Th1-type responses mediate spontaneous ileitis in a novel murine model of Crohn's disease.

We describe here the immunologic characterization of a new mouse strain, SAMP1/Yit, which spontaneously develops a chronic intestinal inflammation localized to the terminal ileum. The resulting ileitis bears a remarkable resemblance to human Crohn's disease. This strain of mice develops discontinuous, transmural inflammatory lesions in the terminal ileum with 100% penetrance by 30 weeks of age. The intestinal inflammation is characterized by massive infiltration of activated CD4+ and CD8alpha(+)TCRalphabeta(+) T cells into the lamina propria and is accompanied by a dramatic decrease in the intraepithelial lymphocyte CD8alpha(+)TCRgammadelta(+)/CD8alpha(+)TCRalphabeta(+) ratio. The results of adoptive transfer experiments strongly suggest that CD4+ T cells that produce a Th1-like profile of cytokines, e.g., IFN-gamma and TNF, mediate the intestinal inflammation found in SAMP1/Yit mice. In addition, pretreatment of adoptive transfer recipients with a neutralizing anti-TNF antibody prevents the development of intestinal inflammation, suggesting that TNF plays an important role in the pathogenesis of intestinal inflammation in this model. To our knowledge, these data provide the first direct evidence that Th1-producing T cells mediate intestinal inflammation in a spontaneous animal model of human Crohn's disease.

Sphingosine-1-phosphate receptor-1 (S1P1) is expressed by lymphocytes, dendritic cells, and endothelium and modulated during inflammatory bowel disease.

The sphingosine-1-phosphate receptor-1 (S1P1) agonist ozanimod ameliorates ulcerative colitis, yet its mechanism of action is unknown. Here, we examine the cell subsets that express S1P1 in intestine using S1P1-eGFP mice, the regulation of S1P1 expression in lymphocytes after administration of dextran sulfate sodium (DSS), after colitis induced by transfer of CD4+CD45RBhi cells, and by crossing a mouse with TNF-driven ileitis with S1P1-eGFP mice. We then assayed the expression of enzymes that regulate intestinal S1P levels, and the effect of FTY720 on lymphocyte behavior and S1P1 expression. We found that not only T and B cells express S1P1, but also dendritic (DC) and endothelial cells. Furthermore, chronic but not acute inflammatory signals increased S1P1 expression, while the enzymes that control tissue S1P levels in mice and humans with inflammatory bowel disease (IBD) were uniformly dysregulated, favoring synthesis over degradation. Finally, we observed that FTY720 reduced T-cell velocity and induced S1P1 degradation and retention of Naïve but not effector T cells. Our data demonstrate that chronic inflammation modulates S1P1 expression and tissue S1P levels and suggests that the anti-inflammatory properties of S1PR agonists might not be solely due to their lymphopenic effects, but also due to potential effects on DC migration and vascular barrier function.

Validated gene expression biomarker analysis for biopsy-based clinical trials in ulcerative colitis.

BACKGROUND: Accurate and reproducible measurement of expression of pro-inflammatory cytokines in colonic biopsies from patients with ulcerative colitis (UC) is essential for proof-of-concept and mechanism-of-action studies. Few studies have rigorously established the number of biopsies required for accurate and reproducible biomarker measurements. AIM: To validate methods for measuring changes in gene expression in colonic biopsy samples. METHODS: Twelve colonic biopsies were obtained from each of six healthy controls, six patients with inactive UC and seven patients with active UC. Mayo endoscopic scores were used as a clinical reference standard. Quantitative PCR was used to assess mRNA expression of eight known inflammatory genes. The power to detect a reduction in gene expression in active vs. inactive UC was calculated using a linear mixed effect model. RESULTS: mRNA analysis of colonic biopsies is a sensitive and feasible approach for measuring inflammatory gene expression in colonic biopsies. Inflammatory biomarkers correlate with Mayo endoscopic subscores for each colonic region. For most genes, three rectal biopsies from two to four patients are required to detect changes in gene expression corresponding to active vs. inactive UC to achieve a power of 80% with an alpha of 0.05. CONCLUSION: Our data suggest that systematic measurement of inflammatory biomarkers at the mRNA level can be a valuable tool for hypothesis testing, and assessment of clinical activity and response to therapy in ulcerative colitis.

Role of beta7 integrins in intestinal lymphocyte homing and retention.

Lymphocytes involved in intestinal immune response are found in organized immune inductive sites of the gut-associated lymphoid tissues (GALT) such as Peyer's patches (PP), mesenteric lymph nodes (MLN) and diffuse effector sites of gut epithelium and lamina propria (LP). beta(7) integrins are responsible for efficient trafficking and retention of lymphocytes in these sites. Naïve and effector lymphocytes use alpha(4)beta(7) integrin to extravasate from blood to gut mucosal tissues of GALT, MLN and LP via interactions with Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1). The alpha(E)beta(7) integrin facilitates retention of effector and memory lymphocytes in the gut epithelial layer via interactions with E-cadherin. Mucosal dendritic cells (DCs) regulate the expression of the gut homing receptors alpha(4)beta(7) integrin and the chemokine receptor CCR9 on activated effector and regulatory lymphocytes in a retinoic acid-dependent manner. CD103 (alpha(E) integrin) identifies a subset of mucosal DCs in MLN and small intestine LP that have an enhanced ability to induce gut-tropic receptors on responding lymphocytes. The interactions between beta(7) integrin and their ligands are also implicated in the pathogenesis and progression of inflammatory bowel diseases (IBDs), intestinal parasitic infections and graft-versus-host diseases. During intestinal inflammation, beta(7) integrin-dependent and -independent pathways contribute to lymphocytes recruitment to the intestinal tissues and disease pat-hogenesis. Recent works have explored the potential of therapeutic targeting of alpha(4) and beta(7) integrins in IBDs. Here, we review the current understanding of the role of beta(7) integrins in intestinal lymphocyte trafficking and retention in health and disease.

Thiols mediate superoxide-dependent NADH modification of glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is covalently modified by NAD in the presence of nitric oxide (NO) and dithiothreitol. Replacement of NAD with NADH in the presence of SIN-1 (3-morpholinosydnonimine) and dithiothreitol increased modification 25-fold. We now demonstrate that in contrast to NO-mediated attachment of NAD, covalent attachment of NADH to GAPDH proceeds in the presence of low molecular weight thiols, independent of NO. Removal of oxygen and transition metal ions inhibited modification, consistent with a role for reactive oxygen species; inhibition by superoxide dismutase, stimulation by xanthine oxidase/hypoxanthine, and the lack of an effect of catalase supported the hypothesis that superoxide, generated from thiol oxidation, was involved. Electrospray mass spectrometry showed covalent linkage of the NADH molecule to GAPDH. Characterization of the product of phosphodiesterase cleavage demonstrated that linkage to GAPDH occurred through the nicotinamide of NADH. Lys-C digestion of GAPDH, followed by peptide isolation by high performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight analysis, and Edman sequencing, demonstrated that NADH attachment occurred at Cys-149, the active-site thiol. This thiol linkage was stable to HgCl2. Thus, linkage of GAPDH to NADH, in contrast to NAD, occurs in the presence of thiol, is independent of NO, and is mediated by superoxide.

Antibody blockade of ICAM-1 and VCAM-1 ameliorates inflammation in the SAMP-1/Yit adoptive transfer model of Crohn's disease in mice.

BACKGROUND & AIMS: Integrins (alpha(4) and beta(2)) and their endothelial ligands vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) play key roles in leukocyte recruitment to areas of inflammation. ICAM-1 and VCAM-1 are expressed in inflamed intestinal tissues. This study investigates a possible causative role of adhesion molecules ICAM-1, VCAM-1, and alpha(4) integrins in mediating the inflammatory response in a murine model of Crohn's disease (CD). METHODS: CD4+ mesenteric lymph node cells from SAMP-1/Yit donor mice were adoptively transferred into major histocompatibility complex-matched severe combined immunodeficiency disease mice. Six weeks later, these mice were left untreated or treated for 3 days with monoclonal antibodies (mAbs) to ICAM-1, VCAM-1, or both, and alpha(4), or both ICAM-1 and alpha(4), dexamethasone, or nonblocking isotype control antibodies. On day 4 after treatment, tissues were investigated for expression of ICAM-1, VCAM-1, and for severity of inflammation using a semiquantitative inflammatory score. Dexamethasone treatment resolved all measures of intestinal inflammation. RESULTS: Blocking either ICAM-1, VCAM-1, or alpha(4) integrins had no significant beneficial effect. However, blocking ICAM-1 and alpha(4), or blocking ICAM-1 and VCAM-1, showed a 70% resolution of the active inflammation, but not chronic inflammation. CONCLUSIONS: These findings suggest that blocking ICAM-1 and VCAM-1 may have therapeutic benefit for the acute inflammatory component of Crohn's disease.