L. Öhman, Department of Microbiology and Immunology, Göteborg University, Box 435, 40530 Göteborg, Sweden. Tel.: +46 31 773 6214; fax: +46 31 773 6205; e-mail: email@example.com
Abstract The aetiology of the irritable bowel syndrome (IBS) is incompletely understood. A low-grade colonic inflammation is frequently seen, but it is unclear to what extent this phenomenon contributes to the pathophysiology of IBS. CD4+CD25+ regulatory T cells (Treg) are implicated to play an important role in suppressing intestinal inflammation. We, therefore, examined whether the intestinal inflammatory process in IBS patients is the result of an altered function and/or frequency of CD25+ Treg cells. Patients with IBS (n = 34), fulfilling the Rome II criteria, were compared with controls (n = 26). The suppressive activity of blood CD25+ Treg cells was determined and the frequency of colonic and blood CD25+ Treg cells was analysed by flow cytometry. The expression of the Treg marker, FOXP3 mRNA, in colonic biopsies was determined by reverse transcription-polymerase chain reaction. Blood CD25+ Treg cells from IBS patients suppressed the proliferation of blood CD4+CD25low/− T cells. Similar frequencies of CD25+ Treg cells were recorded in mucosa and blood of IBS patients and controls. FOXP3 mRNA was equally expressed in the colonic mucosa of patients with IBS and controls. In conclusion, the low-grade intestinal inflammation recorded in patients with IBS is not associated with an altered function or frequency of CD25+ Treg cells.
Irritable bowel syndrome (IBS) is a common bowel disorder in Western society1 and is defined according to the Rome II criteria.2 Despite the absence of identifiable organic disease explaining the sometimes severe symptoms, patients suffer from chronic abdominal pain, bloating and altered bowel habits. Abnormal gastrointestinal (GI) motility,3,4 visceral hypersensitivity,5,6 altered colonic fermentation7 and abnormal GI responses to stress8 and nutrients9 are factors, which may contribute to symptom generation; but no omnipresent aetiological mechanisms have so far been identified.
A low-grade inflammatory process in various compartments of the small and the large bowel has been suggested to be associated with gut dysfunction in at least a subpopulation of IBS patients.10–12 Several recent publications reported increased number of T cells in various lymphoid compartments of the small and/or large intestine.11–15 Irritable bowel syndrome patients have also been found to be genetically predisposed to produce low amounts of the anti-inflammatory cytokine IL-10,16 and to be more prone to express the proinflammatory cytokine IL-1β mRNA after infectious gastroenteritis, when compared with non-IBS gastroenteritis patients.17
The low-grade mucosal inflammation in IBS may theoretically be due to an unregulated mild inflammation or reflect a suppressed potent inflammatory process. Several different T cell subsets with regulatory properties have been described, among which the naturally occurring CD4+CD25+ regulatory T cells (Treg) have attracted most attention during the last decade.18 Thymus-derived human Treg cells are defined by a high expression of CD25,19 the alpha chain of the IL-2 receptor, whereas low/intermediate expression of CD25 is found on virtually all effector T cells. Novel data show that the FOXP3 gene is important in the development and function of Treg cells,20 and the majority of FOXP3 expressing cells in blood and colonic mucosa are contained within the fraction of T cells with high expression of CD25 in both healthy subjects and ulcerative colitis patients.21,22 The Treg cells are known to suppress many T-cell-mediated immune responses; and changes in Treg cell function and/or frequency are suggested to participate in various inflammatory reactions like, for example, multiple sclerosis, type 1 diabetes mellitus and Helicobacter pylori infections.23–25
Studies on mouse models of experimental colitis have demonstrated that intestinal inflammation can not only be prevented but also cured by the presence of CD4+ CD25+ T cells with regulatory properties in the gut mucosa.26–28 Recent reports have also described the occurrence of functional CD25+ Treg cells in the colonic mucosa of healthy individuals and patients with inflammatory bowel disease (IBD).21,22 Chadwick et al.12 have shown that IBS patients have an increased number of colonic lamina propria T cells expressing CD25, and it has been speculated that these cells may indeed have regulatory properties, which affect the intestinal inflammatory activity in these patients.29 The aim of the current study was, therefore, to determine whether the low-grade intestinal inflammation recorded in IBS patients is associated with an altered function and/or frequency of CD4+CD25+ Treg cells. Colonic biopsies and blood were collected from patients scheduled for colonoscopy, with or without IBS symptoms, and we compared the suppressive capacity and frequency of CD4+ CD25+ T regulatory cells and mucosal FOXP3 expression between the two groups.
Materials and methods
Study subjects were recruited among patients referred for colonoscopy at the Sahlgrenska University Hospital, Göteborg, Sweden. Individuals with known celiac disease or food allergy were excluded from the study. Venous blood samples and biopsies from the ascending (5 cm above the ileocecal valve) and the sigmoid colon, respectively, were assessed from each patient. The study was performed after receiving written informed consent from all subjects and approval by the ethics committee of the University of Göteborg.
The IBS patients were defined according to the Rome II Criteria2 and underwent routine diagnostic colonoscopy to exclude organic bowel disease. No evidence for collagenous or lymphocytic colitis was detected based on standard criteria.30 A total of 34 IBS patients were included [24 females, mean age of 40 ± 13 years; body mass index (BMI) 25.0 ± 5.4 kg m−2], and 21 had diarrhoea-predominant IBS (IBS-d), 2 had constipation-predominant IBS (IBS-c) and 11 had alternating bowel habits (IBS-a).2 One of the IBS patients had less than 1-year duration of symptoms, 18 of the patients had symptom duration of 1–5 years and 15 of the patients included reported IBS symptoms for more than 5 years. Acute onset after supposed infectious diarrhoea was described by three IBS-d patients and oneIBS-c patient.
As control subjects we included 26 individuals (13 females, mean age 53 ± 10 years; BMI 24.6 ±3.6 kg m−2), who underwent colonoscopy for investigation of anaemia, rectal bleeding or polyp surveillance. These subjects were free from GI symptoms and the colonoscopy was normal, both macroscopically and microscopically. The control subjects were older than patients with IBS (P = 0.02). Data from the IBS and control subjects included in the flow cytometry analysis have previously been published by our group, where we examined the frequency of other colonic non regulatory T cell populations.15
Proliferation and suppression assays
Peripheral blood lymphocytes were isolated from venous blood by density-gradient centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden). Peripheral CD4+ T cells were separated with Dynabeads CD4 Positive Isolation Kit (Dynal Biotech ASA, Oslo, Norway). The CD4+ T cells were then incubated with Dynabeads CD25 Positive Isolation Kit and the enriched CD25low/− and CD25+ fractions were recovered. As antigen presenting cells (APC), we used monocytes isolated from peripheral blood lymphocytes by adherence of the remaining cell population after CD4 and CD8 cell depletion using Dynabeads Positive Isolation Kit. 1 × 105 CD4−CD8− mononuclear cells were incubated in round-bottomed 96-well plates for 2 h at 37 °C in 5% CO2. Wells were washed and the remaining adherent cells were used as APC in the following experiment. The suppressive capacity of CD25+ cells was determined by adding CD4+CD25low/− and/or CD4+CD25+ T cells at various ratios in Iscove's medium supplemented with 5% AB+ serum, 1% gentamycin (Sigma, St Louis, MO, USA) and 1%l-glutamine (Sigma). Cultures were stimulated with 1 μg mL−1 soluble anti-CD3mAb (BD Pharmingen, San Diego, CA, USA). The cultures were incubated for 5 days and the T cell proliferation was measured by pulsing the cells with 0.5 μCi of [3H] thymidine/well (Amersham, Arlington Heights, IL, USA) for 16 h and the incorporated radioactivity was analysed in a scintillation counter. The purity of enriched cell populations was determined by flow cytometry using the following monoclonal antibodies: anti-CD3-FITC, anti-CD4-PerCP, anti-CD8-APC and anti-CD25-PE all from BD Pharmingen. The purity of enriched CD4+ T cells was approximately 95%. The frequency of CD25+ T cells among CD4+ T cells in the total peripheral blood monocyte was approximately 2%, and the frequency of CD25+ T cells among CD4+ T cells in the enriched CD4+ T cell population was approximately 6% and the frequency of CD25+ T cells among CD4+ T cells in the enriched CD25+CD4+ T cell population was approximately 32% (Fig. 1A). Blood samples from three IBS and four control subjects were used for proliferation and suppression assays.
Isolation of intestinal and peripheral blood lymphocytes
Lamina propria lymphocytes were isolated as previously described.25 Briefly, intraepithelial lymphocytes were isolated from the biopsies by incubation in Hank's balanced salt solution (HBSS), without calcium or magnesium, containing 1 mmol L−1 EDTA and 1 mmol L−1 DTT (Sigma), 4× for 15 min. After washing the biopsies 2× for 15 min in HBSS, lamina propria lymphocytes were isolated by stirring remaining tissue in Iscove's complete medium containing 100 U mL−1 collagenase (Sigma) and 0.1 mg mL−1 DNAse (Sigma) at 37 °C for 2 h. The obtained cell suspension was filtered through a mesh and the number of lymphocytes was counted under the microscope.
Flow cytometric analysis
Freshly isolated cells, 1 × 105 cells per sample, were stained for flow cytometry analysis of various surface markers using combinations of the following antibodies: anti-CD3-FITC, anti-CD4-PerCP, anti-CD8-APC, anti-CD25-PE (BD Pharmingen). All cells were fixed in cellfix (BD Pharmingen) before fluorescence-activated cell sorter analysis, which was performed using a LSR II (BD Pharmingen). At least 10 000 live lymphocytes per sample were analysed, as defined by forward and side scatter. The data were analysed using Flow Jo software (Treestar Inc, Ashland, OR, USA). Biopsies from 19 IBS and 8 control subjects were used for flow cytometric analysis.
Analysis of FOXP3 mRNA levels by quantitative real-time PCR
Total RNA was extracted from mucosal biopsies using the RNeasy Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Contaminating DNA was removed by treatment with DNA-free (Ambion, Austin, TX, USA). cDNA was prepared in a random hexamer-primed superscript RT reaction (Invitrogen, Stockholm, Sweden), according to the manufacturer's protocol. FOXP3 mRNA levels were measured in duplicate using the LightCycler instrument (Roche Diagnostics, Mannheim, Germany) with reagents from the LighCycler FastStart DNA Master SYBR Green I kit and the following primers: 5′-CAG CAC ATT CCC AGA GTT CCT-3′ (forward) and 5′-GCG TGT GAA CCA GTG GTA GAT-3′ (reverse). PBGD was used as endogenous reference gene for relative quantification and detected by the primers: 5′-GAA ACC CTG CCA GAG AAG A-3′ (forward) and 5′-CTG GCC CAC AGC ATA CAT-3′. All primers were designed not to amplify genomic DNA, and were ordered from TIB MOLBIOL (Berlin, Germany). Polymerase chain reaction (PCR) cycling conditions consisted of 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s, 65 °C for 7 s and 72 °C for 10 s. Cycle threshold values for each gene were compared against a standard curve to estimate starting amounts of RNA and received values for target gene were normalized to threshold values for reference gene. A melting curve analysis was performed in each run to ensure the specificity of the primers, and data were obtained using the LightCycler data analysis software; and the LightCycler relative quantification software was used for calibrator normalized relative quantification. Biopsies from 12 IBS patients and 14 control subjects were used for reverse transcription-PCR analysis.
All statistical evaluations were performed with the StatView Software (SAS Institute, Cary, NC, USA). The non-parametric Mann–Whitney test was used to evaluate significance of differences.
CD4+CD25+ T cells from IBS patients suppress CD4+CD25low/− T cell activity
Naturally occurring CD4+CD25+ Treg cells exert suppressive effects on CD4+CD25low/− T cells. We evaluated the suppressive activity of blood CD4+CD25+ T cells on CD4+CD25low/− T cells of IBS patients. Enriched CD4+CD25+ T cells were cocultured with autologous CD4+CD25low/− T cells. The CD25+ T cells proliferated moderately, whereas CD4+ T cells proliferated vigorously in response to anti-CD3 stimulation (Fig. 1B). Depletion of CD25+ T cells increased the proliferative capacity of the CD4+ T cells even further and the proliferation of the colonic CD25low/− T cells was reduced by coculture of colonic CD25+ T cells at a ratio of 1 : 1 (Fig. 1B). Furthermore, the CD25+ T cells from IBS patients suppressed the proliferation of the cocultured CD25low/− T cells in a dose-dependent fashion similar to the suppressive pattern recorded in healthy controls (Fig. 1C). Thus, blood CD4+CD25+ T cells isolated from IBS patients suppress the proliferation of autologous CD4+CD25low/− T cells and are truly CD25+ Treg cells.
Similar frequencies of blood and colonic CD4+CD25+ regulatory T cells in IBS and controls
To determine whether IBS patients have an altered frequency of Treg cells in blood and the colonic mucosa, we investigated the presence of Treg cells of IBS patients and control subjects by flow cytometric analysis. The Treg frequency in the lamina propria of both the ascending colon and sigmoid colon was determined, in order to evaluate the existence of colonic segmental variations. The Treg cells were defined as CD4+ T cells with a high expression of CD25 as shown in a dot plot (Fig. 2A).19 We first compared the Treg cell frequencies among CD4+ T cells in the blood of our subjects groups. The frequencies of Treg cells among CD4+ T cells in peripheral blood were comparable in IBS patients and control subjects (Fig. 2B). We then compared the frequency of lamina propria Treg cells from the ascending colon and the sigmoid colon, respectively, between our subject groups. We found that IBS patients had equivalent frequencies of Treg cells among CD4+ T cells when compared with control subjects in the ascending colon lamina propria and in the sigmoid colon lamina propria (Fig. 2B). Furthermore, the Treg cell frequencies did not differ between diarrhoea, constipation or alternating IBS patients in either blood or colonic mucosa. In conclusion, similar frequencies of colonic lamina propria and blood Treg cells among CD4+ T cells were recorded in IBS patients when compared with control subjects.
Normal mucosal expression of FOXP3 in IBS patients
The expression of FOXP3, a transcription factor previously shown to be exclusively expressed in human and mouse Treg cells,31 is a relevant marker for Treg cells in healthy and inflamed human colonic mucosa.21,22 We, therefore, determined the expression of FOXP3 in colonic biopsies in IBS patients and control subjects with quantitative real-time PCR. The FOXP3 mRNA levels in colonic biopsies from the ascending colon of IBS patients did not differ from FOXP3 mRNA levels from the ascending colon of control subjects (Fig. 3). In addition, the FOXP3 mRNA levels in colonic biopsies from the sigmoid colon of IBS patients were comparable to FOXP3 mRNA levels from the sigmoid colon of control subjects (Fig. 3). Furthermore, the dominating IBS symptom did not affect FOXP3 expression. Thus, the Treg cell marker FOXP3 is expressed to a similar degree in the colonic mucosa of patients with IBS, irrespective of the dominating symptom, and control subjects.
Several recent reports have described a low-grade intestinal inflammation in IBS patients. One possible explanation for this phenomenon is a dysfunction of the Treg cell system. A reduced CD4+CD25+ Treg cells function and/or frequency could result in an insufficient suppression of gut inflammatory activity. It is also theoretically possible that there is an increased frequency of functional Treg cells trying to restrain an underlying potent proinflammatory activity, as was recently shown to be the case in ulcerative colitis.21 One of the aims of the present study was to test whether T cell activity is indeed suppressed by Treg cells from patients with IBS. We found that blood Treg cells from IBS patients indeed are able to suppress the proliferative response of anti-CD3-stimulated autologous T cells. Thus, our results indicate that IBS Treg cells do have a grossly normal suppressive activity and that effector T cells from IBS patients are likewise able to respond to the suppression. These data are in accordance with findings in patients with ulcerative colitis and Crohn's syndrome, showing that these patients also have functional Treg cells suppressing the activity of autologous T cells.21,22,32
The frequencies of Treg cells in the colonic mucosa and blood of IBS patients and healthy controls were found to be comparable as determined by flow cytometry. The finding of normal levels of colonic Treg cells in IBS patients was also confirmed using real-time PCR analysis, and equivalent expression of FOXP3 mRNA was found in colonic biopsies from IBS patients and control subjects. FOXP3 mRNA is a relevant marker for mucosal Treg cells, because we and others previously have shown that most FOXP3 expressing cells in normal and inflamed colonic mucosa are contained within the CD25+ Treg cell fraction.21,22 The normal frequencies of colonic Treg cells found in IBS patients stand in sharp contrast to the numerous reports of accumulation of Treg cells to the site of inflammation in chronic inflammatory diseases like ulcerative colitis, Crohn's syndrome and rheumatoid arthritis.21,22,32–34 Our group has also demonstrated that the frequency of colonic Treg increases with colonic and systemic disease activity. Therefore, the absence of an increased accumulation of Treg cells in the colonic mucosa of IBS patients likely reflects the modest increase in recruitment of lymphoid cells to the gut mucosa and the low-grade intestinal inflammation recorded in this patient group.
The reason for the intestinal low-grade inflammation seen in IBS patients in spite of normal levels of apparently functionally suppressive colonic Treg pool still remains unclear. However, we cannot be certain that the Treg cells present in the colonic mucosa of IBS patients are fully functional, i.e. indeed have a suppressive effect on colonic effector T cells, as this has not been investigated. To evaluate whether colonic Treg cells from IBS patients are suppressive, large numbers of isolated colonic Treg cells and colonic effector T cells are needed to be able to perform coculture assays. The needed numbers of enriched colonic Treg cells can only be obtained from resected colonic tissue. Thus, it is currently not possible to examine whether colonic Treg cells of IBS patients are functionally active, for ethical considerations. It is, however, less likely that the intestinal immune activation of IBS patients is due to non-functional Treg cells, as colonic Treg cells of IBD patients with severe intestinal inflammation have been demonstrated to be functional at least ex vivo.21,22
The control subjects were older than patients with IBS in our study, although both groups were middle aged. The efficacy of Treg cell suppression has been reported to decline with age; the functional activity of Treg cells decreased after the mid twenties and was then constant during later stages in life.35 On the contrary, the frequency of Treg cells in blood has been shown to increase with age, although the Treg frequency did not vary substantially within the group of middle-aged subjects.36 We, therefore, conclude that it is unlikely that the age difference between the groups have influenced our results in this study. No gender differences in Treg cell frequencies or suppressive functions are presently reported in the literature to the best of our knowledge.
In conclusion, our study shows that Treg cells from IBS patients exert suppressive function and that IBS patients have normal frequencies of Treg cells in both the colonic lamina propria and peripheral blood. This indicates that IBS is not associated with an altered function or frequency of Treg cells. Still, our finding does not rule out the possibility that the colonic Treg cells are dysfunctional in vivo or that alterations of other mucosal populations with suppressive properties are associated with the intestinal inflammatory process in patients with IBS.
This study was supported by the Swedish Medical Research Council, AstraZeneca R & D, Mölndal, Sweden, Nanna Swartz Foundation, Sahlgren's Academy Hospital Foundation, Tore Nilsson Foundation, Wilhelm & Martina Lundgren Foundation, Nio Meter Liv Foundation, Goljes Foundation, Swedish Foundation for Strategic Research ‘The Mucosal Immunobiology and Vaccine Center’ (MIVAC) and The Swedish Society of Medicine.