The role of interleukin 17 in Crohn’s disease-associated intestinal fibrosis
© Biancheri et al.; licensee BioMed Central Ltd. 2013
Received: 11 May 2013
Accepted: 13 June 2013
Published: 8 July 2013
Interleukin (IL)-17A and IL-17E (also known as IL-25) have been implicated in fibrosis in various tissues. However, the role of these cytokines in the development of intestinal strictures in Crohn’s disease (CD) has not been explored. We investigated the levels of IL-17A and IL-17E and their receptors in CD strictured and non-strictured gut, and the effects of IL-17A and IL-17E on CD myofibroblasts.
IL-17A was significantly overexpressed in strictured compared with non-strictured CD tissues, whereas no significant difference was found in the expression of IL-17E or IL-17A and IL-17E receptors (IL-17RC and IL-17RB, respectively) in strictured and non-strictured CD areas. Strictured CD explants released significantly higher amounts of IL-17A than non-strictured explants, whereas no difference was found as for IL-17E, IL-6, or tumor necrosis factor-α production. IL-17A, but not IL-17E, significantly inhibited myofibroblast migration, and also significantly upregulated matrix metalloproteinase (MMP)-3, MMP-12, tissue inhibitor of metalloproteinase-1 and collagen production by myofibroblasts from strictured CD tissues.
Our results suggest that IL-17A, but not IL-17E, is pro-fibrotic in CD. Further studies are needed to clarify whether the therapeutic blockade of IL-17A through the anti-IL-17A monoclonal antibody secukinumab is able to counteract the fibrogenic process in CD.
KeywordsCollagen Interleukin 25 Myofibroblast Stricture TIMP-1
Fibrosis is the common end-stage of chronic inflammatory diseases in many tissues. There is excessive accumulation of extracellular matrix (ECM) components, including collagen and fibronectin, in the damaged tissue, leading to scarring and organ malfunction . ECM-producing myofibroblasts are the key players of fibrotic tissue remodeling,and consequently, much of the fibrosis research has focused on elucidating the molecular and immunological mechanisms underlying myofibroblast activation, proliferation, and migration .
A number of cytokines can modulate myofibroblast function, including those belonging to the interleukin (IL)-17 family [3, 4]. In particular, IL-17A stimulates proliferation and migration of cardiac fibroblasts , promotes hepatic stellate cell activation into fibrogenic myofibroblasts , induces collagen production by skin fibroblasts , and promotes epithelial-mesenchymal transition of lung epithelial cells . IL-17E, also known as IL-25, upregulates pro-inflammatory cytokine expression and collagen production by lung fibroblasts [9, 10].
We recently showed that IL-17A is increased in the inflamed areas of patients with inflammatory bowel disease (IBD) . By contrast, expression of IL-17E, which exerts an anti-inflammatory action by inhibiting T helper (Th)1 and Th17 responses, is markedly reduced in the inflamed mucosa of patients with IBD . However, there is much less clarity about the influence that these cytokines exert on intestinal fibrosis, which is a frequent complication in Crohn’s disease (CD) .
Intestinal myofibroblasts are central to CD fibrosis [14, 15]. Myofibroblasts isolated from strictured gut tissue of patients with CD overexpress collagen and transforming growth factor (TGF)-β1, produce excessive amounts of tissue inhibitor of metalloproteinases (TIMP)-1, and display reduced migratory ability compared with myofibroblasts isolated from non-strictured intestinal areas in CD . Although intestinal myofibroblasts are the target of a number of pro-inflammatory cytokines, including IL-1β and tumor necrosis factor (TNF)-α , no information is available on expression of the IL-17A and IL-17E receptors (IL-17RC and IL-17RB respectively), or on the effect of IL-17A and IL-17E on the release of pro-fibrogenic mediators by these cells.
Thus, in this study, we assessed expression of IL-17A and IL-17E in CD strictured and non-strictured gut, and investigated IL-17A and IL-17E production by CD strictured tissue explants cultured ex vivo. In addition, we assessed IL-17A and IL-17E receptor expression in CD strictured and non-strictured tissue and on myofibroblasts from the same areas, and performed in vitro experiments to determine the effects of IL-17A and IL-17E on CD myofibroblasts.
In vivo IL-17A and IL-17E tissue expression
In parallel, we analyzed IL-17A and IL-17E expression by ELISA in the same samples. IL-17A was significantly upregulated in strictured CD (mean 98.5 ± 13.7 pg/μg total protein) compared with non-strictured CD (mean 42.3 ± 11.0 pg/μg total protein, P<0.005) and control (mean 46.5 ± 8.3 pg/μg total protein, P<0.005) areas (Figure 1B). There was no significant difference in IL-17A expression between non-strictured CD areas and control gut. IL-17E did not significantly differ between strictured CD (mean 222.5 ± 32.4 pg/μg total protein), non-strictured CD (mean 178.1 ± 30.3 pg/μg total protein), and control areas (mean 188.2 ± 35.3 pg/μg total protein).
In vivo IL-17RC and IL-17RB tissue expression
Ex vivo tissue production of IL-17A, IL-17E, IL-6, TNF-α, collagen and TGF-β1
IL-17RC and IL-17RB expression on myofibroblasts
In vitro effect of IL-17A and IL-17E on matrix metalloproteinase (MMP)-3, MMP-12, and TIMP-1 production by myofibroblasts
In vitro effect of IL-17A and IL-17E on myofibroblast collagen production
In vitro effect of IL-17A and IL-17E on myofibroblast migration
In the present study, we found that IL-17A was overexpressed in CD strictures compared with non-strictured CD areas and control gut. We also found that myofibroblasts from CD strictures, which express the IL-17A receptor(IL-17RC), responded to IL-17A by producing more collagen and TIMP-1, and had reduced migratory ability. By contrast, IL-17E was not upregulated in CD strictures, and it did not influence collagen and TIMP-1 production or migration of myofibroblasts from CD strictures.
IL-17A, which is produced mainly by Th1 and Th1/Th17 cells [18, 19], mediates autoimmunity and immune defense against pathogens , and is increased in the intestinal mucosa of patients affected by chronic inflammatory bowel disorders, such as celiac disease , CD, and ulcerative colitis [22, 23]. Moreover, experimental studies have shown an important role for IL-17A in tissue remodeling and fibrosis in a number of different tissues. In particular, IL-1β-induced and bleomycin-induced lung fibrosis seems to depend on the action of IL-17A, and in addition, IL-17A−/− mice are less susceptible to experimental skin fibrosis [7, 24]. As opposed to acute experimental colitis, which is mainly characterized by a Th1 immune response, chronic trinitrobenzenesulfonic acid (TNBS)-induced murine colitis, which is accompanied by intestinal fibrosis, is driven predominantly by IL-17A-producing Th17 cells . The observation that targeting IL-23 (a key cytokine in Th17 cell development) with a p40 peptide-based vaccine ameliorates chronic TNBS-induced colitis,and reduces IL-17A, TGF-β1 levels, and collagen deposition in the bowel wall , highlights the importance of IL-17A in intestinal experimental fibrosis.
In the current study, we found, using immunoblotting and ELISA, that IL-17A was overexpressed in vivo in strictured CD gut compared with non-strictured CD and control gut. We decided to collect samples from uninflamed areas of patients with fibrostenosing CD so that any confounding effect of inflammation on IL-17A was minimized. Similar to the in vivo data, ex vivo tissue explants from CD strictures produced more IL-17A than explants from non-strictured CD areas and control gut. In keeping with our previous results , collagen content and TGF-β1 transcripts in tissue explants from uninflamed CD strictures were also increased compared with uninflamed non-strictured CD areas and control gut. The recent observation that IL-17A expression is higher in long-standing CD mucosa compared with early mucosal lesions  further strengthens our findings, because intestinal fibrosis is a late-stage process in CD.
On the basis of our findings showing that CD myofibroblasts expressed the IL-17A receptor IL-17RC we decided to stimulate these cells with IL-17A in in vitro experiments. In keeping with the study of Bamba et a l. , we found that IL-17A increased MMP-3 and MMP-12 production by myofibroblasts from CD strictured and non-strictured areas. However, in parallel, IL-17A upregulated TIMP-1 and collagen release, and reduced the migration ability of CD myofibroblasts. In a murine model of intestinal fibrosis, TIMP-1, which is upregulated, effectively inhibited ECM degradation by MMPs , and TIMP-1 was found to be increased in collagenous colitis and in CD strictures [16, 29]. Taken together, our data support a pro-fibrogenic role for IL-17A in CD intestinal fibrosis.
In parallel to studying the role of IL-17A, we also investigated the influence of IL-17E in the fibrogenic process in CD. IL-17E exerts two distinct immunological functions: on the one hand, it promotes Th2 response in allergic diseases including asthma , while on the other hand, it dampens the inflammatory process in immune-mediated disorders including IBD . A pro-fibrogenic role for IL-17E in experimental fibrotic disorders has also been shown. In particular, IL-17E mediates pulmonary collagen deposition in mice exposed to house dust mite , and intestinal TNBS-induced fibrosis was found to be associated in the early phase by a marked increase in IL-17E . When we investigated IL-17E in vivo, we found no difference in the expression of IL-17E in uninflamed CD strictures, uninflamed non-strictured CD areas, and control gut. Likewise, ex vivo tissue explants from the same three groups of patients produced comparable amounts of IL-17E. The IL-17E receptor IL-17RB was expressed at a similar level in strictured, non-strictured CD, and control gut. IL-17RB was found to be expressed by CD myofibroblasts, and stimulation with IL-17E increased MMP-3 and MMP-12 production by myofibroblasts from CD strictured and non-strictured areas and from control subjects. However, we did not observe any influence of IL-17E on collagen or TIMP-1 production by CD myofibroblasts or on their migration ability.
In conclusion, our findings provide evidence that IL-17A, but not IL-17E, may have a pro-fibrogenic role in CD. Clinical studies are needed to ascertain whether therapeutic blockade of IL-17A through the monoclonal anti-IL-17A antibody secukinumab  might offer a chance to counteract the fibrogenic process in patients with CD. However, we must emphasize that the recent findings that anti-IL-17A exacerbates CD  will mean that patient selection for such treatment will be crucial.
This study was approved by the local Ethics Committee (NRES Committee London - City & East). Each patient who took part in the study gave informed consent to participate in the study and to publish the results deriving from this research. All the experiments reported in this study are in compliance with the Helsinki Declaration.
Patients and tissues
Clinical features of patients with fibrostenosing Crohn’s disease (n=29)
Characteristics and parameters
35.8 (23 to 61)
Small bowel and colon
Small bowel only
Duration of disease, months
81.2 (7 to 196)
Number of recurrences
4 (0 to 9)
223 (155 to 426)
Intestinal tissue explants (1 mm3 in size) were placed in 12-well tissue culture plates (BD Biosciences, Oxford, UK; one explant per well) and cultured at 37°C and 5%CO2 in 800 μl serum-free HL-1 medium (Cambrex Bio Science, Wokingham, UK) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, as previously described . After 24 hours ex vivo culture, supernatants were collected and stored at −70°C until used for cytokine measurement by ELISA, and intestinal explants were homogenized for subsequent evaluation of TGF-β1 expression by quantitative reverse transcription (qRT)-PCR.
Myofibroblast isolation and culture
The mucosa was dissected away from full thickness samples of bowel and discarded. The remaining tissue was cut into 1 mm-sized cubes and cultured at 37°C in a humidified CO2 incubator in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, Poole, UK) supplemented with 20% fetal bovine serum (FBS), 1% non-essential amino acids (Invitrogen Ltd., Paisley, UK), 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamycin, and 1 μg/ml amphotericin (Sigma-Aldrich). Myofibroblasts migrated out of the tissue after a few days. Established colonies of myofibroblasts were seeded into 25-cm2 culture flasks and cultured in DMEM supplemented with 20% FBS and antibiotics. At confluence, the cells were passaged using trypsin-EDTA in a 1:2 to 1:3 split ratio. Cells were grown to at least passage 4 before there were enough to use in stimulation experiments, and were characterized by immunocytochemical staining, as previously described . Subconfluent monolayers of myofibroblasts seeded in 12-well plates at 3×105 cells per well were starved in serum-free medium for 24 hours at 37°C and 5%CO2 before culture for 24 hours at 37°C and 5%CO2 with serum-free DMEM containing antibiotics in the absence or presence of 10 ng/ml of rhIL-17A, or rhIL-17E, or rhTNF-α (all R&D Systems, Abingdon, Oxfordshire, UK).
Wound-healing scratch assay
Myofibroblast migration was assessed in accordance with the modified method of Rodriguez et al. [16, 38]. Briefly, cells (2×105) were seeded into cell culture dishes (Nunc; Nalge Nunc International, Rochester, NY, USA) with 2 mm grids, size 35×10 mm, in 2 ml of DMEM supplemented with 20% FBS and antibiotics. The cells were maintained at 37°C and 5% CO2 until confluent. Once confluent, each dish of monolayer cells was given a mechanical wound by scoring with a 200 μl pipette tip, parallel to the grid bars along the central grid line. This permitted easy viewing of the cells growing back together, and ensured that the 2 mm grid could be used as a reference, so that the wound areas could be measured and compared. Wound placement was checked with an inverted microscope (CK2; Olympus UK Ltd, London, UK). The medium was then removed, and the cells were washed five times with HL-1 serum-free medium (Cambrex Bio Science) supplemented with antibiotics, and then replaced with 1.5 ml HL-1 medium with or without 10 ng/ml rhIL-17A or rhIL-17E (both R&D Systems). Photographs of the cells in each grid along the induced wound were taken at 0, 2, 4, 8, 16 and 24 hours, using a digital camera (Camedia; Olympus UK Ltd) with 34 to 40 zoom, and 20× magnification, attached to a light microscope. The computer program Image J was used to measure the area of initial damage (images taken at time 0) and of the remaining damage at subsequent time points. Each grid image was observed separately, and two points per grid at the same position at every time point were measured using imaging software at the same magnification. The percentage of wound repair was then calculated.
Concentrations of IL-17A and IL-17E in tissue sample homogenates were measured using specific ELISA kits (R&D Systems), in accordance with the manufacturer’s instructions, and were normalized to the total protein concentration, determined by a protein assay (Bio-Rad Laboratories, Hemel Hempstead, UK). Concentrations of IL-17A, IL-17E, IL-6, and TNF-α in organ-culture supernatants were assessed using specific ELISA kits (R&D Systems), in accordance with the manufacturer’s instructions.
RNA extraction and analysis of mRNA expression by qRT-PCR
RNA was extracted from cultured tissue explants. cDNAs were synthesized (Improm-II RT system; Promega, Southampton, Hampshire, UK) using random hexamers and 1 μg of RNA in a final volume of 20 μl. RT reactions were performed using the Improm-II reverse transcriptase enzyme from the kit. An RT reaction without reverse transcriptase enzyme was performed for each tissue type as a negative control for qPCR. TGF-β1 primers and probe sets were validated for use with the ∆∆Ct method of quantification. The probe was labeled with a 59-reporter dye FAM (6-carboxy-fluorescein) and the 39-quencher dye TAMRA (6-carboxy-N,N,N’,N9-tetramethyl-rhodamine). RT reactions were diluted 1 in 10 in distilled H2O, and 5 μl of template was added to 6.5 μl of 2× master mix (Eurogentech, Seraing, Belgium) containing 1.2 μmol/l forward and reverse primers and 0.248 μmol/l of probe in a total volume of 12.5 μl. The PCR protocol was as follows: 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, and annealing/extension at 60°C for 1 minute. Thermocycling and real-time detection of PCR products were performed on asequence detection system (iCycler iQ; Bio-Rad Laboratories). Expression levels were normalized against 18S, β-actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and values were calculated using the ∆∆Ct method.
Total soluble forms of collagen were measured in supernatants of tissue explants and myofibroblasts (Sircol Collagen Assay Kit; Biocolor Ltd, Belfast, UK), in accordance with the manufacturers’ instructions. The collagen content in each sample was calculated as an average of three readings.
Western blotting was performed using a modification of a previously described method . Tissue samples or myofibroblasts were homogenized in ice-cold lysis buffer, and the amount of protein was determined by a protein assay (Bio-Rad Laboratories). Equal amounts of protein or 15 μl of cell- culture supernatants were loaded into each lane and run in 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels under reducing conditions. After electrophoresis, protein was transferred onto nitrocellulose membranes (Bio-Rad Laboratories). Membranes were blocked with 5% non-fat dry milk, followed by incubation overnight at 4°C with the following antibodies: goat anti-human IL-17A (1 μg/ml), goat anti-human IL-17E (0.1 μg/ml) (both R&D Systems), mouse anti-human IL-17RB (1:500 dilution; LifeSpan Biosciences, Seattle, WA), mouse anti-human IL-17RC (1:750 dilution), rabbit anti-human MMP-3 (1:200 dilution), rabbit anti-human MMP-12 (1:1000 dilution) (all three from Abcam Ltd, Cambridge, UK), or mouse anti-human TIMP-1 (1 μg/ml; Oncogene Research, Nottingham, UK). Appropriate antibodies conjugated to horseradish peroxidase (Dako, High Wycombe, Buckinghamshire, UK) were used as secondary antibodies, and the reaction was developed with enhanced chemiluminescence (ECL Plus Kit;Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Blots were then stripped and analyzed for β-actin, as an internal loading control, using a rabbit anti-β-actin antibody (1:5000 dilution; Abcam Ltd). Bands were quantified by scanning densitometry (LKB Ultrascan XL Laser Densitometer; Kodak, Hemel Hempstead, UK).
Data were analyzed by the GraphPad Prism statistical PC program (GraphPad Software, San Diego, CA) using the paired t test and the Mann–Whitney U-test. P<0.05 was considered significant.
Dulbecco’s modified Eagle’s medium
Fetal bovine serum
Glyceraldehyde 3-phosphate dehydrogenase
Inflammatory bowel disease
Quantitative reverse transcription
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
Transforming growth factor
Tissue inhibitor of metalloproteinase
Tumor necrosis factor.
Sylvia LF Pender was partially funded by Broad Medical Research Program of The Broad foundation and the EU-FP7 IPODD consortium. There are no sources of funding to declare for any of the other authors. We thank Dr Rebecca Morgan-Walsh (Division of Infection, Inflammation and Immunity, University of Southampton, Southampton, UK) for performing some of the time lapse studies.
Guarantor of the article
Dr. Antonio Di Sabatino
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