Mucosal inflammation is accompanied by an alteration in 5-HT. Intestinal 5-HT synthesis is catalyzed by tryptophan hydroxylase 1 (Tph1) and we have shown that mice deficient in this rate-limiting enzyme have reduced severity of intestinal inflammation in models of chemical-induced experimental colitis. Here, we investigated the effect of blocking peripheral 5-HT synthesis in generation of intestinal inflammation by a using peripheral Tph inhibitor, telotristat etiprate (LX1606), in models of intestinal inflammation. LX1606 was given orally either prophylactically or therapeutically to mice with dextran sulfate sodium (DSS)-induced colitis or with infection with Trichuris muris. Severity of intestinal inflammation was measured by assessment of disease activity scores, histological damage, and MPO and inflammatory cytokine levels. LX1606 significantly reduced intestinal 5-HT levels and delayed onset and severity of DSS-induced acute and chronic colitis. This was associated with decreased MPO and proinflammatory cytokine levels compared with vehicle-treated controls. In the infection-induced inflammation model, treatment with LX1606 enhanced worm expulsion as well as increased IL-10 production and goblet cell numbers. LX1606-treated mice had significantly lower MPO and IL-1β levels compared with controls postinfection. Our results demonstrate that peripheral 5-HT plays an important role in intestinal inflammation and in the generation of immune responses. Pharmacological reduction of peripheral 5-HT may serve as a potential strategy for modulating various intestinal inflammatory disorders.
- enterochromaffin cells
- experimental colitis
- tryptophan hydroxylase
- inflammatory bowel disease
the gastrointestinal (GI) tract is the largest endocrine organ in the body and is made up of an extensive system of endocrine cells (56). Enterochromaffin (EC) cells are the best characterized subset of enteric endocrine cells and synthesize the vast majority of the body's serotonin (5-hydroxytryptamine; 5-HT) (35, 61). Once synthesized, 5-HT is packaged into granules and stored near the basal border of the EC cell, though some studies have also identified granules near the apical membrane (1, 20). In response to various mechanical and chemical stimuli, including bacterial toxins, 5-HT is released from granules in a calcium-dependent manner (55, 61). 5-HT released from EC cells goes into the surrounding tissue and gut lumen, where it plays important roles in GI motor and secretory function or can be transported into surrounding epithelial cells by the 5-HT reuptake transporter (SERT) and degraded by monoamine oxidase A (21, 26).
EC cells synthesize 5-HT from its precursor, l-tryptophan. Tryptophan hydroxylase (Tph) catalyzes the rate-limiting step of this reaction and has been detected prominently in EC cells (19). It is now established that there are two known isoforms of this rate-limiting enzyme. EC cells contain one form of this enzyme, Tph1, mainly present in the gut and spleen, whereas the second isoform, Tph2, is mainly expressed in the brain stem and enteric neurons (67, 68). Thus 5-HT is synthesized independently in EC cells and neurons by two different rate-limiting Tph isoenzymes. Alterations in 5-HT signaling have been observed in various GI disorders, such as IBD (2, 6, 10, 15, 46), functional disorders such as irritable bowel syndrome (IBS) (5, 10, 13, 32, 51), and various enteric infections (4, 39, 64, 65, 71). Several studies have shown that 5-HT plays a key role in the generation of intestinal inflammation. Mice that are deficient in the rate-limiting enzyme of 5-HT biosynthesis in EC cells (Tph1−/−) exhibit reduced severity of colitis in two different models of experimental colitis [induced by dextran sulfate sodium (DSS) and dinitrobenzene sulfonic acid], whereas replenishing 5-HT levels in these animals upregulates colitis severity (24). In turn, studies have also shown that chemical-induced colitis or spontaneous colitis associated with an IL-10 deficiency is increased in severity when coupled with the 5-HT enhancing effects of a knockout of the 5-HT reuptake transporter, SERT (7, 29).
The aforementioned studies illustrate that reduction in 5-HT synthesis may alleviate intestinal inflammation in experimental models of IBD. Prior approaches aimed at blocking 5-HT synthesis by a pharmacological agent through inhibition of Tph, as with parachlorophenylalanine, have been impeded by adverse effects to brain 5-HT synthesis leading to alterations in central nervous system-mediated functions (58). Telotristat etiprate (LX1032/LX1606) is an orally delivered, small-molecule Tph inhibitor that reduces peripheral 5-HT synthesis. LX1606 was designed not to penetrate the blood-brain barrier, in part as a result of its molecular size. Here, we investigated the effect of LX1606 in the generation of intestinal inflammation using both the DSS-induced model of acute and chronic colitis and the Trichuris muris infection-induced inflammation model with a view to validate peripheral Tph as a target for modulating gut inflammation.
MATERIALS AND METHODS
C57BL/6 mice (Taconic, Cambridge City, IN) were kept in sterilized, filter-topped cages under specific pathogen-free conditions and fed autoclaved food. All mice were male aged 8–10 wk. All experiments were approved by the animal ethics committee of McMaster University and conducted under the Canadian guidelines for animal research.
Telotristat etiprate (LX1032/LX1606 hippuric acid) was supplied by Lexicon Pharmaceuticals (The Woodlands, TX). The compound was formulated in 0.25% wt/vol methylcellulose (Sigma, Mississauga, ON, Canada) and administered to mice once daily via oral gavage at 300 mg/kg. Preparations of formulations of LX1606 were corrected for salt content (salt correction factor 1.31).
Induction of DSS-induced colitis and treatment with telotristat etiprate (LX1032/LX1606 hippuric acid).
Acute colitis was induced by giving 5% DSS (wt/vol) (molecular mass 40 kDa; ICN Biomedicals, Solon, OH) in drinking water for 5 days. Mean DSS consumption was noted per cage each day. Chronic DSS-induced colitis was induced by a previously published method (37). LX1606 was administered prophylactically and therapeutically. In the prophylactic treatment protocol, mice received vehicle or LX1606 at a dosage of 300 mg/kg body wt orally every day starting 1 day prior to administration of 5% DSS and were euthanized on day 5 post-DSS to assess disease severity, MPO activity, and inflammatory cytokines. In the therapeutic treatment protocol, mice received vehicle or LX1606 every day starting 48 h after DSS administration. For chronic colitis, mice were treated with vehicle or LX1606 (300 mg/kg body wt per day) for 6 days starting 1 day before the beginning of the 2nd and 3rd DSS cycle and were euthanized on the last day of the 3rd DSS cycle (therapeutic strategy). All control mice received vehicle and water without DSS.
Assessment of colitis severity.
Disease activity index (DAI) is a combined score of weight loss, stool consistency, and fecal bleeding and was blindly assessed by using a previously published scoring system (23). This scoring system was defined as weight loss: 0, no loss; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, 20%+; stool: 0, normal; 2, loose stool; 4, diarrhea; and bleeding: 0, no blood; 2, Hemoccult positive (Hemoccult II, Beckman Coulter, Fullerton, CA); and 4, gross blood (blood around anus). DAI was measured on all 5 days of DSS treatment. To assess macroscopic damage, mice were euthanized 5 days post-DSS or at the end of the last cycle of water for chronic DSS-induced colitis. Upon euthanasia, their abdominal cavities were opened, and observations on colonic distension, fluid content, hyperemia, and erythema were recorded. The colon was removed and macroscopic damage was immediately assessed on the full section of the colon. Macroscopic scores were blindly assessed by using a previously described scoring system for DSS colitis (11, 23). To assess for histological damage, formalin-fixed colon segments were paraffin embedded and 3-μm sections were stained with hematoxylin and eosin (H&E). Colonic damage was blindly scored using a previously published scoring system that considers changes in crypt architecture, cellular infiltration, goblet cell depletion, and crypt abscess (11, 22).
Determination of colonic 5-HT content.
Colon samples were weighed and longitudinal length was recorded. Samples were homogenized as previously described (52). 5-HT content was analyzed by enzyme immunoassay using a commercially available kit (Beckman Coulter, Fullerton, CA) and expressed as a function of wet weight (nanogram per milligram tissue).
Measurement of colonic MPO activity and cytokine levels.
Colonic MPO activity and cytokine levels were measured according to a published protocol (38). In brief, for MPO measurement, colonic tissue samples were homogenized in ice-cold 50 mmol/l potassium phosphate buffer containing 0.5% hexadecyl trimethyl ammonium bromide (pH = 6.0) (Sigma). Homogenates were centrifuged and the supernatant was removed, and an aliquot was then added to a solution containing potassium phosphate buffer, O-dianisidine, and hydrogen peroxide (Sigma-Aldrich). The absorbance was measured at 450 nm by a spectrophotometer (model EL808, BioTek). MPO activity was expressed in units per milligram of colon tissue, where one unit is defined as the quantity of enzyme able to convert 1 μmol of hydrogen peroxide in water per minute at room temperature. Cytokine levels from colon lysates were determined by using commercially available ELISA kits according to the manufacturer's instructions (Quantikine Murine; R&D Systems, Minneapolis, MN).
Trichuris muris parasites were harvested and ova collected and maintained as previously described (66). All infected mice received ∼300 T. muris ova in distilled water (200 μl) by oral gavage. Mice received vehicle or LX1606 at a dose level of 300 mg/kg body wt per day, orally starting 3 days prior to infection, and were euthanized on various days postinfection (PI) to examine changes in inflammatory and immune responses; worm burden was assessed by counting the number of worms present in the cecum.
Histological analysis and immunohistochemistry.
A segment of the proximal colon of infected animals was fixed in 10% neutral buffered formalin and stained with periodic acid-Schiff (PAS) stain to detect intestinal goblet cells. For immunohistochemistry, formalin-fixed, paraffin-embedded colonic segments were sectioned to 5 μm in thickness, deparaffinized by heating at 60°C for 30 min, cleared with CitriSolv (Fisher Scientific, ON, Canada), and rehydrated in a graded ethanol series of decreasing ethanol concentrationss. Sections were subjected to heat-induced epitope retrieval (10 mM sodium citrate buffer-0.05% Tween 20, pH 6.0), blocked with 2% goat serum in PBS containing 1% BSA-0.1% Triton X-100-0.05% Tween-20-0.05% sodium azide, and incubated with a polyclonal antibody raised against Muc2 (2.67 μg/ml; sc-15334; Santa Cruz Biotechnology) overnight at 4°C. Sections were washed with PBS-0.5% Tween-20 and incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (2 μg/ml; no. A-11037; Molecular Probes/Invitrogen). Sections were mounted with ProLong Gold antifade reagent containing 4′,6′-diamidino-2-phenylindole (DAPI) (Molecular Probes/Invitrogen). Images were captured by using a Nikon Eclipse 80i through NIS-Elements Basic Research imaging software. Fluorescence filters used were blue (DAPI, Hoechst), excitation filter 340–380 nm, emission filter 435–485 nm; red (TRITC, RFP), excitation filter 540–580 nm, emission filter 600–660 nm. The number of PAS- and Muc2-positive goblet cells was quantified by analyzing at least 40 crypts from each individual colon cross section and was expressed per 10 villus crypt units. Investigators were blinded to the study groups.
Colons isolated from the mice were homogenized in Tris-buffered saline containing protease inhibitor solution (P8340, Sigma). The total protein concentration in each solution of homogenized tissue was determined using the Bio-Rad DC Protein Assay Kit (cat. no. 500-0116, Bio-Rad). Prior to loading onto SDS-PAGE gels, the samples were diluted 1:1 (vol/vol) with 2× Laemmli sample buffer (Bio-Rad) without reducing agent and heated for 5 min at 100°C with a dry block heater. Equal amounts (15 μg) of protein homogenates from each group were loaded and electrophoresed onto 7.5% SDS-PAGE and transferred to a PVDF membrane at 400-mA constant current for 4 h at 4°C. Membranes were blocked with 5% milk blocking buffer (5% wt/vol nonfat milk in 1× wash buffer containing 50 mM Tris-base, 150 mM NaCl, 1% Tween-20, 1% Triton X-100) overnight at 4°C and then incubated with primary antibodies against: Muc2 (0.2 μg/ml) (sc-15334; Santa Cruz Biotechnology) or Muc5ac (1:1000) (M5293; Sigma) for 2 h at room temperature. Membranes were washed with 1× wash buffer, incubated with either anti-rabbit horseradish peroxidase (HRP)-linked antibody (1:5,000, no. 7074, Cell Signaling Technology) or anti-mouse HRP-linked antibody (0.08 μg/ml, sc-2318, Santa Cruz Biotechnology) for 1 h. Proteins were visualized by use of Clarity Western ECL Blotting Substrate (Bio-Rad). β-Actin was used as a loading control. Densitometric analysis was performed on Western blots with ImageJ software (version 1.48), normalized to total actin.
All data are presented as means ± standard error of the mean. Unpaired t-test or one-way ANOVA followed by Tukey multiple-comparisons post hoc test or Mann-Whitney U-test was performed with GraphPad Prism version 6.0b for Mac OS X (GraphPad Software, La Jolla, CA). An associated P value <0.05 was considered statistically significant.
Peripheral Tph inhibitor, LX1606, significantly reduces intestinal 5-HT levels.
To verify that oral administration of LX1606 was able to deplete intestinal 5-HT, colonic and jejunum 5-HT levels were assessed from mice on day 5 post-DSS-induced colitis. Administration of LX1606 significantly decreased 5-HT concentrations in both the colon and jejunum compared with vehicle-treated controls (Fig. 1, A and B, respectively).
Prophylactic and therapeutic treatment with LX1606 delays onset and decreases severity of DSS-induced colitis.
We investigated the effect of LX1606 in an acute model of DSS-induced experimental colitis using a prophylactic treatment strategy (Fig. 2A). Treatment of colitic mice with LX1606 beginning 1 day prior to DSS administration resulted in significantly lower clinical disease activity scores (based on weight loss, fecal blood, and stool consistency) (Fig. 2B). We also observed significantly lower macroscopic scores (based on fecal blood and consistency, rectal bleeding, and erythema) in LX1606-treated mice compared with vehicle-treated mice on day 5 post-DSS-induced colitis (Fig. 2C). H&E-stained colon cross sections of mice that received DSS and were treated with vehicle displayed distortion of epithelial cell architecture, increased leukocyte infiltration, loss of goblet cells, and significant thickening of the muscularis mucosa layer. In mice that received LX1606, histological damage scores were significantly less severe compared with control mice on day 5 post-DSS induction (Fig. 2D). Decrease in colitis severity was associated with significantly lower colonic MPO activity and lower levels of proinflammatory cytokines (IL-1β, IL-6) (Fig. 2, E–G).
We also evaluated the effect of LX1606 on colitis severity using a therapeutic treatment strategy. Here, LX1606 was administered orally on day 2 after DSS administration (Fig. 3A). LX1606 treatment resulted in significantly reduced whole colon tissue 5-HT levels (Fig. 3B), and a modest decrease in total disease activity scores (Fig. 3C). Treatment with LX1606 also resulted in a modest decrease in macroscopic damage scores (Fig. 3D) and less severe histological damage (Fig. 3E). This was associated with lower MPO activity and colonic IL-1β and IL-6 levels (Fig. 3, F and G). In turn, mice treated with vehicle had significantly higher macroscopic and histological damage on day 5 post-DSS, including crypt distortion, inflammatory cell infiltration, and muscle thickening (Fig. 3E).
Therapeutic treatment with LX1606 decreases disease severity in a chronic DSS-induced colitis model.
To investigate the effect of LX1606 treatment in a chronic model of colitis, mice were administered three cycles of DSS solution and treated with LX1606 for 6 days starting 1 day prior to the beginning of the 2nd and 3rd DSS cycles (Fig. 4A). Treatment with LX1606 significantly reduced whole colon tissue 5-HT content compared with vehicle-treated controls (Fig. 4B). Following treatment with LX1606, there was attenuation of disease severity, indicated by lower disease activity scores during the periods of DSS administration (Table 1). Mice were euthanized on day 37 following the last day of the 3rd cycle of DSS administration and colons were excised to assess for macroscopic damage. There was reduced macroscopic damage in LX1606-treated groups compared with controls on day 37 post-DSS administration (Fig. 4C). DSS-induced colitis is accompanied by a shortening of overall colon length. The colon length of mice treated with LX1606 was significantly increased compared with vehicle-treated mice post-DSS (Fig. 4D). There was also less severe histological damage (less distortion of epithelial cell structure, decreased inflammatory cell infiltrate, less significant thickening of the muscularis mucosa) in LX1606-treated mice compared with vehicle-treated controls (Fig. 4E). Delay in disease onset and disease severity in LX1606-treated mice was associated with lower MPO activity and reduced IL-1β levels (Fig. 4F).
LX1606 administration decreases inflammatory response postinfection with nematode T. muris.
T. muris is a natural nematode parasite of mice that resides in the cecum and colon. Different strains of mice display varying resistance to infection with T. muris whereby resistant strains mount a protective Th2 response and susceptible strains mount a Th1 response and cannot expel the worm (16). During infection, the large intestine becomes inflamed as macrophages and other leukocytes accumulate within the tissue (45). This study evaluated the effect of oral administration of peripheral Tph inhibitor in reducing colonic inflammation in relation to reduction of gut 5-HT content. There was no difference in weight loss between LX1606 and vehicle-treated groups with or without infection (data not shown). We assessed the effect of LX1606 on inflammation by measuring MPO activity and production of cytokines. We observed significantly reduced colonic MPO activity in infected mice treated with LX1606 compared with vehicle-treated controls (Fig. 5A). Investigations on cytokines in colonic tissues revealed lower levels of proinflammatory cytokines, IL-1β and IL-17, and higher levels of immunoregulatory cytokine, IL-10, on day 21 PI (Fig. 5, B–D). There was no significant difference in the levels of IL-13, IL-33, and thymic stromal lymphopoietin (TSLP) between the LX1606-treated mice compared with vehicle treated after infection (Table 2).
LX1606 administration promotes worm expulsion and upregulates goblet cell numbers.
Goblet cells reside throughout the GI tract and are the main source of mucins in the gut (34). Parasitic infections, including T. muris, are associated with changes in mucus composition and stimulation of mucus production by goblet cell hyperplasia and increased mucin expression (28, 31, 33, 50). Changes to mucus secretion and mucin expression are correlated to parasite expulsion. Assessment of worm burden in the large intestine of LX1606-treated mice showed acceleration in worm expulsion compared with vehicle-treated controls on day 14 PI (Fig. 5E). To determine whether this was due to enhanced mucus secretion, we used PAS staining to stain goblet cell mucins on formalin-fixed colon sections from uninfected and T. muris-infected mice at days 14 and 21 PI. We observed no changes in PAS staining in colon sections of samples taken from uninfected mice (Fig. 5F). A significant increase in PAS staining was observed, however, in tissue sections of samples taken at days 14 and 21 PI (Fig. 5F). Muc5ac is a mucin that is induced by T. muris infection and plays an important role in the expulsion of these parasites (27). We assessed Muc5ac expression to see whether LX1606-treatment postinfection induced any changes compared with vehicle-treated controls. As assessed by Western blot, we observed a relative increase in Muc5ac on days 14 and 21 PI compared with uninfected mice. We did not observe, however, any changes in Muc5ac levels between LX1606- and vehicle-treated mice on either day (Fig. 5G). Muc2 is a major secreted mucin within the colon and has previously been shown to be correlated with worm expulsion and protective against enteric infections (28). Using Western blot and immunostaining, we assessed changes in Muc2 expression. Muc2 expression is significantly increased in infected mice compared with uninfected mice on days 14 and 21 PI. On both days, there was an increase in Muc2 expression in LX1606-treated groups compared with vehicle-treated control groups (Fig. 5, H–J).
In this study, we tested the hypothesis that an orally delivered Tph inhibitor would confer protection against both chemical- and infection-induced inflammation. The selectivity of LX1606 for inhibition of Tph1 in the GI tract is based on the distribution of the compound and its failure to cross the blood-brain barrier (which is, in part, a result of its molecular size). The inability of the compound to cross the blood-brain barrier and affect central 5-HT stores is important because deletion of Tph2 in the brain is associated with alterations in behavior (59). In turn, deletion of intestinal Tph2 in the enteric nervous system is followed by significant defects in GI motility as well as abnormal development of enteric neurons (43).
A recently published study by Margolis et al. (48) showed oral administration of LX1606 (AKA LX1032) significantly depleted intestinal 5-HT levels compared with vehicle-treated controls. LX1606 administration also induced a significant decrease in blood 5-HT concentration but not in brain 5-HT level. Margolis et al. also evaluated the effect of LX1606 (LX1032) on neuronal 5-HT stores using immunocytochemical techniques and found that LX1606 did not affect the proportion of myenteric 5-HT-immunoreactive neurons or the area of myenteric plexus occupied by 5-HT-immunoreactive nerve fibers. This suggests that, although LX1606 significantly depletes 5-HT stores from EC cells, entire neuronal 5-HT stores are maintained and, therefore, LX1606 and similarly related peripheral Tph inhibitors appear to fail to enter the myenteric plexus and/or inhibit enteric neuronal Tph2. In addition, the safety and efficacy of LX1606 have been recently evaluated in patients with diarrhea associated with carcinoid syndrome (40, 54). Despite the differences in treatment periods (4 wk vs. 12 wk, respectively), both studies demonstrate that patients that receive LX1606 treatment have improvements in bowel movement frequency and reduction in 5-HT production, as measured by a reduction in the metabolic breakdown product of 5-HT, urinary 5-hydroxyindoleacetic acid (40, 54). These studies suggest the benefit of telotristat etiprate/LX1606 in intestinal conditions such as carcinoid syndrome in addition to colitis. In our study, we provide further evidence of a beneficial role for these small molecule inhibitors in experimental models of intestinal inflammation.
We evaluated the effect of LX1606 using both a prophylactic and therapeutic treatment strategy in DSS-induced colitis. Some of the pertinent features of the DSS model of colitis may include weight loss, blood in stools, diarrhea, and anemia (14, 18, 53, 63). Upon euthanasia, mice that have been administered this DSS regimen have shortened colons and feces that are soft and poorly formed. Microscopically, colonic tissue from DSS-treated mice is characterized by inflammatory cell infiltration of the mucosa/submucosa, distortion of crypt architecture, goblet cell depletion, and thickening of the muscle layers (11). Administration of LX1606 significantly depleted colon and jejunum 5-HT content (which includes both EC cell and neural contributions) and reduced the severity of DSS-induced acute colitis albeit to a lesser degree when administered therapeutically. Disease activity scores were significantly lower in mice treated with LX1606 compared with vehicle-treated controls. LX1606-treated mice had significantly lower macroscopic and histopathological damage scores. This was associated with a decrease in MPO activity (an enzyme released by granulocytes such as neutrophils, serving as a surrogate marker of inflammation) (62), and a decrease in the levels of proinflammatory cytokines IL-1β and IL-6. IL-1β is primarily secreted by monocytes and macrophages upon activation and is upregulated in the inflamed colon of patients with IBD and DSS-induced colitis (44, 53). Secreted IL-1β binds to and signals through the IL-1 receptor to activate the NF-κB signal-transduction pathway, resulting in the upregulation of other proinflammatory mediators, including IL-6 (41, 57). These results corroborate with a recent study that evaluated LX1606 (in addition to a related peripheral Tph inhibitor, LP-920540) in a tri-nitrobenzene sulfonic acid (TNBS)-induced colitis model (48). Administration of LX1606 significantly ameliorated the severity of TNBS-induced colitis and reduced expression of proinflammatory gene expression of IL-6 and IL-1β. We also tested the effect of LX1606 using a therapeutic strategy in a chronic model of DSS-induced colitis whereby mice receive repeated cycles of DSS and water. Mice that received LX1606 and were euthanized 1 day after the last day of DSS administration had significantly reduced colon 5-HT levels and improvements in disease activity scores (an additive score considering weight loss, stool blood, and stool consistency) throughout the DSS cycles in the chronic colitis model. However, when mice were euthanized 11 days after the last day of DSS administration we did not observe a difference in jejunum or colon 5-HT levels in LX1606-treated mice (data not shown). This is consistent with previous observation that 5-HT levels return to baseline after discontinuation of drug treatment, asserting that continuous treatment is necessary to maintain the reduction of 5-HT levels.
We also evaluated the effect of LX1606 in an infection-induced model of inflammation using the nematode T. muris. This parasite is very similar to the human parasite Trichuris trichura and is a commonly used murine model of intestinal nematode infection-induced inflammation. Infection with embryonated T. muris eggs is strain specific whereby strains resistant to chronic infection (such as C57BL/6) expel the parasites beginning on day 14 PI and are able to fully clear the parasite by day 35 PI through the generation of the Th2 type immune response. Susceptible strains (such as AKR) generate Th1 immune response, fail to expel worms, and develop chronic infection (12, 52, 69). Once infected, these eggs reach the cecum, hatch (in the cecum and proximal colon), and embed in the mucosal epithelium (9, 25). Once in their intraepithelial niche, the worms fully mature and affect intraepithelial cell homeostasis, and endocrine cell hyperplasia, resulting in the accumulation of inflammatory cells such as macrophages within the tissue (8, 16, 17, 45). In this study, we treated resistant (C57BL/6) mice with either LX1606 or vehicle once daily starting 3 days prior to infection to assess the effect of LX1606 in infection-induced inflammatory responses. Administration of LX1606 reduced intestinal inflammation in the colon of infected mice as indicated by a reduction in proinflammatory markers MPO, IL-1β, and IL-17. IL-17 has been implicated in the pathogenesis of many inflammatory conditions and IL-17 levels and genes in the Th17 pathway have been shown to be upregulated following nematode infection (42, 70). Treatment with LX1606 also increased immunoregulatory IL-10 production in intestinal tissues in infected mice compared with that in vehicle-treated mice. Therefore, it seems very likely that the treatment with LX1606 modulated inflammation by upregulation of IL-10 production. However, the treatment may have an additional indirect effect on inflammation by reducing worm burden. These findings demonstrate that LX1606 is able to reduce severity not only in chemical-induced colitis but also in enteric parasite-induced inflammation. The intestinal mucus serves as a significant component of the host response to nematode infection. Mucins act as the main structural component of the intestinal mucus layer and are secreted mainly by goblet cells that are dispersed throughout the epithelial layer (36). Changes in goblet cell response and mucin production are observed in many intestinal infections, including T. muris, and play an important role in worm expulsion (28, 49). Previously, we demonstrated that an increase in the major intestinal mucin, Muc2, correlates with worm expulsion following T muris. infection and there is a significant delay in early-stage worm expulsion in its absence (28). Interestingly, an increase in Muc5ac, a mucin normally expressed in the airways and stomach, is observed in the colon following infection and has since been shown to play a critical role in the expulsion of T. muris, in addition to affecting worm viability (27, 28). In addition to reducing intestinal inflammation in infected mice, we wanted to investigate the effect of LX1606 treatment on host defense in enteric infection. Previously, our laboratory has shown that infection of resistant C57BL/6 mice with T. muris upregulates the number of EC cells and 5-HT content in the colon (47, 52, 69). Treatment with LX1606 promoted worm expulsion on day 14 PI and resulted in an increase in PAS-positive goblet cell numbers. There was an increase in Muc2 and Muc5ac expression postinfection compared with uninfected mice. As assessed by immunoblotting, we observed no discernible difference in Muc5ac expression between LX1606 and vehicle-treated controls. Interestingly, we did, however, observe an increase in Muc2 in LX1606-treated mice on days 14 and 21 PI. These results suggest that LX1606 treatment leads to an increase in the expression of Muc2 glycoprotein in infected wild-type mice, perhaps as a host defense mechanism. These findings suggest that although Muc2 and Muc5ac may act in concert to promote worm expulsion, upregulation of Muc2 by LX1606 was sufficient to promote worm expulsion in this infection despite no changes in Muc5ac expression. To understand the mechanism by which LX1606 enhanced goblet cell responses, we investigated other cytokines (IL-10, IL-13, IL-33, TSLP) which are shown to be upregulated in T. muris infection (3, 30, 60) and observed a significant increase in IL-10 production after LX1606 treatment on day 21 PI. In our previous studies, we have shown that IL-10 plays an important role in upregulation of intestinal goblet cells numbers (49). These findings suggest that the increase in goblet cells and Muc2 expression that we observed by LX1606 treatment may be in part mediated by IL-10 and warrants further investigation into the direct mechanisms underlying this observation.
Small molecule Tph inhibitors, such as LX1606, represent a novel class of drugs that has the potential to improve inflammatory conditions associated with increased production of 5-HT. The present observations in this study further confirm the ability of selective pharmacological reduction of peripheral (EC-derived) 5-HT to reduce the severity of intestinal inflammation. Importantly, LX1606 is selective to the periphery and thus central effects are avoided. This class of compounds has potential not only in IBD, but also in other intestinal inflammatory conditions associated with increase in 5-HT, such as IBS and carcinoid diarrhea.
This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and Crohn's and Colitis Canada (to W. I. Khan), by funding from Lexicon Pharmaceuticals, Inc., and by an Ontario Graduate Scholarship (to J. J. Kim). W. I. Khan is a recipient of CIHR New Investigator Award.
B. Zambrowicz and Q. M. Yang are employees of Lexicon Pharmaceuticals, Inc.
J.J.K., H.W., and J.D.T. performed experiments; J.J.K., H.W., and J.D.T. analyzed data; J.J.K. and W.I.K. interpreted results of experiments; J.J.K. prepared figures; J.J.K. drafted manuscript; J.J.K., H.W., J.D.T., B.Z., Q.M.Y., and W.I.K. approved final version of manuscript; B.Z., Q.M.Y., and W.I.K. conception and design of research; B.Z., Q.M.Y., and W.I.K. edited and revised manuscript.
The authors thank Dr. Richard Grencis (University of Manchester, Manchester, UK) for providing T. muris and Ivana Sunjic for technical support.
↵* J. J. Kim and H. Wang are both first authors.
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