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Imerio Angriman2, Luca Maria Saadeh*2, Andromachi Kotsafti1, Claudia Mescoli3, Melania Scarpa1, Francesco Cavallin1, Massimo Rugge3, Romeo Bardini2, Ignazio Castagliuolo4, Marco Scarpa1
1Surgical Oncology Unit, Veneto Institute of Oncology, Padova, Italy; 2Department of Surgery, Oncology and Gastroenterology, Università degli Studi di Padova, Padova, Italy; 3Azienda Ospedaliera - Università di Padova, Padova, Italy; 4Department of molecular medicine, Università degli Studi di Padova, Padova, Italy

Crohn Disease (CD) is a chronic inflammatory disease affecting the gastrointestinal tract with a patchy and transmural involvement. CD complications or unresponsiveness to medical therapy are managed with surgery but recurrence rate is high and burdensome. Re-operation is often required because of the fibrotic stenosis of the anastomosis. This study aims to analyse the relationship between innate immunity mediators and ileal wall fibrosis and to define possible molecular predictors of clinical and endoscopic recurrence.

Materials and methods
Mucosal samples were obtained from both healthy and inflamed ileum of 56 consecutive patients undergoing ileo-colonic resection for CD. Ileal mucosal samples of 14 patients undergoing surgery for cancer were obtained as control tissues. Data on clinical, endoscopic and surgical follow-up were collected. Clinical recurrence has been defined as HBI≥8 (moderate-to-severe activity) while endoscopic recurrence has been defined as Rutgeerts's score ≥3. A pathologist evaluated the fibrosis grade with a specific score. CD68, CD163 and iNOS expression was evaluated with immunohistochemistry through a semi-quantitative scale. TLR2, TLR4, TLR5, HBD1, HBD2, HBD3, HD5 and HD6 mRNA expression was quantified through RT-PCR. Concentrations of BDNF, Eotaxin-1, ICAM-1, IL-1beta, IL-1alpha, IL-1ra, IL-12p40, IL-12p70, IL-15, IL-17, IL-23, MMP-3, SCF, VEGF were determined with ELISA. Statistical analysis was carried out with non-parametric tests.

Fibrosis grade showed a direct correlation with IL-17 concentration (r=0,37; p=0,04) and inverse correlation with HBD1 (r=-0,34; p=0,01), TLR4 (r=-0,41; p<0,01) and IL-12p70 (r=-0,37; p=0,01) levels. HBD1 and TLR4 accurately indicated severe fibrosis (AUC 68%; p=0,02 e AUC 72%; p<0,01 respectively).
During the follow-up, 30% of patients (17/56) developed moderate-to-severe clinical recurrence, while 21% of the patients (12/56) developed endoscopic recurrence. Clinical recurrence-free interval is inversely correlated with expression of CD68 (r=-0,335; p=0,01) and CD163 (r=-0,341; p=0,02) and iNOS expression is inversely correlated with endoscopic recurrence-free interval (r=-0,46; p=0,02). Clinical recurrence-free directly correlated TLR2 (r=0,36; p=0,01), TLR5 (r=0,42; p<0,01), IL-17 (r=0,42; p<0,01), IL-23 (r=0,39; p=0,01) levels.

Pro-inflammatory cytokines perpetuate inflammation and were associated to intestinal fibrosis. HBD1 and TLR4 may be accurate molecular markers of severe fibrosis. M1 macrophages seem to play an important role in the re-activation of the inflammation after surgery.

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