A. Pasini

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

BackgroundInterleukin (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.ResultsIL-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.ConclusionsOur 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.

Serum hepcidin in inflammatory bowel diseases: biological and clinical significance.

Background: Hepcidin, a peptide produced by hepatocytes, regulates body iron homeostasis. Inflammation increases serum hepcidin, and its determination can be useful in the differential diagnosis of anemias during inflammatory diseases. Methods: We measured serum hepcidin-25 and hepcidin-20 isoforms in 54 patients with inflammatory bowel diseases (IBD) and 54 reference subjects (36 healthy controls and 18 anemic patients without inflammation or renal failure). Disease activity, blood counts, iron status, and erythropoiesis-related parameters were obtained for all study subjects. Results: In IBD hepcidin-25, the peptide bioactive isoform correlated positively with C-reactive protein and serum ferritin; an inverse correlation was observed with transferrin, the soluble transferrin receptor, and the soluble transferrin receptor to Log(ferritin) ratio. Similar correlations were found in reference subjects. Patients with anemia of inflammation had higher hepcidin-25 levels than those with iron deficiency anemia or a combination of iron deficiency anemia and inflammation (P = 0.0061). In patients with inflammation and serum ferritin concentration 100 to 200 ng/mL, hepcidin-25 was low, suggesting that these patients had iron deficiency. A serum hepcidin-25 concentration below 2.0 nM differentiated 85% of patients with iron deficiency anemia (with or without inflammation) from patients with anemia of inflammation. In IBD, hepcidin-20 correlated with both hepcidin-25 and C-reactive protein. Conclusions: In IBD, iron stores, inflammation, and iron requirement for erythropoiesis influence serum hepcidin-25. Hepcidin-25 determination can be useful in the differential diagnosis of IBD-associated anemias. Serum hepcidin-20 is linked to hepcidin-25, but inflammation has an independent regulatory role on its concentration, indicating that hepcidin-20 may have a biological function.

Effect of Tumor Necrosis Factor-α Blockade on Mucosal Addressin Cell-adhesion Molecule-1 in Crohn's Disease

Background:Mucosal addressin cell-adhesion molecule (MAdCAM)-1, which is overexpressed on gut endothelium in active Crohns disease (CD), promotes intestinal recruitment of integrin &agr;4&bgr;7+ T cells that sustain chronic inflammation. As tumor necrosis factor alpha (TNF)-&agr;, a cytokine centrally involved in CD, modulates gut endothelial adhesion molecules, we here explored the in vivo and ex vivo effects of TNF-&agr; blockade on MAdCAM-1 expression in CD. Methods:MAdCAM-1 was determined by immunoblotting in colonic biopsies collected before and 10 weeks after either infliximab or adalimumab treatment in CD patients, and in CD biopsies incubated with either infliximab or adalimumab or control IgG1. Integrin &bgr;7+ circulating T cells were analyzed by flow cytometry. Results:MAdCAM-1 significantly decreased after either infliximab or adalimumab treatment in responder but not in nonresponder patients. In parallel, an increase of circulating &bgr;7+ T cells was found in responder patients only. A marked downregulation of MAdCAM-1 was observed in CD biopsies cultured with either infliximab or adalimumab in comparison to IgG1-treated biopsies. Conclusions:Our findings showing that MAdCAM-1 is downregulated by TNF-&agr; blockade point to a novel mechanism of action of anti-TNF-&agr; antibodies in CD.

Increase in neuroendocrine cells in the duodenal mucosa of patients with refractory celiac disease.

OBJECTIVES:Several immune-mediated gastrointestinal disorders, including celiac disease (CD), are associated with neuroendocrine cell hyperplasia. However, neuroendocrine cells have never been explored in refractory CD (RCD).METHODS:Serial duodenal sections from 17 patients with RCD (6 type 1 and 11 type 2), 16 uncomplicated CD patients before and after gluten-free diet, 14 patients with potential CD, 27 patients with non-CD villous atrophy, i.e., common variable immunodeficiency (n=12), Whipples disease (n=10) and giardiasis (n=5), and 16 healthy subjects were processed for the immunohistochemical detection of chromogranin A (CgA), serotonin, and somatostatin. Mucosal tryptophan hydroxylase (TpH)-1 and serotonin-selective reuptake transporter (SERT) transcripts were measured by quantitative reverse transcription-PCR. Serum CgA and 24-h urine 5-hydroxyindoleacetic acid (5-HIAA) were assessed. Biopsies from treated CD patients were cultured with serotonin or peptic tryptic digest of gliadin (PT-gliadin), and interferon (IFN)-γ was detected by ELISA in culture supernatants.RESULTS:Epithelial cells positive for CgA and serotonin, but not somatostatin, were significantly increased in RCD. Raised mucosal transcripts of TpH-1, but not SERT, were found in RCD. On biopsies from treated CD patients, serotonin upregulated IFN-γ production at levels comparable to those induced by PT-gliadin. Serum CgA, but not urine 5-HIAA, was increased in RCD. No significant difference was found between RCD type 1 and type 2 in terms of neuroendocrine cells, mucosal TpH-1 transcripts, and serum CgA.CONCLUSIONS:Serotonin-producing neuroendocrine cells are increased in RCD mucosa. IFN-γ upregulation induced by serotonin suggests that this monoamine may have a role in sustaining the local inflammatory response in CD.

Abnormal thymic stromal lymphopoietin expression in the duodenal mucosa of patients with coeliac disease

Objective The short isoform of thymic stromal lymphopoietin (TSLP), a cytokine constitutively expressed by epithelial cells, is crucial in preserving immune tolerance in the gut. TSLP deficiency has been implicated in sustaining intestinal damage in Crohns disease. We explored mucosal TSLP expression and function in refractory and uncomplicated coeliac disease (CD), a T-cell-mediated enteropathy induced by gluten in genetically susceptible individuals. Design TSLP isoforms—long and short—and receptors—TSLPR and interleukin (IL)-7Rα—were assessed by immunofluorescence, immunoblotting and qRT-PCR in the duodenum of untreated, treated, potential and refractory patients with CD. The ability of the serine protease furin or CD biopsy supernatants to cleave TSLP was evaluated by immunoblotting. The production of interferon (IFN)-γ and IL-8 by untreated CD biopsies cultured ex vivo with TSLP isoforms was also assessed. Results Mucosal TSLP, but not TSLPR and IL-7Rα, was reduced in untreated CD and refractory CD in comparison to treated CD, potential CD and controls. Transcripts of both TSLP isoforms were decreased in active CD mucosa. Furin, which was overexpressed in active CD biopsies, was able to cleave TSLP in vitro. Accordingly, refractory and untreated CD supernatants showed higher TSLP-degrading capacity in comparison to treated CD and control supernatants. In our ex vivo model, both TSLP isoforms significantly downregulated IFN-γ and IL-8 production by untreated CD biopsies. Conclusions Reduced mucosal TSLP expression may contribute to intestinal damage in refractory and untreated CD. Further studies are needed to verify whether restoring TSLP might be therapeutically useful especially in refractory patients with CD.

Altered Expression of Type-1 and Type-2 Cannabinoid Receptors in Celiac Disease

Anandamide (AEA) is the prominent member of the endocannabinoid family and its biological action is mediated through the binding to both type-1 (CB1) and type-2 (CB2) cannabinoid receptors (CBR). The presence of AEA and CBR in the gastrointestinal tract highlighted their pathophysiological role in several gut diseases, including celiac disease. Here, we aimed to investigate the expression of CBR at transcriptional and translational levels in the duodenal mucosa of untreated celiac patients, celiac patients on a gluten-free diet for at least 12 months and control subjects. Also biopsies from treated celiac patients cultured ex vivo with peptic-tryptic digest of gliadin were investigated. Our data show higher levels of both CB1 and CB2 receptors during active disease and normal CBR levels in treated celiac patients. In conclusion, we demonstrate an up-regulation of CB1 and CB2 mRNA and protein expression, that points to the therapeutic potential of targeting CBR in patients with celiac disease.

Innate and adaptive immunity in self-reported nonceliac gluten sensitivity versus celiac disease

BACKGROUND Immune mechanisms have been implicated in nonceliac gluten sensitivity (NCGS), a condition characterized by intestinal and/or extraintestinal symptoms caused by the ingestion of gluten in non-celiac/non-wheat allergic individuals. AIMS We investigated innate and adaptive immunity in self-reported NCGS versus celiac disease (CD). METHODS In the supernatants of ex vivo-cultured duodenal biopsies from 14 self-reported NCGS patients, 9 untreated and 10 treated CD patients, and 12 controls we detected innate cytokines - interleukin (IL)-15, tumor necrosis factor-α, IL-1β, IL-6, IL-12p70, IL-23, IL-27, IL-32α, thymic stromal lymphopoietin (TSLP), IFN-α-, adaptive cytokines - interferon (IFN)-γ, IL-17A, IL-4, IL-5, IL-10, IL-13-, chemokines - IL-8, CCL1, CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL10-, granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF). RESULTS Mucosal innate and adaptive cytokines, chemokines and growth factors did not differ between self-reported NCGS, treated CD and controls. On the contrary, IL-6, IL-15, IL-27, IFN-α, IFN-γ, IL-17A, IL-23, G-CSF, GM-CSF, IL-8, CCL1 and CCL4 were significantly higher in untreated CD than in self-reported NCGS, treated CD and controls, while TSLP was significantly lower in untreated CD than in self-reported NCGS, treated CD and controls. CONCLUSION In our hands, patients with self-reported NCGS showed no abnormalities of the mucosal immune response.

Abnormal anandamide metabolism in celiac disease

The endocannabinoid system has been extensively investigated in experimental colitis and inflammatory bowel disease, but not in celiac disease, where only a single study showed increased levels of the major endocannabinoid anandamide in the atrophic mucosa. On this basis, we aimed to investigate anandamide metabolism in celiac disease by analyzing transcript levels (through quantitative real-time reverse transcriptase-polymerase chain reaction), protein concentration (through immunoblotting) and activity (through radioassays) of enzymes responsible for anandamide synthesis (N-acylphosphatidyl-ethanolamine specific phospholipase D, NAPE-PLD) and degradation (fatty acid amide hydrolase, FAAH) in the duodenal mucosa of untreated celiac patients, celiac patients on a gluten-free diet for at least 12 months and control subjects. Also, treated celiac biopsies cultured ex vivo with peptic-tryptic digest of gliadin were investigated. Our in vivo experiments showed that mucosal NAPE-PLD expression and activity are higher in untreated celiac patients than treated celiac patients and controls, with no significant difference between the latter two groups. In keeping with the in vivo data, the ex vivo activity of NAPE-PLD was significantly enhanced by incubation of peptic-tryptic digest of gliadin with treated celiac biopsies. On the contrary, in vivo mucosal FAAH expression and activity did not change in the three groups of patients, and accordingly, mucosal FAAH activity was not influenced by treatment with peptic-tryptic digest of gliadin. In conclusion, our findings provide a possible pathophysiological explanation for the increased anandamide concentration previously shown in active celiac mucosa.

P.03.4 SERUM HEPCIDIN IN INFLAMMATORY BOWEL DISEASES

20)-untreated) rats. 2) TNBS up-regulated mRNA expression of COX-2, TGF-β, TNF-α, HB-EGF and t-TG in colonic tissue. 3) both concentrations of Hp 2-20 significantly (p<0.05) decreased TNBS-induced mRNA overexpression of COX-2, TNF-α, t-TG and TGF-β, whereas HB-EGF mRNA tissue levels remained unchanged; 4) TNBS up-regulated FPR and FPRL-1 mRNA expression and this was counteracted by Hp(2-20). Conclusions: 1) Peptide Hp(2-20) significantly accelerates mucosal healing in a rat model of TNBS-induced colitis, both at the macroscopic and histological level; 2) This effect is associated with a significant reduction in mRNA colonic tissue levels of COX-2, TGF-β, TNF-α, and tissue transglutaminase; 3) Hp(2-20) decreased FPR and FPRL-1 gene expression induced by TNBS; 4) We postulate that Hp(2-20) down-regulation of pro-inflammatory responses in TNBS-colitis may partly explain the inverse association between H. pylori infection and IBD.

P.05.15 IS ULCERATIVE COLITIS AN ATYPICAL T HELPER 2-MEDIATED DISEASE CHARACTERISED BY EXCESSIVE PRODUCTION OF INTERLEUKIN-13?

The aim of this study was to compare the outcomes of therapy with IFX and ADA in CD patients in daily clinical practice. Material and methods: All CD patients treated with anti TNF-alpha antibodies form July 1999 to September 2011 at “Casa Sollievo della Sofferenza” Hospital were enrolled. Demographical and clinical data were collected in an electronic database for the analysis. Results: A total of 188 patients (113 treated with IFX and 75 with ADA) were enrolled. The mean age at diagnosis was 29±12 (IFX) vs 34±14 (ADA) (p=0.003). The mean duration of disease was 8±6 years for both groups. Ileocolonic disease was significantly more frequent in IFX patients (45 vs 19 pts, p=0.03). Luminal disease (74 IFX vs 61 ADA, p=0.02), fistulizing disease (28 IFX vs 12 ADA, p=0.004), and extraintestinal manifestations (11 IFX vs 2 with ADA, p=0.07) were the indications for therapy. All patients treated with ADA vs 66 (58%) treated with IFX received a maintenance therapy; the mean duration of treatment was 12±10 for ADA vs 6±6 months for IFX (p=0.0003). The response rate was similar (76% IFX and 67% ADA; p=0.17). Five of 6 patients (83%) non-responder to ADA and 7 of 10 patients refractory to IFX responded to the alternative anti-TNF (p=ns). 107 patients were treated with immunosuppressants (76 with IFX and 31 with ADA; p=0.0005). The response rate did not improve with the combo therapy in both groups. 74 patients were active smokers (49 treated with IFX and 25 with ADA; p=0.1). The response rate was significantly lower in smokers treated with IFX (71.4% vs 92%, p=0.005). 15 (13%) patients treated with IFX and 10 (14%) treated with ADA had adverse events (AEs) (p=ns). No differences in AE rate between patients in mono or combo therapy were found. Conclusions: IFX and ADA showed similar efficacy and safety. Current smokers showed a significant lower response rate only in patients treated with IFX. Most patients refractory to an antiTNF may respond to the other. Prospective, randomized head to head studies comparing IFX to ADA are strongly desiderable.