MiR-155 modulates the inflammatory phenotype of intestinal myofibroblasts by targeting SOCS1 in ulcerative colitis.

Abnormal levels of microRNA (miR)-155, which regulate inflammation and immune responses, have been demonstrated in the colonic mucosa of patients with inflammatory bowel diseases (IBD), although its role in disease pathophysiology is unknown. We investigated the role of miR-155 in the acquisition and maintenance of an activated phenotype by intestinal myofibroblasts (IMF), a key cell population contributing to mucosal damage in IBD. IMF were isolated from colonic biopsies of healthy controls, ulcerative colitis (UC) and Crohn's disease (CD) patients. MiR-155 in IMF was quantified by quantitative reverse transcription-PCR in basal condition and following exposure to TNF-α, interleukin (IL)-1ß, lipopolysaccharide (LPS) or TGF-ß1. The effects of miR-155 mimic or inhibitor transfection on cytokine release and suppressor of cytokine signaling 1 (SOCS1) expression were assessed by enzyme-linked immunosorbent assay and western blot, respectively. Regulation of the target gene SOCS1 expression by miR-155 was assessed using luciferase reporter construct. We found that miR-155 was significantly upregulated in UC as compared with control- and CD-derived IMF. Moreover, TNF-α and LPS, but not TGF-ß1 and IL-1ß, significantly increased miR-155 expression in IMF. Ectopic expression of miR-155 in control IMF augmented cytokines release, whereas it downregulated SOCS1 expression. MiR-155 knockdown in UC-IMF reduced cytokine production and enhanced SOCS1 expression. Luciferase reporter assay demonstrated that miR-155 directly targets SOCS1. Moreover, silencing of SOCS1 in control IMF significantly increased IL-6 and IL-8 release. In all, our data suggest that inflammatory mediators induce miR-155 expression in IMF of patients with UC. By downregulating the expression of SOCS1, miR-155 wires IMF inflammatory phenotype.

Innate immune environment in ileal pouch mucosa: α5 defensin up-regulation as predictor of chronic/relapsing pouchitis.

Defensins are small cationic peptides with antibacterial activity expressed in Paneth cells (α-defensins) or generally in intestinal epithelial cells (ß-defensins) that have a profound effect on gut microbiota. Chronic pouchitis, which occurs in 5% of patients after restorative proctocolectomy and can cause pouch failure, is associated to a significant increase of Clostridiaceae spp. The aim of this study was to gain further insight in the pathogenesis of pouch dysbiosis by exploring defensin expression. Thirty-two consecutive patients coming for follow-up endoscopy were recruited. On pouch biopsies, we cultured bacteria adherent to the mucosa and determined α- and ß-defensins and toll-like receptor-4 and -2 mRNA by quantitative real-time RT-PCR. Serum and mucosal levels of IL-1ß, IL-6 and TNF-α were measured with immunometric assays. Faecal lactoferrin was analysed by quantitative ELISA. After a median follow-up of 23 (IQR 20-24) months, the patients were contacted for a reassessment of current and past disease activity. During the follow-up, chronic/relapsing pouchitis was diagnosed in six patients. The mucosal level of α-5 and α-6 defensins correlated with chronic/relapsing pouchitis onset (τ = 0.30, p = 0.034 and τ = 0.28, p = 0.053, respectively). High levels of α-5 defensin resulted to be predictive of chronic/relapsing pouchitis [AUC = 74% (95% CI = 53-89%), p = 0.052]. Patients with high levels of α-5 and α-6 defensins had earlier pouchitis relapses (p = 0.009 and p = 0.034, respectively). High levels of α-5 defensin were associated to a significant risk of chronic/relapsing pouchitis [OR = 10.6 (95% CI = 1.2-97.6), p = 0.027]. At multivariate analysis, the mucosal levels of α-5 defensin and the number of CFU of mucosa-associated Clostridiaceae spp resulted to be independent predictors of chronic/relapsing pouchitis [ß = 0.46 (0.18), p = 0.024 and ß = 0.44 (0.18), p = 0.027, respectively]. In conclusion, chronic/relapsing pouchitis is associated to increased expression of mucosal HD-5 and to increased antimicrobial activity against Escherichia coli. In patients with chronic/relapsing pouchitis, HD-5 and TLR-4 over-expression is likely to create a hostile environment against Enterobacteriaceae, thus favouring Clostridiaceae spp by decreasing competing bacteria families.

Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: involvement of the vascular endothelial growth factor receptor 2.

In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells.