The role of interleukin 17 in Crohn’s disease-associated intestinal fibrosis

Background 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. Results 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. Conclusions 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.


Background
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 [1]. 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 [2].
We recently showed that IL-17A is increased in the inflamed areas of patients with inflammatory bowel disease (IBD) [11]. 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 [12]. 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) [13].
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 [16]. Although intestinal myofibroblasts are the target of a number of pro-inflammatory cytokines, including IL-1β and tumor necrosis factor (TNF)-α [17], 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.

Results
In vivo IL-17A and IL-17E tissue expression We analyzed IL-17A and IL-17E levels by immunoblotting in tissue samples collected from uninflamed areas of strictured and non-strictured gut of 12 patients with fibrostenosing CD and from normal gut of 11 control subjects. IL-17A expression was significantly (P<0.005) upregulated in strictured CD areas compared with nonstrictured CD areas and control gut ( Figure 1A). No significant difference in IL-17A expression was found between non-strictured CD areas and control gut. IL-17E levels did not differ significantly between strictured and non-strictured CD areas and control gut.

In vivo IL-17RC and IL-17RB tissue expression
We then analyzed IL-17RC (receptor of IL-17A) and IL-17RB (receptor of IL-17E) expression by immunoblotting in the same tissue samples. IL-17RC expression was not significantly different between CD strictured, CD nonstrictured, and control gut (Figure 2A). Similarly, IL-17RB expression did not differ between CD strictured, CD nonstrictured and control gut ( Figure 2B).

IL-17RC and IL-17RB expression on myofibroblasts
We then analyzed IL-17RC and IL-17RB expression by immunoblotting in lysates of myofibroblasts isolated from uninflamed areas of strictured and non-strictured gut of six patients with fibrostenosing CD, and from normal gut of seven control subjects. Myofibroblasts isolated from strictured CD areas, non-strictured CD areas, and control gut expressed both IL-17RC ( Figure 4A) and IL-17RB ( Figure 4B). However, no significant difference in IL-17RC and IL-17RB expression was found between strictured, non-strictured CD, and control myofibroblasts.
In vitro effect of IL-17A and IL-17E on matrix metalloproteinase (MMP)-3, MMP-12, and TIMP-1 production by myofibroblasts Matrix metalloproteinase (MMP)-3, MMP-12, and TIMP-1 production was evaluated by immunoblotting in culture supernatants of myofibroblasts isolated from uninflamed areas of strictured or non-strictured gut of six patients with fibrostenosing CD, and from normal gut of six control subjects, and subsequently stimulated in vitro with recombinant human (rh)TNF-α, rhIL-17A, or rhIL-17E. rhIL-17A and rhIL-17E induced a significant (P<0.05) increase in both MMP-3 and MMP-12 production by myofibroblasts from strictured and non-strictured areas of patients with CD and from normal gut of control subjects ( Figure 5A,B). rhTNF-α induced a significant (P<0.05) increase in both MMP-3 and MMP-12 production compared with control (myofibroblasts from the same group of patients cultured with medium alone). No significant difference in MMP-3 or MMP-12 production was found between rhTNF-α, rhIL-17A, and rhIL-17E stimulation in the three conditions studied. Myofibroblasts from CD strictured areas showed a significantly (P<0.05) lower spontaneous release of MMP-12 than myofibroblasts from CD non-strictured areas or control gut. Myofibroblast stimulation with rhIL-17A induced a significant (P<0.05) increase in TIMP-1 production compared with unstimulated cells from the same group of patients ( Figure 5C). rhIL-17E did not induce any significant change in TIMP-1 production compared with myofibroblasts from the same group of patients cultured with medium alone. rhTNF-α induced a significant (P<0.05) increase in TIMP-1 production compared with myofibroblasts from the same group of patients cultured with medium alone. No significant difference in TIMP-1 production was found between rhTNF-α and rhIL-17A stimulation in all the three conditions studied.
In vitro effect of IL-17A and IL-17E on myofibroblast collagen production We cultured myofibroblasts isolated from uninflamed areas of strictured and non-strictured gut of six patients with fibrostenosing CD, and from normal gut of six control subjects, in the presence or absence of rhTNF-α, rhIL-17A, or rhIL-17E, and measured collagen in the culture supernatants ( Figure 6). Myofibroblast stimulation with rhIL-17A induced a significant (P<0.05) increase in mean collagen production (strictured CD myofibroblasts: 412 ± 57 μg/ml; non-strictured CD myofibroblasts: 154 ± 17 μg/ml; control myofibroblasts: 114 ± 12 μg/ml) compared with cells from the same group of patients cultured with medium alone (strictured CD myofibroblasts: 155 ± 23 μg/ml; non-strictured CD myofibroblasts: 76 ± 15 μg/ml; control myofibroblasts: 67 ± 7 μg/ml). rhIL-17E did not induce any significant change in mean collagen production by strictured CD myofibroblasts (181 ± 37 μg/ml), non-strictured CD myofibroblasts (83 ± 7 μg/ml) or control myofibroblasts (61 ± 10 μg/ml) compared with unstimulated cells from the same group of patients. rhTNF-α induced a significant (P<0.05) increase in mean collagen production by strictured CD myofibroblasts (399 ± 62 μg/ ml), non-strictured CD myofibroblasts (175 ± 22 μg/ml) and control myofibroblasts (124 ± 18 μg/ml) compared with unstimulated cells from the same group of patients. No significant difference in collagen production was found between cells cultured with rhTNF-α and rhIL-17A. Strictured CD myofibroblasts produced significantly (P<0.05) higher amounts of collagen than did non-strictured CD and control myofibroblasts cultured under the same were detected by immunoblotting in uninflamed areas of strictured (Strict uninfl) and non-strictured (Non-strict uninfl) gut of 12 patients with fibrostenosing Crohn's disease (CD) and from normal gut of 11 healthy control (HC)subjects. Each example shown in the left panels is representative of experiments performed in all patients with CD and all HC subjects. Blots were stripped and analyzed for β-actin as an internal loading control. In the right panels, densitometry of IL-17RC and IL-17RB expression normalized for β-actin is shown. Results are mean ± SEM. au, Arbitrary units.
conditions. No significant difference in collagen production was found between non-strictured CD and control myofibroblasts cultured under the same conditions.

In vitro effect of IL-17A and IL-17E on myofibroblast migration
To evaluate the role of IL-17A and IL-17E on myofibroblast migration, a wound-healing scratch assay was performed using subconfluent monolayers of myofibroblasts isolated from uninflamed areas of strictured and non-strictured gut of six patients with fibrostenosing CD, and from normal gut of seven control subjects. Cell migration was measured as the percentage of wound repair and results were expressed as mean percentage of wound repair (see Methods section). rhIL-17A significantly (P<0.05) reduced migration of strictured CD, non-strictured CD and control myofibroblasts at 8 hours (3.2 ± 1.1%, 4.6 ± Figure 3 Levels of cytokines and pro-fibrogenic mediators in tissue explant organ culture supernatants. Levels of interleukin (IL)-17A, IL-17E, IL-6, and tumor necrosis factor (TNF)-α, expressed as pg/ml, and collagen, expressed as μg/ml, in the supernatants of tissue explants from uninflamed areas of strictured (Strict uninfl) and non-strictured (Non-strict uninfl) gut of six patients with fibrostenosing Crohn's disease (CD) and from normal gut of seven control subjects (HC), cultured for 24 hours in the absence of stimuli, and levels of transforming growth factor (TGF)-β1, expressed as relative units compared with the median expression in control subjects (which was assigned the value 1), in the same cultured tissue explants. Values are mean ± SEM. r.u., Relative units.

Discussion
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 [20], and is increased in the intestinal mucosa of patients affected by chronic inflammatory bowel disorders, such as celiac disease [21], 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 [25]. 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 [25], 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 [16], 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 [26] 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 al. [27], we found that IL-17A increased MMP-3 and MMP-12 production by myofibroblasts from CD strictured and nonstrictured 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 [28], 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 [30], while on the other hand, it dampens the inflammatory process in immune-mediated disorders including IBD [31]. 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 [10], and intestinal TNBS-induced fibrosis was found to be associated in the early phase by a marked increase in IL-17E [32]. 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.

Conclusions
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 [33] 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 [33] will mean that patient selection for such treatment will be crucial.

Ethics approval
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
Surgical specimens were taken from uninflamed areas of strictured and non-strictured ileum or colon of 29 patients with fibrostenosing CD (Table 1). Because the excessive collagen deposition occurs in the sub-mucosa and outer muscle layers, we discarded the mucosa upon dissection, and used the muscle-enriched fraction. Diagnosis of CD was ascertained by standard clinical criteria [34], and strictured areas were identified by enteroclysis [35]. None of the patients with CD had been treated previously with ciclosporin, tacrolimus, methotrexate, or anti-TNF-α antibodies. Intestinal samples were also collected from macroscopically and microscopically unaffected (at  least 500 mm from the tumoral mass) ileum or colon of 27 patients undergoing intestinal resection for colon cancer (mean age 57 years, range 45-71 years). The intestinal tissue was homogenized and used in immunoblotting experiments, or processed to isolate myofibroblasts, or cultured ex vivo.

Organ culture
Intestinal tissue explants (1 mm 3 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% CO 2 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 [36]. 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 CO 2 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-cm 2 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 [37]. Subconfluent monolayers of myofibroblasts seeded in 12-well plates at 3×10 5 cells per well were starved in serum-free medium for 24 hours at 37°C and 5%CO 2 before culture for 24 hours at 37°C and 5%CO 2 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 (2×10 5 ) 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% CO 2 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.

ELISA
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 ΔΔ C t 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 H 2 O, 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 ΔΔC t method.

Collagen assay
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.

Statistical analysis
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.