Role of neurokinin 1 receptors in dextran sulfate ... - Springer Link

Inflamm. Res. (2014) 63:399–409 DOI 10.1007/s00011-014-0712-x

Inflammation Research

ORIGINAL RESEARCH PAPER

Role of neurokinin 1 receptors in dextran sulfate-induced colitis: studies with gene-deleted mice and the selective receptor antagonist netupitant ´ gnes Keme´ny • Istva´n Szitter • Erika Pinte´r • Aniko´ Perkecz • A • • • Jo´zsef Kun La´szlo´ Kereskai Claudio Pietra John P. Quinn • Andreas Zimmer • Alexandra Berger • Christopher J. Paige • Zsuzsanna Helyes

Received: 22 July 2013 / Revised: 12 January 2014 / Accepted: 15 January 2014 / Published online: 28 January 2014 Ó Springer Basel 2014

Abstract Objective and design The function of the neurokinin 1 (NK1) receptor was investigated in the DSS-induced mouse colitis model using NK1 receptor-deficient mice and the selective antagonist netupitant. Subjects Colitis was induced by oral administration of 20 mg/ml DSS solution for 7 days in C57BL/6 and Tacr1 KO animals (n = 5–7). Treatment During the induction, one-half of the C57BL/6 and Tacr1 KO group received one daily dose of 6 mg/kg netupitant, administered intraperitoneally, the other half of the group received saline, respectively. Methods Disease activity index (DAI), on the basis of stool consistency, blood and weight loss, was determined over 7 days. Histological evaluation, myeloperoxidase (MPO) measurement, cytokine concentrations and receptor expression analysis were performed on the colon samples.

Results NK1 receptors are up-regulated in the colon in response to DSS treatment. DSS increased DAI, histopathological scores, BLC, sICAM-1, IFN-c, IL-16 and JE in wildtype mice, which were significantly reduced in NK1 receptor-deficient ones. NK1 receptor antagonism with netupitant significantly diminished DAI, inflammatory histopathological alterations, BLC, IFN-c, IL-13 and IL-16 in wildtype mice, but not in the NK1-deficient ones. MPO was similarly elevated and netupitant significantly decreased its activity in both groups. Conclusions NK1 receptor antagonism could be beneficial for colitis via inhibiting different inflammatory mechanisms. Keywords Neurogenic inflammation  Tachykinins  Inflammatory bowel disease  Edema  NK1 receptor antagonist

Responsible Editor: Ian Ahnfelt-Rønne. ´ . Keme´ny  J. Kun  I. Szitter  E. Pinte´r  A. Perkecz  A Z. Helyes (&) Department of Pharmacology and Pharmacotherapy, University of Pe´cs, Pe´cs, Hungary e-mail: [email protected] ´ . Keme´ny  J. Kun  I. Szitter  E. Pinte´r  A. Perkecz  A Z. Helyes Ja´nos Szenta´gothai Research Centre, Pe´cs, Hungary E. Pinte´r  Z. Helyes PharmInVivo Ltd., Pe´cs, Hungary L. Kereskai Department of Pathology, University of Pe´cs, Pe´cs, Hungary

J. P. Quinn Department of Molecular and Clinical Pharmacology, Institute of Translation, University of Liverpool, Liverpool University, Liverpool, UK A. Zimmer Institute of Molecular Psychiatry, University of Bonn, Bonn, Germany A. Berger  C. J. Paige Ontario Cancer Institute, University Health Network, Toronto, Canada A. Berger  C. J. Paige Department of Immunology, University of Toronto, Toronto, Canada

C. Pietra Helsinn Healthcare SA, Preclinical R&D, Pambio-Noranco, Lugano, Switzerland

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Introduction Adequate pharmacological treatment of inflammatory bowel diseases is an emerging problem. Current treatment has problematic side effects. The latest improvements in the field of inflammation research resulted in specific antibodies against pro-inflammatory mediators (anti-TNFa antibody infliximab), but the details of regulator mechanisms of the pro- and anti-inflammatory processes in the gut are not fully elucidated. Previous studies have shown that neuropeptides released from the primary sensory neurons are involved the inflammatory bowel diseases [1, 2]. Inflammatory tachykinins, such as Substance P (SP) and other neurokinins, play a pivotal role in these processes [3]. The tachykinins are often considered a link for cross talk between the nervous and the immune systems and participate in the neuro-immune interactions [4, 5]. The tachykinins play a pivotal role in the development of neurogenic inflammation, which is characterized by vasodilatation, plasma extravasation, edema, leukocyte activation and migration. In 2000, the family of tachykinins was expanded by the discovery of hemokinins encoded by the Tac4 gene [4]. There are four products of this gene in humans called endokinins (EKA, B, C, D) produced by alternative splicing. In mice, there is only one variant called hemokinin-1 (HK-1). These new members of the tachykinins and their suggested receptor conformers combined with the tissue specific posttranslational modifications result in a finely controlled tachykinin system involving multiple cell types and pathways, as reviewed here [6, 7]. SP changes in the inflamed human gut are contradictory: SP expression is up-regulated in ulcerative colitis, but not in Crohn’s disease [8]. Michalski et al. [9] showed that the expression of the SP-encoding gene was increased in Crohn’s disease. Renzi and co-workers [10] found that SP peptide concentration is decreased in ulcerative colitis. NK1 receptors were shown to be up-regulated in inflammatory bowel diseases [11]. On the basis of these results, the antagonism of the NK1 receptor was investigated in many experiments. Cutrufo and co-workers [12] found that nepadutant has a protective effect in acute colitis in guinea pigs induced by diluted acetic acid. Other NK1 receptor antagonists, CP-96345 and RP67580, significantly reduced the inflammation and oxidative stress in dextran sulfateinduced colitis in rats [13]. Similar results were observed in a hapten-induced (dinitrofluorobenzene, DNFB) colitis model in mice [14], and also the NK1 receptor antagonist SR 140333 was proved to be protective in 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis in rats [15]. NK1 and NK2 receptor antagonism had protective effects in TNBS-induced colitis in rats, while NK2 and NK3 receptor antagonism reduced the inflammatory changes in

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TNBS-induced ileitis in guinea pigs [16]. Furthermore, a centrally (intracerebroventricularly) administered peptidelike NK1 and NK3 receptor agonist exerted a pro-inflammatory role in experimentally induced colitis in the rat [17]. Novel finding that Substance P released from TRPV1 expressing neurons is an aggravating factor in ulcerative colitis [18]. There are two major colitis models in rodents. Dextran sulfate sodium (DSS)-induced colitis was described first [19]. The trinitrobenzene sulfonic acid (TNBS)-induced colitis model was introduced later [20]. Both are wellcharacterized mechanisms [21–23]. DSS-induced murine colitis resembles human ulcerative colitis, while TNBSinduced inflammation, which is a hapten-evoked process, is considered to be a Crohn’s disease model [24]. The aim of the present study was to investigate the expression and inflammation-induced alterations of tachykinin NK1, NK2 and NK3 receptors in the mouse colon, as well as to analyze the role of the NK1 receptor in DSSinduced colitis using gene-deleted mice and the selective antagonist netupitant [25]. An integrative approach was used with functional, histopathological, biochemical and immunological techniques.

Materials and methods Animals Experiments were performed on male NK1 receptor (Tacr1 KO) gene-deficient mice backcrossed for 8–10 generations to C57BL/6 mice. C57BL/6 mice were used as wildtype (WT) controls and the original breeding pairs were purchased from Charles River Ltd. (Hungary). Tacr1 KO mice were generated at the University of Liverpool as previously described [26–29]. The animals were bred and kept in the Laboratory Animal House of the Department of Pharmacology and Pharmacotherapy of the University of Pe´cs at 24–25 °C, provided with standard mouse chow and water ad libitum, and maintained under a 12-h light–dark cycle. Mice were 8–10 weeks old and weighed 22–25 g. Induction of colitis Colitis was induced with 20 mg/ml DSS (MP Biochemical) dissolved in the drinking water and administered for 7 days. The intact control group of animals received only tap water. Then mice were killed in deep ketamine-xylazine anaesthesia (100 mg/kg ketamine i.p.; Richter Gedeon Ltd, Budapest, 5 mg/kg xylazine i.p.; Eurovet Animal Health BV, the Netherlands) after fasting overnight. The distal colon samples (one-third of the colon from anus to cecum) were dissected to determine receptor expression,

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myeloperoxidase enzyme activity, cytokine measurement and histopathological evaluation [30]. Treatment protocol The selective synthetic NK1 receptor antagonist netupitant (Helsinn SA, Lugano, Switzerland) was administered i.p. (6 mg/kg, 100 ll from the 0.6 mg/ml solution) once a day from the beginning of the experiment for a total duration of 1 week. The dosage, administration route and frequency were chosen on the basis of a previous study, where 6 mg/ kg provided adequate NK1 receptor blockade [25]. Mice in the control group of the DSS treatment received the vehicle in the same volume according to the same treatment paradigm. A group of receptor-deficient mice also received the netupitant treatment to identifying any effect caused by netupitant beside Tacr1 receptor blockade. Netupitant stock solution was prepared according to the instruction of the manufacturer. Briefly, 100 mg was dissolved in 2 ml propylene glycol, 100 ll, 1 mol/dm3 hydrochloric acid was added which yielded a 50 mg/ml stock solution. Stock solution was stored in 2 °C. This stock solution was diluted in saline to prepare the final 0.6 mg/ml solution freshly every day. Disease activity index (DAI) assessment The clinical symptoms of colitis, such as body weight change, stool consistency and fecal blood content, were scored on a daily basis. Fecal blood content was assessed with the Hemocare test which uses a modified guaiac method (Care diagnostica, Austria). The detailed scoring system is shown in Table 1. Scores for the three parameters were averaged for each mouse to obtain the disease activity index [30, 31].

Receptor expression Colon samples were stored in RNAlater (Sigma, USA) and homogenized in 1 ml of TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). Isolation of total RNA was carried out according to the manufacturer’s protocol. The quantity and purity of the extracted RNA was assessed on a NanoDrop ND-1000 Spectrophotometer V3.5 (NanoDrop Technologies, Inc., Wilmington, DE, USA). From total RNA, 0.5 lg was reverse-transcribed into cDNA using the Quick Protocol of RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas/Thermo Fisher Scientific, Waltham, MA, USA) with oligo(dT) primers. The obtained cDNA samples were amplified with PCR using specific primers. b-actin served as the reference housekeeping gene. PCR products were run on agarose gels containing DNA specific dye and visualized under UV light. Histological evaluation The distal colon samples were fixed in 40 mg/ml buffered formaldehyde, embedded in paraffin, sectioned (5 lm), and stained with haematoxylin and eosin. Digital micrographs were taken by an Olympus BX51 microscope and Olympus DP50 camera. Inflammatory alterations were evaluated and scored by an expert pathologist blinded from the experimental design. A detailed description of the parameters is given in Table 2 [30, 31]. Additionally, quantitative assessment was also performed to evaluate the histopathological severity of inflammation: the number of inflamed foci was counted and percentage of the damaged area was calculated on the digital micrographs (400 9 magnifications, analySIS image analyzer software, Germany).

Table 1 Disease activity index scoring chart Score

0

1

2

3

4

Body weight loss

0–0.9 %

1–5 %

5.1–10 %

10.1–20 %

[20.1 %

Stool consistency

Normal

Normal/soft

Soft

Soft/watery

Watery

Fecal blood content (Hemocare test)

Negative

Light blue stains

Blue stain

Bloody patches/blue stain

Gross bleeding

Table 2 Histopathological semiquantitative scoring chart (severity of inflammation and extent of inflammation does not have grade 4 for the purpose of simplifying the ranges of categories) Score

0

1

2

3

4

Severity of inflammation

Normal

Mild

Extent of inflammation

Not inflamed

Just mucosa

Moderate

Severe

–

Submucosa

Whole gut wall

Damage to crypts

None

One-third of basal

Two-thirds of basal

Crypts disappeared

– Mucosa disappeared

Percent of damaged area

0%

1–25 %

26–50 %

51–75 %

76–100 %

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MPO measurement

Statistics

Colon samples were snap-frozen immediately after removal in liquid nitrogen and stored at -80 °C. For MPO measurement, the frozen samples were weighed and placed into 1 mL phosphate buffer containing 5 mg/ ml hexadecyltrimethyl ammonium bromide (HTAB). Homogenization was performed at 35,000 rpm for 1 min on ice, and then centrifuged at 4,000 rpm in a cooled centrifuge for 10 min; supernatants were collected for MPO assay and stored at -20 °C. The spectrophotometric MPO assay was performed from the thawed samples on a 96-well plate. Briefly, 25 ll assay buffer, 25 ll samples were pipetted in triplicates onto the plate, then 100 ll H2O2-3,30 ,5,50 -tetramethyl-benzidine (TMB/H2O2; Sigma-Aldrich Ltd, Budapest) was pipetted into each well. Absorbances were read two times: first immediately after the TMB solution was added and second time after 5 min (kinetic reaction) at 620 nm wavelength on a Multiskan plate reader (Labsystems, Sweden). MPO activity was calculated from the absorbance changes with a calibration curve. MPO activities were expressed in unit/ mg wet tissue.

Results are expressed as mean ± SEM. For the evaluation of the data, two-way ANOVA (DAI) followed by Bonferroni’s post-test, unpaired t test with Welch correction (MPO activity, cytokine profile, quantitative histopathological data) or Mann–Whitney U test (semiquantitative histopathological scores) was used. Statistical analysis was done using GraphPad Prism 5.02 for Windows (GraphPad Software, USA). Probability values \0.05 were accepted as significant. Ethical considerations All experimental procedures were carried out according to the 1998/XXVIII Act of the Hungarian Parliament on Animal Protection and Consideration Decree of Scientific Procedures of Animal Experiments (243/1988) and complied with the recommendations of the International Association for the Study of Pain and the Helsinki Declaration. The studies were approved by the Ethics Committee on Animal Research of University of Pe´cs according to the Ethical Codex of Animal Experiments, and license was given (license number: BA 02/2000-2/2012).

Cytokine panel assay Results The cytokine assay was performed according to the manufacturer’s instructions [R&D Systems Mouse Cytokine Array Panel A (ARY006)]. Briefly, the excised and frozen tissues were thawed and weighed, and immediately placed in PBS containing 10 mg/ml phenylmethylsulfonyl fluoride (PMSF) protease inhibitor, and homogenized as described above. Then Triton X-100 was added to the samples to a final concentration of 10 mg/ml and centrifuged at 10,000 g for 5 min to remove cell debris. Total protein concentrations were determined prior to cytokine measurement (BioRad DC protein assay). Captured antibodies of selected cytokines were spotted in duplicate on nitrocellulose membranes. The unknown samples were diluted as required according to their total protein content and mixed with a cocktail of biotinylated detection antibodies and incubated on the membranes overnight. All cytokines which were present in the tissue homogenates formed immune complexes with their specific detection antibodies and bound to the immobilized capture antibodies on the membrane surface. Following a washing step to remove the unbound materials, chemiluminescent detection was performed. The intensity of the emitted light at each spot was proportional to the amount of bound cytokine. Results were calculated by densitometry using ImageJ freeware.

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Receptor expression The receptor expression study revealed that the Tac1 gene (encoding Substance P and NKA) was expressed in the distal colon both in the intact control and DSS-treated mice. We could not detect the Tac3 and Tac4 genes of NKB and HK-1, respectively. Inflammation did not alter this expression pattern. The Tacr1 gene expressing the NK1 tachykinin receptor was significantly up-regulated after 7 days of oral DSS administration. There was no expression of the Tacr2 and Tacr3 genes of the NK2 and NK3 tachykinin receptors in the intact colon, but they were moderately or minimally up-regulated in response to DSS administration (Fig. 1). Disease activity index DAI was calculated daily on the basis of body weight, stool consistency and fecal blood content, which was significantly reduced from day 6 both by genetic deletion and selective antagonism of the NK1 tachykinin receptor with 6 mg/kg i.p. netupitant administration (Fig. 2). Netupitanttreated NK1 receptor-deficient mice showed similar disease activity index compared to vehicle-treated receptordeficient animals.

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Fig. 2 Disease activity index (DAI) in mice. DAI was calculated daily on the basis of body weight, stool consistency and fecal blood content (see Table 1). Mice were drinking 20 mg/ml dextran sulfate (DSS) for 7 days. The selective NK1 tachykinin receptor antagonist netupitant was injected once a day (6 mg/kg i.p.) in wildtype (WT) mice, solvent-treated animals served as controls. Mice lacking the NK1 tachykinin receptors (Tacr1 KO) were also investigated for comparison. Scores of water consuming animals are not shown as those were practically zero. Data points represent mean ± SEM. Two-way ANOVA with Bonferroni‘s post-test (n = 5–7 per group, *p \ 0.05 **p \ 0.01, ****p \ 0.0001 vs. DSS WT veh) Fig. 1 Representative RT-PCR of genes of tachykinins and their receptors. The Tac1 gene (encoding Substance P and NKA), but not the Tac3 and Tac4 genes, is expressed in the distal colon both in the intact control (C1, C2, C3) and DSS-treated (DSS1, DSS2, DSS3, DSS4) mice. Meanwhile the Tacr1 gene expressing the NK1 tachykinin receptor is up-regulated after 7 days of oral DSS treatment. There is no expression of the Tacr2 and Tacr3 genes of the NK2 and NK3 tachykinin receptors in the intact colon but they are moderately up-regulated in response to DSS administration. b-actin served as the reference housekeeping gene, ntc represents no template controls as negative controls, pos shows the positive controls

Histological analysis Compared to a histopathological picture of the noninflamed colon structure showing intact crypts and normal mucosal epithelial layer (Fig. 3a, b), drinking 20 mg/ml DSS resulted in remarkable inflammation and tissue damage in the C57BL/6 wildtype group without netupitant treatment. There was a significant mucosal and submucosal neutrophil infiltration, loss of crypts and disintegration of the mucosal structure (Fig. 3c). The severity and the extent of these characteristic histopathological alterations were reduced by both NK1 receptor deletion (Fig. 3d) and daily administration of the NK1 receptor antagonist netupitant (Fig. 3e). Meanwhile, in the Tacr1 KO animals netupitant treatment did not influence the severity of inflammation compared to the vehicle-treated group (Fig. 3f). These findings were confirmed by the significantly diminished semiquantitative score values determined in the NK1 receptor-deficient and netupitant-treated groups in

comparison with the solvent-treated wildtype animals. The quantitative results describing the number of inflamed foci and the percentage values of the damaged areas also showed an anti-inflammatory effect of netupitant (Fig. 4). Myeloperoxidase enzyme activity assay DSS administration induced an approximately twofold increase in MPO activity of the colon homogenates compared to the respective intact samples of waterdrinking wildtype and NK1 receptor-deficient mice. This elevation was significantly decreased by 6 mg/kg i.p. daily netupitant treatment. Surprisingly, netupitant decreased the MPO activity in DSS-treated NK1 receptor-deficient mice as well compared to their vehicle-treated controls, similar to what was observed in wildtypes (Fig. 5). Murine inflammation cytokine array Among the 40 investigated cytokines, 11 (BLC, sICAM1, IFN-c, IL-1a,IL-1ra, IL-13, IL-16, IP-10, JE, MIG, and TIMP-1) were significantly increased in the colon homogenates in response to DSS administration in wildtype mice compared to their intact controls. In contrast, in the NK1 receptor-deficient group, BLC, sICAM-1, IFN-c, IL-1a, IL-16, JE did not increase significantly while IL-1ra, IP-10, MIG and TIMP were

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Fig. 3 a–f Representative light micrographs of distal colon samples at 9200 magnification, haematoxylin-eosin staining. Arrows show a intact crypts, b mucosal neutrophil infiltration, c submucosal neutrophil infiltration. Asterisk shows the colonic lumen. After 7 days of 2 % dextran sulfate administration, remarkable tissue damage can

be observed in the wildtype mice. These histopathological changes were significantly reduced in the NK1 receptor-deficient animals. Netupitant treatment (6 mg/kg i.p. once a day) also decreased the mucosal damage in wildtype mice

similarly elevated in wildtype animals. There was no difference between the expressions of these cytokines in the intact colon samples of the two groups. Netupitant treatment significantly decreased the DSS-induced elevation of BLC, IFN-c, IL-13 and IL-16 levels compared to vehicle administration. These changes were similar to the results found in NK1 receptor-deficient mice (Fig. 6).

Discussion

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Tachykinins play a pivotal role in several neurogenic inflammatory processes in the gut, with special emphasis on SP released from the capsaicin-sensitive sensory nerves. SP, the first member of the tachykinin system, was discovered in 1931, when it was found to induce peripheral vasodilation and stimulated intestinal muscle contraction

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Fig. 4 Semiquantitative and quantitative histological evaluation. Semiquantitative scores of the histopathological changes in the distal colon samples on the basis of severity of inflammation, extent of inflammation, damage to crypts and percent of damaged areas (see Table 2). Bars represent the minimum to maximum values. Mann–

Whitney U test (n = 5–7 per group, *p \ 0.05, **p \ 0.01 vs. watertreated, #p \ 0.05, ##p \ 0.01 vs. WT DSS). Quantitative results were obtained by the image analysis of the corresponding micrographs (n = 5–7 per group, *p \ 0.05, ***p \ 0.001 vs. water WT veh, # p \ 0.05, ##p \ 0.05 vs DSS veh)

[32]. In the 1980s, further members of the mammalian tachykinin family were discovered [33–36], and parallel to these mediators, the receptors were also described and characterized [37–39]. Selective antagonists, such as osanetant and aprepitant, were developed and investigated in different experimental models as well as clinical studies

[40, 41]. SP has a broad range of effects on the gastrointestinal tract including actions on the motility and inflammatory processes [42]. There are several lines of evidence about their role in maintaining gastrointestinal motility [43]. In vitro studies showed that NK1 and NK2 receptors maintain intestinal peristalsis when cholinergic

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Fig. 5 Myeloperoxidase (MPO) activity measured in distal colon homogenates. MPO content of the samples is increased after DSS administration in wildtype mice and NK1 receptor-deficient mice. Netupitant treatment (6 mg/kg) significantly decreased the MPO level compared to non-treated wildtype animals. Columns represent the mean ± SEM. Unpaired t test with Welch correction (n = 5–7 per group, *p \ 0.05, **p \ 0.01 vs. water, ##p \ 0.0001 vs. DSSveh)

transmission via nicotinic receptors is blocked [44]. Later it was found that the controlling mechanism is more complex, as NK1 receptor activation can trigger myogenic excitatory motor response in addition to the neurogenic inhibitory motor response in guinea pig small intestine [45]. There are two possible ways to modify pathophysiological pathways: either the transmitter synthesis is upregulated or the (tissue specific) expression of a receptor increased [11, 46]. Therefore, we initially investigated the dynamics of the transmitters and their receptors in intact and inflamed colon samples obtained from mice treated with 2 % DSS, which represents a well-established mouse colitis model. Our results revealed that among the measured genes only the Tac1 gene (encoding SP and NKA) is expressed in the intact and also the inflamed colon with no significant quantitative difference. Regarding the receptors, only the NK1 tachykinin receptor (Tacr1) is present in the intact colon, and it is up-regulated in response to the inflammation. Although the other two tachykinin receptors are not detected in the intact colon, they show a minimal expression in the inflamed tissue. Based on these findings, the role of the NK1 receptor was examined with genedeleted mice and also with a selective NK1 receptor antagonist netupitant in the DSS-induced colitis model. SP preferentially binds to NK1 receptors, NKA to NK2 and NKB to NK3, but all tachykinins can bind to all receptors with much lower affinities [47]. Moreover, the tachykinin system turned out to be much more complex than might be expected based on: (1) Radiolabelled NKA showed good binding capability to NK1 receptors in the guinea pig airways, where both NK1 and NK2 receptors are present [48]. (2) While SP and NKA are extensively expressed, NK2 receptors are not as abundant [49]. (3) Septide, which is a SP analogue peptide, evoked strong

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NK1-like responses, while it did not displace SP from NK1 receptor binding suggesting different binding sites [50, 51]. There might be distinct binding sites for the different tachykinins on the NK1 receptor, but the NK1 receptor might exist in two active conformational forms. A general tachykinin-binding conformer is likely to be able to bind most tachykinins while a SP-preferring conformer might be selective for SP [51]. Unfortunately, breakthroughs in clinical trials have not yet been achieved with NK1 receptor antagonists despite huge efforts, which might be explained by suspected receptor conformers or multiple binding sites [52]. However, aprepitant is used in therapy against chemotherapy-induced nausea and vomiting and a netupitant–palonosetron combination is in clinical phase III for the same indication. Both the genetic deletion and pharmacological blockade of the NK1 receptor similarly and significantly decreased the disease activity index, the histopathological changes, some important inflammatory cytokine production and MPO activity in our colitis model. The inhibitory effects of netupitant on the overall histopathological and clinical severity of the inflammatory reactions proved to be selectively mediated by blocking the NK1 receptor, but surprisingly, the MPO activity decreasing action was independent of this receptor antagonism. Although we do not have a precise explanation for this latter finding, since MPO concentration in the colon homogenate was also significantly decreased by netupitant in Tacr1 gene-deleted mice, another, presumably nonreceptor mediated mechanism can be suggested for this compound. There are previous studies about the temporal and spatial analysis of cytokine profiles in DSS-induced colitis but they measured the mRNA levels of certain cytokines with real-time polymerase chain reaction (PCR) and not the translated proteins themselves. Egger et al. [23] had found that IL-12, IFN-c, IL-1, TNF-a and IL-10 are elevated. Yan et al. [22] discovered that also IL-6, MIP-2 and KC are increased and the peak levels of mRNAs were between day 3 and day 9 of DSS treatment. One focus of the present study was to determine cytokine levels in colon samples of the DSS-induced colitis model. The analysis clearly showed which pro-inflammatory cytokines and chemokines increase in response to the inflammation and which of these are influenced by NK1 receptor activation. Significant elevation of BLC, sICAM1, IFN-c, IL-1ra, IL-1a, IL-13, IL-16, JE and MIG were detected in colitis in wildtype mice. The cytokine profile suggest that DSS colitis is mainly influenced by T-cells, but other immune cell types (such as macrophages) are also involved [21]. NK1 receptors are located in enteric neurons, interstitial cells of Cajal, intestinal muscle, epithelium and granulocytes [53]. Since the absence of NK1 receptors prevents the elevation of BLC, sICAM-1, IFN-c, IL-16 and JE, we can conclude that expression of these cytokines is

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Fig. 6 Local cytokine profile in distal colon homogenates. Arrows indicate the cytokines which are not increased upon 20 mg/ml dextran sulfate (DSS) treatment in the NK1 receptor-deficient animals compared to wildtype animals. Columns represent mean ± SEM, two-way ANOVA with Bonferroni’s post-test (n = 5–7, *p \ 0.05, **p \ 0.01, ****p \ 0.0001 vs. water, #p \ 0.05, ##p \ 0.01,

####

related to NK1 receptor activation. It seems that NK1 receptors mainly mediate pro-inflammatory signals via these cytokines, as BLC is responsible for attracting B-cells, sICAM-1 plays a role in the adherence and transmigration of immune cells, IFN-c and IL-16 stimulate each other’s secretion by CD8 ? (cytotoxic T cells) and CD4 ? (helper T cells), which leads to general chemotaxis of macrophages and Th1 differentiation. Induction of JE completes these processes by attracting monocytes, activated T-cells, basophil granulocytes, natural killer cells and naı¨ve dendritic cells. Netupitant decreased the DSSinduced elevation of BLC, IFN-c, IL-13 and IL-16 levels, which is likely to explain, at least partially, the potent antiinflammatory action of this NK1 receptor antagonist.

Since complex sensory-immune interactions are involved in the development of colitis including a network of cytokines, sensory neuropeptides released from peptidergic capsaicin-sensitive afferents, as well as inflammatory cells, the NK1 receptor can be considered as an important component of this inflammatory mechanism. On the basis of the present results we can conclude that NK1 receptor antagonism could be a promising pharmacological approach in the treatment of inflammatory bowel diseases.

p \ 0.0001 vs. DSS WT veh). BLC B lymphocyte chemoattractant, sICAM-1 soluble intracellular adhesion molecule 1, IFN-c interferon gamma, IP-10 interferon gamma-induced protein 10, JE monocyte chemotactic protein 1, MIG monokine induced by gamma interferon, TIMP-1 tissue inhibitor of metallopeptidase 1)

Acknowledgments This work was supported by SROP-4.2.2.A-11/ 1/KONV-2012-0024, SROP-4.2.1.B-10/2/KONV-2010-0002, SROP4.2.2.B-10/1/2010-0029. Alexandra Berger and Christopher J. Paige were supported by Terry Fox Program Project Grant (National Cancer

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408 Institute of Canada #015005), Canadian Institute of Health Research (#9862).

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