Lael Werner

CXCL12 Is a constitutive and inflammatory chemokine in the intestinal immune system

Background: Inflammatory bowel disease (IBD) is characterized by increased lymphocytic infiltrate to the lamina propria (LP) and upregulation of inflammatory chemokines and receptors. CXCL12 is a constitutive chemokine involved in lung, brain, and joint inflammation. We hypothesized that CXCL12 and its receptor, CXCR4, would have a constitutive and inflammatory role in the gut. Methods: Intestinal epithelial cells (IECs) and T lymphocytes were isolated from intestinal mucosa of IBD and control patients undergoing bowel resection. Autologous T cells were isolated from peripheral blood (PB). CXCL12 and CXCR4 expression by IECs was assessed by polymerase chain reaction and immunohistochemistry, lymphocyte phenotype by flow cytometry, and migration by Transwells. Results: IECs expressed CXCL12 and expression was increased and more diffuse in IBD compared to normal crypts (ulcerative colitis [UC] > Crohns disease [CD], inflamed > noninflamed). CXCR4 was expressed by IECs, LP T cells (LPTs), and PB T cells (PBTs), and CXCR4+ cells were increased in IBD LP in situ. PBTs and LPTs from all patients had a high and comparable migration toward CXCL12 (P < 0.0001 and P < 0.05 vs. medium, respectively). Migration toward IBD‐IEC‐derived supernatant was significantly higher compared to normal. Antibodies against CXCR4 and CXCL12 blocked migration. Conclusions: CXCL12 is expressed by normal IECs and upregulated and differentially distributed in IBD IECs. CXCR4 is expressed by IECs and LPTs, and CXCR4+ cells are significantly increased in IBD LP. CXCL12 is chemotactic for both PBTs and LPTs. Thus, CXCL12 and CXCR4 have a constitutive and inflammatory role in the intestinal mucosa and their selective therapeutic manipulation may be considered in IBD management. (Inflamm Bowel Dis 2009;)

Involvement of CXCR4/CXCR7/CXCL12 Interactions in Inflammatory Bowel Disease

Directional movement of cells in the human body is orchestrated via chemokines. This migration was initially identified in pathological and immunological processes but quickly extended to homeostatic cell trafficking. One such chemokine is the ubiquitous CXCL12 (initially called SDF1-α) which signals via the chemokine receptors CXCR4 and CXCR7. In the last decade CXCL12 was recognized to participate not only in embryonic development and homeostatic maintenance, but also in progression of inflammation. A role for CXCL12 and its receptors CXCR4 and CXCR7 in inflammatory bowel diseases was recently shown. The current review discusses up to date knowledge of CXCL12 in inflammation, focusing on the involvement of CXCL12 and its receptors, CXCR4 and CXCR7, in inflammatory bowel diseases.

Antibodies against glycoprotein 2 are novel markers of intestinal inflammation in patients with an ileal pouch

BACKGROUND AND AIMS The Crohns disease (CD)-specific pancreatic auto-antibodies (PAB), have been recently identified to target glycoprotein 2 (GP2). Pouchitis is an inflammation of the small bowel developing in up to 60% of ulcerative colitis patients undergoing proctocolectomy and ileal pouch anal anastomosis. Occurrence of CD-specific antibodies was reported to be a predictor of pouchitis. We aimed to assess the prevalence of anti-GP2 antibodies (anti-GP2) in the serum and feces of pouch patients and to correlate them with clinical parameters. Furthermore, we examined mucosal expression of the GP2 protein in the pouch. METHODS Pouch patients were prospectively recruited and checked for clinical, endoscopic, and laboratory markers of inflammation. IgG and IgA anti-GP2 levels in serum and fecal samples were determined using ELISA. GP2 protein was assessed by immunohistochemistry. RESULTS Anti-GP2 was elevated in both serum and fecal samples of patients with inflamed compared to those with non-inflamed pouches and patients with familial-adenomatous polyposis after surgery (p<0.05, respectively). Moreover, patients with CD-like complications exhibited significantly higher anti-GP2 titers than those without CD-like complications (p≤0.01). High levels of anti-GP2 correlated with more frequent bowel movements per day and with the presence of at least one anti-glycan antibody (p≤0.05). GP2 itself was more abundant in the mucosa of patients with chronic pouchitis. CONCLUSIONS Anti-GP2 exists in the serum and feces of pouch patients and correlates with pouch inflammation, and presence of other serological markers. Thus, anti-GP2 may contribute to better stratification of pouchitis, more-so when the inflammation exhibits CD-like complications.

Reciprocal regulation of CXCR4 and CXCR7 in intestinal mucosal homeostasis and inflammatory bowel disease

IBDs are characterized by increased influx of immune cells to the mucosa of genetically susceptible persons. Cellular migration to injury sites is mediated by chemokines. CXCL12 is a ubiquitous, constitutive chemokine that participates in stem cell proliferation and migration and mediates T lymphocyte migration to inflamed tissues. We have recently reported that CXCL12 and its receptor, CXCR4, are expressed in normal and more prominently, inflamed human intestinal mucosa. However, the interactions and roles of CXCL12 and its receptors, CXCR4 and the recently discovered CXCR7, in intestinal inflammation have not been defined. In the present study, we further dissected the effects of CXCL12 on lymphocytes in intestinal homeostasis and inflammation and delineated the interplay between CXCL12 and its receptors CXCR4 and CXCR7. To that end, fresh mononuclear cells were isolated from mucosa and PB of healthy or IBD patients. Phenotypical and functional assays were conducted using flow cytometry, Transwell migration chambers, and ELISA. The data show that CXCL12‐mediated migration of T cells is CXCR4‐ but not CXCR7‐dependent. T cell activation reciprocally regulates CXCR7 and CXCR4 expression and migratory capacity. IBD PBTs expressed more CXCR7 than normal PBTs. Finally, T cells attracted by CXCL12 are mostly of a memory phenotype. In conclusion, the present study suggests that the interplay between CXCL12 and its receptors affects homeostasis and inflammation in the intestinal mucosa.

TNFα inhibitors restrict T cell activation and cycling via Notch-1 signalling in inflammatory bowel disease

Background Tumour necrosis factor α (TNFα) inhibitors such as adalimumab and infliximab are frequently prescribed for inflammatory bowel disease (IBD). Despite the clinical success of TNFα inhibitors, their physiological mode of action is not fully understood. The aim of this study was to investigate the mode of action of anti-TNFα agents in IBD. Methods It was hypothesised that Notch mediates anti-TNFα action in T cells. A study was carried out to identify Notch-1 as a link by which anti-TNFα antibodies mediate their inhibitory functions. Results TNFα inhibitors induced T cell apoptosis, inhibited activation, reduced cytokine secretion and restricted cell cycling. TNFα blockade at several levels showed that TNFα is responsible for inducing apoptosis by anti-TNFα but not for cell cycle restriction. By linking Notch and TNFα it was shown that (1) Notch-1 mucosal expression differs in inflamed and non-inflamed mucosa and increases in response to anti-TNFα treatment; (2) Notch-1 function is regulated by TNFα inhibitors; (3) Notch-1 binds to TNFα; and (4) Notch-1 inhibition prevents anti-TNFα-induced T cell cycle arrest but not apoptosis. Conclusions TNFα inhibitors potently inhibit T cell function. By demonstrating for the first time that Notch-1 mediates the inhibitory effects of adalimumab and infliximab on T cell cycling, this study reveals a new mode of action and also an underlying signalling pathway by which biological agents act in IBD.

Enhanced TH17 Responses in Patients with IL10 Receptor Deficiency and Infantile-onset IBD

Background: IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4+ T-cell function. Methods: Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4+ T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. Results: Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4+ T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1&bgr; leads to enhanced production of IL17A. Conclusions: IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

Su1165 Serum Alpha-1-Antitrypsin: A Non-Invasive Marker of Pouchitis

106 DBEs were performed in 61 patients. (47 oral , 59 anal) with total enteroscopy achieved in 30%; 69% had history of previous surgical resection. Among those with established DBE, findings led to escalation of medical therapy in 30%, maintenance of therapy in 25%, and referral to surgery (25% resection, 8% bowel preservating). 4 patients underwent repeat DBE for disease monitoring after therapeutic escalation. Among 61 patients with known CD, 17 underwent dilation of 67 strictures during 32 endoscopic sessions. Of these patients, 65% were surgery-free during the study period. In those with CD diagnosis made at time of index DBE, 23 DBEs were performed in 18 patients (11 oral, 12 anal). Of these, 1 had total enteroscopy and 2/2 had successful capsule retrieval. All patients had previously undergone ileocolonoscopy and small bowel imaging without an established diagnosis (Table). Overall, there was one perforation in a patient with CD and a prior surgical resection; no patients developed DBE-associated pancreatitis or died during immediate followup period. Conclusions: Diagnostic and therapeutic DBE is safe and effective in patients with suspected or established CD, even in high risk patients with previous small bowel resection. DBE identified small bowel stricturing disease missed on prior ileocolonoscopy and small bowel imaging studies, and offers therapeutic potential for stricturing disease that might otherwise require surgery. Operating Characteristics of Small Bowel Imaging and Ileocolonoscopy for the Diagnosis of Isolated Small Bowel Crohns Disease Using DBE as Reference

Intestinal epithelial cells and T cells differentially recognize and respond to Candida albicans yeast and hypha

Inflammatory bowel diseases (IBD) are a multifactorial disorder. Our understanding of the role of bacteria in the pathogenesis of IBD has increased substantially; however, only scarce data exist regarding the role of commensal fungi in maintaining intestinal homeostasis and triggering IBD. Candida albicans (C. albicans) is a member of the intestinal mycobiome and proposed to contribute to IBD pathogenesis. We aimed to investigate the influence of the two morphologies of C. albicans, yeast and hypha, on epithelial cells and T cells from IBD patients versus healthy controls. We found that C. albicans was recognized by both epithelial cells lines and T cells. In the intestinal epithelial cell line, Caco‐2, response to hypha was different than to yeast cells, and this was mimicked by synthetic β‐glucans and Pam3CSK4. Unstimulated T cells exhibited increased activation and pro‐inflammatory cytokine secretion upon exposure, while there was no effect on apoptosis or proliferation. In contrast, C. albicans‐challenged CD3‐stimulated T‐cells exhibited decreased activation, cytokine secretion, apoptosis, and proliferation, suggesting reciprocal responsiveness to C. albicans. Glycans alone did not mimic abovementioned influences on T cells, suggesting alternative modes of recognition. In conclusion, we provide evidence for glycan dependent and independent recognition of C. albicans by epithelial cells and T cells.