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Inflammatory bowel disease
Differential regulation of interleukin 17 and interferon γ production in inflammatory bowel disease
  1. L Rovedatti1,2,
  2. T Kudo1,
  3. P Biancheri1,2,
  4. M Sarra3,
  5. C H Knowles4,
  6. D S Rampton5,
  7. G R Corazza2,
  8. G Monteleone3,
  9. A Di Sabatino1,2,
  10. T T MacDonald1
  1. 1
    Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK
  2. 2
    First Department of Medicine, Fondazione IRCCS Policlinico S. Matteo, Centro per lo Studio e la Cura delle Malattie Inflammatorie Croniche Intestinali, University of Pavia, Pavia, Italy
  3. 3
    Dipartimento di Medicina Interna e Centro di Eccellenza per lo Studio delle Malattie Complesse e Multifattoriali, Università Tor Vergata, Rome, Italy
  4. 4
    Centre for Academic Surgery, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK
  5. 5
    Centre for Gastroenterology, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK
  1. Correspondence to Professor T T MacDonald, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London E1 2AT, UK; t.t.macdonald{at}qmul.ac.uk

Abstract

Background and Aims: Interleukin 17 (IL17) is now known to be involved in a number of chronic inflammatory disorders. However, the mechanisms regulating its production in inflammatory bowel disease (IBD) are still unclear.

Methods: Endoscopic biopsies or surgical specimens were taken from inflamed and uninflamed colonic mucosa of 72 patients with IBD (38 with Crohn’s disease and 34 with ulcerative colitis), and normal colon of 38 control subjects. IL17 and interferon γ (IFNγ) were detected by ELISA in the supernatants of biopsies cultured ex vivo, and anti-CD3/CD28-stimulated lamina propria mononuclear cells (LPMCs) incubated with IL12, IL23, IL1β plus IL6, transforming growth factor β1 (TGFβ1), or anti-IL21 neutralising antibody. Intracellular flow cytometry was performed to analyse mucosal Th17 and Th1/Th17 cells.

Results: IL17 production by organ culture biopsies was higher in IBD inflamed mucosa than IBD uninflamed mucosa and controls, and was equivalent in amount to IFNγ. Anti-CD3/CD28-stimulated IBD LPMCs produced higher IL17 amounts compared to controls. The percentages of Th17 and Th1/Th17 cells were increased in patients with IBD. IL23 and IL1β plus IL6 had no effect on IBD LPMC production of IL17; however, IL12 markedly increased IFNγ production and decreased IL17 production. TGFβ1 dose-dependently decreased IFNγ, but had no significant inhibitory effect on IL17 production. Blocking IL21 significantly downregulated IL17 production.

Conclusions: Our findings support a role for IL12, TGFβ and IL21 in modulating IL17/IFNγ production in IBD. The abundant IL17 in inflamed IBD mucosa may help explain the relative lack of efficacy of anti-IFNγ antibodies in clinical trials of Crohn’s disease.

https://doi.org/10.1136/gut.2009.182170

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    Crohn’s disease and ulcerative colitis are chronic inflammatory bowel diseases (IBDs) thought to be caused by an abnormal immune response directed against normal constituents of the luminal flora.1 Treatment strategies are largely focused on the suppression or modulation of the excessive immune response in the gut. However, due to the involvement of different cytokines and individual variability, the main challenge is to identify inflammatory pathways and develop disease-specific therapies that can be tailored for each patient.2

    It is well known that Crohn’s disease is characterised by a preponderance of T helper cell type (Th)1-associated cytokines, including interferon γ (IFNγ), tumour necrosis factor α (TNFα) and interleukin 12 (IL12), while Th2 cytokines, such as IL5 and IL13, are considered to be dominant in ulcerative colitis.3 Nonetheless, both Crohn’s disease and ulcerative colitis share crucial end-stage pathways, as shown by the enhanced production of IL21 and IL23 in both the disorders.4 5

    During the past few years, two novel subsets of CD4+ T cells have been identified, namely Th17 cells, which produce IL17, and Th1/Th17 cells, which produce both IFNγ and IL17.6 7 Both subsets can originate from a CD161+ CD4+ natural killer (NK) T cell precursors present in the umbilical cord blood, in the presence of IL1β and IL23 as polarising cytokines, but whether this pathway is important in vivo in man is not known.8 Many chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes,9 and IL17 over-expression has been found in a number of inflammatory disorders, including IBD.10 11 12 13 14 15 16 17 However, the relative roles of Th1/Th17 cells in chronic gut inflammation is still under debate.

    IL17 is a pleiotropic cytokine which acts on both immune and non-immune cells. IL17 receptor is expressed on a wide range of cells, including osteoblasts, fibroblasts, epithelial and endothelial cells.18 Ligation of IL17 with its receptor induces IL6 and IL8 production in vitro through mitogen-activated protein kinase pathways, thus favouring the recruitment of neutrophils at sites of inflammation,19 and triggers T cell proliferation and upregulation of a number of pro-inflammatory molecules, such as inducible nitric oxide synthase and IL1β.20 Additionally, IL17 induces pro-inflammatory cytokine production by macrophages, thus creating a link between innate and adaptive immunity,21 and plays a key role in host defence against bacteria and fungi, particularly at mucosal surfaces, by favouring the local recruitment of immune cells.22

    Contrasting data are available on the role of IL17 in Crohn’s disease and ulcerative colitis. Kobayashi et al23 detected high levels of IL17 transcripts in both Crohn’s disease and ulcerative colitis mucosa compared to normal gut. This group also found that high IL23p19 transcript levels correlated with IL17 levels in ulcerative colitis and IFNγ in Crohn’s disease, thus concluding that IL23 differentially regulates the Th1/Th17 balance in ulcerative colitis and Crohn’s disease. In contrast, Fujino et al10 showed by immunohistochemistry that there was higher IL17 expression in Crohn’s disease than ulcerative colitis mucosa. The demonstration by Fuss et al24 that IL23 is upregulated in Crohn’s disease but not ulcerative colitis further confuses the picture, and makes it difficult to understand the control of IL17 production in ulcerative colitis. A number of studies have examined the role of different cytokines in inducing IL17 by virgin CD4+ T cells. A role for IL1β, IL6 and TGFβ in polarising Th17 cells in man is clear,25 26 27 but the contribution of these cytokines to IL17 production in the gut is not known. Again, it is well known that IL12 is upregulated in Crohn's disease and induces an increase of IFNγ production by Th1 cells.28 However, its influence on IL17-producing LPMCs has not been investigated. IL21 also seems to be implicated in the regulation of IL17 production by lamina propria lymphocytes.29

    In order to clarify these issues, we have analysed lamina propria Th17 and Th1/Th17 populations in patients with IBD, and investigated the control of IL17 production using ex vivo and in vitro experiments of tissues and cells from IBD mucosa.

    Materials and methods

    Patients and tissues

    Colonic biopsies or surgical specimens were taken from macroscopically and microscopically inflamed and uninflamed areas of the mucosa of 38 patients affected by Crohn’s disease (table 1) and 34 patients affected by ulcerative colitis (table 2). Diagnosis of Crohn’s disease and ulcerative colitis was ascertained according to the usual clinical criteria, and the site and extent of the disease were confirmed by endoscopy and histology. In patients with Crohn’s disease, disease activity was assessed by the Crohn’s Disease Activity Index. Patients with scores below 150 were classified as being in remission, whereas those with scores over 450 had severe disease.30 In patients with ulcerative colitis, disease activity was assessed according to the Clinical Activity Index of Rachmilewitz.31 Clinical remission was defined as a score below 4. None of the patients with IBD had been ever treated with cyclosporine, tacrolimus or anti-TNF antibodies. Mucosal samples were also collected from the colon of 27 subjects who had functional diarrhoea at the end of their diagnostic work-up (mean age 38.1 years, range 29–68), and from macroscopically and microscopically unaffected colonic areas of 11 patients undergoing colectomy for colon cancer (mean age 51 years, range 39–70). Biopsy specimens taken from the inflamed mucosa of a patient with radiation colitis, two patients with diverticulitis and two patients with ischaemic colitis were used as disease controls. Some of the mucosal samples were used to isolate LPMCs, some others for organ culture experiments. Informed consent was obtained in all cases.

    View this table:
    Table 1

    Clinical features of patients with Crohn’s disease (n = 38)

    View this table:
    Table 2

    Clinical features of patients with ulcerative colitis (n = 34)

    Organ culture

    Biopsy specimens were placed on iron grids in serum-free HL-1 medium (Cambrex BioScience, Wokingham, UK) with 100 U/ml penicillin and 100 μg/ml streptomycin, in the central well of an organ culture dish, and the dishes placed in a tight chamber containing 95% O2/5% CO2 at 37°C.32 Spontaneous cytokine production by biopsies grown ex vivo was assessed. After 24 h culture, supernatants were snap frozen and stored at −70°C.

    Cell isolation

    LPMCs were isolated and purified from freshly resected surgical specimens or endoscopic biopsies as previously described.32 Briefly, the epithelial layer was removed with 1 mmol/l EDTA (Sigma-Aldrich, Poole, UK). After stirring for 1 h at 37°C, the supernatant was removed and the remaining tissue was treated with type 1A collagenase (1 mg/ml; Sigma-Aldrich) for 2 h with stirring at 37°C. The resulting crude cell suspension was preferentially enriched for intestinal LPMCs using a Ficoll–Paque Plus gradient (Amersham Pharmacia Biotech, Uppsala, Sweden) following the manufacturer’s protocol. Cells from the supernatant were washed twice, resuspended in 1 ml RPMI-1640 medium (Sigma-Aldrich) containing 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin, and kept on ice until used. Cells were not used if viability did not exceed 90%.

    Cell culture

    Freshly isolated LPMCs (2×105 cells/well) were stimulated in anti-CD3-coated 96-well plates (BD Biosciences, Oxford, UK) with soluble anti-CD28 antibody (0.5 μg/ml; eBioscience, San Diego, California, USA), and incubated for 48 h with medium containing IL1β (10 ng/ml) plus IL6 (20 ng/ml), IL23 (10 ng/ml), IL12 (5 ng/ml), TGFβ1 (10, 1, 0.1 and 0.01 ng/ml), a mouse anti-human IL21 neutralising antibody (20 μg/ml; kindly provided by Giovanni Monteleone) or its isotype control (mouse IgG). All the above-mentioned recombinant human cytokines were from R&D System (Minneapolis, Minnesota, USA). For intracellular cytokine staining, purified LPMCs were stimulated for 4 h in RPMI complete medium with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich) in the presence of monensin (2 μmol/l; eBioscience).

    Enzyme-linked immunosorbent assay

    Concentrations of IL17 and IFNγ in organ culture and LPMC supernatants were measured using specific ELISA kits (R&D System), according to the manufacturer’s instructions.

    Flow cytometry

    Surface-staining of LPMCs was performed at 4°C for 30 min with FITC-conjugated anti-CD3 and APC-conjugated anti-CD161 antibodies (BD Biosciences). After fixation with 100 μl Leucoperm A, and permeabilisation with 100 μl Leucoperm B (Serotec, Oxford, UK), PE-conjugated anti-IL17 and PE-Cy5-conjugated anti-IFNγ antibodies (BD Biosciences) were added for 30 min. Appropriate isotype-matched control antibodies were purchased from BD Biosciences and included in all experiments. After washing twice with 250 μl fluorescence-activated cell sorter buffer (phospate buffer containing 1 mmol/l EDTA and 0.02% sodium azide), cells were fixed with 2% paraformaldehyde and analysed by flow cytometry using a FACSAria II Flow Cytometer (BD Biosciences).

    Statistical analysis

    Data were analysed in the GraphPad Prism statistical PC program (GraphPad Software, San Diego, California, USA) using the paired t test and the Mann–Whitney U test. A level of p<0.05 was considered statistically significant.

    Results

    Ex vivo production of IL17 and IFNγ

    First, we aimed to determine the spontaneous production of IL17 and IFNγ by organ culture biopsies from the inflamed and uninflamed mucosa of 17 patients with IBD (seven with Crohn’s disease and 10 with ulcerative colitis) and normal mucosa of 10 control subjects (fig 1). The concentration of IL17 was significantly higher in the supernatants of both Crohn’s disease (mean 188 (SD 50) pg/ml, p<0.01) and ulcerative colitis organ culture biopsies (mean 130 (SD 47) pg/ml, p<0.05) in comparison to controls (mean 27 (SD 5) pg/ml). Uninflamed mucosa from patients with Crohn’s disease and ulcerative colitis showed significantly lower levels of IL17 in comparison to inflamed mucosa (Crohn’s disease: mean 36 (SD 11) pg/ml, p<0.01; ulcerative colitis: mean 48 (SD 14) pg/ml, p<0.05). Likewise, the concentration of IFNγ was significantly higher in the supernatants of both Crohn’s disease (234 (SD 53) pg/ml, p<0.01) and ulcerative colitis organ culture biopsies (201(SD 49) pg/ml, p<0.01) in comparison to uninflamed areas (Crohn’s disease: mean 79(SD 24) pg/ml, p<0.01; ulcerative colitis: mean 69(SD 18) pg/ml, p<0.01) and controls (23 (SD 6) pg/ml). No significant difference was observed in the production of IL17 and IFNγ between patients with Crohn’s disease and those with ulcerative colitis.

    Figure 1
    Figure 1

    Levels of interleukin 17 (IL17) and interferon γ (IFNγ), expressed in pg/ml, in the supernatants of biopsies taken from the inflamed and uninflamed colon of seven patients with Crohn’s disease (CD) and 10 patients with ulcerative colitis (UC), and from the normal colon of 10 healthy controls (HC), cultured for 24 h in the absence of stimuli. Results are mean with the SD. *p<0.01 versus HC and uninflamed areas of Crohn’s disease and ulcerative colitis; **p<0.05 versus HC and uninflamed areas of Crohn’s disease and ulcerative colitis.

    In vitro production of IL17 and IFNγ

    We then determined the production of both IL17 and IFNγ from unstimulated and anti-CD3/CD28-stimulated LPMCs isolated from the inflamed mucosa of 28 patients with IBD (14 with Crohn’s disease and 14 ulcerative colitis), the inflamed mucosa of five patients without IBD (one with radiation colitis, two with diverticulitis and two with ischaemic colitis), and the normal mucosa of 14 control subjects (fig 2). In unstimulated conditions, LPMCs from Crohn’s disease and ulcerative colitis patients produced significantly (p<0.05) higher amounts of IL17 (mean 862 (SD 372) pg/ml and 644 (SD 385) pg/ml, respectively) in comparison to control LPMCs (mean 92 (SD 11) pg/ml) and LPMCs from non-IBD patients (mean 124 (SD 25) pg/ml). No difference was found between control subjects and non-IBD patients, and between Crohn’s disease and ulcerative colitis. Activation with anti-CD3/CD28 antibodies significantly enhanced the LPMC production of IL17 in comparison to unstimulated conditions in all the four groups, particularly in patients with IBD (Crohn’s disease: mean 6421 (SD 1869) pg/ml, p<0.001; ulcerative colitis: mean 4696 (SD 2049) pg/ml, p<0.001; control subjects: 1795 (SD 583) pg/ml, p<0.01; non-IBD patients: 2099 (SD 497) pg/ml, p<0.01). No significant difference was found in CD3/CD28-stimulated LPMC production of IL17 between control subjects and non-IBD patients, or between patients with Crohn’s disease and those with ulcerative colitis. Unstimulated LPMCs from Crohn’s disease patients, ulcerative colitis patients and disease control patients produced significantly (p<0.05) higher amounts of IFNγ (mean 710 (SD 212) pg/ml; 544 (SD 161) pg/ml; and 503 (SD 143) pg/ml, respectively) in comparison to control LPMCs (mean 188 (SD 109) pg/ml). After stimulation with anti-CD3/CD28 antibodies, IFNγ production was significantly enhanced in comparison to unstimulated conditions in all the four groups, particularly in IBD patients and non-IBD patients (Crohn’s disease: mean 7101 (SD 2099) pg/ml, p<0.001; ulcerative colitis: mean 5764 (SD 2038) pg/ml, p<0.001; non-IBD patients: 5532 (SD 2391) pg/ml; p<0.001; control subjects: 2848 (SD 851) pg/ml, p<0.01).

    Figure 2
    Figure 2

    Levels of interleukin 17 (IL17) and interferon γ (IFNγ), expressed in pg/ml, in the supernatants of unstimulated and anti-CD3/CD28-stimulated lamina propria mononuclear cells (LPMCs) isolated from the inflamed colon of 14 patients with Crohn’s disease (CD), 14 patients with ulcerative colitis (UC), and five non-IBD patients patients (one with radiation colitis, two with diverticulitis and two with ischaemic colitis), and from the normal colon of 14 healthy controls (HC), and cultured for 48 h. Results are mean with the SD. †p<0.05 versus unstimulated HC LPMCs; §p<0.01 versus unstimulated HC LPMCs; *p<0.001 versus anti-CD3/CD28-stimulated HC LPMCs and versus all unstimulated LPMCs.

    Analysis of Th17 and Th1/Th17 cells

    To determine the percentage of mucosal T cells producing IL17 and IFNγ, we cultured LPMCs isolated from inflamed mucosa of 17 patients with IBD (10 with Crohn’s disease and seven with ulcerative colitis) and from normal mucosa of nine control subjects for 4 h with PMA and ionomycin in the presence of monensin. As shown in fig 3A, the percentage of CD3+ LPMCs producing IL17 was significantly (p<0.05) higher in Crohn’s disease patients (mean 5.6 (SD 3.3)%) and ulcerative colitis patients (mean 6.7 (SD 3.1)%) in comparison to controls (mean 2.4 (SD 1.9)%). Likewise, the percentage of CD3+ LPMCs producing IFNγ was significantly higher (p<0.05) in Crohn’s disease patients (mean 18.8 (SD 6.0)%) and ulcerative colitis patients (mean 17.5 (SD 2.6)%) in comparison to controls (mean 11.8 (SD 2.7)%). Finally, the percentage of CD3+ LPMCs producing both IL17 and IFNγ was significantly (p<0.05) higher in Crohn’s disease patients (mean 1.9 (SD 1.4)%) and ulcerative colitis patients (mean 2.6 (SD 1.6)%) in comparison to controls (mean 0.7 (SD 0.4)%). No significant difference was found between Crohn’s disease and ulcerative colitis in terms of production of IFNγ alone, IL17 alone, and both IL17 and IFNγ. In fig 3B, representative dot plots show the flow cytometric analysis of gated CD3+ LPMCs producing IL17 and IFNγ in a control subject, a Crohn’s disease and a ulcerative colitis patient. A higher proportion of Th1/Th17 cells were observed in the CD3+CD161+ pool than in the CD3+CD161− pool of LPMCs (fig 3C).

    Figure 3
    Figure 3

    (A) Percentages of CD3+ lamina propria mononuclear cells (LPMCs) producing interferon γ (IFNγ) only (Th1 cells), interleukin 17 (IL17) only (Th17 cells), or both (Th1/Th17 cells), assessed by flow cytometry after cytokine intracellular staining in nine control subjects (HC), 10 patients with Crohn’s disease (CD) and seven patients with ulcerative colitis (UC). Cells were analysed after 4 h stimulation with PMA and ionomycin. Results are mean with the SD. *p<0.05 versus HC LPMCs. (B) Flow cytometric analysis of PMA/ionomycin-stimulated LPMCs producing IL17 and IFNγ in gated CD3+ cells. Numbers within the dot plots represent the percentages of Th1 cells (lower-right quadrant), Th17 cells (upper-left quadrant), and Th1/Th17 cells (upper-right quadrant). The example is representative of experiments performed in nine control subjects, 10 patients with Crohn’s disease and seven patients with ulcerative colitis. (C) Flow cytometric analysis of PMA/ionomycin-stimulated LPMCs producing IL17 and IFNγ in gated CD3+CD161+ and CD3+CD161− cells. Numbers within the dot plots represent the percentages of Th1 cells (lower-right quadrant), Th17 cells (upper-left quadrant), and Th1/Th17 cells (upper-right quadrant). The example is representative of experiments performed in six control subjects, six patients with Crohn’s disease and six patients with ulcerative colitis. PMA, phorbol 12-myristate 13-acetate.

    Control of IL17 and IFNγ production by pro-inflammatory cytokines

    Anti-CD3/CD28-stimulated LPMCs from 11 patients with Crohn’s disease and eight with ulcerative colitis were cultured for 48 h with IL1β plus IL6, IL23 or IL12. No effect was exerted by IL1β plus IL6, or IL23 alone on the production of IL17 and IFNγ both in Crohn’s disease and ulcerative colitis patients (table 3). In contrast, IL12 significantly (p<0.05) decreased IL17 production (from mean 6810 (SD 2200) pg/ml to 2201 (SD 1069) pg/ml; fig 4A), while it significantly (p<0.005) upregulated IFNγ production (from mean 6979 (SD 419) pg/ml to 43 341 (SD 9086) pg/ml; fig 4B). We then investigated the effect of IL21 blockade on IL17 production by culturing anti-CD3/CD28-stimulated LPMCs from four patients with Crohn’s disease with an anti-IL21 blocking antibody (fig 4C). The anti-IL21 antibody significantly (p<0.05) decreased the IL17 production in comparison to IgG-treated cells (from mean 700 (SD 291) pg/ml to 450 (SD 105) pg/ml). A significant (p<0.005) difference was also found in the production of IL17 between anti-CD3/CD28-stimulated cells cultured with IgG and unstimulated conditions (20 (SD 8) pg/ml).

    Figure 4
    Figure 4

    Levels (pg/ml) of interleukin 17 (IL17) (A) and interferon γ (IFNγ) (B) in the supernatants of anti-CD3/CD28-stimulated lamina propria mononuclear cells (LPMCs) from the inflamed colon of three patients with ulcerative colitis (UC) and three with Crohn’s disease (CD), cultured for 48 h with 5 ng/ml IL12. Horizontal bars are mean. *p<0.05 versus anti-CD3/CD28-stimulated LPMCs; **p<0.005 versus anti-CD3/CD28-stimulated LPMCs. (C) Levels (pg/ml) of IL17 in the supernatants of LPMCs isolated from the inflamed colon of four patients with Crohn’s disease stimulated with anti-CD3/CD28 antibodies and cultured for 48 h with 20 μg/ml anti-IL21 blocking antibody or its isotype control (mouse IgG). Results are mean with the SD. *p<0.005 versus unstimulated LPMCs; **p<0.05 versus IgG-treated anti-CD3/CD28-stimulated LPMCs.

    View this table:
    Table 3

    Levels of interleukin 17 and interferon γ (pg/ml) in the supernatants of anti-CD3/CD28-stimulated (αCD3/CD28) lamina propria mononuclear cells isolated from the inflamed colon of 11 patients with Crohn’s disease and eight with ulcerative colitis, and cultured for 48 h with 10 ng/ml interleukin 23 (IL23) or 10 ng/ml IL1β plus 20 ng/ml IL6

    Control of IL17 and IFNγ production by TGFβ1

    To investigate the role of TGFβ in modulating IL17 and IFNγ production, we cultured anti-CD3/CD28-stimulated LPMCs from four patients with Crohn’s disease and three with ulcerative colitis with different concentrations of TGFβ1. As shown in fig 5, at different concentrations of TGFβ1 (10, 1, 0.1 and 0.01 ng/ml) the mean production of IL17 was, respectively, 67.7%, 61.2%, 89.6% and 86.2% of that measured in the presence of medium only, while the mean production of IFNγ was respectively 45.9%, 41.6%, 69.9% and 92.1% of that measured in the presence of medium only. When we compared the inhibitory effects of TGFβ1 on IFNγ with those on IL17, we found a significantly (p<0.05) greater effect on IFNγ production in comparison to IL17 production at the TGFβ1 concentrations of 10 and 1 ng/ml. No significant difference was found at the TGFβ1 concentrations of 0.1 and 0.01 ng/ml.

    Figure 5
    Figure 5

    Production of interleukin 17 (IL17) and interferon γ (IFNγ) by anti-CD3/CD28-stimulated laminal propria mononuclear cells (LPMCs) isolated from the inflamed colon of four patients with Crohn’s disease (CD) and three with ulcerative colitis (UC), and cultured for 48 h with different concentrations of transforming growth factor β1 (TGFβ1) (10, 1, 0.1 and 0.01 ng/ml). Cytokine levels in the supernatants of LPMCs cultured with TGFβ1 are expressed as percentage of the cytokine production in the presence of medium only. Horizontal bars are mean.

    Discussion

    In the present study we provide evidence that IL17 production by gut biopsies grown ex vivo and LPMCs cultured in vitro is higher in IBD patients than in control subjects, and is associated with an increased percentage of Th17 and Th1/Th17 cells. Moreover, we show that a differential regulation is played by pro- and anti-inflammatory cytokines on IL17 and IFNγ production by IBD LPMCs, the former downregulated by IL12 but not TGFβ, and the latter upregulated by IL12 and downregulated by TGFβ. Finally, our findings support a crucial role for IL21 in controlling IL17 production by Crohn’s disease LPMCs.

    We first observed that IL17 production from organ culture biopsies is increased in IBD patients in comparison to controls almost at the same levels of IFNγ. In contrast with Kobayashi et al,23 who found a higher in vivo mucosal production of IL17 in ulcerative colitis than in Crohn’s disease, we did not observe any significant difference between these two disorders. When we measured the concentration of IL17 in the supernatants of isolated LPMCs, we found a higher production of IL17 and IFNγ in IBD patients in comparison to controls, both in unstimulated and stimulated conditions. Again, no difference was found between Crohn’s disease and ulcerative colitis LPMCs. The fact that IL17 production by organ culture biopsies was not increased in uninflamed areas of patients with IBD further strengthens the key role of IL17 in mediating inflammation. Of note, the absence of IL17 upregulation in both unstimulated and stimulated LPMCs from inflamed areas of non-IBD patients suggests the specific contribution of IL17 to the mucosal damage in Crohn’s disease and ulcerative colitis.

    IL17 had been already investigated in the gut of patients with IBD by immunohistochemistry which showed that IL17 is markedly expressed by CD3+ and CD68+ cells in the inflamed mucosa.10 Here we used dual parameter intracellular flow cytometry to identify IL17 and IFNγ producing cells. We examined three cellular subsets, namely IFNγ-producing T cells (Th1), IL17-producing T cells (Th17), and IL17/IFNγ co-producing T cells (Th1/Th17). All were increased in patients with Crohn’s disease and those with ulcerative colitis, without any difference between ulcerative colitis and Crohn’s disease. Interestingly, we observed that the majority of Th1/Th17 cells expressed CD161, a well known marker of NK T cells recently identified on IL17-producing memory T cells.8

    There is good evidence that IL17 production is differentially regulated in mouse and human. In mice, IL1β, IL6 and TGFβ appear to be responsible for Th17 cell differentiation,33 34 while IL23 is required for their expansion and/or maintenance.35 In contrast, human studies showed that TGFβ, IL1β and IL6 are critical for the priming of Th17 responses.34 36 37 More recent studies have shown that IL21, IL23, TGFβ, and IL1β plus IL6 are able to induce IL17 production by naïve human CD4+ T cells isolated from umbilical cord blood.25 26 Hence, it seems that IL1β and IL6 induce IL17 secretion by central memory T cells, while TGFβ and IL21 are required to promote the differentiation of naïve CD4+ T cells into Th17 cells.27 In our hands, neither IL1β plus IL6 nor IL23 alone were able to induce IL17 and IFNγ production by anti-CD3/CD28-stimulated IBD LPMCs. The discrepancy between our data and that of Yang et al27 might depend on the different source of cells used – peripheral blood or inflamed mucosa – which could affect the effector-to-memory T cell ratio in the cell sample. In contrast, IL12 greatly enhanced IFNγ production, while it reduced IL17 secreted by anti-CD3/CD28-stimulated IBD LPMCs. Given that it is known that IFNγ in Crohn’s disease is driven by IL12 and accessory cytokines such as IL18 and IL21, the notion that IL12 suppresses IL17 production is interesting because it suggests that perhaps a component of the rather weak efficacy of anti-IL12/IL23 p40 antibody in active Crohn’s disease is that it allows Th17 differentiation and a second effector function activated by the therapy itself. However, it must be also noted that IFNγ, induced by IL12, appears to play a role in limiting IL17 production.38

    Interestingly, TGFβ, a cytokine known to have a pleiotropic function in controlling gut inflammation,39 significantly reduced IFNγ production by anti-CD3/CD28-stimulated IBD LPMCs, while it did not exert any significant downregulatory effect on IL17 production. This might shed light on the role of IL17 in IBD as a key cytokine in sustaining chronic gut inflammation unable to be regulated by immunomodulatory factors like TGFβ. We have previously shown that IFNγ transcripts are not as well inhibited by active TGFβ in cells from patients with Crohn’s disease as they are in cells from controls.40 We confirmed this here in that TGFβ did not shut off IFNγ production, only reducing it by about 50%. The lack of effect of TGFβ is due to the presence of Smad7, the intracellular inhibitor of TGFβ signalling in cells from IBD patients.41 Critically, however, to understand why TGFβ only barely inhibited IL17 production in IBD is the fact that TGFβ appears to be important for Th17 cell generation. It has been proposed by Zhou et al42 that T cells receiving a TGFβ signal can develop into either the T regulatory cell or Th17 lineage. Foxp3 induction may restrain the differentiation of inflammatory Th17 cells in response to TGFβ in the absence of other pro-inflammatory cytokines by inhibiting RORγt activity. In the presence of pro-inflammatory cytokines, the suppression of Foxp3 expression and inhibitory function, together with the concurrent upregulation or stabilisation of RORγt expression, may favour the development into Th17 lineage. Thus, a fine balance between RORγt and Foxp3 might be critical for immune homeostasis in the gut.

    IL21 is another pro-inflammatory cytokine overproduced in the inflamed intestine of patients with Crohn’s disease. CD4+ T cells infiltrating Crohn’s disease mucosa are the main source of IL21. In Crohn’s disease LPMC cultures, IL21 blockade reduces the expression of p-Stat4 and T-bet and the production of IFNγ.4 In keeping with the findings by Fina et al,29 we did find that IL21 blockade significantly reduces IL17 secretion by Crohn’s disease LPMCs.

    In conclusion, this study demonstrates the importance of Th17 and Th1/Th17 cells in the pathogenesis of IBD, and further clarifies which stimuli can modulate and maintain IL17 production. The differential effect of TGFβ on IL17 and IFNγ production suggests that IL17 plays a crucial role in sustaining chronic inflammation. Our findings support a role for IL21 and IL12 but not TGFβ in modulating IL17 production in IBD. The multiple faces of the pathogenesis of IBD will lead soon to develop disease-specific therapies and hopefully to recognise which patients will better respond to a particular therapy. In this context, our study suggests that the abundant IL17 in inflamed mucosa may also help explain the relative lack of efficacy of anti-IFNγ antibodies in clinical trials, suggesting the existence of a Th1/Th17 population which may represent an intermediate phenotype between Th1 and Th17 cells.

    Acknowledgments

    We would like to thank G Warnes, N Ahmad and A Stagg for their help in the flow cytometric analysis.

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    Footnotes

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Ethics approval Each patient who took part in the study was recruited after appropriate local Ethics Committee approval.

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