Irene Marafini

Distinct Profiles of Effector Cytokines Mark the Different Phases of Crohn’s Disease

Objective Crohn’s Disease (CD)-associated inflammation is supposed to be driven by T helper (Th)1/Th17 cell-derived cytokines, even though there is evidence that the mucosal profile of cytokine may vary with the evolution of the disease. We aimed at comparing the pattern of effector cytokines in early and established lesions of CD. Design Mucosal samples were taken from the neo-terminal ileum of CD patients undergoing ileocolonic resection, with (early lesions) or without post-operative recurrence, and terminal ileum of CD patients with long-standing disease undergoing intestinal resection (established lesions). Inflammatory cell infiltrate was examined by immunofluorescence and cytokine expression was analysed by real-time PCR, flow-cytometry and ELISA. Results Before the appearance of endoscopic lesions, the mucosa of the neo-terminal ileum contained high number of T cells and macrophages, elevated levels of Th1-related cytokines and TNF-α and slightly increased IL-17A expression. Transition from this stage to endoscopic recurrence was marked by abundance of Th1 cytokines, marked increase in IL-17A, and induction of IL-6 and IL-23, two cytokines involved in the control of Th17 cell responses. In samples with established lesions, there was a mixed Th1/Th17 response with no TNF-α induction. Expression of IL-4 and IL-5 was up-regulated in both early and established lesions even though the fraction of IL-4-producing cells was lower than that of cells producing either interferon-γ or IL-17A. Conclusions Distinct mucosal profiles of cytokines are produced during the different phases of CD. A better understanding of the cytokines temporally regulated in CD tissue could help optimize therapeutic interventions in CD.

Th17 Cytokines in Inflammatory Bowel Diseases: Discerning the Good from the Bad

T helper (Th) 17 cells are a branch of the CD4+ T cell compartment involved in host protection against bacterial and fungal infections as well as in orchestration of chronic inflammation and autoimmunity in many organs. Th17 cells produce interleukin (IL)-17A and variable amounts of IL-17F, IL-21, IL-22 and IL-26, under the regulation of retinoic-acid-related orphan receptors (ROR)-γt and ROR-α. Accumulating evidence supports the involvement of Th17 cells in the tissue-damaging immune response occurring in patients with Crohns disease and patients with ulcerative colitis, the two major forms of inflammatory bowel disease (IBD) in human beings. However in the gut, Th17 cells can also have tissue-protective effects, mostly depending on their ability to enhance epithelial barrier function and counter-regulatory mechanisms. In this article, we summarize the available data on the dual role of Th17 cells in gut homeostasis and inflammation and discuss whether and how Th17 cytokine blockers can enter into the therapeutic armamentarium of IBD.

The Dual Role of Smad7 in the Control of Cancer Growth and Metastasis

Smad7 was initially identified as an inhibitor of Transforming growth factor (TGF)-β due mainly to its ability to bind TGF-β receptor type I and prevent TGF-β-associated Smad signaling. More recently, it has been demonstrated that Smad7 can interact with other intracellular proteins and regulate also TGF-β-independent signaling pathways thus making a valid contribution to the neoplastic processes in various organs. In particular, data emerging from experimental studies indicate that Smad7 may differently modulate the course of various tumors depending on the context analyzed. These observations, together with the demonstration that Smad7 expression is deregulated in many cancers, suggest that therapeutic interventions around Smad7 can help interfere with the development/progression of human cancers. In this article we review and discuss the available data supporting the role of Smad7 in the modulation of cancer growth and progression.

Defective expression of SIRT1 contributes to sustain inflammatory pathways in the gut

In inflammatory bowel disease (IBD), tissue damage is driven by an excessive immune response, poorly controlled by counter-regulatory mechanisms. SIRT1, a class III NAD+-dependent deacetylase, regulates negatively the expression of various proteins involved in the control of immune-inflammatory pathways, such as Stat3, Smad7, and NF-κB. Here we examined the expression, regulation, and function of SIRT1 in IBD. SIRT1 RNA and protein expression was less pronounced in whole biopsies and lamina propria mononuclear cells (LPMCs) of IBD patients in comparison with normal controls. SIRT1 expression was downregulated in control LPMC by tumor necrosis factor (TNF)-α and interleukin (IL)-21, and upregulated in IBD LPMC by neutralizing TNF-α and IL-21antibodies. Consistently, SIRT1 expression was increased in mucosal samples taken from IBD patients successfully treated with Infliximab. Treatment of IBD LPMC with Cay10591, a specific SIRT1 activator, reduced NF-κB activation and inhibited inflammatory cytokine synthesis, whereas Ex527, an inhibitor of SIRT1, increased interferon (IFN)-γ in control LPMC. SIRT1 was also reduced in mice with colitis induced by 2,4,6-trinitrobenzenesulphonic acid or oxazolone. Cay10591 prevented and cured experimental colitis whereas Ex527 exacerbated disease by modulating T cell-derived cytokine response. Data indicate that SIRT1 is downregulated in IBD patients and colitic mice and suggest that SIRT1 activation can help attenuate inflammatory signals in the gut.

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.

Plasma Cells in the Mucosa of Patients with Inflammatory Bowel Disease Produce Granzyme B and Possess Cytotoxic Activities

In both Crohn’s disease (CD) and ulcerative colitis (UC), the gut is massively infiltrated with B cells and plasma cells, but the role of these cell types in the pathogenesis of gut tissue damage remains largely unknown. Human B cells express granzyme B (GrB) when cultured with IL-21, a cytokine overproduced in CD and UC mucosa. We therefore examined whether mucosal B cells express GrB and have cytotoxic activity in inflammatory bowel disease (IBD). GrB-expressing CD19+ and IgA+ cells were seen in the normal intestinal mucosa, but they were significantly more frequent in both CD and UC. In contrast, only a minority of CD19+ and IgA+ cells expressed perforin with no difference between IBD and controls. GrB-producing CD19+ cells expressed CD27 and were CD38high and CD20 negative. CD19+ B cells from IBD patients induced HCT-116 cell death. IL-21 enhanced GrB expression in control CD19+ B cells and increased their cytotoxic activity. These data indicate that IBD-related inflammation is marked by mucosal accumulation of cytotoxic, GrB-expressing CD19+ and IgA+ cells, suggesting a role for these cells in IBD-associated epithelial damage.

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.

Analysis of the cytokine profile in the duodenal mucosa of refractory coeliac disease patients

RCD [refractory CD (coeliac disease)] is characterized by severe symptoms/signs of malabsorption and mucosal damage unresponsive to a GFD (gluten-free diet). The pathogenesis of RCD is not fully understood. In the present paper, we have characterized the mucosal profile of effector cytokines in RCD. Duodenal biopsies were taken from patients with RCD, patients with active CD and normal controls and were analysed for inflammatory cytokines by real-time PCR and ELISA. IFN (interferon)-γ and IL (interleukin)-21 transcripts were increased in active CD patients but not in RCD patients as compared with normal controls, whereas IL-17A RNA was up-regulated in both active CD and RCD. No significant increase in IL-15 transcripts was observed in both active CD and RCD, whereas IL-15 protein was increased in active CD. IL-6 and TNF (tumour necrosis factor)-α were up-regulated only in RCD. As a proof, we present the case of a woman affected by RCD who responded to anti-TNF-α treatment with improvement of malabsorptive symptoms/signs but no healing of mucosal lesions. The findings indicate that the profile of mucosal effector cytokines differs between RCD and active CD and suggest that TNF-α, IL-6 and IL-17A, but not Th1-type cytokines, could drive the detrimental response in this condition.

TGF-Beta signaling manipulation as potential therapy for IBD.

Crohns disease (CD) and ulcerative colitis (UC), two chronic and relapsing inflammatory bowel diseases (IBD), are supposed to develop in genetically-predisposed individuals as a result of an excessive immune mucosal response directed against normal components of the gut microbiota. There is also evidence that defects in counter-regulatory mechanisms play a major role in the pathogenesis of IBD. One such a defect involves TGF-β1, a cytokine produced by multiple cells types and able to inhibit pathogenic responses in the gut. In both CD and UC, TGF-β1 is highly produced but unable to signal through the TGF-β receptor-associated Smad pathway and suppress production of inflammatory molecules. Abrogation of TGF-β1 activity has been related to Smad7, an intracellular protein that binds to TGF-β receptor and inhibits TGF-β1-driven Smad-dependent signalling. Indeed, silencing of Smad7 with a specific antisense oligonucleotide restores TGF-β1/Smad signalling, thereby down-regulating inflammatory cytokine production and ameliorating experimental colitis in mice. Altogether these observations led to the development of an oral pharmaceutical compound containing the specific Smad7 antisense oligonucleotide (herein termed GED0301), which seems to be safe and well tolerated in CD patients. In this article we summarize the data supporting the pathogenic role of Smad7 in IBD and discuss the recent results of the use of GED0301 in CD.

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.