Jonas Mudter

CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific blockade of Rac1 activation through binding of azathioprine-generated 6-thioguanine triphosphate (6-Thio-GTP) to Rac1 instead of GTP. The activation of Rac1 target genes such as mitogen-activated protein kinase kinase (MEK), NF-kappaB, and bcl-x(L) was suppressed by azathioprine, leading to a mitochondrial pathway of apoptosis. Azathioprine thus converts a costimulatory signal into an apoptotic signal by modulating Rac1 activity. These findings explain the immunosuppressive effects of azathioprine and suggest that 6-Thio-GTP derivates may be useful as potent immunosuppressive agents in autoimmune diseases and organ transplantation.

The Transcription Factor T-bet Regulates Mucosal T Cell Activation in Experimental Colitis and Crohn's Disease

The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-γ/interleukin (IL)-4 and transforming growth factor (TGF)-β activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models of chronic intestinal inflammation by taking advantage of mice that lack T-bet and retroviral transduction techniques, respectively. Whereas retroviral transduction of T-bet in CD62L+ CD4+ T cells exacerbated colitis in reconstituted SCID mice, T-bet–deficient T cells failed to induce colitis in adoptive transfer experiments suggesting that overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis in vivo. Furthermore, T-bet–deficient CD62L− CD4+ T cells showed enhanced protective functions in Th1-mediated colitis and exhibited increased TGF-β signaling suggesting that a T-bet driven pathway of T cell activation controls the intestinal balance between IFN-γ/IL-4 and TGF-β responses and the development of chronic intestinal inflammation in T cell–mediated colitis. Furthermore, TGF-β was found to suppress T-bet expression suggesting a reciprocal relationship between TGF-β and T-bet in mucosal T cells. In summary, our data suggest a key regulatory role of T-bet in the pathogenesis of T cell–mediated colitis. Specific targeting of this pathway may be a promising novel approach for the treatment of patients with Crohns disease and other autoimmune diseases mediated by Th1 T lymphocytes.

High definition colonoscopy combined with i-Scan is superior in the detection of colorectal neoplasias compared with standard video colonoscopy: a prospective randomized controlled trial

INTRODUCTION Colonoscopy is the accepted gold standard for the detection of colorectal cancer. The aim of the current study was to prospectively compare high definition plus (HD+) colonoscopy with I-Scan functionality (electronic staining) vs. standard video colonoscopy. The primary endpoint was the detection of patients having colon cancer or at least one adenoma. METHODS A total of 220 patients due to undergo screening colonoscopy, postpolypectomy surveillance or with a positive occult blood test were randomized in a 1 : 1 ratio to undergo HD+ colonoscopy in conjunction with I-Scan surface enhancement (90i series, Pentax, Tokyo, Japan) or standard video colonoscopy (EC-3870FZK, Pentax). Detected colorectal lesions were judged according to type, location, and size. Lesions were characterized in the HD+ group by using further I-Scan functionality (p- and v-modes) to analyze pattern and vessel architecture. Histology was predicted and biopsies or resections were performed on all identified lesions. RESULTS HD+ colonoscopy with I-Scan functionality detected significantly more patients with colorectal neoplasia (38 %) compared with standard resolution endoscopy (13 %) (200 patients finally analyzed; 100 per arm). Significantly more neoplastic (adenomatous and cancerous) lesions and more flat adenomas could be detected using high definition endoscopy with surface enhancement. Final histology could be predicted with high accuracy (98.6 %) within the HD+ group. CONCLUSIONS HD+ colonoscopy with I-Scan is superior to standard video colonoscopy in detecting patients with colorectal neoplasia based on this prospective, randomized, controlled trial.

Functional Relevance of T Helper 17 (Th17) Cells and the IL-17 Cytokine Family in Inflammatory Bowel Disease

&NA; The recent discovery and characterization of T helper 17 cells (Th17) and their signature cytokines (IL‐17) represents a hallmark in T‐cell immunobiology by providing a new distinctive pathway for the communication between adaptive and innate immunity. From the six members of the IL‐17 cytokine family presently known, at least two have evident proinflammatory qualities and are involved in several chronic inflammatory disorders, including inflammatory bowel disease (IBD). IL‐17A and IL‐17F are abundantly found in inflamed IBD mucosa, suggesting their pivotal role in IBD. However, the precise implication of IL‐17 cytokine family members in IBD pathogenesis and the mechanisms regulating their secretion are incompletely understood. Importantly, recent findings suggest that beyond IL‐17 production‐Th17 cells may secret a plethora of other effector cytokines such as IL‐21, IL‐22, and IL‐9‐ which is in part induced by its own IL‐9 production. However, the use of anti‐IL‐17 therapeutic strategies in experimental models of chronic inflammation results in disease‐ameliorating effects suggesting their potential use in IBD patients. In this review article we discuss the latest findings on the role of Th17 cells and IL‐17 family members in IBD immunopathology, as well as research perspectives. (Inflamm Bowel Dis 2011;)

The transcription factor IFN regulatory factor–4 controls experimental colitis in mice via T cell–derived IL-6

The proinflammatory cytokine IL-6 seems to have an important role in the intestinal inflammation that characterizes inflammatory bowel diseases (IBDs) such as Crohn disease and ulcerative colitis. However, little is known about the molecular mechanisms regulating IL-6 production in IBD. Here, we assessed the role of the transcriptional regulator IFN regulatory factor-4 (IRF4) in this process. Patients with either Crohn disease or ulcerative colitis exhibited increased IRF4 expression in lamina propria CD3+ T cells as compared with control patients. Consistent with IRF4 having a regulatory function in T cells, in a mouse model of IBD whereby colitis is induced in RAG-deficient mice by transplantation with CD4+CD45RB(hi) T cells, adoptive transfer of wild-type but not IRF4-deficient T cells resulted in severe colitis. Furthermore, IRF4-deficient mice were protected from T cell-dependent chronic intestinal inflammation in trinitrobenzene sulfonic acid- and oxazolone-induced colitis. In addition, IRF4-deficient mice with induced colitis had reduced mucosal IL-6 production, and IRF4 was required for IL-6 production by mucosal CD90+ T cells, which it protected from apoptosis. Finally, the protective effect of IRF4 deficiency could be abrogated by systemic administration of either recombinant IL-6 or a combination of soluble IL-6 receptor (sIL-6R) plus IL-6 (hyper-IL-6). Taken together, our data identify IRF4 as a key regulator of mucosal IL-6 production in T cell-dependent experimental colitis and suggest that IRF4 might provide a therapeutic target for IBDs.

New endoscopic approaches in IBD.

Recent advances in endoscopic imaging techniques have revolutionized the diagnostic approach of patients with inflammatory bowel disease (IBD). New, emerging endoscopic imaging techniques visualized a plethora of new mucosal details even at the cellular and subcellular level. This review offers an overview about new endoscopic techniques, including chromoendoscopy, magnification endoscopy, spectroscopy, confocal laser endomicroscopy and endocytoscopy in the face of IBD.

IL-9 and its receptor are predominantly involved in the pathogenesis of UC

Objective Several pathogenic roles attributed over the past two decades to either T helper (Th)1 or Th2 cells are increasingly becoming associated with interleukin (IL)-17 and most recently IL-9 signalling. However, the implication of IL-9 in IBD has not been addressed so far. Design We investigated the expression of IL-9 and IL-9R by using peripheral blood, biopsies and surgical samples. We addressed the functional role of IL-9 signalling by analysis of downstream effector proteins. Using Caco-2 cell monolayers we followed the effect of IL-9 on wound healing. Results IL-9 mRNA expression was significantly increased in inflamed samples from patients with UC as compared with controls. CD3+ T cells were major IL-9-expressing cells and some polymorphonuclear leucocytes (PMN) also expressed IL-9. IL-9 was co-localised with the key Th9 transcription factors interferon regulatory factor 4 and PU.1. Systemically, IL-9 was abundantly produced by activated peripheral blood lymphocytes, whereas its receptor was overexpressed on gut resident and circulating PMN. IL-9 stimulation of the latter induced IL-8 production in a dose-dependent manner and rendered PMN resistant to apoptosis suggesting a functional role for IL-9R signalling in the propagation of gut inflammation. Furthermore, IL-9R was overexpressed on gut epithelial cells and IL-9 induced STAT5 activation in these cells. Moreover, IL-9 inhibited the growth of Caco-2 epithelial cell monolayers in wound healing experiments. Conclusions Our results provide evidence that IL-9 is predominantly involved in the pathogenesis of UC suggesting that targeting IL-9 might become a therapeutic option for patients with UC.

Assessment of Crohn's disease activity by confocal laser endomicroscopy

Background: Confocal laser endomicroscopy (CLE) allows microscopic imaging within the mucosal layer of the gut during ongoing endoscopy. Different studies have addressed the potential of CLE for in vivo diagnosis of ulcerative colitis and microscopic colitis. However, there are no data on the utility of CLE for in vivo diagnosis of Crohns disease (CD). The aim was to assess the clinical utility of CLE in patients with CD and to determine whether disease activity can be graded using CLE. Methods: Consecutive patients with and without CD were enrolled. The colonic mucosa was examined by standard white‐light endoscopy followed by CLE. The features seen on CLE were compared between CD patients and controls. Results: In all, 76 patients with CD were screened, of whom 54 patients were included in the present study. Eighteen patients without inflammatory bowel disease (IBD) served as controls. A significantly higher proportion of patients with active CD had increased colonic crypt tortuosity, enlarged crypt lumen, microerosions, augmented vascularization, and increased cellular infiltrates within the lamina propria. In quiescent CD, a significant increase in crypt and goblet cell number was detected compared with controls. Based on our findings, we propose a Crohns Disease Endomicroscopic Activity Score (CDEAS) for assessing CD activity in vivo. Conclusions: CLE has the potential to significantly improve diagnosis of CD compared with standard endoscopy. These findings should be evaluated in future prospective trials to assess the value of this newly developed CLE score for prediction of disease course and therapeutic responses. (Inflamm Bowel Dis 2012;)

High-definition endoscopy with i-Scan and Lugol's solution for more precise detection of mucosal breaks in patients with reflux symptoms.

BACKGROUND AND STUDY AIMS Patients with gastroesophageal reflux disease are subdivided into non-erosive (NERD) and erosive reflux disease (ERD). The newly available EPKi processor enables high-definition resolution above HDTV standard (HD+). The aim of the study was to test the efficacy of HD+ esophagogastroduodenoscopy alone and in conjunction with i-Scan (newly developed postprocessing digital filter) and chromoendoscopy (Lugols solution) for differentiation of reflux patients. METHODS The distal esophagus of patients with heartburn was inspected with three imaging modalities. HD+ was followed by i-Scan and 15-mL Lugols solution (1.5 %). The esophagus was evaluated for mucosal breaks (Los Angeles Classification [LA]). Small visible changes were also characterized, and targeted biopsies were performed. End points of the study were the presence and grade of esophagitis and the number of circumscribed changes. RESULTS A total of 50 patients were included (female 29; mean age 54.7 years). HD+ identified nine patients with mucosal breaks (LA 7A; 2C), i-Scan was able to detect 12 patients (LA 8A; 2B; 2C; 0D) ( P = n. s.) and chromoendoscopy identified 25 patients (LA 16A; 7B; 1C, 1D) ( P < 0.01). Furthermore, a higher grade of esophagitis was recognized by using i-Scan and Lugols solution in 19 patients. The number of circumscribed lesions could be increased from 21 (HD+) to 58 (i-Scan) ( P < 0.01), and up to 85 after Lugol spraying ( P < 0.01). CONCLUSIONS Lugols solution in conjunction with HD+ endoscopy significantly improves the identification of patients with esophagitis and reduces misclassification. The i-Scan filter and chromoendoscopy help to identify reflux-associated lesions.

IRF4 regulates IL-17A promoter activity and controls RORγt-dependent Th17 colitis in vivo.

Background: The transcription factor IRF4 is involved in several T‐cell‐dependent chronic inflammatory diseases. To elucidate the mechanisms for pathological cytokine production in colitis, we addressed the role of the IRF transcription factors in human inflammatory bowel disease (IBD) and experimental colitis. Methods: IRF levels and cytokine production in IBD patients were studied as well as the effects of IRF4 deficiency in experimental colitis. Results: In contrast to IRF1, IRF5, and IRF8, IRF4 expression in IBD was augmented in the presence of active inflammation. Furthermore, IRF4 levels significantly correlated with IL‐6 and IL‐17 mRNA expression and to a lesser extent with IL‐22 mRNA expression in IBD. To further explore the role of IRF4 under in vivo conditions, we studied IRF4‐deficient and wildtype mice in experimental colitis. In contrast to DSS colitis, IRF4 deficiency was protective in T‐cell‐dependent transfer colitis associated with reduced ROR&agr;/&ggr;t levels and impaired IL‐6, IL‐17a, and IL‐22 production, suggesting that IRF4 acts as a master regulator of mucosal Th17 cell differentiation. Subsequent mechanistic studies using database analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assays identified a novel IRF4 binding site in the IL‐17 gene promoter. Overexpression of IRF4 using retroviral infection induced IL‐17 production and IL‐17 together with IL‐6 induced ROR&ggr;t expression. Conclusions: IRF4 can directly bind to the IL‐17 promotor and induces mucosal ROR&ggr;t levels and IL‐17 gene expression thereby controlling Th17‐dependent colitis. Targeting of this molecular mechanism may lead to novel therapeutic approaches in human IBD. (Inflamm Bowel Dis 2011;)