Florian Obermeier

Neutralization of tumour necrosis factor (TNF) but not of IL-1 reduces inflammation in chronic dextran sulphate sodium-induced colitis in mice.

The cytokines TNF and IL‐1 have been implicated as mediators of the inflammatory processes in patients with inflammatory bowel disease (IBD). To investigate the role of these cytokines in mucosal inflammation we used anti‐cytokine strategies in a mouse model of acute and chronic colitis. Mice which received 5% dextran sulphate sodium (DSS) in their drinking water showed signs of acute colitis on day 4, with severe weight loss and bloody diarrhoea. Chronic colitis was established after four cycles of feeding 5% DSS for 7 days and water for 10 days, with the mice showing diarrhoea but no weight loss. In acute colitis, treatment with anti‐IL‐1 reagents, anti‐TNF MoAb, or dexamethasone (DEX) led to aggravation. By contrast, in chronic colitis, treatment of mice with several IL‐1 activity‐inhibiting reagents failed to show significant effects, whereas anti‐TNF MoAb or DEX significantly reduced the colitis. We conclude that in acute colitis IL‐1 and TNF are beneficial, whereas in chronic colitis, TNF but not IL‐1 seems to play a major role in perpetuation of chronic inflammation.

Interferon-gamma (IFN-γ)- and tumour necrosis factor (TNF)-induced nitric oxide as toxic effector molecule in chronic dextran sulphate sodium (DSS)-induced colitis in mice

Excess nitric oxide formation caused by the activity of the inducible nitric oxide synthase has been implicated as a toxic effector molecule in the pathogenesis of experimental colitis and inflammatory bowel disease. It was therefore investigated whether inhibition of this synthase or the cytokines TNF and IFN‐γ, inducers of nitric oxide synthase, had effects on chronic colitis in mice. Chronic colitis was induced in mice by repeated feeding of DSS. Cytokines were neutralized by treatment with MoAbs and nitric oxide synthase was inhibited by aminoguanidine. The degree of colonic inflammation was assessed by a histological score and colon length. Aminoguanidine treatment reduced nitric oxide activity by 60% (P = 0.0004), the histological score by 31% (P = 0.005) and increased colon length by 1.4 cm (P = 0.002). Neutralization of TNF and IFN‐γ resulted in increased colon length (0.7 cm, P = 0.07 and 0.8 cm, P = 0.03), improved histological score (19%, P = 0.045 and 25%, P = 0.013), and reduced nitric oxide activity (31%, P = 0.07 and 54%, P = 0.004) compared with controls. The combination of anti‐cytokine treatments had additive effects. TNF and IFN‐γ are involved in perpetuation of chronic DSS‐induced colitis, and induction of excessive nitric oxide activity could be their common effector mechanism.

Influence of intestinal bacteria on induction of regulatory T cells: lessons from a transfer model of colitis

Background: The resident flora plays a critical role in initiation and perpetuation of intestinal inflammation, as demonstrated in experimental models of colitis where animals fail to develop disease under germ free conditions. However, the importance of exposure to commensal bacteria before the onset of colitis is unclear. Our aim was to investigate the influence of previous exposure of donor animals to bacterial antigens on colitis development using a transfer model. Methods: Clinical course and histology were evaluated after transfer of CD4+CD62L+ lymphocytes from germ free and conventionally housed donor mice into SCID recipients. Cotransfer of CD4+CD62L+ cells with CD4+CD62L− lymphocytes from both groups of mice was initiated. Lymphocytes were analysed by FACS, polarisation potential of cells determined, and cytokines measured within the supernatant by enzyme linked immunosorbent assay. Results: Animals that received cells from germ free donors developed an earlier onset of colitis compared with mice reconstituted with lymphocytes from conventionally housed animals. Additionally, CD4+CD62L− cells from germ free mice were not able to abrogate colitis induced by cotransfer with CD4+CD62L+ lymphocytes whereas CD4+CD62L− T cells from normal mice ameliorated disease. The higher percentage of CD4+GITR+ expressing lymphocytes and the production of interleukin 10 after priming by dendritic cells suggests the presence of Treg cells within the CD4+CD62L+ lymphocyte subset derived from conventional housed mice and assumes a lack of Treg cells within germ free mice. Conclusion: The results indicate that bacterial antigens are crucial for the generation and/or expansion of Treg cells in a healthy individual. Therefore, bacterial colonisation is of great importance in maintaining the immunological balance.

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‐γ. 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‐γ and TNF was elevated (200‐ and 150‐fold, respectively) and IFN‐γ 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 asignificant role in the initiation and perpetuation of inflammation in IBD.

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

ABSTRACT 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-γ], 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-γ, 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.

Contrasting activity of cytosin-guanosin dinucleotide oligonucleotides in mice with experimental colitis

Intestinal inflammation in inflammatory bowel disease (IBD) and experimental models of colitis is characterized by a dysregulated intestinal immune response with elevated levels of Th1 cytokines. The luminal flora has been implicated as a major factor contributing to the initiation and perpetuation of inflammation in experimental colitis by mechanisms not known. Bacterial DNA contains unmethylated cytosin–guanosin dinucleotides (CpG) which strongly activate Th1‐mediated immune responses. To test whether these CpG‐motifs modulate intestinal inflammation we treated mice with dextran sulphate sodium (DSS)‐induced colitis with CpG‐containing oligodeoxynucleotides (CpG‐ODN). CpG‐ODN given after the onset of DSS colitis aggravated the disease, as indicated by a significantly increased loss of body weight and a 30% increase of the histological score. Further, we found a severe increase of proinflammatory cytokines (interleukin (IL)‐6: 40‐fold; interferon (IFN)‐γ : 11‐fold). In a pretreatment setting CpG‐ODN reduced weight loss significantly and reduced intestinal inflammation by 45%. Colonic IFN‐γ and IL‐6 mRNA levels were reduced by 75%, and IL‐10 was elevated by 400% compared to controls. The prophylactic CpG‐effect was not imitated by IL‐12 because IL‐12 pretreatment was not protective. In time‐course experiments, CpG‐ODN pretreatment over 5 days resulted in a tolerance effect concerning its IFN‐γ‐inducing quality, and during the following days of colitis induction IL‐10 secretion from mesenterial lymph node cells was elevated compared to controls. Therefore, the prophylactic effect of CpG‐ODN might be explained by its tolerizing effect and/or the increased ability for IL‐10 production during the consecutive intestinal inflammation.

Early life stress enhances the vulnerability to chronic psychosocial stress and experimental colitis in adult mice.

Early life stress enhances the vulnerability to both mood and chronic inflammatory disorders, suggesting a link between these stress-related disorders. To study this, we exposed male C57BL/6 mice to early life stress [maternal separation (MS), 3 h/d, d 1-14] and to adult chronic psychosocial stress [chronic subordinate colony housing (CSC)] and measured changes in neuroendocrine parameters and in the severity of a chemically induced colitis. In both unseparated and MS mice, 19 d of CSC exposure resulted in a transient decrease in body weight gain, increased anxiety-related behavior, and decreased vasopressin mRNA expression in the hypothalamic paraventricular nucleus compared with respective nonstressed mice. However, only CSC-stressed MS mice showed elevated CRH mRNA expression in the paraventricular nucleus and reduced plasma corticosterone. Subsequent treatment with dextran sulfate sodium (1%, 7 d) resulted in a more severe colonic inflammation in MS compared with unseparated mice. This was indicated by an increased histological damage score and increased TNF secretion (nonstressed MS mice), more severe body weight loss and inflammatory reduction in colon length (CSC-stressed MS mice), and increased interferon-gamma secretion (nonstressed and CSC-stressed MS mice). In conclusion, early life stress and subsequent exposure to chronic psychosocial stress in adulthood induced neuroendocrine abnormalities, which likely contributed to enhanced vulnerability to chemically induced colitis. The combined use of MS and CSC represents a potential animal model providing novel (patho)physiological insights into the complex interactions between neuroendocrine and inflammatory actions upon chronic stress exposure. These findings may further help to reveal mechanisms of hypocortisolemic disorders.

DSS induced colitis increases portal LPS levels and enhances hepatic inflammation and fibrogenesis in experimental NASH

BACKGROUND & AIMS Intestinal bacterial overgrowth and increased permeability are features of non alcoholic steatohepatitis (NASH). Bacterial endotoxin has been shown to promote NASH progression. Application of dextran sulfate sodium (DSS) is a colitis model in mice characterized by damage of the intestinal barrier. This study was designed to investigate if application of DSS aggravates experimental NASH. METHODS Male C57bl/6 mice were allocated into four experimental groups receiving either (I) standard chow (SC), (II) a high fat (HF) diet, (III) SC+DSS (1% in the drinking water), and (IV) HF+DSS for 12 weeks. RESULTS DSS treatment caused inflammation and proinflammatory gene expression (IL-1β, IL-17, TNF) in the colon. Expression of colonic antimicrobial peptide Cramp was significantly induced in SC+DSS mice, whereas expression was blocked in the HF+DSS group. Endotoxin levels were elevated in SC+DSS and HF mice but further augmented in the HF+DSS group. In line with this, increased hepatic TLR4 and TLR9 mRNA levels were detected in HF+DSS mice. The histological analysis revealed hepatic steatosis in both HF groups. Hepatic inflammation was more severe in HF+DSS mice, reflected by histology and analysis of proinflammatory gene expression (TNF and MCP-1). HF+DSS mice showed increased hepatic fibrosis by sirius red staining, hepatic collagen I expression, and α-SMA positive cells accompanied by higher p47(phox), TIMP-1, TGF-β, Pai-1, and α-SMA mRNA expression. CONCLUSIONS Induction of an intestinal inflammation in experimental NASH promotes LPS translocation, hepatic inflammation, and fibrogenesis probably due to inhibition of intestinal antimicrobial peptides. These findings underscore the pathophysiological role of the gut-liver axis in the progression of NASH.

Anti-Inflammatory Role of Sympathetic Nerves in Chronic Intestinal Inflammation

Background: Substance P (SP) is a pro-inflammatory neuropeptide in colitis, whereas sympathetic neurotransmitters are anti-inflammatory at high concentrations. Aim and methods: In all layers of the colon, nerve fibre densities of SP+ and sympathetic nerve fibres were investigated (22 Crohn’s disease, six diverticulitis, and 22 controls). In addition, the nerve fibre repellent factor semaphorin 3C (SEMA3C) was studied. The functional role of the sympathetic nervous system was tested in dextran sodium sulfate (DSS) and Il10−/− colitis. Results: In all layers, Crohn’s disease patients demonstrated a loss of sympathetic nerve fibres. Sprouting of SP+ nerve fibres was particularly observed in the mucosa and muscular layer in Crohn’s disease. SEMA3C was detected in epithelial cells, and there was a marked increase of SEMA3C-positive crypts in the mucosa of Crohn’s disease patients compared to controls. In Crohn’s disease, the number of SEMA3C-positive crypts was negatively related to the density of mucosal sympathetic nerve fibres. Sympathectomy reduced acute DSS colitis but increased chronic DSS colitis. Sympathectomy also increased chronic colitis in Il10−/− mice. Conclusions: This study demonstrated a loss of sympathetic and an increase of SP+ nerve fibres in Crohn’s disease. SEMA3C, a sympathetic nerve repellent factor, is highly expressed in the epithelium of Crohn’s disease patients. In chronic experimental colitis, the sympathetic nervous system confers an anti-inflammatory influence. Thus, the loss of sympathetic nerve fibres in the chronic phase of the disease is most probably a pro-inflammatory signal, which might be related to repulsion of these fibres by SEMA3C and other repellents.

In vivo treatment with the herbal phenylethanoid acteoside ameliorates intestinal inflammation in dextran sulphate sodium-induced colitis.

Recently we demonstrated that in inflammatory bowel disease (IBD) macrophage‐oxidative burst activity is increased and NADPH oxidase mRNA is induced. The herbal phenylethanoid acteoside isolated from Plantago lanceolata L. was shown to exhibit anti‐oxidative potential. Using the dextran sulphate sodium (DSS)‐induced colitis model, in this study we have assessed whether systemic application of acteoside affects colitis. Colitis was induced by DSS in Balb/c mice. Treatment with acteoside (120, 600 µg/mouse/day) was performed intraperitoneally. The colon lengths were determined. Colonic tissue was scored histologically (max. score 8) by a blinded investigator. T cells isolated from mesenteric lymph nodes (MLN) were stimulated with anti‐CD3 antibody in the presence of interleukin (IL)‐2 (final concentration 10 U/ml). After incubation for 24 h, IL‐1β, IL‐6, IL‐12 tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ levels in supernatants were analysed by the beadlyte® cytokine detection system. Histological scoring of colonic tissue revealed that application of acteoside was followed by a significantly improved histological score. In acute colitis the histological score was 3·2 with acteoside versus 5·2 with phosphate‐buffered saline (PBS) (P < 0·02). In chronic colitis both 120 µg (3·3 versus 5·2) or 600 µg acteoside (3·0 versus 5·2) significantly ameliorated colitis (both P < 0·02). Stimulated MLN from mice with chronic DSS‐induced colitis treated with acteoside showed a significant down‐regulation of IFN‐γ secretion (195 pg/ml with 600 µg acteoside versus 612 pg/ml with PBS, P < 0·02). Inhibition of oxidative burst activity with acteoside reduced mucosal tissue damage in DSS colitis and could be a therapeutic alternative for IBD treatment. Further studies of this agent are warranted.