge frequency of type 2 units. In addition, the excitatory effectsof leptin on type 1 units were blocked by Il-1ra. These resultssuggested that the excitatory effects of leptin are mediatedthrough the release of Il-1�. When leptin was administered afterCCK we noticed that its excitatory effects were enhanced and thatits inhibitory effects were blocked. In addition, we demonstratedthat Il-1� induced excitatory responses in both types of units.After pre-treatment with CCK, these excitatory effects were en-hanced in type 1 units and blocked in type 2 units.

In this study, we demonstrated that TNF-� induced excitatoryresponses in the type 1 units whereas it had no effects on the type2 units. After CCK pre-treatment, TNF-� induced an excitatoryresponse in the type 2 units and enhanced the excitatory effectson the type 1 units. Our results suggest that TNF-� may be involvedin the regulation of food intake via the intestinal vagal afferentnerve fibers during inflammation.

Effects of TNF-� on the intestinal vagal afferent nerve fibersThere is strong evidence that pathological states (nausea, eme-

sis, anorexia) are correlated with high levels of circulating TNF-�,which indicates that TNF-� may act on neuronal circuits that con-trol gastrointestinal functions (Hermann et al. 2005; Hermannand Rogers 2008). It has been demonstrated that TNF-� potenti-ates vagal afferent signaling through a ryanodine-based, calcium-induced calcium release mechanism (Rogers et al. 2006). Asdemonstrated for leptin and Il-1� in previous studies (Gaige et al.2002, 2004), the effects of peripherally generated TNF-� are notrelated to the arterial distension that can be associated with druginjection. Moreover, these effects are also not due to motor acti-vation, because the TNF-� injection did not affect the intestinalintraluminal pressure and atropine, which is generally used toinhibit motor activity, did not modify the TNF-� effects.

Our results show that injection of Il-1ra, an endogenous peptidethat is a specific Il-1� receptor antagonist, inhibits the excitatoryeffects of TNF-� on type 1 units. This finding indicates that thisdrug, like leptin (Gaige et al. 2004), does not act directly on sen-sory nerve endings but instead induces a release of Il-1�.

In this study, all of the effects of TNF-� are specific, because allof them disappear after pre-treatment with anti-TNF-�. The excit-atory effects of TNF-� on the intestinal vagal afferent fibers areconsistent with the numerous studies showing that peripheralproinflammatory cytokine signaling is transmitted via a vagalpathway to the brain. For example, peripheral proinflammatorycytokine signaling serves to depress food-motivated behavior(Bret-Dibat et al. 1995), induce fever (Watkins et al. 1995b; Gordon2000; Hansen et al. 2001) and hyperalgesia (Watkins et al. 1994),and increase the extracellular noradrenaline concentration in theparaventricular nucleus in the hypothalamus (Fleshner et al. 1995;Hansen and Krueger 1997; Ishizuka et al. 1997). Peripheral proin-flammatory cytokine signaling also increases plasmatic ACTH and

Fig. 4. Statistical analysis of the effects of tumour necrosis factor � (TNF-�, 1 �g, intra-arterial (i.a.)) on the discharge frequency of intestinalvagal mechanoreceptors, before and after administering sulfated cholecystokinin-8 (CCK) (10 �g, i.a.). (A) Type 1 intestinal vagalmechanoreceptors; (B) type 2 intestinal vagal mechanoreceptors. Column C shows the mean value of the control responses (100%). Values forthe effects of TNF-� are the mean compared with the control value. The vertical bars at the top of the columns represent the SEM. *, P < 0.05;**, P < 0.0001; ns, not significant; §, P < 0.05 from Fisher's PLSD post-hoc test refers to the comparison between the effects of TNF-� before andafter administering CCK. The number of units (n) is indicated under the columns.

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corticosterone; this effect is abolished by subdiaphagmatic vagot-omy (Wieczorek et al. 2005).

Interactions between TNF-� and CCKIn the type 1 units, the excitatory effects of TNF-� were signifi-

cantly enhanced by CCK. In the type 2 units, the excitatory effectsof TNF-� were induced by CCK. These results could not have beendue to a tachyphylactic disorder, since 2 injections of TNF-�, given20 min apart, had resulted in identical effects on both types ofintestinal vagal afferent fibers.

In addition, the excitatory effects of TNF-� administration aloneon type 1 units, and after CCK on the type 1 and type 2 units,demonstrated similar latencies, which indicates that similar acti-vation mechanisms are involved. In addition, these effects wereperipheral processes since they persisted under atropine-treatedconditions.

It is well-known that CCK has excitatory effects on the basalactivity of vagal afferent fibers (Mei et al. 1996; van de Wall et al.2005). The enhancement of the TNF-� effects observed after CCKpre-treatment to type 1 units could not have been due to CCK'sown effects. Nor was it due to motor activation by CCK, which hadrecovered to basal level before the TNF-� injection was performed.

In addition, the mechanism by which CCK enhances the effects ofTNF-� could not have been due to the effects of CCK on the vagalafferent discharge frequency because TNF-� was injected 20 minafter CCK, the effects of which lasted for approximately 8 min.The discharge frequency of the mechanoreceptors was thereforesimilar before the injection of TNF-� (alone) and prior to the in-jection of TNF-� given 20 min after CCK pre-treatment. Changes inthe effects of either leptin or Il-1� were able to activate vagalmechanoreceptors in response to CCK, which have also been de-scribed in the intestine (Gaige et al. 2003) and the stomach (Wanget al. 2000). In the stomach, these changes involved a mode ofinteraction similar with those for TNF-� in the type 2 units. Thesemechanoreceptors that were not initially affected by leptin be-came leptin sensitive.

Interactions between Il-1� and TNF-�TNF-� induces an excitatory response, which is enhanced by

CCK in type 1 units. Il-1ra pre-treatment blocks these excitatoryeffects and provides evidence for the existence of a functionalrelationship between TNF-� and Il-1�. It has been shown a corre-lation between both TNF-� and IL-� secretion during local mucosalinflammation demonstrating an important role of proinflammatory

Fig. 5. Effects of tumour necrosis factor � (TNF-�, 1 �g, intra-arterial (i.a.)) on the discharge frequency of the type 1 intestinal vagalmechanoreceptors, before and after administration of atropine (250 �g, i.a.). (A) Control; (B) TNF-� administration (arrow) induced activationof the receptor; (C) prior administration of atropine had no effect on the activation induced by TNF-�; (D) prior administration of atropine hadno effect on the strong activation induced by TNF-� after pre-treatment with sulfated cholecystokinin-8 (CCK) (10 �g, i.a.). *, Indicates anexample of an analyzed vagal afferent unit.

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cytokines in the pathogenesis of IBD (Reinecker et al. 1993;Dionne et al. 2003). TNF-� may act on the vagal afferent terminalsdirectly on specific receptors (Hughes et al. 2012) or by a signaltransduction cascade inducing Il-1� release (Watkins et al. 1995a).This last mechanism is consistent with the observation that otherbrain-mediated consequences of intraperitoneal injection ofTNF-�, such as hyperalgesia (Watkins et al. 1995a) and plasmacorticosterone elevation (Fleshner et al. 1998), can be blocked orattenuated by the intraperitoneal administration of Il-1ra. Theseobservations support the idea that TNF-� signals to the brain, atleast in part, via an Il-1� receptor-dependent signaling pathway.

The excitatory effects of TNF-� on type 2 units after CCK pre-treatment were not due to the release of Il-1�, since Il-1ra failed tomodify this response. Therefore, it is possible that TNF-�, as dem-onstrated for leptin in some cases, requires the presence of CCK(Wang et al. 2000; Lewin and Bado 2001).

The results of these experiments support the idea that vagalafferent fibers provide an important pathway of brain signaling inthe presence of peripheral immune substances.

Because type 1 and type 2 units are CCK and leptin sensitive,they may be involved in the control of food intake. They are alsoIl-1� (Gaige et al. 2003) and TNF-� sensitive and might thereforealso be involved in the transmission of information regardingperipheral inflammation to the CNS. So these units may generatean acute satiety signal under physiological conditions but mayalso control food intake under pathological conditions (i.e., dur-ing inflammation). Nevertheless we cannot exclude the implica-tion of the vago–vagal anti-inflammatory reflex that represents anadditional regulatory system controlling the intestinal inflamma-tory response. Indeed recent studies have indicated that the vagusnerve negatively regulates immune responses (Tracey 2002; Ulloa2005; Matteoli and Boeckxstaens 2013). Efferent signals from the

Fig. 6. Effects of tumour necrosis factor � (TNF-�, 1 �g, intra-arterial (i.a.)) on the discharge frequency of the type 2 intestinal vagalmechanoreceptors, before and after administration of atropine (250 �g, i.a.). (A) Control; (B) administering TNF-� (arrow) after pre-treatmentwith sulfated cholecystokinin-8 (CCK, 10 �g, i.a.) induced activation of the receptor; (C) prior administration of atropine had no effect on theexcitatory responses induced by TNF-� after pre-treatment with CCK. *, Indicates an example of an analyzed vagal afferent unit.

Fig. 7. Effects of tumour necrosis factor � (TNF-�, 1 �g, intra-arterial(i.a.)) on the discharge frequency of the type 1 intestinal vagalmechanoreceptors, before and after administration of Il-1ra (250 �g,i.a.). Column C shows the mean value of the control responses (100%).Values for the effects of TNF-� are the mean compared with the controlvalue. Error bars represent the SEM. *, P < 0.05; ns, not significant. Thenumber of units (n) is indicated under the histograms.

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vagus nerve inhibit cytokine production via pathways dependenton nicotinic AchR on immune cells such as macrophages (Wanget al. 2003; de Jonge et al. 2005; Tracey 2007).

Our results demonstrate that the activation of vagal afferentpathway by proinflammatory cytokines such as TNF-� and Il-1�may be involved in the short-term control of food intake. Thisactivation may also induce a vago–vagal anti-inflammatory reflexthat reduces intestinal inflammation and improve pathologicalstatus.

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