Giorgos Bamias

Expression, Localization, and Functional Activity of TL1A, a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease

TL1A is a novel TNF-like factor that acts as a costimulator of IFN-γ secretion through binding to the death domain-containing receptor, DR3. The aim of this study was to test the hypothesis that TL1A may play an important role in inflammatory bowel disease (IBD) by functioning as a Th1-polarizing cytokine. The expression, cellular localization, and functional activity of TL1A and DR3 were studied in intestinal tissue specimens as well as isolated lamina propria mononuclear cells from IBD patients and controls. TL1A mRNA and protein expression was up-regulated in IBD, particularly in involved areas of Crohn’s disease (CD; p < 0.03 vs control). TL1A production was localized to the intestinal lamina propria in macrophages and CD4+ and CD8+ lymphocytes from CD patients as well as in plasma cells from ulcerative colitis patients. The amount of TL1A protein and the number of TL1A-positive cells correlated with the severity of inflammation, most significantly in CD. Increased numbers of immunoreactive DR3-positive T lymphocytes were detected in the intestinal lamina propria from IBD patients. Addition of recombinant human TL1A to cultures of PHA-stimulated lamina propria mononuclear from CD patients significantly augmented IFN-γ production by 4-fold, whereas a minimal effect was observed in control patients. Our study provides evidence for the first time that the novel cytokine TL1A may play an important role in a Th1-mediated disease such as CD.

New Concepts in the Pathophysiology of Inflammatory Bowel Disease

Clinical Principles The inflammatory bowel diseases (IBDs), that is, Crohn disease and ulcerative colitis, affect approximately 1 million persons in North America and several million persons worldwide. Approximately 30% of patients present between 10 and 30 years of age. Current therapeutic options are limited and include nonspecific anti-inflammatory and immunosuppresive medications. Surgery is required for 50% to 80% of patients with Crohn disease, while only 20% of patients with ulcerative colitis have surgery. Novel biological therapeutics have greatly improved the quality of life of patients with IBD. Pathophysiologic Principles Both genetic and environmental factors play important roles in disease pathogenesis. New hypotheses implicate the innate immune system and the intestinal epithelium in the pathogenesis of the disease. Lymphocytes, cytokines, and adhesion molecules are dysregulated and have been targeted for therapeutic intervention. Based on a new understanding of the complicated mechanisms that underlie the disease process, combination therapies are currently being pursued. A better understanding of the pathophysiologic mechanisms will aid in prevention and more effective maintenance of remission of IBDs. Ulcerative colitis and Crohn disease are collectively called the inflammatory bowel diseases (IBDs) because of such similarities as a chronic remitting and relapsing course, their inflammatory nature, and their unknown causes. Nevertheless, these 2 disorders are clearly separated by distinct clinicopathologic features, including different locations within the gastrointestinal tract, diverse histologic patterns of inflammation, and the various disease-specific complications. Data that diverge from the traditionally accepted view of the pathogenesis of IBDs have recently been published (1). This new information has substantially challenged our conception on the pathophysiology of IBD and has complicated what was originally believed to be a simple dichotomy between Crohn disease and ulcerative colitis. According to the currently accepted hypothesis, ulcerative colitis and Crohn disease result from a dysregulated response of the mucosal immune system toward intraluminal antigens of bacterial origin in genetically predisposed persons (2-4). However, this hypothesis has been challenged by unexpected results from animal models of intestinal inflammation, which have led investigators to reject traditional pathogenetic concepts of these diseases (5, 6). Such animal models allow experimental manipulations that cannot be done in humans and are frequently used to test the efficacy of candidate therapies. In this review, we present emerging pathophysiologic concepts and discuss their effect on the classical paradigms for IBD. The Role of the Innate Immune System in IBD The innate immune system is the bodys nonspecific defense against pathogens; it responds immediately or within the first few hours after a challenge. This is commonly considered the first line of defense and includes such physical barriers as the skin and the intestinal mucosa as well as immune cells that identify and remove foreign bodies. The innate immune system reacts to the chemical properties of the antigen rather than to the specific antigen itself. The acquired immune system, however, responds specifically to antigens. The antigen is processed and recognized, and immune cells that are specific to that antigen are then selectively proliferated. Memory is also a part of adaptive immunity, which improves the efficiency of future immunologic responses. The traditional view of the pathogenesis of inflammatory bowel disease is that intestinal inflammation is mediated by cells of the acquired immune system (Figure 1). The chronic inflammation could result from overly aggressive activity of effector lymphocytes and proinflammatory cytokines, which overcome the control mechanisms. Alternatively, IBD may result from a primary failure of regulatory lymphocytes and cytokines, such as interleukin-10 and transforming growth factor-, to control inflammation and effector pathways (7, 8). In addition, a central pathogenic mechanism in Crohn disease is the resistance of T cells to undergoing apoptosis after activation. The exact cause of this phenomenon has not yet been fully elucidated; nonetheless, the ability of anti-tumor necrosis factor and anti-interleukin-12 antibodies to efficiently prevent or reverse clinical and experimental IBD is largely mediated by their ability to restore mucosal homeostasis and redirect mucosal effector T cells into apoptotic pathways. In both scenarios, lymphocytes are considered to be the major culprits. However, there is emerging evidence that defects in the innate immune system may play an equal or even more important role in IBD (9, 10). Figure 1. The traditional paradigm for the pathogenesis of inflammatory bowel disease ( IBD ). APCs left Teff Treg right Evidence of the role of the innate immune system comes from the recently discovered association between Crohn disease and loss-of-function mutations in the caspase-activating and recruitment domain 15 gene (card15so named because the protein it encodes contains a CARD proteinprotein interaction domain), which is also known as nod2. The NOD2 protein is an intracellular receptor for a component of the bacterial cell wall and plays an important role in triggering cells of the innate immune system. The nod2 Gene Genetic factors play an important role in the pathogenesis of IBD, with 5% to 10% of patients reporting a positive family history (11). Although family and twin concordance studies support a stronger genetic influence in Crohn disease than in ulcerative colitis, both diseases represent complex polygenic traits (12). Genome-wide searches have identified at least 7 loci that confer susceptibility to Crohn disease or ulcerative colitis or both (Table 1) (13). Table 1. Genetic Associations in Inflammatory Bowel Diseases The first locus to be identified and best characterized is IBD1, which is located at chromosome position 16q12 (14, 15). Because several genes are included within this locus, converging techniques have been used to identify predisposing genes. These techniques have singled out nod2 as playing an important role in the predisposition to IBDs associated with this locus (16-18). Mutations in the nod2 gene are present in as many as one third of individuals with Crohn disease. Three common single nucleotide polymorphisms that independently associate with Crohn disease have been identified in the nod2 gene. Carriage of 1 pathologic allele increases the risk for Crohn disease by 2- to 4-fold, compared with a 15- to 40-fold increase when 2 risk alleles are present (11). Despite this genedose effect, fewer than 2% of individuals with 2 risk alleles eventually develop Crohn disease. Indeed, 20% of healthy white controls carry 1 risk allele and 1% carry 2 risk alleles. Examination of genotype-phenotype relationships in Crohn disease showed an association of mutations in nod2 with ileal disease rather than colonic disease, an earlier age of disease onset, and possibly fibrostenosis (19). In contradistinction to Crohn disease, mutations in nod2 do not seem to be a significant risk factor in ulcerative colitis. NOD2 is an intracellular protein that senses bacterial products and activates components of the innate immune system. The functional significance of Crohn disease-associated mutations in NOD2 is currently being investigated, with several controversies remaining to be resolved (20). However, the current observations indicate that an impaired inflammatory response rather than an overly aggressive inflammatory response by a defective intestinal innate immune system may underlie the initial phase of IBD. In this context, the lack of appropriate secretion of defensins (peptides that are produced by enterocytes to control the levels of commensal microbes) may be relevant to the pathogenesis of IBD (21). Figure 2 summarizes the current theories regarding the role of NOD2 in IBD. Figure 2. Proposed functional significance of NOD2 mutations in Crohn disease. TLRs NF-B right left left right The Role of the Epithelium The intestinal epithelium, which is considered to be part of the innate immune system, plays an active role in the maintenance of mucosal homeostasis (22). Consequently, dysfunction of epithelial cells can contribute to and may even be the primary defect in IBD. Epithelial cells form a tight, highly selective barrier between the body and the intraluminal microenvironment. Failure of this barrier may result in intestinal inflammation, most likely through exposure to fecal antigens leading to inappropriate activation of the mucosal immune system. Indeed, mice with genetically introduced defects in intestinal permeability develop intestinal inflammation (23, 24). Within the intestinal mucosa, there is constant cross-talk between the epithelium and cells of the immune system (25). Epithelial cells can act as antigen-presenting cells because they are able to take up and process antigens and present them to cells of the immune system, along with appropriate activating stimuli. Aberrant communication, therefore, has the potential to create inappropriate signals that activate effector cells and lead to inflammation. Epithelial cells are avid producers of chemokines, which regulate recruitment of acute and chronic inflammatory cells within the intestinal mucosa. In addition, many cytokines that are considered central to the pathogenesis of IBD, such as tumor necrosis factor, interleukin-1, and interleukin-6, are expressed in the intestinal epithelium. Aberrant secretion of these proinflammatory chemokines and cytokines by epithelial cells is an integral part of the dysregulated immune response that initiates or perpetuates intestinal inflammation (26, 27). The Effector Immune Response in IBD In addition to being thought to result from dysfunction of the acquire

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.

Immunopathogenesis of inflammatory bowel disease: current concepts

Purpose of review According to the current paradigm, ulcerative colitis and Crohns disease occur in genetically predisposed individuals because of dysregulated immune responses against intraluminal bacterial antigens. Data have recently accumulated supporting alternative hypotheses for the pathogenesis of inflammatory bowel disease. Here, we present novel immunogenetic pathways and discuss their impact on traditional understanding of inflammatory bowel disease. Recent findings In the gastrointestinal tract the innate immune system contains intraluminal bacteria locally, avoiding invasion of the deeper layers and preventing induction of long-standing proinflammatory responses. Failure of this protective function of the innate immune system appears to be the primary defect in inflammatory bowel disease, as a result of impairment of NOD2 signaling or other unidentified deficiencies. The adaptive immune response that ensues was thought to be strictly differentiated between T-helper-1 mediated in Crohns disease and T-helper-2 mediated in ulcerative colitis. This concept is rapidly changing, however, in light of recent evidence suggesting that tissue injury in inflammatory bowel disease is mediated by novel effector pathways, the most prominent of which is the interleukin-23/Th17 axis. Summary Elucidation of the pathways that underlie chronic intestinal inflammation will facilitate the development of new treatments with increased specificity and probably with decreased toxicity.

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.

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.

Commensal Bacteria Exacerbate Intestinal Inflammation but Are Not Essential for the Development of Murine Ileitis

The pathogenesis of Crohn’s disease has been associated with a dysregulated response of the mucosal immune system against intraluminal Ags of bacterial origin. In this study, we have investigated the effects of germfree (GF) conditions in the SAMP1/YitFc murine model of Crohn’s disease-like ileitis. We show that the bacterial flora is not essential for ileitis induction, because GF SAMP1/YitFc mice develop chronic ileitis. However, compared with disease in specific pathogen-free (SPF) mice, ileitis in GF mice is significantly attenuated, and is associated with delayed lymphocytic infiltration and defective mucosal expression of Th2 cytokines. In addition, we demonstrate that stimulation with purified fecal Ags from SPF, but not GF mice leads to the generation of IL-4-secreting effector lymphocytes. This result suggests that commensal bacteria drive Th2 responses characteristic of the chronic phase of SAMP1/YitFc ileitis. Finally, adoptive transfer of CD4-positive cells from GF, but not SPF mice induces severe colitis in SCID recipients. These effects were associated with a decreased frequency of CD4+CD25+Foxp3+ T cells in the mesenteric lymph nodes of GF mice compared with SPF mice, as well as lower relative gene expression of Foxp3 in CD4+CD25+ T cells in GF mice. It is therefore apparent that, in the absence of live intraluminal bacteria, the regulatory component of the mucosal immune system is compromised. All together, our results indicate that in SAMP1/YitFc mice, bacterial flora exacerbates intestinal inflammation, but is not essential for the generation of the chronic ileitis that is characteristic of these mice.

Resistin-like molecule β (RELMβ/FIZZ2) is highly expressed in the ileum of SAMP1/YitFc mice and is associated with initiation of ileitis

SAMP1/Fc mice develop spontaneous ileitis that shares many features with human Crohn’s disease. One of the earliest features of ileitis in SAMP1/Fc mice is an increase in the number of ileal goblet and intermediate cells. Resistin-like molecule β (RELMβ) is a goblet cell-specific, cysteine-rich peptide previously shown to function as part of the innate immune response. In this study, we examined the role of expression of RELMβ in the initiation of ileal inflammation in SAMP1/Fc mice. RELMβ was highly induced in the ilea of SAMP1/Fc mice beginning at age 5 wk, coincident with the histological appearance of inflammation. RELMβ was found in ileal goblet cells and some intermediate and Paneth cells. Surprisingly, RELMβ mRNA levels were significantly increased in the ilea of 80% of germ-free SAMP1/Fc mice examined compared with specific pathogen-free AKR control mice of similar age. Ileitis was observed in germfree SAMP1/Fc mice, although it was attenuated relative to specific pathogen-free SAMP1/Fc mice. These data suggest that neither the early induction of RELMβ expression nor ileal inflammation requires the presence of viable intestinal flora. Neither was the induction of RELMβ dependent on the major Th1 or Th2 cytokines. However, RELMβ stimulated naive bone marrow-derived macrophages to secrete significant amounts of TNF-α, IL-6, and RANTES. Our data suggest that RELMβ is involved in the initiation of ileitis in SAMP1/Fc mice and may act through the induction of proinflammatory cytokines from resident immune cells within the mucosa.

Prevalence and Characteristics of Extra-intestinal Manifestations in a Large Cohort of Greek Patients with Inflammatory Bowel Disease

BACKGROUND AND AIMS Extraintestinal manifestations [EIMs] are common in inflammatory bowel disease [IBD]. Data on epidemiology and risk factors of EIMs in IBD patients are limited. The aim of this study was to investigate the prevalence of EIMs in a large cohort of Greek IBD patients and identify risk factors for their development. METHODS The study population consisted of IBD patients, who were followed in eight tertiary Greek hospitals. Demographic and clinical characteristics of patients were analysed. The diagnosis of EIMs was based on standard criteria and on specialist consultation. RESULTS In total, 1860 IBD patients (1001 with Crohns disease [CD], 859 with ulcerative colitis [UC]) were registered. Among them 615 [33.1%] exhibited at least one EIM; 238 patients [38.6%] developed an EIM before IBD diagnosis. An association between active IBD and presence of an EIM was established in 61.1% of the patients. Arthritic [peripheral arthritis], mucocutaneous [erythema nodosum], and ocular [episcleritis] were the most common manifestations. EIMs were more prevalent in females, patients with CD, smokers [for all p <0.0001], patients with extensive UC [p = 0.007], and patients with a previous appendectomy [p < 0.0001] or a major IBD-related surgery [p = 0.012]. CONCLUSIONS About one-third of Greek IBD patients developed at least one EIM. Of those, more than one-third had their EIM diagnosed before IBD, and in about two-thirds it was related to disease activity. EIMs were more frequently present in females and patients with extensive UC in multivariate analysis.