Inflammatory Cytokine Gene Expression in Mesenteric Adipose Tissue during Acute Experimental Colitis

Background

Production of inflammatory cytokines by mesenteric adipose tissue (MAT) has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Animal models of colitis have demonstrated inflammatory changes within MAT, but it is unclear if these changes occur in isolation or as part of a systemic adipose tissue response. It is also unknown what cell types are responsible for cytokine production within MAT. The present study was designed to determine whether cytokine production by MAT during experimental colitis is depot-specific, and also to identify the source of cytokine production within MAT.

Methods

Experimental colitis was induced in 6-month-old C57BL/6 mice by administration of dextran sulfate sodium (2% in drinking water) for up to 5 days. The induction of cytokine mRNA within various adipose tissues, including mesenteric, epididymal, and subcutaneous, was analyzed by qRT-PCR. These adipose tissues were also examined for histological evidence of inflammation. The level of cytokine mRNA during acute colitis was compared between mature mesenteric adipocytes, mesenteric stromal vascular fraction (SVF), and mesenteric lymph nodes.

Results

During acute colitis, MAT exhibited an increased presence of infiltrating mononuclear cells and fibrotic structures, as well as decreased adipocyte size. The mRNA levels of TNF-α, IL-1β, and IL-6 were significantly increased in MAT but not other adipose tissue depots. Within the MAT, induction of these cytokines was observed mainly in the SVF.

Conclusions

Acute experimental colitis causes a strong site-specific inflammatory response within MAT, which is mediated by cells of the SVF, rather than mature adipocytes or mesenteric lymph nodes.     

IL-23 in inflammatory bowel diseases and colon cancer

Studies in recent years have identified a pivotal role of the cytokine IL-23 in the pathogenesis of inflammatory bowel diseases (IBD: Crohn´s disease, ulcerative colitis) and colitis-associated colon cancer. Genetic studies revealed that subgroups of IBD patients have single nucleotide polymorphisms in the IL-23R gene suggesting that IL-23R signaling affects disease susceptibility. Furthermore, increased production of IL-23 by macrophages, dendritic cells or granulocytes has been observed in various mouse models of colitis, colitis-associated cancer and IBD patients. Moreover, in several murine models of colitis, suppression of IL-12/IL-23 p40, IL-23 p19 or IL-23R function led to marked suppression of gut inflammation. This finding was associated with reduced activation of IL-23 target cells such as T helper 17 cells, innate lymphoid cells type 3, granulocytes and natural killer cells as well as with impaired production of proinflammatory cytokines. Based on these findings, targeting of IL-23 emerges as important concept for suppression of gut inflammation and inflammation-associated cancer growth. Consistently, neutralizing antibodies against IL-12/IL-23 p40 and IL-23 p19 have been successfully used in clinical trials for therapy of Crohn´s disease and pilot studies in ulcerative colitis are ongoing. These findings underline the crucial regulatory role of IL-23 in chronic intestinal inflammation and colitis-associated cancer and indicate that therapeutic strategies aiming at IL-23 blockade may be of key relevance for future therapy of IBD patients.     

The role of activated cytotoxic T cells in inflammatory bowel disease

The role of cell-mediated cytotoxicity in the pathogenesis of ulcerative colitis and Crohn's disease has been controversial since reports indicating either a decreased, or an increased, activity of cytotoxic T cells in active stages of inflammatory bowel disease exist. Some of these discrepancies may be attributed to the fact that so far mostly peripheral blood lymphocytes rather than intestinal T cells have been examined. To overcome some of these limitations we performed in situ hybridizations for the detection of perforin and granzyme A mRNA expressing cells, i.e. of cytotoxic cells activated in situ, in the affected intestinal mucosa. These studies revealed increased frequencies of activated, cytotoxic T cells in active stages of ulcerative colitis and Crohn's disease. Interestingly, activated perforin mRNA expressing T cells are present both in the CD4 and in the CD8 T cell subsets. In the latter T cell subset up to 60% of the mucosal T cells isolated from the affected sites express perforin mRNA at detectable levels. The elevated frequency of activated cytotoxic cells and their histological distribution also in close proximity to the epithelial cells may thus indicate an important role for cytotoxic cells in the pathogenesis of inflammatory bowel disease since activated cytotoxic T cells may further exacerbate the inflammatory process through the production of pro-inflammatory cytokines such as interferon-gamma or tumor necrosis factor-alpha, but also through the release of pro-inflammatory cytokines and chemokines upon lysis of epithelial cells and the increased influx of luminal antigens at the site of epithelial erosions.     

Prevention of experimental colitis in SCID mice reconstituted with CD45RBhigh CD4+ T cells by blocking the CD40-CD154 interactions

Increased expression of CD40 and CD40 ligand (CD40L or CD154) has been found in inflamed mucosa of human inflammatory bowel disease (IBD), and interactions between these molecules seem to be involved in local cytokine production by macrophages. However, the precise role of CD40 signaling in the pathogenesis of IBD is still poorly understood. The aim of the present study was to investigate the in vivo relevance of CD40 signaling in experimental colitis in SCID mice reconstituted with syngeneic CD45RBhighCD4+ T cells. The results demonstrated that CD40+ and CD40L+ cells as well as their mRNA levels were significantly increased in inflamed mucosa. Administration of anti-CD40L neutralizing mAb over an 8-wk period starting immediately after CD45RBhighCD4+ T cell reconstitution completely prevented symptoms of wasting disease. Intestinal mucosal inflammation was effectively prevented, as revealed by abrogated leukocyte infiltration and decreased CD54 expression and strongly diminished mRNA levels of the proinflammatory cytokines IFN-gamma, TNF, and IL-12. When colitic SCID mice were treated with anti-CD40L starting at 5 wk after T cell transfer up to 8 wk, this delayed treatment still led to significant clinical and histological improvement and down-regulated proinflammatory cytokine secretion. These data suggest that the CD40-CD40L interactions are essential for the Th1 inflammatory responses in the bowel in this experimental model of colitis. Blockade of CD40 signaling may be beneficial to human IBD.     

Histone hyperacetylation is associated with amelioration of experimental colitis in mice

Inhibitors of histone deacetylases (HDAC) are being studied for their antiproliferative effects in preclinical cancer trials. Recent studies suggest an anti-inflammatory role for this class of compounds. Because inflammatory bowel disease is associated with an increased risk of malignancies, agents with antiproliferative and anti-inflammatory properties would be of therapeutic interest. HDAC inhibitors from various classes were selected and evaluated for their in vitro capacity to suppress cytokine production and to induce apoptosis and histone acetylation. Valproic acid (VPA) and suberyolanilide hydroxamic acid (SAHA) were chosen for further studies in dextran sulfate sodium- and trinitrobenzene sulfonic acid-induced colitis in mice. In vitro, inhibition of HDAC resulted in a dose-dependent suppression of cytokine synthesis and apoptosis induction requiring higher concentrations of HDAC inhibitors for apoptosis induction compared with cytokine inhibition. Oral administration of either VPA or SAHA reduced disease severity in dextran sulfate sodium-induced colitis. The macroscopic and histologic reduction of disease severity was associated with a marked suppression of colonic proinflammatory cytokines. In parallel to the beneficial effect observed, a dose-dependent increase in histone 3 acetylation at the site of inflammation was shown under VPA treatment. Furthermore, SAHA as well as VPA treatment resulted in amelioration of trinitrobenzene sulfonic acid-induced colitis, which was associated with an increase of apoptosis of lamina propria lymphocytes. Inhibitors of HDAC reveal strong protective effects in different models of experimental colitis by inducing apoptosis and suppressing proinflammatory cytokines, thereby representing a promising class of compounds for clinical studies in human inflammatory bowel disease.     

Th17 cells induce colitis and promote Th1 cell responses through IL-17 induction of innate IL-12 and IL-23 production

Both Th1 and Th17 cells have been implicated in the pathogenesis of inflammatory bowel disease and experimental colitis. However, the complex relationship between Th1 and Th17 cells and their relative contributions to the pathogenesis of inflammatory bowel disease have not been completely analyzed. Although it has been recently shown that Th17 cells can convert into Th1 cells, the underlying in vivo mechanisms and the role of Th1 cells converted from Th17 cells in the pathogenesis of colitis are still largely unknown. In this study, we report that Th17 cells from CBir1 TCR transgenic mice, which are specific for an immunodominant microbiota Ag, are more potent than Th1 cells in the induction of colitis, as Th17 cells induced severe colitis, whereas Th1 cells induced mild colitis when transferred into TCRβxδ(-/-) mice. High levels of IL-12 and IL-23 and substantial numbers of IFN-γ(+) Th1 cells emerged in the colons of Th17 cell recipients. Administration of anti-IL-17 mAb abrogated Th17 cell-induced colitis development, blocked colonic IL-12 and IL-23 production, and inhibited IFN-γ(+) Th1 cell induction. IL-17 promoted dendritic cell production of IL-12 and IL-23. Furthermore, conditioned media from colonic tissues of colitic Th17 cell recipients induced IFN-γ production by Th17 cells, which was inhibited by blockade of IL-12 and IL-23. Collectively, these data indicate that Th17 cells convert to Th1 cells through IL-17 induction of mucosal innate IL-12 and IL-23 production.     

Topical antisense oligonucleotide therapy against LIX, an enterocyte-expressed CXC chemokine, reduces murine colitis

Epithelial neutrophil-activating peptide-78 (ENA-78), a member of the CXC chemokine subfamily, is induced by inflammatory cytokines in human colonic enterocyte cell lines and increased in the colon of patients with inflammatory bowel disease (IBD). Lipopolysaccharide-induced CXC-chemokine (LIX) was recently identified as the murine homolog of ENA-78. Here we show that, similar to ENA-78, inflammatory cytokine stimulation of a murine colonic epithelial cell line, MODE-K, results in increased LIX expression. Consistent with the expression pattern of ENA-78 in IBD, LIX expression is significantly increased in mice with colitis induced by the ingestion of dextran sodium sulfate (DSS). Treating mice with antisense oligonucleotides to LIX via rectal enema delivery before DSS treatment results in colonic enterocyte uptake and a significant reduction in neutrophil infiltration and severity of colitis. These findings indicate that LIX plays an integral role in the pathogenesis of DSS-induced colitis. Similarly, enterocyte-derived CXC chemokines may play a key role in regulating neutrophil recruitment and intestinal injury in IBD. The intracolonic administration of ENA-78 antisense oligonucleotides may be effective in treating distal ulcerative colitis in humans.     

A novel murine model of inflammatory bowel disease and inflammation-associated colon cancer with ulcerative colitis-like features

Mutations that increase susceptibility to inflammatory bowel disease (IBD) have been identified in a number of genes in both humans and mice, but the factors that govern how these mutations contribute to IBD pathogenesis and result in phenotypic presentation as ulcerative colitis (UC) or Crohn disease (CD) are not well understood. In this study, mice deficient in both TNF and IL-10 (T/I mice) were found to spontaneously develop severe colitis soon after weaning, without the need for exogenous triggers. Colitis in T/I mice had clinical and histologic features similar to human UC, including a markedly increased risk of developing inflammation-associated colon cancer. Importantly, development of spontaneous colitis in these mice was prevented by antibiotic treatment. Consistent with the known role of Th17-driven inflammation in response to bacteria, T/I mice had elevated serumTh17-type cytokines when they developed spontaneous colitis and after systemic bacterial challenge via NSAID-induced degradation of the mucosal barrier. Although TNF production has been widely considered to be be pathogenic in IBD, these data indicate that the ability to produce normal levels of TNF actually protects against the spontaneous development of colitis in response to intestinal colonization by bacteria. The T/I mouse model will be useful for developing new rationally-based therapies to prevent and/or treat IBD and inflammation-associated colon cancer and may further provide important insights into the pathogenesis of UC in humans.     

Significant increase of interleukin 6 production in blood mononuclear leukocytes obtained from patients with active inflammatory bowel disease

In the present study, we compared the potency of interleukin 6 production in peripheral blood mononuclear leukocytes between paired patients with active stage and inactive stage of inflammatory bowel disease. Subjects included nine patients with ulcerative colitis, ten patients with Crohn's disease and sex-matched nine healthy volunteers. Mononuclear leukocytes were stimulated with concanavalin A for 24 h to induce interleukin 6 production. Interleukin 6 content in the culture medium was assayed by using specific ELISA and interleukin 6 dependent cell line MH-60. Interleukin 6 production was found to be significantly increased in mononuclear leukocytes from both active ulcerative colitis and Crohn's disease as compared to that from control subjects. There was no significant difference in interleukin 6 production between ulcerative colitis and Crohn's disease. The potency of interleukin 6 production was returned to the control level when the diseases became inactive. The present results, therefore, may indicate some important role of interleukin 6 in the pathogenesis of inflammatory bowel disease and also the potency of interleukin 6 production in mononuclear leukocytes can be an indicator of the activity of inflammatory bowel disease.     

Tumor necrosis factor and lymphotoxin in regulation of intestinal inflammation

Ulcerative colitis and Crohn’s disease are the major forms of inflammatory bowel disease. Cytokines of the tumor necrosis factor (TNF) family play an important role in the regulation of intestinal inflammation. In this review, we discuss the function of key cytokines of this family–TNF and lymphotoxin (LT)–in mucosal healing, IgA production, and in control of innate lymphoid cells (ILCs), novel regulators of mucosal homeostasis in the gut. TNF plays a central role in the pathogenesis of inflammatory bowel diseases (IBD). LT regulates group 3 of ILCs and IL-22 production and protects the epithelium against damage by chemicals and mucosal bacterial pathogens. In addition, we discuss major mouse models employed to study the mechanism of intestinal inflammation, their advantages and limitations, as well as application of TNF blockers in the therapy for IBD.     

Inflammatory bowel disease: the role of inflammatory cytokine gene polymorphisms

The mechanisms responsible for development of inflammatory bowel disease (IBD) have not been fully elucidated, although the main cause of disease pathology is attributed to up-regulated inflammatory processes. The aim of this study was to investigate frequencies of polymorphisms in genes encoding pro-inflammatory and anti-inflammatory markers in IBD patients and controls. We determined genotypes of patients with IBD (n= 172) and healthy controls (n= 389) for polymorphisms in genes encoding various cytokines (interleukin (IL)-1beta, IL-6, tumour necrosis factor (TNF), IL-10, IL-1 receptor antagonist). Association of these genotypes to disease incidence and pathophysiology was investigated. No strong association was found with occurrence of IBD. Variation was observed between the ulcerative colitis study group and the control population for the TNF-alpha-308 polymorphism (p= 0.0135). There was also variation in the frequency of IL-6-174 and TNF-alpha-308 genotypes in the ulcerative colitis group compared with the Crohn's disease group (p= 0.01). We concluded that polymorphisms in inflammatory genes are associated with variations in IBD phenotype and disease susceptibility. Whether the polymorphisms are directly involved in regulating cytokine production, and consequently pathophysiology of IBD, or serve merely as markers in linkage disequilibrium with susceptibility genes remains unclear.     

白介素23 及Th17 细胞在炎症性肠病的作用

炎症性肠病(Inflammatory Bowel Diseases,IBD),是一组病因未明的累及胃肠道的慢性炎症性疾病,一般指克罗恩病(Crohn’sdisease,CD)和溃疡性结肠炎(ulcerative colitis,UC)。目前认为它是由多种因素相互作用所致的一种自身免疫性疾病,主要包括免疫、环境以及遗传等因素,其中免疫在IBD的发生过程中起着极其重要的作用。以往研究认为与T辅助细胞(T Helper cells)Th1或Th2细胞反应的增强或减弱有关。然而最近研究发现一类新细胞亚群,称为Th17细胞,与之相关的细胞因子可导致包括肠道在内的多脏器病变。Th17细胞分化过程中又需要IL-23的参与,因此IL-23/Th17细胞在炎症性肠病患者肠道内过度表达可以解释肠组织损伤的新途径,并为制定新的治疗策略提出依据。本文就IL-23/Th17轴在炎症性肠病中的作用的研究进展作一综述。     

Cutting edge: The IkappaB kinase (IKK) inhibitor, NEMO-binding domain peptide, blocks inflammatory injury in murine colitis

Inflammatory mediators such as TNF-alpha, IL-6, and IL-1 are important in the pathogenesis of inflammatory bowel diseases and are regulated by the activation of NF-kappaB. The aim of the present study was to investigate whether the NF-kappaB essential modulator (NEMO)-binding domain (NBD) peptide, which has been shown to block the association of NEMO with the IkappaB kinasebeta subunit (IKKbeta) and inhibit NF-kappaB activity, reduces inflammatory injury in mice with colitis. Two colitis models were established by the following: 1) inclusion of dextran sulfate sodium salt (DSS) in the drinking water of the mice; and 2) a trinitrobenzene sulfonic acid enema. Marked NF-kappaB activation and expression of proinflammatory cytokines were observed in colonic tissues. The NBD peptide ameliorated colonic inflammatory injury through the down-regulation of proinflammatory cytokines mediated by NF-kappaB inhibition in both models. These results indicate that an IKKbeta-targeted NF-kappaB blockade using the NBD peptide could be an attractive therapeutic approach for inflammatory bowel disease.     

A knowledgebase resource for interleukin-17 family mediated signaling

Interleukin-17 (IL-17) belongs to a relatively new family of cytokines that has garnered attention as the signature cytokine of Th17 cells. This cytokine family consists of 6 ligands, which bind to 5 receptor subtypes and induce downstream signaling. Although the receptors are ubiquitously expressed, cellular responses to ligands vary across tissues. The cytokine family is associated with various autoimmune disorders including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma and psoriasis in addition to being implicated in the pathogenesis of cancer. In addition, this family plays a role in host defense against bacterial and fungal infections. The signaling mechanisms of the IL-17 family of proinflammatory cytokines are not well explored. In this study, we present a resource of literature-annotated reactions induced by IL-17. The reactions are catalogued under 5 categories, namely; molecular association, catalysis, transport, activation/inhibition and gene regulation. A total of 93 molecules and 122 reactions have been annotated. The IL-17 pathway is freely available through NetPath, a resource of signal transduction pathways previously developed by our group.     

A novel role for defensins in intestinal homeostasis: regulation of IL-1beta secretion

Impaired expression of alpha-defensin antimicrobial peptides and overproduction of the proinflammatory cytokine IL-1beta have been associated with inflammatory bowel disease. In this study, we examine the interactions between alpha-defensins and IL-1beta and the role of defensin deficiency in the pathogenesis of inflammatory bowel disease. It was found that matrix metalloproteinase-7-deficient (MMP-7(-/-)) mice, which produce procryptdins but not mature cryptdins (alpha-defensins) in the intestine, were more susceptible to dextran sulfate sodium-induced colitis. Furthermore, both baseline and dextran sulfate sodium-induced IL-1beta production in the intestine were significantly up-regulated in MMP-7(-/-) mice compared with that in control C57BL/6 mice. To elucidate the molecular mechanism for the increased IL-1beta production in defensin deficiency in vivo, we evaluated the effect of defensins on IL-1beta posttranslational processing and release. It was found that alpha-defensins, including mouse Paneth cell defensins cryptdin-3 and cryptdin-4, human neutrophil defensin HNP-1, and human Paneth cell defensin HD-5, blocked the release of IL-1beta from LPS-activated monocytes, whereas TNF-alpha expression and release were not affected. Unlike alpha-defensins, human beta-defensins and mouse procryptdins do not have any effect on IL-1beta processing and release. Thus, alpha-defensins may play an important role in intestinal homeostasis by controlling the production of IL-1beta.     

Therapeutic effects of novel sphingosine-1-phosphate receptor agonist W-061 in murine DSS colitis

Although IL-17 is a pro-inflammatory cytokine reportedly involved in various autoimmune inflammatory disorders, its role remains unclear in murine models of colitis. Acute colitis was induced by 2.5% dextran sodium sulfate (DSS) treatment for 5 days. A novel sphingosine-1-phosphate receptor agonist W-061, a prototype of ONO-4641, was orally administered daily, and histopathological analysis was performed on the colon. The number of lymphocytes and their cytokine production were also evaluated in spleen, mesenteric lymph node, Peyer's patch and lamina propria of the colon. Daily administration of W-061 resulted in improvement of DSS-induced colitis, and significantly reduced the number of CD4+ T cells in the colonic lamina propria. Numbers of both Th17 and Th1 cells were reduced by W-061 treatment. W-061, however, had no influence on the number of Treg cells in lamina propria. Thus, Th17 and Th1 cells in lamina propria were thought to be the key subsets in the pathogenesis of DSS-induced colitis. In conclusion, W-061 may be a novel therapeutic strategy to ameliorate acute aggravation of inflammatory bowel diseases.     

Tissue factor-dependent chemokine production aggravates experimental colitis

Tissue factor (TF) is traditionally known as the initiator of blood coagulation, but TF also plays an important role in inflammatory processes. Considering the pivotal role of coagulation in inflammatory bowel disease, we assessed whether genetic ablation of TF limits experimental colitis. To this end, wild-type and TF-deficient (TFlow) mice were treated with 1.5% dextran sulfate sodium (DSS) for 7 d, and effects on disease severity, cytokine production and leukocyte recruitment were examined. Clinical and histological parameters showed that the severity of colitis was reduced in both heterozygous and homozygous TFlow mice compared with controls. Most notably, edema, granulocyte numbers at the site of inflammation and cytokine levels were reduced in TFlow mice. Although anticoagulant treatment with dalteparin of wild-type mice reduced local fibrin production and cytokine levels to a similar extent as in TFlow mice, it did not affect clinical and histological parameters of experimental colitis. Mechanistic studies revealed that TF expression did not influence the intrinsic capacity of granulocytes to migrate. Instead, TF enhanced granulocyte migration into the colon by inducing high levels of the granulocyte chemoattractant keratinocyte-derived chemokine (KC). Taken together, our data indicate that TF plays a detrimental role in experimental colitis by signal transduction-dependent KC production in colon epithelial cells, thereby provoking granulocyte influx with subsequent inflammation and organ damage.     

The immunological and genetic basis of inflammatory bowel disease

The inflammatory bowel diseases (IBDs), Crohn's disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract. Enormous progress has been made recently in understanding the pathogenesis of these diseases. Through the study of patients and mouse models, it has emerged that Crohn's disease is driven by the production of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma), whereas ulcerative colitis is probably driven by the production of IL-13. A second area of progress is in the identification of specific genetic abnormalities that are responsible for disease. The most important finding is the identification of mutations in the gene that encodes NOD2 (nucleotide-binding oligomerization domain 2) protein in a subgroup of patients with Crohn's disease. Here, we discuss these recent findings and the implications for therapy.     

IL-6 is required for the development of Th1 cell-mediated murine colitis

Proinflammatory cytokines have been demonstrated to play a crucial role in the pathogenesis of Crohn's disease. Among those cytokines, strong expression of IL-6 has been repeatedly demonstrated. To examine the role for IL-6 in the pathogenesis of Crohn's disease, we introduced anti-IL-6R mAb to a murine model of colitis. Colitis was induced in C.B-17-scid mice transferred with CD45RBhigh CD4+ T cells from BALB/c mice. Anti-IL-6R mAb or rat IgG was administered weekly after T cell transfer. ICAM-1 and VCAM-1 expression were analyzed by immunohistochemistry. Colonic cytokine expression was determined by RT-PCR. Mice treated with mAb showed normal growth, whereas controls lost weight. The average colitis score was 0.64 for mAb-treated mice and 1.80 for controls. T cell expansion in treated mice was less remarkable than in the controls. Colonic ICAM-1 and VCAM-1 expression were markedly suppressed by mAb. IFN-gamma, TNF-alpha, and IL-1beta mRNA were reduced by the treatment. The results presented here show a crucial role for IL-6 in the pathogenesis of murine colitis and suggest a therapeutic potential of anti-IL-6R mAb for treatment of human Crohn's disease.