Background/aim: 5-Hydroxytryptamine (5-HT) released from enterochromaffin cells influences intestinal homeostasis by altering gut physiology and is implicated in the pathophysiology of various gut disorders. The mechanisms regulating 5-HT production in the gut remain unclear. This study investigated the T helper (Th) 1/Th2-based immunoregulation of enterochromaffin cell function and 5-HT production in a model of enteric infection.
Methods and results: Trichuris muris-infected AKR (susceptible to infection and generates Th1 response), BALB/c (resistant to infection and generates Th2 response), Stat4-deficient (impaired in Th1 response) and Stat6-deficient (impaired in Th2 response) mice were investigated to assess enterochromaffin cells, 5-HT and cytokines. In association with the generation of a Th2 response we observed higher enterochromaffin cell numbers and 5-HT content in the colon of BALB/c mice compared with AKR mice. Numbers of enterochromaffin cells and amount of 5-HT were significantly lower in Stat6-deficient mice after infection compared with Stat4-deficient mice. In addition, enterochromaffin cell numbers and 5-HT content were significantly higher after reconstitution of severe combined immunodeficient mice with in-vitro polarised Th2 cells.
Conclusion: The study demonstrated that enterochromaffin cell and 5-HT responses to the same infectious agent are influenced by Th1 or Th2 cytokine predominance and suggests that the immunological profile of the inflammatory response is important in the regulation of enterochromaffin cell biology in the gut. In addition to new data on enterochromaffin cell function in enteric infection and inflammation, this study provides important information on the immuno–endocrine axis in the gut, which may ultimately lead to improved strategies against gut disorders.
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Enteric endocrine cells are dispersed throughout the gastrointestinal (GI) mucosa. The best characterised subset of enteric endocrine cells is the enterochromaffin cell, which is the main source of the biogenic amine serotonin (5-hydroxytryptamine; 5-HT) in the GI tract.1 5-HT influences intestinal homeostasis by altering gut physiology (motility and secretory function) and has been implicated in the pathophysiology of various GI disorders, which include inflammatory bowel disease and functional disorders such as irritable bowel syndrome (IBS).2–4 In addition, alteration in enterochromaffin cells is also observed in a number of bacterial, viral and parasitic infections of the GI tract.5–7 5-HT is released from enterochromaffin cells into the blood, into the surrounding tissue and into the gut lumen in a regulated and calcium-dependent manner in response to various mechanical and chemical stimuli including bacterial toxins and participates in gut physiology.8 The association between alteration in enterochromaffin cells/5-HT production and various GI diseases greatly emphasises the significance of 5-HT in intestinal homeostasis. The precise mechanisms regulating the alteration of enterochromaffin cell function and 5-HT production in the gut during infection and inflammation remain to be determined.
Considering the strategic location of enterochromaffin cells in the GI mucosa, changes in 5-HT signalling and secretory motor function in enteric infection and inflammation are likely to be modulated by the immune system, and we have recently shown evidence that CD4 T cells play an important role in the development of enterochromaffin cell hyperplasia and in the upregulation of 5-HT production in the colon in intestinal nematode infection.9 There is also evidence of low-grade inflammation and/or immune activation (an increase in CD3-positive T cells) in patients with postinfectious IBS in whom increased numbers of enterochromaffin cells have been reported10–12 and this is reflected in an animal model of postinfectious IBS.13 14 It is therefore very likely that immune activation may increase enterochromaffin cell numbers in the clinically relevant context of functional bowel disease such as IBS. The reduced number of enterochromaffin cells in the colon of mice with targeted disruption of IL215 and T-cell receptor alpha16 and the presence of enterochromaffin cells in contact with, or in very close proximity to, lymphocytes17 further support the presence of an immunological control of enterochromaffin cell function and 5-HT production in the GI tract.
The interaction between each cytokine and its receptor leads to activation of signalling molecules including signal transducer and activator of transcription (Stat) proteins.18 Stat6 is activated by both IL4 and IL13 and is essential in T helper (Th) 2 development, whereas Stat4 is activated by IL12 and is important for a Th1 type response. In our previous studies using infection with the small intestinal nematode Trichinella spiralis, we have demonstrated that Th2 cytokines, IL4 and IL13, acting via Stat6, play an essential role in the alteration of intestinal physiology and host defence in this infection.19 We have also shown that a shift to a Th1 response by gene transfer and overexpression of IL12 (a critical cytokine in the development of Th1 immune response) significantly altered intestinal physiology and inhibited host protective immunity in this Th2-biased enteric infection.20 These observations, along with our recent findings on CD4 T-cell-regulated control of 5-HT production in the gut,9 show an immunological basis for an alteration of gut physiology and prompted further investigations on the precise mechanisms of alteration in enterochromaffin cell function and 5-HT production in the gut.
In a colonic parasitic infection with Trichuris muris, resistant strains (BALB/c, C57BL/6, NIH) expel the parasites through the generation of a Th2 response, whereas susceptible strains (AKR, B10.BR) develop a chronic infection with activation of a Th1 response.21 T muris is thus the only nematode infection that shows a clear dichotomy in strain-dependent T helper responses after infection and allows the analysis of host responses to the same infectious agent in different cytokine environments. Using a T muris model in this study, we investigated how the Th1 versus Th2 immune response to enteric infection influences changes in the function of enterochromaffin cells and 5-HT production in relation to host defence.
MATERIALS AND METHODS
Male BALB/c and AKR mice were obtained from Harlan (Indianapolis, Indiana, USA) and C57BL/6 and severe combined immunodeficient (SCID; C57BL/6 background) mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). Stat4-deficient (Stat4–/–) and Stat6-deficient (Stat6–/–) mice on C57BL/6 background were generated as described previously.22 23 Breeding pairs of Stat6−/− and Stat4−/− mice and their wild-type littermates were obtained from the John Curtin School of Medical Research, Australian National University, Canberra, Australia and Indiana University School of Medicine, Indianapolis, Indiana, USA, respectively. All mice were kept in sterilised, filter-topped cages and fed autoclaved food in the animal facilities of McMaster University. Only 8–10-week-old male mice were used. The protocols employed were in direct accordance with guidelines drafted by the McMaster University Animal Care Committee and the Canadian Council on the Use of Laboratory Animals.
Immunohistochemical studies on 5-HT-expressing enterochromaffin cells and CD3-positive cells were performed on formalin-fixed, paraffin-embedded samples. Sections were deparaffinised in CitriSolv (Fisher Scientific, Ontario, Canada) and rehydrated through a graded series of ethanol and phosphate-buffered saline (PBS). Endogenous peroxide was blocked by incubation in peroxidase-blocking reagent (DakoCytomation, Ontario, Canada) for 15 minutes. After washing, sections were subjected to antigen retrieval in citrate buffer after heating in a microwave or were predigested with proteinase K solution (DakoCytomation) for 15 minutes. After blocking of non-specific binding with 1% bovine serum albumin in PBS, the sections were incubated with rabbit anti-5-HT antibody (ImmunoStar, Inc, Hudson, Wisconsin, USA; 1 : 5000 for 1 h at room temperature) or with polyclonal rabbit anti-CD3 antibody (DakoCytomation Inc; 1 : 500 for 1 h at room temperature). After washing, sections were incubated with Envision (horseradish peroxidase-coupled anti-rabbit secondary reagent; DakoCytomation) for 30 minutes. The sections were developed with 3,3′-diaminobenzidine and counterstained with Meyer’s haematoxylin. The numbers of CD3-positive and 5-HT-expressing enterochromaffin cells were counted by an investigator blinded to conditions and the numbers of CD3-positive and enterochromaffin cells were expressed per high power field and per 10 glands, respectively.
Determination of colonic 5-HT content
Segments of colon were homogenised in 0.5 ml 0.2 mol perchloric acid and centrifuged at 10 000g for five minutes. The supernatants were neutralised with 0.5 ml 1.0 mol borate buffer (pH 9.25) and centrifuged at 10 000g for one minute. The 5-HT content in the supernatant was analyzed by enzyme immunoassay with a commercially available kit (Beckman Coulter, California, USA). The 5-HT content of the tissue was expressed as a function of wet weight (in mg).
Measurement of cytokines in colonic tissues
Frozen colonic tissues were homogenised in lysis buffer containing protease inhibitor cocktail (Sigma, St Louis, Missouri, USA). The homogenates were freeze-thawed three times and centrifuged and the supernatants were collected and stored at −20°C until analyzed.
The homogenised colonic samples were analyzed using a mouse IL13 and IFN-γ ELISA kit (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s instructions. Results are corrected for protein concentration, which was measured by DC Protein Assay kit (Bio-Rad Laboratories, Hercules, California, USA).
Evaluation of in-vitro cytokine production from splenocytes
Single-cell suspensions of spleen were prepared in RPMI 1640 containing 10% fetal calf serum, 5 mmol l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 25 mmol Hepes, 0.05 mmol 2-ME (all Gibco BRL, Gaithersburg, Maryland, USA). Cells (107) were incubated in the presence of 5 μg/ml concanavalin A (Con A). IL4 and IL13 levels in the supernatant were measured by enzyme immunoassay using a commercially available kit (R&D Systems; Minneapolis, Minnesota, USA).
In-vitro Th1 and Th2 cell polarisation
Splenocytes from non-infected C57BL/6 mice were collected in Hank’s balanced salt solution containing 10% fetal bovine serum and 1% antibiotic/antimycotic. CD4 T cells were isolated from the mixed splenocytes by negative selection using EasySep mouse CD4 T-cell enrichment cocktail with magnetic nanoparticles (Stem Cell Technologies, Vancouver, BC, Canada). Cell purity, as determined by flow cytometry using antimouse CD4 (LT34) monoclonal antibody (BD Pharmingen, California, USA), was 90%. CD4 T cells (2.5 × 106 cells/ml) were resuspended in RPMI 1640 containing 10% fetal bovine serum and 1% antibiotic/antimycotic and activated with plate-bound 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for 24 h. After 24 h, cells were incubated with 5 ng/ml recombinant IL12, 10 μg/ml anti-IL4 and 50 U/ml rIL2 for Th1 differentiation, whereas for Th2 differentiation cells were incubated with 10 ng/ml rIL4 and 10 μg/ml anti-IL12 in the presence of 50 U/ml of rIL2. On day 7, cells were harvested, washed and re-stimulated with plate-bound 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for 24 h. After 24 h supernatants were collected and frozen for analyses of cytokines, the cells were harvested and re-suspended in PBS and each SCID mouse received 2.5 × 106 cells intraperitoneally.
Data were analyzed using unpaired Student’s t test, one-way analysis of variance with Dunnett’s post-hoc test for comparisons with a control, two-way one-way analysis of variance followed by Tukey’s test and Pearson’s correlation analysis as appropriate, when p<0.05 was considered significant. All results are expressed as the mean ± SEM.
Numbers of enterochromaffin cells and 5-HT content in the colon differ in resistant and susceptible mice after T muris infection
BALB/c mice are resistant to T muris infection and expelled almost all worms by day 21 postinfection, whereas AKR mice failed to clear the infection and we recovered substantial numbers of worms from AKR mice on day 21 postinfection (fig 1A). This was associated with significantly higher levels of IL13 in colonic tissues of BALB/c mice compared with AKR mice (fig 1B), whereas tissue IFN-γ levels were significantly higher in AKR mice compared with those in BALB/c mice (fig 1C). We also observed significantly higher levels of IL4 and IL13 from in-vitro ConA-stimulated spleen cells from resistant BALB/c mice (fig 2).
Th1 and Th2 environments in AKR and BALB/c mice, respectively, in T muris infection had a significant influence on enterochromaffin cells and 5-HT content in the colon. We observed significantly higher numbers of enterochromaffin cells in the colon of BALB/c mice compared with those in AKR mice after infection (fig 3A). Consistent with increased enterochromaffin cell numbers, we also observed significantly higher amounts of 5-HT in the colon of BALB/c mice compared with those in AKR mice after infection (fig 3B). There were significant correlations between the ability to expel the parasite (as measured by worm burden) and both the enterochromaffin cell numbers and amount of 5-HT (Pearson’s correlation coefficient −0.93 and −0.80, respectively; p<0.0001 and p<0.01, respectively).
Numbers of enterochromaffin cells and 5-HT content in colon differ in Stat4−/− and Stat6−/− mice after T muris infection
We next examined enterochromaffin cells and 5-HT content in the colon of Stat4−/− and Stat6−/− mice, which are impaired in Th1 and Th2 responses, respectively. As shown in fig 4A the enterochromaffin cell numbers were significantly lower in Stat6−/− mice compared with those in Stat4−/− mice after infection with T muris. We also observed a significantly lower amount of 5-HT in colon tissues of Stat6−/− mice compared with Stat4−/− mice after infection (fig 4B). The reduction in enterochromaffin cell numbers and amount of 5-HT after infection was associated with significant impairment in Th2 cytokines in the colon of Stat6−/− mice. In contrast, the higher enterochromaffin cell numbers and amount of 5-HT in Stat4−/− mice after infection was associated with an upregulation of Th2 cytokines in colonic tissue (IL4 levels (in pg/mg protein): 0 (SD 0), 14.8 (SD 4.1) and 0 (SD 0), in wild-type control mice, Stat4−/−mice and Stat6−/− mice, respectively; IL13 levels (in pg/mg protein): 0 (SD 0), 61.2 (SD 13.6) and 0 (SD 0), in wild-type control mice, Stat4−/−mice and Stat6−/− mice, respectively, on day 15 postinfection). In addition, in association with the enhanced Th2 response we observed faster worm expulsion in Stat4−/− mice compared with Stat6−/− mice. Almost all worms were expelled from Stat4−/− mice by day 15 postinfection, whereas we recovered substantial numbers of worms from Stat6−/− mice on day 15 postinfection (fig 4E). There were significant correlations between the ability to expel the parasite (as measured by worm burden) and both the enterochromaffin cell numbers and amount of 5-HT (Pearson’s correlation coefficient −0.88 and −0.83, respectively; p<0.005 and p<0.05, respectively).
Numbers of enterochromaffin cells and amount of 5-HT in colon differ after reconstitution of SCID mice with in-vitro polarised Th1 and Th2 cells
In previous studies we have demonstrated significantly lower colonic enterochromaffin cell numbers and 5-HT content in the immunodeficient SCID mice compared with those in wild-type mice in T muris infection.9 In the present study we observed a difference in the numbers of enterochromaffin cells and 5-HT content in the colon in a Th1 and Th2 environment in T muris-infected AKR and BALB/c mice, respectively. Therefore, we next investigated the role of in-vitro polarised Th1 and Th2 cells in the regulation of enterochromaffin cell function and 5-HT production in the gut using SCID mice. As shown in fig 5 the numbers of enterochromaffin cells were significantly greater after reconstitution of SCID mice with purified CD4 T cells polarised to the Th2 type compared with those mice reconstituted with cells polarised to the Th1 type. Colonic 5-HT content was also markedly higher in SCID mice reconstituted with Th2 cells compared with that in SCID mice reconstituted with Th1 cells (fig 5B).
To investigate the effectiveness of Th1 and Th2 polarisation we investigated the IL4 and IFN-γ levels in the supernatant of the polarised cells after in-vitro ConA stimulation and observed a higher level of IL4 in the supernatant of cells polarised to the Th2 type and a higher level of IFN-γ in the supernatant of cells polarised to the Th1 type (IFN-γ: 9.3 pg/ml versus 0 pg/ml in the supernatant of Th1 versus Th2 polarised cells, respectively; IL4: 35.8 pg/ml versus 0 pg/ml in the supernatant of Th2 versus Th1 polarised cells, respectively). To evaluate the effectiveness of the reconstitution of SCID mice we investigated tissue cytokine levels in the reconstituted mice and observed significantly greater amounts of IFNγ and IL4 in the colon of SCID mice that received Th1 and Th2 cells, respectively, on day 21 postreconstitution (fig 6). There were significant correlations between Th2-biased (as measured by the ratio between IL4 and IFN-γ levels) and both the enterochromaffin cell numbers and amount of 5-HT (Pearson’s correlation coefficient −0.76 and −0.89, respectively; p<0.05 and p<0.001, respectively). Immunohistochemical studies on colonic CD3-positive T cells revealed a significant upregulation of colonic CD3-positive T cells in the SCID mice reconstituted with both Th1 and Th2 polarised cells (fig 7).
Our study demonstrates a difference in enterochromaffin cell numbers and colonic 5-HT content in the Th1 and Th2 environment and provides further evidence for the immunological control of endocrine function in the gut in enteric infection and inflammation. We observed greater numbers of enterochromaffin cells and a higher amount of 5-HT in the colon of mice that are resistant to T muris infection and generate a Th2 type immune response compared with those in the colon of mice that are susceptible to infection with the generation of a Th1 type immune response. There were also significantly lower numbers of enterochromaffin cells in mice deficient in Stat6, which are impaired in generating a Th2 response. In contrast, mice deficient in Stat4, which are impaired in Th1 cytokine production, exhibited greater numbers of enterochromaffin cells. In addition, adoptive transfer studies in immunodeficient SCID mice with in-vitro polarised Th1 and Th2 cells show a significantly higher number of enterochromaffin cells and higher 5-HT content in the colon of mice that received Th2 cells compared with those in the colon of mice that received Th1 cells. Taken together, the present study provides evidence for the first time that enterochromaffin cell response and 5-HT production to the same infectious agent are influenced by Th1 or Th2 cytokine predominance.
Enterochromaffin cells have specialised microvilli that project into the lumen and contain enzymes and transporters known to be present in the apical parts of the enterocytes.26 Enterochromaffin cells function as sensors for the contents of the gut lumen and respond to luminal stimuli directly via these transporters and/or indirectly by mediators from the surrounding cells.26 5-HT produced from enterochromaffin cells is implicated in the neuro–immuno–endocrine networks of both humans and rodents.27–29 5-HT is an important enteric mucosal signalling molecule and by virtue of their structural location enterochromaffin cells are likely to play a strategic role in the maintenance of gut homeostasis. 5-HT has a confounding range of effects in the GI tract and has been implicated in the pathophysiology of a number of GI disorders such as enteritis, IBS, inflammatory bowel disease and colon carcinoma. It has recently been shown that T spiralis infection-induced upregulation of enterochromaffin cells is attenuated in T-cell receptor (β × δ) knockout mice.30 The role of the host’s immune response underlying changes in enterochromaffin cells and 5-HT has also been demonstrated in mice infected with the bacterial pathogen Citrobacter rodentium.31 The number of enterochromaffin cells and amount of 5-HT in the colon was significantly reduced in C rodentium-infected immunocompetent mice. This C rodentium infection-induced alteration in enterochromaffin cells and 5-HT was not, however, evident in SCID mice. In our previous study we have shown that CD4 T cells play a critical role in the development of enterochromaffin cell hyperplasia and 5-HT production in nematode infection.9 In the present study we observed that the T muris infection-induced upregulation of enterochromaffin cells and 5-HT was significantly higher in the Th2 environment of resistant mice compared with that in the Th1 environment in susceptible mice. The influence of Th1 and Th2 type immune responses on enterochromaffin cell function and 5-HT production was further illustrated by the studies using Stat4−/− and Stat6−/− mice. Infection-induced changes in enterochromaffin cells and 5-HT were significantly lower in Stat6−/− mice compared with those in Stat4−/− mice. This was associated with an attenuated and enhanced Th2 response in infected Stat6−/− mice and Stat4−/− mice, respectively. In addition, transfer experiments with in-vitro polarised Th1 and Th2 cells in SCID mice provide direct evidence on the differential regulation of Th1 and Th2 cells in enterochromaffin cell and 5-HT response in the gut. These observations suggest that infection and inflammation of the GI tract cause significant alteration in enterochromaffin cell function and 5-HT production and the immunological profile of the inflammatory response may be an important determinant of the changes that occur in the 5-HT response in the gut. Our findings on Th1/Th2 regulation of enterochromaffin cell biology corroborate with recent studies on lower numbers of enterochromaffin cells and a lower amount of 5-HT in Th1-biased bacterial infection with C rodentium31 and on the inhibitory effect of IFN-γ on the proliferation of BON tumour cells (model of human enterochromaffin cells).32 Related to this it has recently been shown that the rate of epithelial cell turnover in the large intestine was higher in resistant BALB/c mice in T muris infection compared with that in susceptible AKR mice and that the rate of epithelial cell movement is under immune control by IL13.33 By laser capture microdissection-based molecular and immunofluorescence techniques we have recently demonstrated the presence of the IL13 receptor on colonic enterochromaffin cells.9 As enterochromaffin and epithelial cells arise from common totipotent stem cells in the gut it is surmised that Th2 cytokines play an important role in enterochromaffin cell biology in this infection by influencing cell turnover and the release of 5-HT.
Enterochromaffin cells are located in close proximity to the mucosal sensory nerve endings and interganglionic neurons, which synapse on motor neurons. When secreted from enterochromaffin cells, 5-HT activates intrinsic and extrinsic neural pathways affecting GI motor function.34–36 Alteration in GI motor function occurs in diverse clinical settings, including infections by a variety of infectious agents. Data obtained from previous studies in nematode models including the T muris model show that Th2 cells play a pivotal role in the regulation of intestinal muscle function and mucin production and this is associated with host resistance in enteric parasitic infection.37 38 The present study shows that the extent of enterochromaffin cell response and 5-HT production is dependent on the immunological profile (Th1 or Th2) in the same infection. The results of this study corroborate well with the findings on the amount of 5-HT in Crohn’s disease and cholera, which generates predominant Th1 and Th2 responses, respectively.5 39–41 In addition, it has recently been shown that the reduced expression of phospho-MEK, a downstream target of c-Raf, in neuroendocrine cells in the colonic biopsies correlates with clinical responses in Crohn’s disease as a result of treatment with the anti-inflammatory small molecule semapimod, suggesting that neuroendocrine cells that are important regulators of gut physiology might be involved in the pathogenesis of human colonic inflammation.42
Taken together, in addition to providing novel data on the mechanisms of 5-HT production, these observations give new insights into the mechanisms of gut immuno–endocrine interaction in relation to gut physiology, which may ultimately lead to improved therapeutic strategies in various GI infections and inflammatory conditions in which alterations of 5-HT signalling are seen in association with immune activation in the GI tract.
The authors would like to thank Dr Richard Grencis (University of Manchester, UK) for providing T muris, Dr Mark H Kaplan (Indiana University School of Medicine, USA) for Stat4−/− mice, Dr Klaus I Matthaei (Australian National University) for Stat6−/− mice and Dr Stephen M Collins (McMaster University) for his valuable input and J Steeds and Y Deng (McMaster University) for technical support.
Funding: This work is supported by grants from the Canadian Institutes of Health Research (CIHR) and the Canadian Association of Gastroenterology (CAG)/Crohn’s and Colitis Foundation of Canada (CCFC) to Dr Waliul I Khan. Dr Khan holds a McMaster University Department of Medicine Internal Career Research Award.
Competing interests: None.
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