Davide Di Fusco

TRAF3IP2 gene is associated with cutaneous extraintestinal manifestations in Inflammatory Bowel Disease

BACKGROUND AND AIMS Genome-wide association (GWA) studies recently identified a novel gene, TRAF3IP2, involved in the susceptibility to psoriasis. Common immune-mediated mechanisms involving the skin or the gut have been suggested. We therefore aimed to assess the role of TRAF3IP2 gene in IBD, with particular regard to the development of cutaneous extraintestinal manifestations (pyoderma gangrenosum, erythema nodosum). The association with psoriasis was also assessed in a secondary analysis. METHODS The analysis included 267 Crohns disease (CD), 200 ulcerative colitis (UC) patients and 278 healthy controls. Three TRAF3IP2 SNPs were genotyped by allelic discrimination assays. A case/control association study and a genotype/phenotype correlation analysis have been performed. RESULTS All three SNPs conferred a high risk to develop cutaneous manifestations in IBD. A higher risk of pyoderma gangrenosum and erythema nodosum was observed in CD patients carrying the Rs33980500 variant (OR 3.03; P=0.026). In UC, a significantly increased risk was observed for both the Rs13190932 and the Rs13196377 SNPs (OR 5.05; P=0.02 and OR 4.1; P=0.049). Moreover, association of TRAF3IP2 variants with ileal (OR=1.92), fibrostricturing (OR=1.91) and perianal CD (OR=2.03) was observed. CONCLUSIONS This is the first preliminary report indicating that TRAF3IP2 variants increase the risk of cutaneous extraintestinal manifestations in IBD suggesting that the analysis of the TRAF3IP2 variants may be useful for identifying IBD patients at risk to develop these manifestations.

A multilocus genetic study in a cohort of Italian SLE patients confirms the association with STAT4 gene and describes a new association with HCP5 gene.

Background Systemic lupus erythematosus (SLE) is an autoimmune disease with complex pathogenesis in which genes and environmental factors are involved. We aimed at analyzing previously identified loci associated with SLE or with other autoimmune and/or inflammatory disorders (STAT4, IL10, IL23R, IRAK1, PSORS1C1, HCP5, MIR146a, PTPN2, ERAP1, ATG16L1, IRGM) in a sample of Italian SLE patients in order to verify or confirm their possible involvement and relative contribution in the disease. Materials and methods Two hundred thirty-nine consecutive SLE patients and 278 matched healthy controls were enrolled. Study protocol included complete physical examination, and clinical and laboratory data collection. Nineteen polymorphisms were genotyped by allelic discrimination assays. A case-control association study and a genotype-phenotype correlation were performed. Results STAT4 was the most associated gene [P = 3×10−7, OR = 2.13 (95% CI: 1.59–2.85)]. IL10 confirmed its association with SLE [rs3024505: P = 0.02, OR = 1.52 (95% CI: 1.07–2.16)]. We describe a novel significant association between HCP5 locus and SLE susceptibility [rs3099844: P = 0.01, OR = 2.06 (95% CI: 1.18–3.6)]. The genotype/phenotype correlation analysis showed several associations including a higher risk to develop pericarditis with STAT4, and an association between HCP5 rs3099844 and anti-Ro/SSA antibodies. Conclusions STAT4 and IL10 confirm their association with SLE. We found that some SNPs in PSORS1C1, ATG16L1, IL23R, PTPN2 and MIR146a genes can determine particular disease phenotypes. HCP5 rs3099844 is associated with SLE and with anti-Ro/SSA. This polymorphism has been previously found associated with cardiac manifestations of SLE, a condition related with anti-Ro/SSA antibodies. Thus, our results may provide new insights into SLE pathogenesis.

Association between CYP2B6 polymorphisms and Nevirapine-induced SJS/TEN: a pharmacogenetics study.

PurposeNevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor, widely prescribed for type 1 human immunodeficiency virus infection. A small proportion of individuals treated with NVP experience severe cutaneous adverse events, including Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Our aim was to verify whether genetic variability in NVP-metabolizing cytochromes or in transporter genes could be involved in susceptibility to SJS/TEN.MethodsTwenty-seven patients with NVP-induced SJS/TEN and 78 controls, all from Mozambique, were genotyped for the ABCB1 and ABCC10 transporter genes and for CYP2B6, CYP3A4 and CYP3A5 cytochrome gene variants. A case–control and a genotype–phenotype analysis were performed.ResultsCYP2B6 G516T and T983C single nucleotide polymorphisms (SNPs) were found to be associated with SJS/TEN susceptibility. The 983C allele in particular was found to be highly associated with a higher risk to develop SJS/TEN [odds ratio (OR) 4.2, P = 0.0047]. The GT haplotype (wildtype for both SNPs) showed a protective effect, with an OR = 0.33 (P = 0.0016).ConclusionsThis is the first study showing that genetic variability in a metabolizing enzyme can also contribute to NVP-induced SJS/TEN susceptibility.

Sodium chloride–enriched Diet Enhanced Inflammatory Cytokine Production and Exacerbated Experimental Colitis in Mice

Background and Aim: Environmental factors are supposed to play a decisive role in the pathogenesis of inflammatory bowel diseases [IBDs]. Increased dietary salt intake has been linked with the development of autoimmune diseases, but the impact of a salt-enriched diet on the course of IBD remains unknown. In this study, we examined whether high salt intake alters mucosal cytokine production and exacerbates colitis. Methods: Normal intestinal lamina propria mononuclear cells [LPMCs] were activated with anti-CD3/CD28 in the presence or absence of increasing concentrations of sodium chloride [NaCl] and/or SB202190, a specific inhibitor of p38/MAP Kinase. For in vivo experiments, a high dose of NaCl was administered to mice 15 days before induction of trinitrobenzene-sulfonic acid [TNBS]-colitis or dextran sulfate sodium [DSS]-colitis. In parallel, mice were given SB202190 before induction of TNBS-colitis. Transcription factors and effector cytokines were evaluated by flow-cytometry and real-time PCR. Results: IL-17A, IL-23R, TNF-&agr;, and Ror-&ggr;T were significantly increased in human LPMCs following NaCl exposure, while there was no significant change in IFN-&ggr;, T-bet or Foxp3. Pharmacologic inhibition of p38/MAPK abrogated the NaCl-inducing effect on LPMC-derived cytokines. Mice receiving the high-salt diet developed a more severe colitis than control mice, and this effect was preventable by SB202190. Conclusions: Our data indicated that exposure of intestinal mononuclear cells to a high-NaCl diet enhanced effector cytokine production and contributed to the exacerbation of experimental colitis in mice.

Antisense Approach to Inflammatory Bowel Disease: Prospects and Challenges

Despite the great success of anti-tumour necrosis factor-based therapies, the treatment of Crohn’s disease (CD) and ulcerative colitis (UC) still remains a challenge for clinicians, as these drugs are not effective in all patients, their efficacy may wane with time, and their use can increase the risk of adverse events and be associated with the development of new immune-mediated diseases. Therefore, new therapeutic targets are currently being investigated both in pre-clinical studies and in clinical trials. Among the technologies used to build new therapeutic compounds, the antisense oligonucleotide (ASO) approach is slowly gaining space in the field of inflammatory bowel diseases (IBDs), and three ASOs have been investigated in clinical trials. Systemic administration of alicaforsen targeting intercellular adhesion molecule-1, a protein involved in the recruitment of leukocytes to inflamed intestine, was not effective in CD, even though the same compound was of benefit when given as an enema to UC patients. DIMS0150, targeting nuclear factor (NF) κB-p65, a transcription factor that promotes pro-inflammatory responses, was very promising in pre-clinical studies and is currently being tested in clinical trials. Oral mongersen, targeting Smad7, an intracellular protein that inhibits transforming growth factor (TGF)-β1 activity, was safe and well tolerated by CD patients, and the results of a phase II clinical trial showed the efficacy of the drug in inducing clinical remission in patients with active disease. In this leading article, we review the rationale and the clinical data available regarding these three agents, and we discuss the challenge of using ASOs in IBD.

Aryl hydrocarbon receptor-driven signals inhibit collagen synthesis in the gut

Fibrostrictures (FS) are a major complication of Crohns disease (CD). Pathogenesis of FS is not fully understood, but activation of fibroblasts and excessive collagen deposition are crucial in the development of FS. Here, we investigated the role of aryl hydrocarbon receptor (AhR) in intestinal fibrosis. AhR RNA and protein expression were evaluated in intestinal fibroblasts of CD patients and controls. CD fibroblasts were stimulated with TGF‐β1 or TNF‐α in the presence or absence of the AhR activator Ficz, an AhR antagonist CH223191, or a specific AhR‐silencing RNA. In CD fibroblasts, TGF‐β1 and TNF‐α increased Col1A1, Col3A1 and α‐SMA transcripts and collagen secretion and this effect was reduced by Ficz and upregulated by CH22319. TGF‐β1 or TNF‐α induced activation of p38 and ERK1/2 MAP kinases was decreased by Ficz and increased by CH223191. The inhibitory effect of Ficz on Map kinase activation and collagen induction was abolished by AhR silencing. To assess the role of AhR in vivo, mice with trinitrobenzene‐sulfonic‐acid induced colonic fibrosis were given Ficz or CH223191. Mice given either Ficz or CH223191 produced less or more collagen respectively as compared with control mice. Our results indicate that AhR is a negative regulator of profibrotic signals in the gut.

TNF-α Producing Innate Lymphoid Cells (ILCs) Are Increased in Active Celiac Disease and Contribute to Promote Intestinal Atrophy in Mice

Innate lymphoid cells (ILCs) are an emerging family of innate hematopoietic cells producing inflammatory cytokines and involved in the pathogenesis of several immune-mediated diseases. The aim of this study was to characterize the tissue distribution of ILCs in celiac disease (CD), a gluten-driven enteropathy, and analyze their role in gut tissue damage. ILC subpopulations were analyzed in lamina propria mononuclear cells (LPMCs) isolated from duodenal biopsies of CD patients and healthy controls (CTR) and jejunal specimens of patients undergoing gastro-intestinal bypass by flow cytometry. Cytokines and Toll-like receptors (TLR) were assessed in ILCs either freshly isolated or following incubation of control LPMC with peptidoglycan, poly I:C, or CpG, the agonists of TLR2, TLR3, or TLR9 respectively, by flow cytometry. The role of ILCs in gut tissue damage was evaluated in a mouse model of poly I:C-driven small intestine atrophy. Although the percentage of total ILCs did not differ between CD patients and CTR, ILCs producing TNF-α and IFN-γ were more abundant in CD mucosa compared to controls. ILCs expressed TLR2, TLR3 and TLR9 but neither TLR7 nor TLR4. Stimulation of LPMC with poly I:C but not PGN or CpG increased TNF-α and IFN-γ in ILCs. RAG1-deficient mice given poly I:C exhibited increased frequency of TNF-α but not IFN-γ/IL17A-producing ILCs in the gut and depletion of ILCs prevented the poly I:C-driven intestinal damage. Our data indicate that CD-related inflammation is marked by accumulation of ILCs producing TNF-α and IFN-γ in the mucosa. Moreover, ILCs express TLR3 and are functionally able to respond to poly I:C with increased synthesis of TNF-α thus contributing to small intestinal atrophy.

IL-21 as a therapeutic target in inflammatory disorders

Introduction: IL-21, a cytokine produced by activated CD4+ cells, activated natural killer T cells and T helper cells in the germinal centers, is involved in the control of the function of both immune and parenchymal cells. Areas covered: IL-21 is overproduced in many chronic inflammatory disorders, including inflammatory bowel diseases, psoriasis, rheumatoid arthritis, type I diabetes and systemic lupus erythematosus, and studies in experimental models indicate that IL-21 plays an important role in sustaining tissue-damaging immune responses in such pathologies. However, genetic deficiency of IL-21 associates with inflammatory bowel diseases and blockade of IL-21 in the early phases exacerbates the disease progression in some models of rheumatoid arthritis and systemic lupus erythematosus, thus suggesting a dual role of IL-21 in the control of immune-mediated diseases. IL-21 can exert additional protective functions for the host as it promotes cytotoxic responses against tumors and viruses. Expert opinion: We here review the available data on the role of IL-21 in chronic inflammatory diseases and discuss the therapeutic benefit of IL-21 inhibitors in such diseases as well as the potential risks of such treatments.

CCL20 Is Negatively Regulated by TGF-β1 in Intestinal Epithelial Cells and Reduced in Crohn’s Disease Patients With a Successful Response to Mongersen, a Smad7 Antisense Oligonucleotide

Background and Aims The chemokine CCL20 is over-produced in epithelium of Crohns disease [CD] patients and contributes to recruiting immune cells to inflamed gut. Tumour necrosis factor-α [TNF-α] is a powerful inducer of CCL20 in intestinal epithelial cells. In CD, high levels of Smad7 block the activity of transforming growth factor-β1 [TGF-β1], a negative regulator of TNF signalling. We investigated whether intestinal epithelial cell-derived CCL20 is negatively regulated by TGF-β1 and whether Smad7 knock-down reduces CCL20 in CD. Methods CCL20 was evaluated in NCM460, a normal colonic epithelial cell line, stimulated with TGF-β1 and TNF-α, and in Smad7 over-expressing NCM460 cells. CCL20 and Smad7 expression were assessed in sections of CD intestinal specimens by immunochemistry, and in CD colonic explants treated with mongersen, a Smad7 antisense oligonucleotide. CCL20 was examined in serum samples taken from 95 of 166 active CD patients receiving mongersen or placebo for 2 weeks and participating in a phase II, multicentre, double-blind, placebo-controlled study. Results CCL20 expression was increased by TNF-α, and this effect was inhibited by TGF-β1 in NCM460 cells, but not in Smad7 over-expressing NCM460 cells. In CD, epithelium CCL20 and Smad7 co-localised, and treatment of CD explants with mongersen reduced CCL20 production. During follow-up, in responders to mongersen, serum CCL20 levels significantly decreased, whereas patients without response/remission to mongersen and placebo patients did not have change in CCL20. Conclusions TGF-β1 reduces intestinal epithelial cell-derived CCL20 production, an effect abrogated by Smad7. CD patients responding to mongersen demonstrated a reduction in serum CCL20.

The TGF-β/Smad System in IBD Pathogenesis

Abstract:In Crohns disease and ulcerative colitis, the tissue-damaging destructive immune response is sustained by defects of counterregulatory mechanisms, which normally attenuate inflammatory pathways and promote repair of mucosal injury. One such mechanism involves transforming growth factor-&bgr;1 (TGF-&bgr;1), a cytokine that is produced by multiple cell types and targets both immune and nonimmune cells. Both in vitro and in vivo studies strongly support the role of TGF-&bgr;1 as a negative regulator of mucosal inflammation and indicate that defective production/activity of this cytokine can lead to the development of or exacerbate colitis. Interestingly, in the inflamed intestine of patients with inflammatory bowel disease, TGF-&bgr;1 expression is upregulated but TGF-&bgr;1–mediated immunosuppression is markedly impaired because of high Smad7, an intracellular inhibitor of TGF-&bgr;1–associated signaling. Consistently, knockdown of Smad7 with a specific antisense oligonucleotide restores TGF-&bgr;1 activity, thus leading to decreased production of inflammatory cytokines in both colitic mice and inflammatory bowel disease patients and attenuates clinical activity in Crohns disease patients. In this article, we review data supporting the role of Smad7 in the pathogenesis of inflammatory bowel disease and discuss whether inhibition of Smad7 is therapeutically useful in Crohns disease and how the benefit/risk of such an intervention should be monitored in the patients.