Jesus Rivera-Nieves

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 Crohns 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 Crohns disease.

Role of β7 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 Peyers 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.

TNF-αneutralization ameliorates the severity of murine Crohn's-like ileitis by abrogation of intestinal epithelial cell apoptosis

Tumor necrosis factor α (TNF-α) is an important mediator of programmed cell death, and TNF-α blockade significantly improves disease severity in several inflammatory conditions, including Crohns disease (CD), one of the idiopathic inflammatory bowel diseases. However, the precise mechanism(s) of action of anti-TNF-α therapy in CD remains poorly understood. SAMP1/YitFc mice develop a spontaneous ileitis with similarities to human CD in regard to histological features as well as response to conventional treatments. In this report, we tested the novel hypothesis that the beneficial effects of anti-TNF-α therapy in CD are mediated by a mechanism that involves down-regulation of intestinal epithelial cell (IEC) apoptosis. Similar to the efficacy of monoclonal anti-TNF-α antibodies in human CD, a single injection of a chimeric anti-murine TNF-α antibody into SAMP1/YitFc mice resulted in a marked suppression of intestinal inflammation and epithelial cell damage compared with mice injected with an isotype control antibody. These effects were associated with a significant reduction in apoptosis of freshly isolated IEC as assessed by propidium iodide staining and DNA laddering. In contrast, an increase in lamina propria mononuclear cell apoptosis was observed in anti-TNF-α-treated mice compared with control. These results were confirmed in vivo by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-assay. In addition, neutralization of TNF-α reduced membrane bound FAS/CD95 expression in IEC from SAMP1/YitFc mice compared with control antibody. These data demonstrate a novel mechanism of action of anti-TNF-α therapy that involves homeostatic regulation of mucosal cell apoptosis, which results in the net decrease of chronic inflammation typically found in CD.

L-Selectin, α4β1, and α4β7 Integrins Participate in CD4+ T Cell Recruitment to Chronically Inflamed Small Intestine

CD4+ T cells are essential for development and perpetuation of Crohn’s disease, a chronic immune-mediated condition that affects primarily the small intestine. Using novel models of Crohn’s disease-like ileitis (i.e., SAMP1/YitFc and CD4+ T cell transfer models), we have begun to understand the adhesive pathways that mediate lymphocyte trafficking to the chronically inflamed small bowel. Expansion of the CD4/β7+ population and increased mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression were observed within the intestinal lamina propria with disease progression. However, Ab blockade of the β7 integrin, the α4β7 heterodimer, MAdCAM-1, or L-selectin did not attenuate inflammation. Blockade of two pathways (L-selectin and MAdCAM-1 or α4 integrins) was required to improve ileitis. Further analyses showed that 55 ± 7% of the mesenteric lymph node α4β7+CD4 expressed L-selectin. These L-selectin+ T cells were the main producers of TNF-α and the predominant ileitis-inducing subpopulation. Mechanistically, combined blockade of L-selectin and MAdCAM-1 depleted the intestinal lamina propria of CD4+ T cells that aberrantly coexpressed α4β7 and α4β1 integrins, markedly decreasing local production of TNF-α and IFN-γ. Thus, pathogenic CD4+ T cells not only use the physiologic α4β7/MAdCAM-1 pathway, but alternatively engage α4β1 and L-selectin to recirculate to the chronically inflamed small intestine.

Integrin-based therapeutics: biological basis, clinical use and new drugs

Integrins are activatable molecules that are involved in adhesion and signalling. Of the 24 known human integrins, 3 are currently targeted therapeutically by monoclonal antibodies, peptides or small molecules: drugs targeting the platelet αIIbβ3 integrin are used to prevent thrombotic complications after percutaneous coronary interventions, and compounds targeting the lymphocyte α4β1 and α4β7 integrins have indications in multiple sclerosis and inflammatory bowel disease. New antibodies and small molecules targeting β7 integrins (α4β7 and αEβ7 integrins) and their ligands are in clinical development for the treatment of inflammatory bowel diseases. Integrin-based therapeutics have shown clinically significant benefits in many patients, leading to continued medical interest in the further development of novel integrin inhibitors. Of note, almost all integrin antagonists in use or in late-stage clinical trials target either the ligand-binding site or the ligand itself.

Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease

SAMP1/YitFc mice develop discontinuous, transmural inflammatory lesions in the terminal ileum, similar to what is found in human Crohn disease. Compared with the mesenteric lymph nodes (MLNs) of AKR control mice, SAMP1/YitFc MLNs contain a 4.3-fold expansion in total B cell number and a 2.5-fold increased percentage of CD4(+) T cells expressing the alpha(E)beta(7) integrin. Although alpha(E)beta(7)(+)CD4(+) T cells possess a regulatory phenotype (CD25(+), L-selectin(lo), and CD45RB(lo)), express IL-10, and suppress effector T cell proliferation in vitro, they cannot prevent ileitis development in SCID mice adoptively transferred with effector CD4(+) T cells, although the CD4(+)CD25(+) subset, which overlaps with the alpha(E)beta(7)(+)CD4(+) subset, prevents colitis. The alpha(E)beta(7)(+)CD4(+) T cells express high levels of ICOS, a costimulatory molecule that augments B cell function, suggesting their involvement in the increase in B cells, IgA(+) cells, and soluble IgA found within the MLNs and ileum of SAMP1/YitFc mice. MLN B cell numbers correlate with ileitis severity in SAMP1/YitFc mice, and cotransfer of SAMP1/YitFc MLN B cells along with CD4(+) T cells increases ileitis severity in SCID mice compared with transfer of CD4(+) T cells alone. SAMP1/YitFc B cells prevent alpha(E)beta(7)(+)CD4(+) T cells from suppressing effector T cell proliferation. We conclude that SAMP1/YitFc MLN B cells contribute to the development of SAMP1/YitFc ileitis.

Interaction of the selectin ligand PSGL-1 with chemokines CCL21 and CCL19 facilitates efficient homing of T cells to secondary lymphoid organs

P-selectin glycoprotein ligand 1 (PSGL-1) is central to the trafficking of immune effector cells to areas of inflammation through direct interactions with P-selectin, E-selectin and L-selectin. Here we show that PSGL-1 was also required for efficient homing of resting T cells to secondary lymphoid organs but functioned independently of selectin binding. PSGL-1 mediated an enhanced chemotactic T cell response to the secondary lymphoid organ chemokines CCL21 and CCL19 but not to CXCL12 or to inflammatory chemokines. Our data show involvement of PSGL-1 in facilitating the entry of T cells into secondary lymphoid organs, thereby demonstrating the bifunctional nature of this molecule.

Critical role of endothelial P-selectin glycoprotein ligand 1 in chronic murine ileitis

L-selectin ligands might be relevant for inflammatory cell trafficking into the small intestine in a spontaneous model of chronic ileitis (i.e., SAMP1/YitFc mice). Immunoblockade of peripheral node addressin or mucosal addressin cell adhesion molecule 1 failed to ameliorate ileitis, whereas P-selectin glycoprotein ligand 1 (PSGL-1) neutralization attenuated both the adoptively transferred and spontaneous disease. PSGL-1 was detected in venules of mesenteric lymph node and small intestine by immunohistochemistry and confirmed by real-time reverse transcription polymerase chain reaction and flow cytometry. In addition, reconstitution of wild-type mice with PSGL-1−/− bone marrow demonstrated that PSGL-1 messenger RNA and PSGL-1 protein expression remained on endothelium, localized within mesenteric lymph node and small intestine. Endothelial PSGL-1 bound P-selectin–IgG and its blockade or genetic deletion altered the recruitment of lymphocytes to the small intestine, as revealed by intravital microscopy and homing studies. Endothelial expression of PSGL-1 adds a new dimension to the various cellular interactions involved in small intestinal recruitment. Thus, the multiple roles of PSGL-1 may explain why targeting this single adhesion molecule results in attenuation of chronic murine ileitis, a disease previously resistant to antiadhesion molecule strategies.

Down-Regulation of Intestinal Lymphocyte Activation and Th1 Cytokine Production by Antibiotic Therapy in a Murine Model of Crohn’s Disease

Resident intestinal bacteria likely play an important role in the pathogenesis of Crohn’s disease through their interaction with the gut immune system. SAMP1/YitFc mice spontaneously develop chronic, discontinuous, transmural ileitis with many features similar to Crohn’s disease. The aim of this study was to determine the effects and elucidate the mechanisms of action of antibiotic treatment in the SAMP1/YitFc mouse model of ileitis. Mice were treated orally with ciprofloxacin and metronidazole before the development of ileitis (prevention protocol) or after ileitis was fully established (treatment protocol). Terminal ilea were harvested for histological scoring, and lamina propria and mesenteric lymph node cells were isolated for analysis of activation markers and cytokine production. Antibiotic therapy significantly decreased the severity of ileitis both in the prevention (40% reduction, p < 0.05) and the treatment (25% reduction, p < 0.01) protocols, compared with untreated, control mice. These effects were associated with a decreased percentage of CD4+/CD45RBhigh lymphocytes in mesenteric lymph nodes of antibiotic-treated mice, as well as decreased production of IFN-γ (prevention: 0.53 ± 0.21 vs 1.84 ± 0.04 ng/ml, p < 0.05; treatment: 8.4 ± 0.4 vs 12.4 ± 0.7 ng/ml, p < 0.005) and TNF (prevention: 61.5 ± 13 vs 134 ± 19 pg/ml, p < 0.01; treatment: 333.5 ± 11 vs 496 ± 20 pg/ml, p < 0.001). The number of activated lamina propria lymphocytes was also reduced after antibiotic treatment. In conclusion, antibiotic therapy significantly ameliorates the severity of ileitis in SAMP1/YitFc mice by a mechanism involving down-regulation of activated gut lymphocytes and inhibition of intestinal Th1 cytokine production.

Neuronal guidance molecule netrin-1 attenuates inflammatory cell trafficking during acute experimental colitis

Background Inflammatory bowel diseases, encompassing Crohns disease and ulcerative colitis, are characterised by persistent leucocyte tissue infiltration leading to perpetuation of an inappropriate inflammatory cascade. The neuronal guidance molecule netrin-1 has recently been implicated in the orchestration of leucocyte trafficking during acute inflammation. We therefore hypothesised that netrin-1 could modulate leucocyte infiltration and disease activity in a model of inflammatory bowel disease. Design DSS-colitis was performed in mice with partial genetic netrin-1 deficiency (Ntn-1+/− mice) or wild-type mice treated with exogenous netrin-1 via osmotic pump to examine the role of endogenous and therapeutically administered netrin-1. These studies were supported by in vitro models of transepithelial migration and intestinal epithelial barrier function. Results Consistent with our hypothesis, we observed induction of netrin-1 during intestinal inflammation in vitro or in mice exposed to experimental colitis. Moreover, mice with partial netrin-1 deficiency demonstrated an exacerbated course of DSS-colitis compared to littermate controls, with enhanced weight loss and colonic shortening. Conversely, mice treated with exogenous mouse netrin-1 experienced attenuated disease severity. Importantly, permeability studies and quantitative assessment of apoptosis reveal that netrin-1 signalling events do not alter mucosal permeability or intestinal epithelial cell apoptosis. In vivo studies of leucocyte transmigration demonstrate suppression of neutrophil trafficking as a key function mediated by endogenous or exogenously administered netrin-1. Finally, genetic studies implicate the A2B adenosine receptor in netrin-1-mediated protection during DSS-colitis. Conclusions The present study identifies a previously unrecognised role for netrin-1 in attenuating experimental colitis through limitation of neutrophil trafficking.