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Inflammatory Bowel Diseases:
doi: 10.1097/MIB.0b013e3182a85709
Original Basic Science Articles

Increased Prevalence of Circulating Novel IL-17 Secreting Foxp3 Expressing CD4+ T Cells and Defective Suppressive Function of Circulating Foxp3+ Regulatory Cells Support Plasticity Between Th17 and Regulatory T Cells in Inflammatory Bowel Disease Patients

Ueno, Aito PhD*; Jijon, Humberto MD, PhD*,†; Chan, Ronald MSc*; Ford, Kim BPE*; Hirota, Christina PhD*; Kaplan, Gilaad G. MD, MPh*,†; Beck, Paul L. MD, PhD*,†; Iacucci, Marietta MD; Fort Gasia, Miriam MD*,†; Barkema, Herman W. DVM PhD*; Panaccione, Remo MD*,†; Ghosh, Subrata MD*,†

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Author Information

*Alberta IBD Consortium, and

Department of Medicine, University of Calgary, Calgary, Alberta, Canada.

Reprints: Aito Ueno, The Alberta IBD Consortium, University of Calgary, 3330 Hospital Drive North West, Calgary, Alberta, Canada T2N1N4 (e-mail: aueno@ucalgary.ca).

The Alberta IBD Consortium supported by the Alberta Innovates-Health Solutions.

The authors have no conflicts of interest to disclose.

Supported and funded by the Alberta Inflammatory Bowel Disease Consortium, which is funded by an Alberta Heritage Foundation for Medical Research Interdisciplinary Team Grant (Alberta Heritage Foundation for Medical Research is now Alberta Innovates - Health Solutions).

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.ibdjournal.org).

Received July 04, 2013

Accepted August 6, 2013

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Abstract

Background: IL-17 and Foxp3 double-expressing (DE) CD4+ T lymphocytes are novel crossover immune cell population, but the presence and role of these cells in human intestinal inflammation is unclear. The aim of this study was to investigate the circulating IL-17 and Foxp3 DE CD4+ T lymphocytes in patients with inflammatory bowel disease (IBD).

Methods: The entire cohort consisted of 79 subjects: 31 patients with Crohn's disease, 28 patients with ulcerative colitis, and 20 healthy control subjects (HC). IBD patients with evidence of active disease at endoscopy were entered into the study. Peripheral blood mononuclear cells were used for ex vivo and in vitro studies to assess the characteristics and generation of these novel cells and the function of circulating Foxp3+ CD4+ regulatory T lymphocytes (Treg) in patients with IBD compared with HC.

Results: Patients with IBD had significantly higher prevalence of IL-17 and Foxp3 DE CD4+ T lymphocytes compared with age- and gender-matched HC. These cells expressed RORγt. The ability of Treg cells to suppress autologous T-cell proliferation was reduced by approximately 60% in patients with IBD compared with HC. Increased generation of these DE cells was demonstrated by the modulation of cytokine environment of CD4+ lymphocytes in vitro in patients with Crohn's disease.

Conclusions: Prevalence of circulating IL-17 and Foxp3 DE CD4+ T cells is increased in patients with IBD. Coexpression of RORγt and Foxp3 in these cells implies conversion from Treg cells to Th17 cells. This is associated with a decreased suppressive function of Foxp3+ CD4+ T lymphocytes in patients with IBD.

Inflammatory bowel disease (IBD), which comprises Crohn's disease (CD) and ulcerative colitis (UC), is characterized by chronic intestinal inflammation leading to damage of the intestinal mucosa. The chronic inflammation may result in the activation of circulating T lymphocytes.1 Indeed, a number of studies have demonstrated the existence of distinct subpopulations of circulating T lymphocytes in patients with IBD when compared with healthy individuals.2–4 Various reports have indicated that an imbalance between regulatory T cells (Treg) and IL-17–secreting CD4+ T cells (Th17 cells) may be associated with T-cell–mediated chronic inflammation and autoimmune diseases.5–7

Th17 lymphocytes are a recently discovered T-cell subset that may be of prime importance in the pathogenesis of chronic inflammatory diseases.8,9 Aberrant expression of Th17 cells in peripheral blood has been reported in UC.10 Conversely, CD4+, CD25+ Foxp3+ Treg cells maintain immune tolerance by suppressing inflammation resulting from aberrant immune responses to self-antigens and commensal bacteria. Treg cells can be derived from thymic precursors through IL-2 signaling (naturally developed Treg [nTreg]) or from naive CD4+ T cells (Th0) in the organs in the presence of TGF-β and all-trans retinoic acid (inducible Treg [iTreg]). Numbers of circulating Foxp3+ Treg cells have been shown to be inversely related to levels of C-reactive protein in patients with IBD treated with infliximab, suggesting the possibility of decreased tolerance during active inflammation.11,12 Despite very distinct phenotypes, Treg and Th17 cells are developed from the same precursor lineage under the influence of distinct cytokine microenvironments.13,14 In addition to TGF-β1, which is a common requirement for both Treg and Th17 generation, the development of Treg cells from Th0 cells requires retinoic acid, whereas the development of Th17 cells requires IL-21 signaling in humans.15–17

Recent studies have reported that CD4+ T cells may demonstrate more “plasticity” between the T-cell subsets, rather than concrete committed as previously thought. For instance, interferon (IFN)-γ and IL-17 double-expressing (DE) cells are considered a crossover transition of Th17 into Th1 lymphocytes.18 This cell population represents an efficient local host defense system, which shifts host defense from targeting extracellular pathogens to intracellular microbial infections19 and may contribute to autoimmune pathogeneses in both mouse models and in human diseases.20,21 Furthermore, under proinflammatory conditions, Treg cells may differentiate into Th17 cells, a paradigm shift involving altered cytokine patterns and having as yet largely unknown consequences for human disease initiation or progression.22 This novel and recently described IL-17 and Foxp3 DE CD4+ T-cell population characterizes a more unusual subpopulation of CD4+ T cells than IFN-γ and IL-17 DE T cells. These cells are thought to develop in the context of reciprocal regulation between Treg and Th17 cells23 resulting from concomitant but contradictory expressions of their signature transcription factors: Foxp3 for Treg and RORγt for Th17.24 Under the influence of proinflammatory cytokines, such as IL-1β, IL-6, IL-21, IL-23, and TGF-β, Th17 cells can be generated from CD4+ naive T cells and from the memory/effector CD4+ T-cell population.24 The current concept of Th17 plasticity and development is illustrated in Figure 1.

Figure 1
Figure 1
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The importance of these crossover immune cells in T-lymphocyte biology is just a beginning to be appreciated.25 The functional properties of these DE cells (ie, whether they are pathogenic or protective) are poorly understood and may underlie contradictory functions carried out by what was originally thought to be the same T-lymphocyte subset, such as Th17 cells.26 Because the IL-23/Th17 axis is emerging as a therapeutic target for inflammatory diseases, our understanding of this area is crucial. In the context of IBD, regulatory T-cell therapy has been well-tolerated and demonstrated some dose-related efficacy.27 With this in mind, it is now vitally important to determine whether these cells will demonstrate any plasticity under inflammatory conditions to fully validate these regulatory cells as a potential therapy.28

In this study, we investigated the prevalence and function of circulating IL-17+ Foxp3+ CD4+ T cells in peripheral blood from both the patients with CD and UC. The investigation of this novel DE “crossover” phenotype in peripheral blood may further establish the concept of T-lymphocyte plasticity in patients with IBD.

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MATERIALS AND METHODS

Participant Cohort

Patients with documented IBD and evidence of active disease at endoscopy were entered into the study. Peripheral blood was collected within 10 days of the qualifying endoscopy. Age- and gender-matched healthy control subjects (HC) were recruited from a cohort of patients with normal colonoscopic finding at the time of colon cancer screening. Baseline characteristics collected included disease categories (ie, CD, UC, or HC), duration of the disease, and medication utilization. Blood (40 mL) was collected from each subject using standard phlebotomy methods and placed in heparinized tubes. The cohort consisted of 79 subjects: 31 CD, 28 UC, and 20 HC. Demographic information of each cohort is summarized in Table 1.

Table 1
Table 1
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Ex Vivo Peripheral Blood Mononuclear Cell Culture

Peripheral blood mononuclear cells (PBMCs) were separated by standard Ficoll-Histopaque density gradient centrifugation. PBMCs (500,000 cells/mL) were cultured in the presence or absence of phorbol-12-myristate-acetate (PMA, 5 μg/mL; Sigma-Aldrich, Burlington, Canada) and ionomycin (5 nM; Sigma-Aldrich, St Louis, MO) for 15 hours plus addition of monensin solution (GolgiStop; BD Biosciences, Mississauga, Canada) according to the manufacturer’s instructions for the last 12 hours of culture preparation to assess the proportion of CD4+ T-cell subsets, defined as Th17, Th1, Th2, Treg, and Foxp3/IL-17 double-positive cells, in peripheral blood. Serum-free X-VIVO 10 medium (Lonza, Allendale, NJ) containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, gentamicin, 2-mercaptoethanol, sodium pyruvate, and L-glutamine (all additives from Life Technologies, Burlington, Canada) was used for cell cultures throughout this study. Supernatants were collected from cell cultures after 16 hours of culture with or without PMA and ionomycin in the absence of monensin for IL-17A and F measurements by Luminex (Eve Technologies, Calgary, Canada).

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Flow Cytometry Analysis

To determine CD4+ helper T (Th) cell subsets, harvested cells were washed with phosphate-buffered saline and stained with fluorochrome-conjugated monoclonal antibodies against the following cell-surface markers: CD3 (clone OKT3; eBioscience, San Diego, CA), CD4 (clone SK3; BD Biosciences), CD25 (clone M-A251; BD Biosciences), CD45RA (clone HI100; eBioscience), and CD45RO (clone UCHL1; eBioscience). Cells were then fixed and permeabilized using a Foxp3 staining kit (eBioscience) according to the manufacturer’s instructions. After washing with 1% fetal bovine serum containing phosphate buffered saline, pH7.4 buffer, cells were stained with fluorochrome-conjugated antibodies against the following intracellular cytokines or transcription factors: IFN-γ (clone 4S.B3; eBioscience), IL-4 (clone 8D4-8; BD Biosciences), IL-17A (clone N49-653; BD Biosciences), Foxp3 (clone 259D/C7; BD Biosciences), and RORγt (clone AFKJS-9; eBioscience). T-cell proliferation and CD4+ T-cell polarization were measured by the 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; BioLegend, San Diego, CA) fading method and by the intracellular expression of cytokines and Foxp3, respectively. All samples were acquired on an LSR II flow cytometer (BD Biosciences), and postacquisition analyses were performed using the FlowJo software (TreeStar, Inc, Ashland, OR).

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CD25+ Regulatory T Cell Isolation and Treg Generation

Th cells were magnetically enriched (BD Bioscience) from the PBMC fraction. The naive T cells were further selected by CD45RA expression using magnetic particles (BD Bioscience). The enriched cells were labeled with fluorochrome-conjugated anti-CD4 and anti-CD25 antibodies. The CD25-positive and CD25-negative populations were sorted from the total Th cell population on a FACSAria flow cytometer and sorter (BD Biosciences). After sorting, the purity of each population was verified by flow cytometric analysis for Foxp3+ regulatory T cells (see Fig., Supplemental Digital Content 1, http://links.lww.com/IBD/A289). The sorted cells were labeled with CFSE and cultured (50,000 cells/well) in a round-bottom 96-well plate for 10 days with anti-CD3 and anti-CD28 stimulation beads (Miltenyi, Auburn, CA) plus recombinant human IL-2 (25 ng/mL; R&D Systems, Minneapolis, MN) in the presence of human recombinant TGF-β (10 ng/mL; R&D Systems). PMA (5 μg/mL), ionomycin (5 nM), and monensin solution were added to the culture medium for the last 6 hours before harvesting cells.

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Treg Suppressive Function

CFSE-labeled CD25 CD4+ T cells were cultured (50,000 cells/well) in round-bottom 96-well plates for 5 days with plate-bound anti-CD3 (10 μg/mL plate bound) and soluble anti-CD28 (10 μg/mL) antibodies. Autologous CD25+ cells were added to half of the cultured cells in a 1:2 ratio (25,000 CD25+ cells plus 50,000 cells CD25 cells/well). Proliferation of CD25 cells was assessed by the CFSE fading method. Suppression ability was calculated by the following formula: [CD25 only]−[CD25 and CD25+ coculture]/[CD25 only] × 100.

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CD4+ T Cells Isolation and Th17 Polarization

CD4+ T helper (Th) cells were magnetically enriched (BD Bioscience) from the PBMC fraction. After magnetic selection, the purity of the naive T-cell population (>95% CD3+ CD4+) was verified by flow cytometric analysis. The enriched cells (50,000 cells/well) were labeled with CFSE and cultured in round-bottom 96-well plates for 10 days with anti-CD3 (1 μg/mL, plate bound) and soluble anti-CD28 (2 μg/mL) in the presence or absence of one of the following cytokine cocktails: (1) IL-1β (25 ng/mL), IL-6 (25 ng/mL), and IL-23 (100 ng/mL) or (2) TGF-β (5 ng/mL) and IL-21 (25 ng/mL). PMA (5 μg/mL), ionomycin (5 nM), and monensin solution were added to the culture medium for the last 6 hours before harvesting cells. T-cell proliferation and polarization were then assessed within cytokine cocktail–treated and untreated groups.

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Statistical Analyses

Data among different cohorts were compared using a 1-tailed nonpaired t test. Data within the same cohort were analyzed through 1-tailed paired t tests. For both analyses, P < 0.05 was considered significant.

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ETHICAL CONSIDERATIONS

This study was approved by the Conjoint Health Research Ethics Board of the University of Calgary. All research subjects consented to participate in this study before blood collection and chart review.

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RESULTS

Foxp3 and IL-17 DE CD4+ T-Cell Populations Are Increased in PBMC from Patients With IBD

First, we investigated the prevalence of IL-17–secreting CD4+ T-cell (Th17) and regulatory T-cell (Treg) subsets in peripheral blood of research subjects. A greater percentage of IL-17–secreting CD4+ T cells (Th17) and Foxp3+ CD4+ T cells (Treg) was found in samples from patients with IBD than in samples from HC subjects (Fig. 2A). Overall, the prevalence of the Treg cell population was increased in both CD (mean, 7.57%; SD, 5.57) and UC (mean, 5.93%; SD, 4.60) compared with HC (mean, 3.37%; SD, 2.90; HC versus CD, P = 0.005; HC versus UC, P = 0.04; Fig. 2B, left panel). The circulating Th17 population was increased in patients with CD (mean, 5.88%; SD, 4.62) and in patients with UC (mean, 6.68%; SD, 8.30) compared with HC (mean, 3.37%; SD, 2.90; HC versus CD, P = 0.01; HC versus UC, P = 0.03; Fig. 2B, middle panel). Conversely, the prevalence of IFN-γ–secreting CD4+ (Th1) T cells was not different among cohorts (CD: mean, 5.76%; SD, 8.27; UC: mean, 4.61%; SD, 8.39; and HC: mean, 1.84%; SD, 2.22; CD versus UC, P = 0.28; CD versus HC, P = 0.13; UC versus HC, P = 0.31; Fig. 2B, right panel). We then examined IL-17+ Foxp3+ DE CD4+ T cells present in PBMCs. The circulating DE cell population was increased in both patients with CD (mean, 1.16%; SD, 1.26) and those with UC(mean, 0.97%; SD, 1.14), whereas this cell population was barely detectable in HC (mean, 0.24%; SD, 0.30; HC versus CD, P = 0.005; HC versus UC, P = 0.0009, Fig. 2C), suggesting that the circulating DE cells are generated more frequently in patients with IBD compared with HC.

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IL-17 and Foxp3 DE T Cells Express the Transcription Factor RORγ

Next, we explored whether these IL-17 and Foxp3 DE cells also express the Th17 signature transcription factor RORγt. Expression of RORγt was found in IL-17 single-positive cells and in IL-17 and Foxp3 DE cells but not in Foxp3 single-positive cells, regardless of the cohort (Fig. 3). DE cells expressed 2 different T-cell transcription factors, RORγt and Foxp3, suggesting that these “crossover” DE cells may be converting from Treg to Th17 cells. This population was also found more abundantly in patients with IBD than in HC subjects, implying that this conversion may be due to dysregulated immunity in patients with IBD.

Figure 3
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Exogenous TGF-β Increased the Generation of Treg Cells in UC Samples

To examine specific cytokines that could be increasing the generation of Th17, Treg, and/or DE cells in patients with IBD, we first evaluated the involvement of TGF-β in Treg generation using blood samples. We selected CD25 CD45RA+ CD4+ T cells to perform a Treg generation assay in vitro, assessing the ability of non–Treg naive Th cells polarizing into Treg cells under the influence of exogenous TGF-β (Fig. 4A). The purity of our cell population was confirmed by flow cytometry to contain <0.1% of Foxp3+ cells and <1% of RORγt+ cells (Fig., Supplemental Digital Content 1, http://links.lww.com/IBD/A289), followed by the measurement of intracellular IL-17 and Foxp3 expressions to define the Treg, Th17, and DE cell populations. Under these conditions, exogenous TGF-β generated significantly more Treg cells in patients with UC (mean, 23.8%; SD, 15.7) compared with other cohorts (CD: mean, 5.9%; SD, 4.9; CD versus UC, P = 0.03; HC: mean, 3.8%; SD, 5.8; HC versus UC, P = 0.04), but there was no significant effect of TGF-β on the generation of Th17 cells (CD: mean, 2.1%; SD; 2.2; UC: mean, 17.8%; SD, 37.9; HC: mean, 7.4%; SD, 12.1) and DE cells (CD: mean, 0.47%; SD, 0.45; UC: mean, 8.3%; SD, 18.0; HC: mean, 3.3%; SD, 5.7) among cohorts, although there was variability in the generation of these 2 cell populations in samples from patients with UC. These results suggest an increased sensitivity of patients with UC to TGF-β in the generation of Treg cells.

Figure 4
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Impaired Suppressive Function of CD25+ Foxp3+ Regulatory T Cells in Patients With IBD

It is very difficult to investigate Foxp3 and IL-17 DE T cells for subsequent in vitro functional assays including T-cell suppression because of its rare predominance. Therefore, we isolated CD25+ cells using this as a surrogate marker for Foxp3 from peripheral blood of patients with IBD. Specifically, we examined the ability of Treg cells to reduce the proliferative capacity of a non-Treg T-cell population using a coculture system of autologous CD25+ and CD25 cells (Fig. 5A). Postsorting analysis confirmed that more than 80% of the CD25+ population and less than 0.1% of the CD25 population expressed Foxp3 before initiating the coculture (Fig., Supplemental Digital Content 1, http://links.lww.com/IBD/A289). There was no significant difference in the proliferation of the CD25 population in the absence of a CD25+ population (CD: mean, 51.9%; SD, 22.7; UC: mean, 44.6%; SD, 11.22; HC: mean, 40.6%; SD, 19.59; CD versus UC, P = 0.42; CD versus HC, P = 0.27; UC versus HC, P = 0.19), suggesting that the proliferative ability of pan CD4+ T cells was equivalent among cohorts. Coculture with CD25+ cells led to a decrease in CD25 cell proliferation by 20% to 30% in HC samples (HC: mean, 16.51%; SD, 11.11); however, CD25+ cells from patients with IBD were not as effective in reducing autologous CD25 cell proliferation (CD: mean, 40.5%; SD, 20.8; UC: mean 33.1%, SD 21.40; CD versus UC, P = 0.27; CD versus HC, P = 0.05; UC versus HC, P = 0.13). Overall, the ability of Treg cells to suppress autologous T-cell proliferation was reduced by approximately 60% in IBD samples (CD: mean, 23.6%; SD, 16.5; UC: mean, 30.3%; SD, 15.3) compared with that of Treg cells from HC samples (mean 61.1%, SD 8.8; HC versus CD, P = 0.01; HC versus UC, P = 0.008), indicating that the suppressor function of Treg cells is significantly impaired in patients with IBD (Fig. 5C).

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Increased Sensitivity to Th17-Generating Cytokines in IBD Samples

To assess the influence of cytokines on T-cell polarization upon activation, Th cells were isolated from PBMC and cultured for 10 days with CD3 and CD28 antibodies in the presence or absence of TGF-β/IL-21 cytokine cocktail (Fig. 6A) or IL-1β/IL-6/IL-23 cytokine cocktail (Fig. 6C), which are required for the conversion of iTreg and nTreg into Th17 cells, respectively (Fig. 1). The addition of TGF-β and IL-21 significantly increased Foxp3+ Treg generation in both IBD cohorts (Fig. 7B; CD: P = 0.007; UC: P = 0.05) and also increased generation of both Th17 cells and DE cells in CD samples (P = 0.03 and P = 0.009, respectively). Of note, under these culture conditions, CD samples displayed a significantly higher proportion of Th17 cells both before and after cytokine treatment (Fig. 6B, D, top right panel in both) when compared with HC samples. Furthermore, after treatment with TGF-β and IL-21, CD samples contained a significantly higher proportion of Th1 cells compared with UC samples (Fig. 6B; P = 0.03). As shown in Fig. 6D, we further observed that the addition of IL-1β, IL-6, and IL-23 significantly increased the Foxp3+ Treg population in both IBD cohorts without significantly affecting the Treg population in HC samples (Fig. 6D top left panel; CD: P = 0.03; UC: P = 0.03). This cocktail also increased the generation of Th17 cells in UC samples (Fig. 6D top right panel; P = 0.04) and the generation of DE cells in CD samples (Fig. 6D bottom left panel; P = 0.003), although having no effect on Th1 generation in either IBD cohort, despite its ability to promote Th1 generation in HC samples (Fig. 6D bottom right panel; P = 0.05). Furthermore, it led to a higher proportion of Th17 cells in UC samples compared with HC samples (Fig. 6D, top right panel; P = 0.002) and to a higher proportions of Th1 cells in CD and HC compared with UC samples (Fig. 6D bottom right panel; UC versus CD, P = 0.04 and UC versus HC, P = 0.02). Overall, the addition of either cytokine cocktail led to similar outcomes, namely, an increase in the generation of Treg and DE cell populations in patients with CD and an increase in the generation of Treg cell populations in patients with UC. Furthermore, under these conditions, both IBD cohorts displayed generally higher proportions of Th17 cells when compared with the HC group. DE cells significantly increased with either cytokine cocktail only in patients with CD.

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Figure 7
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Impaired IL-17A and F Secretion From PBMC in UC Samples
Figure 6
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Finally, we investigated whether Th17 function was altered in patients with IBD by measuring levels of IL-17A and F, signature cytokines secreted by Th17 cells. Ex vivo assays identified that PBMC of patients with CD demonstrated significantly increased levels of IL-17A secretion compared with UC and HC samples and also significantly increased levels of IL-17F compared with UC samples on PMA and ionomycin stimulation (Fig. 7). These results suggest that despite a 3-fold increase in the percentage of Th17 cells within PBMCs from patients with UC compared with HC (Fig. 2B), secretion of IL-17A and F was impaired in the UC samples.

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DISCUSSION

In this study, we have reported on the prevalence and development of IL-17+ Foxp3+ DE CD4+ T-cell subsets in peripheral blood from patients with IBD. We made several observations: (1) An increased prevalence of IL-17 and Foxp3 DE CD4+ T cells was observed in PBMCs of patients with CD and UC, and this cell population also expressed RORγt in addition to Foxp3 (Figs. 2C and 3). (2) Two Th17-driving cytokine cocktails,29 TGF-β/IL-21 and IL-1β/IL-6/IL-23, increased IL-17 and Foxp3 DE cells in patients with CD but not in those with UC and in HC (Fig. 6B, D). (3) Impaired suppressive function of Treg was observed in both CD and UC patient populations (Fig. 5C). In contrast, impaired IL-17 secretion by PBMC was found only in patients with UC (Fig. 7).

In PBMC, patients with CD and UC demonstrated a higher prevalence of conventional Treg and Th17 cells (Fig. 2B). Previous studies using CD25 as a Treg marker showed that there was a reduction in the prevalence of Treg cells among PBMCs from patients with IBD, whereas studies using Foxp3 as a Treg marker, including our study, have demonstrated an increase in Treg prevalence among PBMCs from patients with IBD compared with HCs (Fig. 2B).30,31 Recently, Hovhannisyan et al32 reported an increased prevalence of IL-17+ Foxp3+ DE CD4+ T cells in the lamina propria of inflamed colonic tissues from patients with CD compared with normal tissues from patients with colon cancer. Their observations also suggested that DE cells exist in CD patients with active disease but not in CD patients in remission. It was further shown that lamina propria T cells could be converted to IL-17+ Foxp3+ DE cells using exogenous cytokine cocktails.32 Similarly, we also demonstrate Treg/Th17 DE T cells using PBMC in this study (Fig. 2C) expressing both RORγt and Foxp3. The previously mentioned study32 did not find differences in peripheral blood as we have shown here. This may be explained by differences in our stimulation and/or culture conditions because we have used higher concentrations of PMA and ionomycin and longer incubation times compared with the previous study.

In vitro, we have observed the generation of IL-17 and Foxp3 DE cells in response to either TGF-β/IL-21 or IL-1β/IL-6/IL-23 in patients with CD (Fig. 6B, D). This contrasts with the results in UC, where this population failed to generate in response to either cocktail. These findings suggest that PBMC from patients with CD are more predisposed to polarize toward DE cells under the influence of Th17-polarizing cytokines. In contrast, UC-derived PBMC showed a greater ability than CD- and HC-derived cells to generate Foxp3+ Treg cells from CD25 Treg-depleted PBMC (presumably all non-Treg cells) in the presence of supplemental TGF-β, which may suggest that UC-derived PBMC are more sensitive to TGF-β for the generation of Treg cells than CD-derived samples (Fig. 4B). The addition of the IL-1β/IL-6/IL-23 cocktail to UC-derived samples increased the generation of Th17 and Treg populations, whereas the addition of TGF-β/IL-21 only increased the Treg population in these samples (Fig. 6B, D). Interestingly, supplementation with TGF-β induced Treg but not Th17 cells regardless of the presence of IL-21 (Figs. 4B and 6B). However, these observations fail to explain the high prevalence of IL-17+ Foxp3+ DE cells found in peripheral blood from patients with UC. Future experiments looking at genotype, receptor abundance, and downstream signaling events in precursor cell populations under the influence of the various cytokine cocktails will be required to understand why a high prevalence of circulating IL-17+ Foxp3+ DE cells is found in patients with UC.

A lesser amount of TGF-β together with IL-6 or IL-21 would be expected to promote Th17 differentiation, whereas a larger amount of TGF-β would be expected to induce Treg differentiation under the control of the vitamin A metabolite all-trans retinoic acid in the periphery.33 Generally, these 2 cell populations are clearly distinct from each other and demonstrate counterregulatory functions. For instance, Foxp3, which is the signature transcription factor of Treg cells, can bind the promoter region of the transcription factor, RORγt, which is expressed in Th17 cells, thus inhibiting the expression of this transcription factor and blocking RORγt-dependent differentiation pathways and IL-17 expression.33 To date, CD45RA+ CD25+ Foxp3+ nTreg cells comprise the only identified naive T-cell population that constitutively expresses a lineage-specific transcription factor, namely, Foxp3. As a result, Treg cells are precommitted to differentiate into a specific lineage even before priming. Several recent studies have documented similarities in the development of the Foxp3+ Treg and Th17 lineages.34–36 The stimulation of human Foxp3+ Treg cells in the presence of lipopolysaccharide-activated monocytes and IL-2 converts them into Th17 cells,34 and recently, a subpopulation of memory Treg cells that coexpress RORγt and secrete high levels of IL-17 ex vivo has been identified.35 Together, these findings suggest that human Th17 cells could preferentially originate from Treg cells rather than from conventional naive CD4+ T-cell precursors.37

Regarding the function of Treg, both CD and UC PBMCs demonstrated impairment in Treg suppressive function (Fig. 5B). Buckner38 has discussed that chronic inflammation can result from failures of Treg-mediated regulation by inadequate Treg numbers, defective Treg function, or resistance of effector T cells. Our findings suggest that IBD pathogenesis can be associated with defective Treg function. UC-derived PBMCs demonstrated a downregulation in IL-17A and F secretion (Fig. 7). These results suggest that aberrant Th differentiation and function may contribute to disease pathogenesis in these conditions. A recent study has shown that human CD4+ Foxp3+ CCR6+ Treg cells can differentiate into IL-17–producing cells following the stimulation of T-cell receptor in the presence of IL-1β, IL-2, IL-21, IL-23, and human serum. This, together with the finding that the human thymus does not contain IL-17–producing Treg cells, suggests that IL-17+ Foxp3+ Treg cells are generated in the periphery. It has been proposed that IL-17–producing Treg cells play a critical role in antimicrobial defense, while controlling autoimmunity and inflammation.36 However, the increased presence of this population in the both UC and CD relative to HC might suggest that this population either results from or contributes to the derangement of immune homeostasis that is evident in IBD.

Reactivity of T lymphocytes was once considered pathogen specific, committed, and deliberate. Recently, this concept is challenged by the discovery of crossover cells such as innate lymphocyte cells, which possess the same role as adaptive immune cells but act immediately after pathogen invasion similar to other innate cells.25 The apparent plasticity of T cells can be proposed as another form of crossover because their commitment to T-cell polarization can be modified by culture conditions. Studies have recently revealed that another crossover DE population, namely, IFN-γ+ IL-17+ DE T cells, is more abundant in patients with IBD than in healthy individuals.39 The low suppressive ability of Treg cells isolated from patients with IBD may be the result of increased IL-17+ Foxp3+ cells in the disease, as Treg cells from patients with IBD could modify their phenotypes leading to inflammation. However, due to the paucity of this IL-17+ Foxp3+ population, it is difficult to prove this hypothesis beyond the general impairment of Treg suppressive function observed in IBD (Fig. 5B). Furthermore, failure of anti–IL-17 antibody administration in clinical trials suggests that the Th17 population may play a protective role in CD.40 Further understanding of the function of these “crossover” T lymphocytes can provide a missing link in the pathogenesis of T-cell–mediated immune dysregulation disorders, such as IBD. Using Foxp3+ regulatory T cells for therapy in inflammatory diseases requires an understanding of how the generation of DE T lymphocytes in the local inflammatory environment may interfere with Treg suppressive function.41 Such an understanding may contribute to the future development of personalized medicine in that the success of Treg administration therapy could be predicted from the cell propensity in blood of the patients whether the administrated Treg cells will convert into pro-inflammatory Th17 cells. The transcription control of Th17 and Treg cells is complex and depends on the interaction between Runx1, RORγt, and Foxp3 and the cytokines these cells are exposed to.42

In conclusions, the induction of Foxp3 and IL-17 DE cells probably occurs through different pathways in both patients with CD and those with UC. Both IBD patient cohorts demonstrate an increased prevalence of circulating Foxp3 and IL-17 DE cells, yet only PBMCs from patients with CD possess the potential to develop into Foxp3 and IL-17 DE cells under the influence of exogenous cytokines. The increased prevalence of Foxp3 and IL-17 DE cells may be a result of aberrant Th17 generation under the influence of several cytokines in patients with CD, but this mechanism fails to explain the increased prevalence of this cell population in the peripheral blood of patients with UC. The paradigm of an imbalance between Treg and Th17 cells leading to inflammation in IBD can no longer be explained solely by these 2 populations individually, but rather, we must also take into account the prevalence of intermediate Foxp3 and IL-17 DE cells.

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ACKNOWLEDGMENTS

All researchers would like to thank Dr. Karen Poon, the Microbe Core Laboratory in Snyder Institute for chronic disease in University of Calgary for technical assistance on flow cytometry.

Author Contributions: Designing and planning study: A. Ueno and S. Ghosh. Clinical aspects: H. Jijon, S. Ghosh, R. Panaccione, and G. G. Kaplan. Patient recruitments: K. Ford, G. G. Kaplan, R. Panaccione, S. Ghosh, P. L. Beck, M. Iacucci, and M. Fort Gasia. Sample coordination: K. Ford. Experimental performance: A. Ueno, R. Chan, and H. Jijon. Writing manuscript: A. Ueno, H. Jijon, C. Hirota, S. Ghosh, H. W. Barkema, and R. Panaccione. Financial support: S. Ghosh and H. W. Barkema.

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Keywords:

IL-17 and Foxp3 double-expressing cells; inflammatory bowel disease; Th17 cells; regulatory T cells

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