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. 2013 Jul 8;6(1):13.
doi: 10.1186/1755-1536-6-13.

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

Paolo Biancheri  1 , Sylvia Lf Pender, Francesca Ammoscato, Paolo Giuffrida, Gianluca Sampietro, Sandro Ardizzone, Amir Ghanbari, Renata Curciarello, Alessandra Pasini, Giovanni Monteleone, Gino R Corazza, Thomas T Macdonald, Antonio Di Sabatino
Affiliations

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

Paolo Biancheri et al. Fibrogenesis Tissue Repair. .

Abstract

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.

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Figures

Figure 1
Figure 1
In vivo expression of interleukin (IL)-17A and IL-17E. IL-17A and IL-17E were detected by both (A) immunoblotting and (B) ELISA 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. (A) Each example shown in the upper panel is representative of experiments performed in 12 patients with CD and 11 HC subjects. Blots were stripped and analyzed for β-actin as an internal loading control. In the lower panel, densitometry of IL-17A and IL-17E expression normalized for β-actin is shown. Results are mean ± SEM. au, Arbitrary units. (B) Results, expressed as pg/100 μg of total protein, are mean ± SEM. *P<0.005 versus Non-strict uninfl and HC tissue samples.
Figure 2
Figure 2
In vivo expression of interleukin (IL)-17A and IL-17E receptors. (A) IL-17RC (receptor of IL-17A), and (B) IL-17RB (receptor of IL-17E) 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.
Figure 3
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.
Figure 4
Figure 4
Expression of interleukin (IL)-17A and IL-17E receptors on intestinal myofibroblasts. (A) IL-17RC and (B) IL-17RB were detected by immunoblotting on lysates of myofibroblasts isolated 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 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.
Figure 5
Figure 5
Effect of interleukin (IL)-17A and IL-17E on the production of matrix metalloproteinase (MMP)-3, MMP-12, and tissue inhibitor of metalloproteinase (TIMP)-1 by intestinal myofibroblasts. (A) MMP-3, (B) MMP-12, and (C) (TIMP-1 in culture supernatants of myofibroblasts isolated 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 six healthy control (HC) subjects, cultured for 24 hours with medium alone or recombinant human (rh) tumor necrosis factor (TNF)-α, or rhIL-17A, or rhIL-17E. Blots are representative of experiments performed in all patients with CD and HC subjects. Lower panels show densitometry of western blots. Values are mean ± SEM. *P<0.05 versus myofibroblasts from the same study group cultured with medium alone. §P<0.05 versus Non-strict uninfl CD and HC myofibroblasts cultured under the same conditions.
Figure 6
Figure 6
Effect of interleukin (IL)-17A and IL-17E on the production of collagen by intestinal myofibroblasts. Levels of collagen, expressed as μg/ml, in the supernatants of myofibroblasts isolated 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 six healthy control (HC) subjects, cultured for 24 hours with medium alone or recombinant human (rh)tumor necrosis factor (TNF)-α, or rhIL-17A, or rhIL-17E. Values are mean ± SEM. *P<0.05 versus myofibroblasts from the same study group cultured with medium alone. §P<0.05 versus Non-strict uninfl CD and HC myofibroblasts cultured under the same conditions.
Figure 7
Figure 7
Wound-healing scratch assay. Effect of recombinant human (rh)IL-17A and rhIL-17E on the migration, assessed by an in vitro wound-healing scratch assay, of myofibroblasts isolated 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 healthy control (HC) subjects. Myofibroblasts were cultured with rhIL-17A or rhIL-17E or medium alone. Results, expressed as percentage of wound repair, are mean ± SEM. *P<0.05 versus myofibroblasts cultured with medium only at 8, 16, and 24 hours.
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