Anthocyanins Prevent Colorectal Cancer Development in a Mouse Model.

BACKGROUND: Colorectal cancer is the main leading cause of cancer-related deaths worldwide. Present data suggest that plant-derived anthocyanins have anti-inflammatory and chemopreventive properties. This study was aimed at evaluating the effect of an anthocyanin-rich extract from bilberries on colorectal tumour development and growth in the administration of azoxymethan (AOM)/dextran sodium sulfate (DSS) mouse model. METHODS: Colonic carcinogenesis was induced by AOM and DSS 3 or 5%, respectively, in 50 female Balb/c mice. Mice received either normal food (controls) or a diet containing either 10 or 1% anthocyanin-rich bilberry extract. Colonoscopy took place at week 4 and 9 after initiation of carcinogenesis. After termination at week 9, colon samples were analysed macroscopically and microscopically. RESULTS: Mice receiving 10% anthocyanins showed significantly (p < 0.004) less reduced colon length (12.1 cm [8.5-14.4 cm]) as compared to controls (11.2 cm [9.8-12.3]) indicating less inflammation. Mice fed with 10% anthocyanin-rich extract revealed significantly less mean tumour numbers (n = 1.2) compared to control (n = 14) and anthocyanin 1% treated mice (n = 10.6, p < 0.001). CONCLUSION: Anthocyanins prevented the formation and growth of colorectal cancer in AOM/DSS-treated Balb/c mice. Further studies should investigate the mechanisms of how anthocyanins influence the development of colorectal cancer.

Galectin-3 Modulates Experimental Colitis.

BACKGROUND: We recently identified galectin-3 (gal-3) as a new and strong fibroblast activator produced by colonic epithelial cells. Very little is known about the influence of gal-3 in inflammatory bowel disease. We, therefore, investigated the impact of gal-3 on dextran sodium sulfate (DSS)-induced colitis in a mouse model. METHODS: Colonic lamina propria fibroblasts of healthy controls were used for co-incubation studies of gal-3 with gal-1, TGF-ß1, IFNγ, IL-4 and IL-10. Acute and chronic DSS colitis was induced by 3% DSS in drinking water in female Balb/c mice weighing 20-22 g. Recombinant gal-3 was expressed by the pET vector system and used for a 5-day treatment in different concentrations intraperitoneally. The distal third of the colon was used for histologic analysis. Colonic cytokine expression was determined by quantitative RT-PCR. RESULTS: In vitro, gal-3 induced IL-8 secretion was significantly reduced by co-incubation with IL-10 (5 ng/ml) and IL-4 (10 ng/ml). Acute DSS-induced colitis was ameliorated by gal-3 treatment as indicated by increased colonic length and reduced weight loss compared to that of controls. In acute and chronic colitis, gal-3 treatment resulted in a significant suppression of colonic IL-6. CONCLUSION: Gal-3 significantly reduces inflammation in acute and chronic DSS colitis in mice indicating a potential role in intestinal inflammation.

Physiologic TLR9-CpG-DNA interaction is essential for the homeostasis of the intestinal immune system.

BACKGROUND: Cytosine-guanosine dinucleotide (CpG) motifs are immunostimulatory components of bacterial DNA and activators of innate immunity through Toll-like receptor 9 (TLR9). Administration of CpG oligodeoxynucleotides before the onset of experimental colitis prevents intestinal inflammation by enforcement of regulatory mechanisms. It was investigated whether physiologic CpG/TLR9 interactions are critical for the homeostasis of the intestinal immune system. METHODS: Mesenteric lymph node cell and lamina propria mononuclear cell (LPMC) populations from BALB/c wild-type (wt) or TLR9 mice were assessed by flow cytometry and proteome profiling. Cytokine secretion was determined and nuclear extracts were analyzed for nuclear factor kappa B (NF-κB) and cAMP response-element binding protein activity. To assess the colitogenic potential of intestinal T cells, CD4-enriched cells from LPMC of wt or TLR9 donor mice were injected intraperitoneally in recipient CB-17 SCID mice. RESULTS: TLR9 deficiency was accompanied by slight changes in cellular composition and phosphorylation of signaling proteins of mesenteric lymph node cell and LPMC. LPMC from TLR9 mice displayed an increased proinflammatory phenotype compared with wt LPMC. NF-κB activity in cells from TLR9 mice was enhanced, whereas cAMP response-element binding activity was reduced compared with wt. Transfer of lamina propria CD4-enriched T cells from TLR9 mice induced severe colitis, whereas wt lamina propria CD4-enriched T cells displayed an attenuated phenotype. CONCLUSIONS: Lack of physiologic CpG/TLR9 interaction impairs the function of the intestinal immune system indicated by enhanced proinflammatory properties. Thus, physiologic CpG/TLR interaction is essential for homeostasis of the intestinal immune system as it is required for the induction of counterregulating anti-inflammatory mechanisms.

Glycogen synthase kinase 3-ß: a master regulator of toll-like receptor-mediated chronic intestinal inflammation.

BACKGROUND: A disturbed regulation of Toll-like receptor (TLR) signal transduction resulting in the exclusive activation of proinflammatory signaling pathways may be critical for the perpetuation of established chronic colitis. Glycogen synthase kinase 3-ß (GSK3-ß) was recently identified as an important regulator of TLR signaling mediating excessive inflammatory responses. The aim of this study was to assess the role of GSK3-ß activity in chronic intestinal inflammation. METHODS: Chronic colitis was induced by dextran sodium sulfate (DSS) treatment. Mice were treated intraperitoneally with phosphate-buffered saline (PBS), CpG-ODN, or GSK3-ß inhibitors (SB216763, LiCl). Intestinal inflammation was evaluated by histologic analysis and cytokine secretion of mesenteric lymph node cells (MLC). Nuclear extracts of MLC and lamina propria mononuclear cells (LPMC) were analyzed for nuclear factor kappaB (NF-κB) and CREB activity. Murine and human intestinal immune cells were stimulated in vitro with CpG-ODN, lipopolysaccharide (LPS), or anti-CD3 with or without LiCl. RESULTS: GSK3-ß blockade significantly reduced chronic intestinal inflammation and even abolished the colitis-intensifying effects of CpG-ODN treatment. In vitro inhibition of GSK3-ß reduced the proinflammatory phenotype of both murine and human intestinal immune cells from chronic inflamed tissue. In vivo blockade of GSK3-ß resulted in a shift from NF-κB activity toward CREB activity in murine MLC and LPMC. CONCLUSIONS: Blockade of GSK3-ß attenuates excessive proinflammatory TLR-mediated immune responses. GSK3-ß inhibition therefore constitutes a promising therapeutic option for selectively reducing exaggerated intestinal immune reactions toward the luminal flora in inflammatory bowel disease.

T cell-dependent protective effects of CpG motifs of bacterial DNA in experimental colitis are mediated by CD11c+ dendritic cells.

BACKGROUND: Oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanosine (CpG) sequence motifs constitute the immunostimulatory components of bacterial DNA which potently activate innate immunity. Administration of CpG-ODNs before the onset of experimental colitis prevents intestinal inflammation by induction of colitis-suppressing T cells. AIMS: To identify the interplay between innate and adaptive immune cells finally leading to protective CpG-ODN effects in intestinal inflammation. METHODS: Total splenic cells or purified selected cell types (CD4(+)CD62L(+) T cells alone or with B cells or dendritic cells (DCs)) from BALB/c mice were (co)-incubated in vitro with CpG-ODN for 5 days and CD4(+)CD62L(+) cells were injected intraperitoneally into C.B.-17 SCID (severe combined immunodeficiency) mice. Splenic CD4(+)CD62L(+) T cells were isolated from transgenic donor mice in which CD11c(+) DCs were depleted by diphtheria toxin administration during CpG-ODN treatment and injected into C57BL/6 Rag2(-/-) recipients. Intestinal inflammation was evaluated by histological scoring and cytokine secretion of mesenteric lymph node cells. RESULTS: CpG-ODN treatment of total splenic cells but not of purified CD4(+)CD62L(+) cells reduced the colitogenic potential of transferred T cells. While CpG-ODN stimulation of co-cultured CD4(+)CD62L(+) and B-cells did not alter the colitogenic potential of T cells, co-incubation of CpG-ODN-stimulated DCs and CD4(+)CD62L(+) cells reduced the colitogenic potential of the T cell population. Depletion of CD11c(+) DCs during CpG-ODN administration in vivo abolished the protective CpG-ODN effects. CONCLUSIONS: CpG-ODN-dependent protective effects in experimental colitis act indirectly on CD4(+)CD62L(+) T cells. While the involvement of B cells could be excluded, CD11c(+) DCs were identified as key mediators of CpG-ODN-induced protection in experimental colitis.

Calcitriol analog ZK191784 ameliorates acute and chronic dextran sodium sulfate-induced colitis by modulation of intestinal dendritic cell numbers and phenotype.

AIM: To investigate the effects of ZK1916784, a low calcemic analog of calcitriol on intestinal inflammation. METHODS: Acute and chronic colitis was induced by dextran sodium sulfate (DSS) according to standard procedures. Mice were treated intraperitoneally with ZK1916784 or placebo and colonic inflammation was evaluated. Cytokine production by mesenterial lymph node (MLN) cells was measured by ELISA. Immunohistochemistry was performed to detect intestinal dendritic cells (DCs) within the colonic tissue, and the effect of the calcitriol analog on DCs was investigated. RESULTS: Treatment with ZK191784 resulted in significant amelioration of disease with a reduced histological score in acute and chronic intestinal inflammation. In animals with acute DSS colitis, down-regulation of colonic inflammation was associated with a dramatic reduction in the secretion of the proinflammatory cytokine interferon (IFN)-gamma and a significant increase in intereleukin (IL)-10 by MLN cells. Similarly, in chronic colitis, IL-10 expression in colonic tissue increased 1.4-fold when mice were treated with ZK191784, whereas expression of the Th1-specific transcription factor T-beta decreased by 81.6%. Lower numbers of infiltrating activated CD11c+ DCs were found in the colon in ZK191784-treated mice with acute DSS colitis, and secretion of proinflammatory cytokines by primary mucosal DCs was inhibited in the presence of the calcitriol analog. CONCLUSION: The calcitriol analog ZK191784 demonstrated significant anti-inflammatory properties in experimental colitis that were at least partially mediated by the immunosuppressive effects of the derivate on mucosal DCs.

IL-15 protects intestinal epithelial cells.

IL-15, a T-cell growth factor, has been shown to be increased in inflammatory bowel disease (IBD). It has been suggested that neutralization of IL-15 could protect from T cell-dependent autoimmune inflammation. On the other hand, an anti-apoptotic effect of IL-15 has been demonstrated in kidney epithelial cells during nephritis. We therefore tested the role of IL-15 in two different experimental models of colitis in vivo, and in models of intestinal epithelial cell (IEC) apoptosis in vitro. IL-15 blockade in chronic dextran sulphate sodium-induced colitis resulted in aggravation of the disease with a significantly 2.1-fold increased epithelial damage score compared to controls. TUNEL staining clearly revealed increased apoptosis. IL-6, TNF and IFN-gamma secretion by mesenteric lymph node cells were increased. In the T cell-dependent SCID transfer model of colitis IL-15 neutralization reduced the inflammatory infiltration and proinflammatory cytokine production. Despite that, the intestinal epithelial damage was not reduced. In vitro, IL-15 pre-incubation prevented up to 75% of CH11 antibody-induced apoptosis in SW-480 cells and reduced caspase-3 activity. According to this, endogenously produced IL-15 in chronic colitis does not only act as a proinflammatory cytokine but has at the same time the potential to reduce mucosal damage by preventing IEC apoptosis.

CpG motifs of bacterial DNA essentially contribute to the perpetuation of chronic intestinal inflammation.

BACKGROUND & AIMS: Recently, we demonstrated a proinflammatory effect of cytosin-guanosin dinucleotide (CpG)-oligodeoxynucleotide (ODN) treatment in established dextran sulphate sodium (DSS)-induced colitis. Here, we investigated whether DNA derived from luminal bacteria plays a role in the perpetuation of chronic intestinal inflammation. METHODS: Toll-like receptor (TLR9)-deficient and wild-type (wt) control mice were used for the induction of chronic DSS colitis. Moreover, mice with established chronic colitis using different experimental models were treated with adenoviral ODN (AV-ODN) known to block CpG effects. Colonic inflammation was scored and cytokine production was quantified both in colonic tissue and draining mesenteral lymph node cells (MLC). RESULTS: Eight weeks after induction of chronic DSS colitis in TLR9-deficient mice, intestinal inflammation was significantly lower (-68%), and proinflammatory cytokine production was drastically reduced. Treatment of wt mice with chronic DSS-induced colitis with AV-ODN resulted in a significant amelioration of disease with a reduced histologic score (-43%) and reduced cytokine production of MLC (interleukin [IL]-6: -68%; interferon [IFN]-gamma: -48%) and RNA expression of the T helper (Th)1-specific transcription factor T-bet (-62%) in colonic tissue. Qualitatively, the same results were obtained in the severe combined immunodeficiency disease (SCID) transfer model of colitis and in spontaneous colitis in IL-10-deficient mice. CONCLUSIONS: Bacterial DNA derived from luminal bacteria contributes significantly to the perpetuation of chronic intestinal inflammation. Inhibition of the immune-stimulating properties of bacterial DNA using AV-ODN may offer a novel and specific tool for the treatment of inflammatory bowel disease.

Preventive effects of Escherichia coli strain Nissle 1917 on acute and chronic intestinal inflammation in two different murine models of colitis.

Escherichia coli strain Nissle 1917 (EcN) is as effective in maintaining remission in ulcerative colitis as is treatment with mesalazine. This study aims to evaluate murine models of acute and chronic intestinal inflammation to study the antiinflammatory effect of EcN in vivo. Acute colitis was induced in mice with 2% dextran-sodium sulfate (DSS) in drinking water. EcN was administered from day -2 to day +7. Chronic colitis was induced by transfer of CD4(+) CD62L(+) T lymphocytes from BALB/c mice in SCID mice. EcN was administered three times/week from week 1 to week 8 after cell transfer. Mesenteric lymph node (MLN) cytokine secretion (of gamma interferon [IFN-gamma], interleukin 5 [IL-5], IL-6, and IL-10) was measured by enzyme-linked immunosorbent assay. Histologic sections of the colon were analyzed by using a score system ranging from 0 to 4. Intestinal contents and homogenized MLN were cultured, and the number of E. coli-like colonies was determined. EcN was identified by repetitive extragenic palindromic (REP) PCR. EcN administration to DSS-treated mice reduced the secretion of proinflammatory cytokines (IFN-gamma, 32,477 +/- 6,377 versus 9,734 +/- 1,717 [P = 0.004]; IL-6, 231 +/- 35 versus 121 +/- 17 [P = 0.02]) but had no effect on the mucosal inflammation. In the chronic experimental colitis of the transfer model, EcN ameliorated the intestinal inflammation (histology score, 2.7 +/- 0.2 versus 1.9 +/- 0.3 [P = 0.02]) and reduced the secretion of proinflammatory cytokines. Translocation of EcN and resident E. coli into MLN was observed in the chronic colitis model but not in healthy controls. Administration of EcN ameliorated acute and chronic experimental colitis by modifying proinflammatory cytokine secretion but had no influence on the acute DSS-induced colitis. In this model, preexisting colitis was necessary for translocation of EcN and resident E. coli into MLN.

OX40/OX40L interaction induces the expression of CXCR5 and contributes to chronic colitis induced by dextran sulfate sodium in mice.

Interactions between APC and T lymphocytes have been implicated as a major factor contributing to inflammatory bowel disease. To test whether OX40/OX40L interaction plays a role in chronic intestinal inflammation, we induced chronic colitis using dextran sulfate sodium and treated the mice with a murine fusion protein (OX40-IgG). Treatment resulted in a dose-dependent and significant reduction of intestinal inflammation (46%) as measured by a histologic score. IL-10 and IL-5 production from mesenteric lymph node cells increased 20-fold and 18-fold, respectively. In colonic tissue, IL-10 mRNA levels increased and the expression of T-bet was decreased to 30%. IL-10 neutralization partly inhibited the beneficial effects of OX40-IgG treatment. Surprisingly, despite the reduction of inflammation we found the number and size of colonic lymphoid follicles increased, with an accumulation of CD4(+) cells in the mantle area. In contrast, the number of CD4(+) cells infiltrating the mucosa was significantly reduced, as was their CXCR5 expression (24-fold). We conclude that OX40/OX40L interaction contributes to the perpetuation of chronic colitis partly by suppressing IL-10 production. Furthermore, our data suggest that the OX40/OX40L-induced CXCR5 expression on CD4(+) cells may be important for the inflammatory process by allowing migration to the germinal center for further differentiation of CD4(+) cells before they infiltrate the chronically inflamed mucosa.

CpG motifs of bacterial DNA exacerbate colitis of dextran sulfate sodium-treated mice.

Inflammatory bowel disease (IBD) is characterized by a dysregulated intestinal immune response with elevated levels of the Th1 cytokines TNF, IL-12 and IFN-gamma. The luminal flora has been implicated as a major factor contributing to the initiation and perpetuation of chronic intestinal inflammation by as yet unknown mechanisms. Bacterial DNA contains unmethylated cytosine-guanosine dinucleotides (CpG) which strongly activate Th1-mediated immune responses. To test whether these CpG-motifs contribute to intestinal inflammation we treated mice with dextran-sulfate-sodium (DSS)-induced acute or chronic colitis for 5 days with CpG-containing oligodeoxynucleotides (CpG-ODN). Colonic inflammation was assessed by histological scoring. Colonic cytokine RNA was quantified by reverse transcription-PCR and cytokine secretion from mesenterial lymph node cells by ELISA. In chronic colitis, CpG-ODN treatment severely aggravated inflammation by 50%. Colonic expression of IFN-gamma and TNF was elevated (200- and 150-fold, respectively) and IFN-gamma and IL-12 secretion from lymph node cells was increased 5,000- and 8-fold, respectively, compared to GpG-ODN-treated controls. Similar effects were obtained in acute colitis. In conclusion, CpG-motifs of bacterial DNA have proinflammatory activity by strengthening the Th1 arm of immunity in DSS-induced colitis, and might therefore play a significant role in the initiation and perpetuation of inflammation in IBD.