Indoleamine 2,3-Dioxygenase: Expressing Cells in Inflammatory Bowel Disease—A Cross-Sectional Study

Aim. To characterise and enumerate IDO+ cells, Tregs, and T cell subsets in patients with ulcerative colitis (UC) and Crohn's disease (CD) with regard to their clinical activity. Methods. Ten active UC (aUC), 10 inactive UC (iUC), 6 aCD, and 8 iCD patients and 10 healthy individuals were included in the study. Circulating Foxp3-, IDO-, IL-17A-, IL-4-, IFN-γ-, and IL-10-expressing CD4+ T cells were quantitated by flow cytometry. Interleukin-17-expressing cells, CD25+/Foxp3+ Tregs, and CD123+/IDO+ plasmacytoid dendritic cells were evaluated in intestinal biopsies from 10 aUC, 6 aCD, and 10 noninflamed tissues. Results. All CD4+ T subsets were increased in aIBD patients compared with healthy donors. Meanwhile, frequency of CD8α +/CD16+/IDO+, CD8α +/CD56+/IDO+, CD8α +/CD80+/IDO+, CD8α +/CD123+/IDO+ large granular nonlymphoid cells, and CCR6+/CD123+/IDO+ plasmacytoid dendritic cells was higher in aIBD patients versus healthy donors or iIBD patients. Tissue IL-17A+ cells were present in higher amounts in aIBD versus noninflamed controls. IDO- and Foxp3-expressing cells were increased in aUC versus aCD patients and noninflamed tissues. Conclusions. The findings represent an original work in Mexican Mestizo patients with IBD. It shows that Tregs and IDO-expressing cells are increased with regard to disease activity. These cells could significantly shape inflammatory bowel disease pathophysiology, severity, and tolerance loss.


Introduction
Inflammatory bowel disease (IBD) including ulcerative colitis (UC) and Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract. Thus far, the aetiology is not completely understood. Furthermore, initiation and exacerbation of the inflammatory process seem to be due to massive mucosal immune response [1]. Therefore, it is not preposterous to correlate the chronic relapsing inflammation with a tolerance dysregulation mechanism or an aberrant immune response to intestinal flora in a context of genetic predisposition [2].
In this vein, the catabolism of tryptophan, by the enzyme indoleamine 2,3-dioxygenase (IDO) expressed in plasmacytoid dendritic cells, generates kynurenines, 3-hydroxyanthranilic, and quinolic acids, molecules with the ability to exert cytotoxic action on T, B, and NK cells, but not on DCs themselves [3]. Moreover, deprivation of tryptophan by IDO halts the proliferation of T cells at mid-G 1 phase, which in concert with the proapoptotic activity of kynurenine leads to diminishing T cell-mediated immune responses and the subsequent development of immune tolerance [4]. IDO has a selective sensitivity for Th1 over Th2 cells to tryptophan metabolites, suggesting a potential role for Th2 differentiation [4][5][6][7][8][9][10]. IDO-competent DCs have shown to induce CD4 + /CD25 hi regulatory T cells (Tregs) in vivo, and Tregexpressed glucocorticoid-induced tumour necrosis factor receptor (GITR) which in turn can use IDO + DCs to expand their own population in a positive feedback loop [11,12].
Thus, quantitative and functional modifications of Tregs and IDO-producing cells might play a role in the

Subjects.
For this exploratory, observational, and crosssectional study, 10 active UC (aUC), 10 inactive UC (iUC), 6 active CD (aCD), and 8 inactive CD (iCD) patients were included. UC diagnosis was performed by the presence of the following criteria: a history of diarrhoea or blood in stools, macroscopic appearance by endoscopy, and biopsy compatible with UC. Relevant clinical and demographic information in all UC patients was collected from medical records: gender, age at diagnosis, familial aggregation, smoking history, previous appendectomy, disease evolution, extension, extraintestinal manifestations, medical or surgical treatment, and clinical course of disease. The clinical course of disease was defined as active then inactive (first episode with activity and then longterm remission for more than 5 years); intermittent activity (2 relapses in a year); chronic continual activity (>2 relapses or persistent activity despite of medical conventional therapy) as previously described [16][17][18][19].
In addition, 10 healthy donors (HD) age-matched who volunteered were included as controls. HDs were also interviewed in order to discard any known autoimmune disease, use of immunosuppressors and prednisone, and concurrent infections.
A sample of venous blood (10 mL) was obtained from each subject. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation on Lymphoprep (Axis-Shield PoC AS, Oslo, Norway).
As isotype control, IgG 1 -FITC/IgG 1 -PE/CD45-PeCy5 mouse IgG 1 , k (BD Tritest, BD Biosciences) was used to set the threshold and gates in the cytometer. We ran an unstained (autofluorescence control) and permeabilised PBMCs sample. Autofluorescence control was compared to single stained cell positive controls to confirm that the stained cells were on scale for each parameter. Besides, BD Calibrite 3 beads were used to adjust instrument settings, set fluorescence compensation, and check instrument sensitivity (BD CaliBRITE, BD Biosciences). Fluorescence minus one (FMO) controls were stained in parallel using the panel of antibodies with sequential omission of one antibody, with the exception of the anti-Foxp3 antibody, which was replaced by an isotype control rather than simply omitted.
To determine IDO cell expression, PBMCs were surface labelled with an anti-human CD8 -PE and CD16-PECy5, CD8 -PE and CD56-PECy5, CD8 -PECy5 and CD80-PE, CD8 -PE and CD123-PECy5, CD8 -PECy5 and CCR6-PE, or CCR6-PE and CD123-PECy5 monoclonal antibodies (BD Biosciences) in separated tubes during 20 min at 37 ∘ C in the dark. Cells were permeabilised and stained with a sheep anti-human-IDO (Chemicon, Temecula, CA) and then with FITC-conjugated-rabbit anti-sheep antibody. Cell subset was analysed by flow cytometry. As control of FITC-labelledrabbit anti-sheep specificity staining, cells were incubated with surface antibodies and FITC-conjugated-rabbit antisheep in the absence of sheep anti-human IDO antibody. An electronic gate was made for each and every one of the surface markers employed. Results are expressed as the relative percentage of IDO-expressing cells in each gate.

Tissue Samples.
For in situ study a total of 10 patients with definitive diagnosis of aUC calculated by the Mayo Score Activity Index [16] and 6 patients with definitive diagnosis of aCD calculated by the Crohn's Disease Activity Index (CDAI) were enrolled in the study [18,19]. In IBD patients, colonic mucosal biopsies were sampled from the region with the most severe inflammation, evaluated by colonoscopy. The biopsies from non-inflamed tissue were collected from 10 healthy individuals with constipation and bloating undergoing colonoscopy.

Immunohistochemical Detection of IL-17A-Expressing
Cells. In order to determine IL-17A-expressing cells, 4 m thick sections of available formalin-fixed paraffin embedded tissue were placed on positively charged slides. Sections were deparaffinised and rehydrated through a series of xylene and graded alcohols. Endogenous peroxidase was blocked with 3% H 2 O 2 for 20 min. A 3% normal serum was employed for 30 min as protein blocker. Tissues were incubated for 18 h at 4 ∘ C with rabbit polyclonal anti-human IL-17A antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 10 g/mL. Binding was detected by incubating sections for 60 min at room temperature with biotinylated goat anti-rabbit IgG antibody (ABC Staining System; Santa Cruz Biotechnology). Slides were incubated with horseradish peroxidase (HRP) streptavidin for 45 min, followed by incubation with the peroxidase substrate 3,3 -diaminobenzidine (DAB) (Sigma, St Louis, MO, USA) for 10 min. The sections were counterstained with haematoxylin, dehydrated with alcohol and xylene, and mounted in resin. Negative control staining was performed with normal human serum diluted 1 : 100, instead of primary antibody. The reactive blank was incubated with phosphate buffer saline-egg albumin (Sigma) instead of the primary antibody. Both controls excluded nonspecific staining or endogenous enzymatic activities. Morphometric evaluation of the immunostained sections was performed in a blinded manner by light microscope. In brief, IL-17A-expressing cells were counted in at least three optical fields from each slide in X320 high-power magnifications.
The average values per slide were used for statistical analysis. Results are expressed as the mean ± standard error of the mean (SEM) of cells quantified by the program Image Pro Plus version 5.1.1.

Double-Staining Procedure.
To evaluate CD25 + /Foxp3 + regulatory T cells and CD123 + /IDO + plasmacytoid dendritic cells a simultaneous detection was performed. A second generation of EnVision G|2 Doublestain System (Dako, Glostrup, Denmark) was used for the simultaneous detection of two antigens present at low concentrations within one biopsy. The procedure is a sequential double staining where the first antigen was visualized using horseradish peroxidase (HRP)/3 3 -diaminobenzidine (DAB) and the second antigen was visualized using alkaline phosphatase (AP)/Permanent Red. Briefly, incubation of samples with 200 L of dual endogenous enzyme block was performed for 5 min. This procedure inhibited endogenous AP, peroxidase, and pseudoperoxidase activity present in tissues. After blocking, tissues were incubated with 200 L of normal serum as negative control, primary rabbit polyclonal anti-IDO IgG antibody, or mouse monoclonal anti-Foxp3 IgG 1 antibody (Santa Cruz Biotechnology) at 10 g/mL, for 15 min, at room temperature. The samples were then incubated with 200 L polymer/HRP reagent for 10 min. This reagent is an HRP conjugated dextran polymer that also carries antibodies to mouse and rabbit immunoglobulins. The reaction was visualized by incubation of 200 L DAB plus chromogen for 5-15 min. Tissues were incubated with 200 L Doublestain Block reagent for 3 min and were then incubated with a normal serum as negative control or second primary rabbit polyclonal anti-CD25 IgG antibody (Santa Cruz Biotechnology) or mouse monoclonal anti-CD123 IgG 1 antibody (Abcam) at 10 g/mL for 15 min at room temperature. In the next step, 200 mL rabbit/mouse LINK was added for 10 min. This reagent is a dextran polymer carrying antibodies to mouse and rabbit immunoglobulins. Finally, tissues were incubated with 200 L of polymer/AP reagent for 10 min. Reaction was visualized by incubation with 200 L Permanent Red chromogen for 5-20 min. Tissues were counterstained with haematoxylin and mounted in aqueous mounting medium. At least two different sections and two fields (X320) were examined for each biopsy. Double positive CD25 + /Foxp3 + regulatory T cells and CD123 + /IDO + -producing plasmacytoid dendritic cells were assessed by estimating positively staining cells and results were reported as the percentage of immunoreactive cells. Results are expressed as the mean ± SEM of cells quantified by the program Image-Pro Plus version 5.1.1.

Ethical
Considerations. This work was performed according to the principles expressed in the Declaration of Helsinki. The study was approved by the ethical committee from our institution, and a written informed consent was obtained from all subjects.

Statistics.
Sample size has not been determined for this study; it was merely observational. Descriptive statistics were performed, and categorical variables were compared using the 2 test or Fisher's exact test. One way analysis of variance on ranks by Holm-Sidak Method was performed for all pairwise multiple comparison procedures. Statistical analysis was done using the Sigma Stat 11.2 program (Aspire Software International, Leesburg, VA, USA). Data were expressed as the median, range, and mean ± standard deviation (SD)/standard Error of the mean (SEM). The values smaller than or equal to 0.05 were considered as significant. This study conforms to STROBE statement along with references to STROBE and the broader EQUATOR guidelines [20].

Demographic Features.
We studied a total of 44 individuals who were divided in five groups: aUC ( = 10); iUC ( = 10); aCD ( = 6); iCD ( = 8); healthy individuals ( = 10). The demographic and clinical variables of patients are summarized in Table 1. Laboratory data are condensed in Table 2. There was a statistical difference in ESR between active IBD (CD and UC) patients and inactive IBD patients ( < 0.05; Table 2). aCD had higher levels of CRP versus iCD ( < 0.05; Table 2). Haemoglobin concentration in aCD patients was decreased compared with inactive CD patients ( < 0.05; Table 2).

Frequency of Foxp3 + Regulatory T Cells in IBD Patients.
Regarding lymphocytes that regulate the adaptive immune response and induce peripheral tolerance, CD4 + /CD25 hi / Foxp3 + cell subpopulation was quantified. The frequency of these cells in peripheral blood was higher in clinical active IBD patients compared with healthy donor group (3.9±0.2%). aCD patients had higher frequency of Treg cells compared with iCD patients (10.4 ± 1.1% versus 4.7 ± 0.3%, ≤ 0.001; Figures 1(f) and 2(f)). aUC patients had higher percentage of circulating Tregs cells versus iUC patients (11.1 ± 0.5% versus 6.5 ± 0.3%, ≤ 0.001). Higher amount of Tregs was observed in iUC patients versus iCD patients and healthy donors ( = 0.001).

Percentage of IL-17A-Expressing Cells on Tissue from IBD Patients.
In order to determine in situ IL-17A protein expression from aUC and aCD intestinal biopsies, tissues were immunostained and compared with non-inflamed tissues. The percentage of IL-17A immunoreactive cells was higher in UC and CD patients compared with controls ( < 0.02; Figures 6(a) and 6(b)). Furthermore, IL-17Aproducing cells were conspicuously increased in UC patients compared to CD patients; positive cells were localized mainly in mucosa, lamina propria, submucosa, muscular, and perivascular inflammatory infiltrates but not in goblet cells, crypt lumen, or crypt blanching. IL-17A + cells were lower in adventitia from UC versus CD patients ( < 0.001).

Percentage of CD123/IDO-Expressing Cells on Tissue from UC or CD Patients.
IDO is primarily expressed in CD123 + dendritic cells infiltrating areas of the inflamed lesions. This subpopulation has been shown to exert potent inhibitory effects on T cell-proliferation and depletion of tryptophan. Thus, overexpression of IDO in CD123 + dendritic cells was determined on mucosa, submucosa, muscular, and adventitia from aUC patients compared with aCD patients and noninflamed control tissue ( ≤ 0.001; Figures 7(a) and 7(b)). In turn, aCD patients showed a statistically significant increase in percentage of CD123 + /IDO + double positive cells when compared with control tissue.

Percentage of CD25/Foxp3-Expressing Cells on
Tissue from UC or CD Patients. Foxp3 regulatory T cells are critical for controlling inflammation in the gastrointestinal tract. For this reason, CD25 + /Foxp3 + Treg cells were determined in tissue from IBD patients. The frequency of Tregs on mucosa, submucosa, muscular, and adventitia from aUC patients was higher compared with aCD patients and non-inflamed control tissue ( ≤ 0.006; Figures 8(a) and 8(b)). No differences were found in CD25 + /Foxp3 + immunoreactive cell percentage on mucosa, submucosa, and muscular layers of aCD patients when compared with non-inflamed control tissue.

Discussion
IBD is a chronic inflammatory state of the gastrointestinal tract of unknown aetiology. Classically, tissue injury in IBD is thought to be primarily mediated by Th1 cells in CD or Th2 in UC [21], as we observed in our Mexican Mestizo patients.
Recently, Th17 cells have been introduced as a third and new lineage of Th cells that seem to affect both innate and adaptive immune responses by the release of proinflammatory cytokines and their involvement in autoimmune disorders [22]. The concept of breaking of tolerance is fundamental to the development of autoimmunity. The first and perhaps most vital stage of tolerance induction to self-antigens occurs in the thymus during T cell development. Because proteins with tissue-restricted or peripheral expression are traditionally thought to be unavailable for presentation in the thymus, it has been proposed that tolerance to such proteins can only be achieved through mechanisms of peripheral tolerance [23]. The role of IDO and the specific IDO-expressing cells in normal and disease conditions has not been yet fully characterised. Significant interest has been generated, however, in manipulating IDO expression in transplantation, HIV, tumour resistance, and autoimmunity [24][25][26]. The expression of IDO by different cell types, as well as the capacity of IDO competent DCregs to induce Treg cells through high levels of IDO activity, could have broader immunological significance in tolerance and immunoregulation; for example, it has been suggested that defects in the immunoregulatory mechanism initiated by IDO are involved in the development of autoimmune conditions, such as multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and autoimmune diabetes [27][28][29]. In this vein, our results showed an accumulation of IDO + /CD123 + plasmacytoid dendritic cells in colonic biopsies of IBD patients. The findings are consistent with those of Wolf et al. 's and Zhou et al. 's studies which showed increased expression of IDO mRNA and protein in IBD Austrian and Chinese patients, respectively. They suggest that high local IDO expression may represent an anti-inflammatory mechanism tempting to counterbalance the tissue-damaging effects of activated T cells infiltrating the colonic mucosa in IBD [30,31]. On the other hand, our results also showed that in aUC and aCD patients there was a conspicuously increase in the number of CD8 + / CD16 + /IDO + , CD8 + /CD56 + /IDO + , CD8 + /CD80 + /IDO + , and CD8 + /CD123 + /IDO + large granular cells when percentages were compared to healthy controls. Meanwhile, in Control (n = 10) CD (n = 6) UC (n = 10) iCD and iUC patients, there was a marked decrease of IDOexpressing cell frequency when percentages were compared to active IBD or healthy donors. These IDO-expressing subsets have not been previously described, and we suggest that it could be a plasmacytoid DC, according to blood DC subsets described by Dzionek et al. Indeed one of these pDC subsets displays the expression of CD123 hi and pre-TCRchain [32]. Generally, already small amounts of IDO + DCs are sufficient to promote immune suppression, directly or by means of bystander suppression [10]. In mice, as few as 3% of CD8 + /IDO + DC has been reported to suppress the stimulatory effects of significantly higher amounts of CD40activated CD8 − DC in vivo [33,34]. CD123 + /IDO + cells pDCs constitute only 0.2-0.8% of peripheral blood cells and represent a unique rather plastic, versatile, and important immune cell population capable of producing over 95% of IFN-I synthesized by peripheral blood mononuclear cells in response to viruses as well as nucleic acid-containing complexes from the host. In addition to IFN-I, human pDCs produce proinflammatory cytokines such as TNF-and IL-6 in response to TLR activation [35]. Manlapat [32,36,37]. Human pDCs possess an exclusive TLR repertoire, TLR7 and TLR9, express several unique surface receptors, but do not produce IL-12p70 or IL-10. CpG-activated human pDCs upregulate B7 ligands, HLA-DR, and IFN-I production, through TLR9. IFN-I upregulates IDO expression in CpG-activated human pDCs, contributing to regulatory T cell generation (CD4 + /CD25 + /Foxp3 + ) from CD4 + /CD25 − T cells with potent suppressor cell function [38]. IDO-expressing and Treg peripheral cells increased percentage in active IBD patients may be a compensatory mechanism for functional induction of tolerance in active disease, due to the increase in absolute number of circulating IL-17A-, IL-4-, and IFN--expressing cell percentage. In this case, IDO expression could merely be an indicator of the strength of an immune response or Th17, Th1, and Th2-related inflammation and act as a counter-regulatory response [39,40]. On the other hand, the scant IDO-peripheral cells frequency in inactive IBD patients is consistent with the findings of our group in rheumatoid arthritis and systemic lupus erythematosus patients and in human primary biliary cirrhosis [27,41], and it might be related to the recruitment of blood derived-DC at the tissue in order to induce tolerance [42]. This could be true for CD123/IDO and CD25/Foxp3 cells as we determined in biopsies from clinical active patients. These data suggest that increased IDO expression in the gut may be used as a prognostic factor or diagnostic biomarker.
In summary, tolerogenic mechanisms in aUC patients might be based on the increase of IDO-expressing cells CD8 + /CD16 + /IDO + , CD8 + /CD56 + /IDO + , CD8 + / CD80 + /IDO + , CD8 + /CD123 + /IDO + , CD8 + /CCR6 + / IDO + , IL-10-producing CD4 T cells, and Tregs in a proportional rate of increment of Th17, Th2, and Th1. These IDO + cell subsets might actively participate as a compensatory mechanism to develop peripheral tolerance inducing the polarization of CD4 + T cells to Treg cells and suppressing Th17, Th2, and Th1 differentiation, albeit functional assessments of IDO-expressing cells are required. In contrast, in aCD patients tolerogenic cells are at lower levels when compared with aUC patients and healthy donors. Our results shed further light into the preponderant role of IDO in autoimmune disorders and certainly deserve to be studied in depth in order to evaluate the clinical relevance of these findings.