Silvio Danese

Epithelial NEMO links innate immunity to chronic intestinal inflammation

Deregulation of intestinal immune responses seems to have a principal function in the pathogenesis of inflammatory bowel disease. The gut epithelium is critically involved in the maintenance of intestinal immune homeostasis—acting as a physical barrier separating luminal bacteria and immune cells, and also expressing antimicrobial peptides. However, the molecular mechanisms that control this function of gut epithelial cells are poorly understood. Here we show that the transcription factor NF-κB, a master regulator of pro-inflammatory responses, functions in gut epithelial cells to control epithelial integrity and the interaction between the mucosal immune system and gut microflora. Intestinal epithelial-cell-specific inhibition of NF-κB through conditional ablation of NEMO (also called IκB kinase-γ (IKKγ)) or both IKK1 (IKKα) and IKK2 (IKKβ)—IKK subunits essential for NF-κB activation—spontaneously caused severe chronic intestinal inflammation in mice. NF-κB deficiency led to apoptosis of colonic epithelial cells, impaired expression of antimicrobial peptides and translocation of bacteria into the mucosa. Concurrently, this epithelial defect triggered a chronic inflammatory response in the colon, initially dominated by innate immune cells but later also involving T lymphocytes. Deficiency of the gene encoding the adaptor protein MyD88 prevented the development of intestinal inflammation, demonstrating that Toll-like receptor activation by intestinal bacteria is essential for disease pathogenesis in this mouse model. Furthermore, NEMO deficiency sensitized epithelial cells to tumour-necrosis factor (TNF)-induced apoptosis, whereas TNF receptor-1 inactivation inhibited intestinal inflammation, demonstrating that TNF receptor-1 signalling is crucial for disease induction. These findings demonstrate that a primary NF-κB signalling defect in intestinal epithelial cells disrupts immune homeostasis in the gastrointestinal tract, causing an inflammatory-bowel-disease-like phenotype. Our results identify NF-κB signalling in the gut epithelium as a critical regulator of epithelial integrity and intestinal immune homeostasis, and have important implications for understanding the mechanisms controlling the pathogenesis of human inflammatory bowel disease.

Vedolizumab as Induction and Maintenance Therapy for Ulcerative Colitis

BACKGROUND Gut-selective blockade of lymphocyte trafficking by vedolizumab may constitute effective treatment for ulcerative colitis. METHODS We conducted two integrated randomized, double-blind, placebo-controlled trials of vedolizumab in patients with active disease. In the trial of induction therapy, 374 patients (cohort 1) received vedolizumab (at a dose of 300 mg) or placebo intravenously at weeks 0 and 2, and 521 patients (cohort 2) received open-label vedolizumab at weeks 0 and 2, with disease evaluation at week 6. In the trial of maintenance therapy, patients in either cohort who had a response to vedolizumab at week 6 were randomly assigned to continue receiving vedolizumab every 8 or 4 weeks or to switch to placebo for up to 52 weeks. A response was defined as a reduction in the Mayo Clinic score (range, 0 to 12, with higher scores indicating more active disease) of at least 3 points and a decrease of at least 30% from baseline, with an accompanying decrease in the rectal bleeding subscore of at least 1 point or an absolute rectal bleeding subscore of 0 or 1. RESULTS Response rates at week 6 were 47.1% and 25.5% among patients in the vedolizumab group and placebo group, respectively (difference with adjustment for stratification factors, 21.7 percentage points; 95% confidence interval [CI], 11.6 to 31.7; P<0.001). At week 52, 41.8% of patients who continued to receive vedolizumab every 8 weeks and 44.8% of patients who continued to receive vedolizumab every 4 weeks were in clinical remission (Mayo Clinic score ≤2 and no subscore >1), as compared with 15.9% of patients who switched to placebo (adjusted difference, 26.1 percentage points for vedolizumab every 8 weeks vs. placebo [95% CI, 14.9 to 37.2; P<0.001] and 29.1 percentage points for vedolizumab every 4 weeks vs. placebo [95% CI, 17.9 to 40.4; P<0.001]). The frequency of adverse events was similar in the vedolizumab and placebo groups. CONCLUSIONS Vedolizumab was more effective than placebo as induction and maintenance therapy for ulcerative colitis. (Funded by Millennium Pharmaceuticals; GEMINI 1 ClinicalTrials.gov number, NCT00783718.).

Development of the Crohn's disease digestive damage score, the Lemann score.

Crohns disease (CD) is a chronic progressive destructive disease. Currently available instruments measure disease activity at a specific point in time. An instrument to measure cumulative structural damage to the bowel, which may predict long-term disability, is needed. The aim of this article is to outline the methods to develop an instrument that can measure cumulative bowel damage. The project is being conducted by the International Program to develop New Indexes in Crohns disease (IPNIC) group. This instrument, called the Crohns Disease Digestive Damage Score (the Lémann score), should take into account damage location, severity, extent, progression, and reversibility, as measured by diagnostic imaging modalities and the history of surgical resection. It should not be “diagnostic modality driven”: for each lesion and location, a modality appropriate for the anatomic site (for example: computed tomography or magnetic resonance imaging enterography, and colonoscopy) will be used. A total of 24 centers from 15 countries will be involved in a cross-sectional study, which will include up to 240 patients with stratification according to disease location and duration. At least 120 additional patients will be included in the study to validate the score. The Lémann score is expected to be able to portray a patients disease course on a double-axis graph, with time as the x-axis, bowel damage severity as the y-axis, and the slope of the line connecting data points as a measure of disease progression. This instrument could be used to assess the effect of various medical therapies on the progression of bowel damage. (Inflamm Bowel Dis 2011)

Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 3: special situations.

### 8.1 General Proctocolectomy with ileal pouch-anal anastomosis (IPAA) is the procedure of choice for most patients with ulcerative colitis (UC) requiring colectomy.1 Pouchitis is a non-specific inflammation of the ileal reservoir and the most common complication of IPAA in patients with UC.2–7 Its frequency is related to the duration of follow up, occurring in up to 50% of patients 10 years after IPAA in large series from major referral centres.1–9 The cumulative incidence of pouchitis in patients with an IPAA for familial adenomatous polyposis is much lower, ranging from 0 to 10%.10–12 Reasons for the higher frequency of pouchitis in UC remain unknown. Whether pouchitis more commonly develops within the first years after IPAA or whether the risk continues to increase with longer follow up remains undefined. ### Statement 8A The diagnosis of pouchitis requires the presence of symptoms, together with characteristic endoscopic and histological abnormalities [EL3a, RG B]. Extensive UC, extraintestinal manifestations (i.e. PSC), being a non-smoker, p-ANCA positive serology and NSAID use are possible risk factors for pouchitis [EL3b, RG D] #### 8.1.1 Symptoms After proctocolectomy with IPAA, median stool frequency is 4 to 8 bowel movements,1–4,13,14 with about 700 mL of semiformed/liquid stool per day,2,13,14 compared to a volume of 200 mL/day in healthy people. Symptoms related to pouchitis include increased stool frequency and liquidity, abdominal cramping, urgency, tenesmus and pelvic discomfort.2,15 Rectal bleeding, fever, or extraintestinal manifestations may occur. Rectal bleeding is more often related to inflammation of the rectal cuff (“cuffitis,” Section 1.4),16 than to pouchitis. Faecal incontinence may occur in the absence of pouchitis after IPAA, but is more common in patients with pouchitis. Symptoms of pouch dysfunction in patients with IPAA may be caused by conditions other than pouchitis, including Crohns disease of the pouch,17–19 cuffitis16 and an irritable pouch …

Regulation of leukocyte recruitment by the long pentraxin PTX3

Pentraxins are a superfamily of conserved proteins involved in the acute-phase response and innate immunity. Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a key component of the humoral arm of innate immunity that is essential for resistance to certain pathogens. A regulatory role for pentraxins in inflammation has long been recognized, but the underlying mechanisms remain unclear. Here we report that PTX3 bound P-selectin and attenuated neutrophil recruitment at sites of inflammation. PTX3 released from activated leukocytes functioned locally to dampen neutrophil recruitment and regulate inflammation. Antibodies have glycosylation-dependent regulatory effect on inflammation. Therefore, PTX3, which is an essential component of humoral innate immunity, and immunoglobulins share functional outputs, including complement activation, opsonization and, as shown here, glycosylation-dependent regulation of inflammation.

Treatment of Relapsing Mild-to-Moderate Ulcerative Colitis With the Probiotic VSL#3 as Adjunctive to a Standard Pharmaceutical Treatment: A Double-Blind, Randomized, Placebo-Controlled Study

OBJECTIVES:VSL#3 is a high-potency probiotic mixture that has been used successfully in the treatment of pouchitis. The primary end point of the study was to assess the effects of supplementation with VSL#3 in patients affected by relapsing ulcerative colitis (UC) who are already under treatment with 5-aminosalicylic acid (ASA) and/or immunosuppressants at stable doses.METHODS:A total of 144 consecutive patients were randomly treated for 8 weeks with VSL#3 at a dose of 3,600 billion CFU/day (71 patients) or with placebo (73 patients).RESULTS:In all, 65 patients in the VSL#3 group and 66 patients in the placebo group completed the study. The decrease in ulcerative colitis disease activity index (UCDAI) scores of 50% or more was higher in the VSL#3 group than in the placebo group (63.1 vs. 40.8; per protocol (PP) P=0.010, confidence interval (CI)95% 0.51–0.74; intention to treat (ITT) P=0.031, CI95% 0.47–0.69). Significant results with VSL#3 were recorded in an improvement of three points or more in the UCDAI score (60.5% vs. 41.4%; PP P=0.017, CI95% 0.51–0.74; ITT P=0.046, CI95% 0.47–0.69) and in rectal bleeding (PP P=0.014, CI95% 0.46–0.70; ITT P=0.036, CI95% 0.41–0.65), whereas stool frequency (PP P=0.202, CI95% 0.39–0.63; ITT P=0.229, CI95% 0.35–0.57), physicians rate of disease activity (PP P=0.088, CI95% 0.34–0.58; ITT P=0.168, CI95% 0.31–0.53), and endoscopic scores (PP P=0.086, CI95% 0.74–0.92; ITT P=0.366, CI95% 0.66–0.86) did not show statistical differences. Remission was higher in the VSL#3 group than in the placebo group (47.7% vs. 32.4%; PP P=0.069, CI95% 0.36–0.60; ITT P=0.132, CI95% 0.33–0.56). Eight patients on VSL#3 (11.2%) and nine patients on placebo (12.3%) reported mild side effects.CONCLUSIONS:VSL#3 supplementation is safe and able to reduce UCDAI scores in patients affected by relapsing mild-to-moderate UC who are under treatment with 5-ASA and/or immunosuppressants. Moreover, VSL#3 improves rectal bleeding and seems to reinduce remission in relapsing UC patients after 8 weeks of treatment, although these parameters do not reach statistical significance.

Farnesoid X receptor activation inhibits inflammation and preserves the intestinal barrier in inflammatory bowel disease

Background & aims Inflammatory bowel disease (IBD) is characterised by chronic intestinal inflammation, resulting from dysregulation of the mucosal immune system and compromised intestinal epithelial barrier function. The bile salt, nuclear farnesoid X receptor (FXR), was recently implicated in intestinal antibacterial defence and barrier function. The aim of this study was to investigate the therapeutic potential of FXR agonists in the treatment of intestinal inflammation in complementary in vivo and in vitro models. Methods Colitis was induced in wild-type (WT) and Fxr-null mice using dextran sodium sulfate, and in WT mice using trinitrobenzenesulfonic acid. Mice were treated with vehicle or the FXR agonist INT-747, and colitis symptoms were assessed daily. Epithelial permeability assays and cytokine expression analysis were conducted in mouse colon and enterocyte-like cells (Caco-2/HT29) treated with medium or INT-747. Inflammatory cytokine secretion was determined by ELISA in various human immune cell types. Results INT-747-treated WT mice are protected from DSS- and TNBS-induced colitis, as shown by significant reduction of body weight loss, epithelial permeability, rectal bleeding, colonic shortening, ulceration, inflammatory cell infiltration and goblet cell loss. Furthermore, Fxr activation in intestines of WT mice and differentiated enterocyte-like cells downregulates expression of key proinflammatory cytokines and preserves epithelial barrier function. INT-747 significantly decreases tumour necrosis factor α secretion in activated human peripheral blood mononuclear cells, purified CD14 monocytes and dendritic cells, as well as in lamina propria mononuclear cells from patients with IBD. Conclusions FXR activation prevents chemically induced intestinal inflammation, with improvement of colitis symptoms, inhibition of epithelial permeability, and reduced goblet cell loss. Furthermore, FXR activation inhibits proinflammatory cytokine production in vivo in the mouse colonic mucosa, and ex vivo in different immune cell populations. The findings provide a rationale to explore FXR agonists as a novel therapeutic strategy for IBD.

Inflammation and Coagulation in Inflammatory Bowel Disease: The Clot Thickens

Inflammation and coagulation play crucial roles in the pathogenesis of multiple chronic inflammatory disorders. Growing evidence highlights a tight mutual network in which inflammation, coagulation, and fibrinolysis play closely related roles. Crohns disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD), are chronic inflammatory conditions, characterized by a hypercoagulable state and prothrombotic conditions, and accompanied by abnormalities in coagulation. From a pathophysiological point of view, cells and molecules classically implicated in the physiological process of coagulation have now been shown to behave abnormally in IBD and possibly to also play an active role in disease pathogenesis and/or disease progression. This paper reviews studies performed on the coagulation profile and risk factors for thrombosis in IBD. In particular, an overview is provided of the epidemiology, clinical features, and etiology of thromboembolic complications in IBD. Furthermore, we review hemostatic abnormalities in IBD, as well as the cell types involved in such processes. Finally, we highlight the coagulation system as a dynamic participant in the multifaceted process of chronic intestinal inflammation. Overall, an overview is provided that the coagulation system represents an important, though previously underestimated, component of IBD pathogenesis, and may be a possible target for therapeutic intervention.

New therapies for inflammatory bowel disease: from the bench to the bedside

The mechanisms underlying the chronic intestinal inflammation that is a hallmark of inflammatory bowel diseases (IBD) are complex. Components of the pathological response include the adaptive and innate immune systems, as well as the intestinal epithelium and endothelium. Advances in the understanding of the roles of each of these components have resulted in the development of multiple biological agents that all represent an alternative to the use of current therapies in patients with refractory Crohns disease or ulcerative colitis. This study systematically reviews the mechanisms of action, efficacy and safety of new and emerging therapies that are currently in clinical trials and discusses future directions in the treatment of IBD.

Activated platelets are the source of elevated levels of soluble CD40 ligand in the circulation of inflammatory bowel disease patients

Background: The CD40/CD40L system, a key regulator and amplifier of immune reactivity, is activated in inflammatory bowel disease (IBD) mucosa. Aims: To determine whether plasma levels of sCD40L are elevated in Crohn’s disease (CD) and ulcerative colitis (UC) patients compared with normal controls, to investigate the cellular source of sCD40L, and to explore CD40L induction mechanisms. Patients: CD, UC, and normal control subjects were studied. Methods: The concentration of sCD40L in plasma and supernatants of freshly isolated platelets and autologous peripheral blood T cells (PBT) was measured by ELISA. Surface CD40L expression level was measured by flow cytometry in resting and thrombin activated platelets, and unstimulated and CD3/CD28 stimulated PBT before and after coculture with human intestinal microvascular endothelial cells (HIMEC). Results: Compared with normal controls, plasma sCD40L levels were significantly higher in both CD and UC patients and proportional to the extent of mucosal inflammation. Platelets from IBD patients displayed a significantly higher surface CD40L expression than those from control subjects, and released greater amounts of sCD40L than autologous PBT. Contact with IL-1β activated HIMEC induced significant upregulation of CD40L surface expression and release by platelets. Conclusions: Elevated levels of sCD40L in the circulation of IBD patients reflect enhanced surface expression and release of CD40L by platelets. This phenomenon translates to an increased platelet activation state apparently induced by passage through an inflamed mucosal microvascular bed, a conclusion supported by the positive correlation of plasma sCD40L levels with the extent of anatomical involvement by IBD. These results suggest that platelet-endothelial interactions critically contribute to activation of the CD40 pathway in IBD.