Nadja Dunger

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.

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.

Cathepsins B, L and D in inflammatory bowel disease macrophages and potential therapeutic effects of cathepsin inhibition in vivo.

The cathepsins D (CTSD), B (CTSB) and L (CTSL) are important for the intracellular degradation of proteins. Increased cathepsin expression is associated with inflammatory diseases. We have shown previously an induction of CTSD expression in intestinal macrophages (IMAC) in inflamed mucosa of patients with inflammatory bowel disease (IBD). Here we investigated the regulation of CTSB and CTSL in IMAC during IBD and effects of CTSD and CTSB/CTSL inhibition in vivo. Human IMAC were isolated from normal and inflamed mucosa. Reverse transcription–polymerase chain reaction (RT–PCR) was performed for CTSB and CTSL mRNA. Immunostaining was used to confirm PCR results. Cathepsin inhibition was investigated in the dextran–sulphate–sodium (DSS) colitis model in mice with application of pepstatin A (CTSD inhibitor), CA‐074 (CTSB inhibitor) and Z‐Phe‐Tyr‐aldehyde (CTSL inhibitor). CTSL mRNA was significantly up‐regulated in IMAC isolated from IBD mucosa. Up‐regulated protein expression was found mainly in areas of mucosal damage by immunostaining. Inhibition of CTSD in mouse DSS colitis was followed by an amelioration of the disease. Inhibitor‐treated mice showed a significant lower histological score (HS) and less colon reduction in comparison to controls. Similarly, simultaneous inhibition of CTSB/CTSL was followed by a significant amelioration of colitis. Expression of tissue‐degrading cathepsins is increased in IMAC in IBD. Inhibition of CTSD as well as CTSB/CTSL is followed by an amelioration of experimental colitis. The prevention of mucosal damage by cathepsin inhibition could represent a new approach for the therapy of IBD.

In vivo CpG DNA/toll-like receptor 9 interaction induces regulatory properties in CD4+CD62L+ T cells which prevent intestinal inflammation in the SCID transfer model of colitis

Background and methods: Cytosin-guanosin dinucleotide (CpG) motifs of bacterial DNA are known to be potent activators of innate immunity. We have shown previously that administration of CpG containing oligodeoxynucleotide (CpG-ODN) to mice before the onset of dextran sodium sulphate induced colitis ameliorated colitis and inhibited induction of proinflammatory cytokines. To investigate the possible involvement of CD4+ T cells in the prophylactic CpG-ODN effects, we used the SCID transfer model of colitis. Results: CD4+CD62L+ T cells from CpG-ODN treated donors did not induce significant intestinal inflammation in SCID recipients, in contrast with control cells. Additionally, cotransfer of these cells with CD4+CD62L+ cells from normal mice protected recipient animals from colitis, indicating regulatory activity. Also, CD4+CD62L+ cells from toll-like receptor 9 deficient animals induced a significantly more severe colitis in SCID recipients than cells from wild-type littermate controls, suggesting a similar protective role of “endogenous” bacterial DNA leading to a less “aggressive” phenotype of these cells. There was no detectable difference in regulatory T cell surface markers between aggressive and attenuated cell pools but attenuated cell pools showed reduced proliferation in vitro and in vivo and produced less interferon γ, interleukin (IL)-5, and IL-6 after anti-CD3 stimulation. Conclusions: Collectively, our data support the concept that both endogenous bacterial DNA and exogenously supplied CpG motifs of bacterial DNA induce regulatory properties in CD4+ T cells. Therefore, bacterial DNA derived from the normal gut flora may contribute essentially to the homeostasis between effector and regulatory immune mechanisms in healthy individuals to protect them from chronic intestinal inflammation.

Glycogen synthase kinase 3‐β: 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‐&bgr; (GSK3‐&bgr;) 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‐&bgr; 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‐&bgr; 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‐&kgr;B) 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‐&bgr; blockade significantly reduced chronic intestinal inflammation and even abolished the colitis‐intensifying effects of CpG‐ODN treatment. In vitro inhibition of GSK3‐&bgr; reduced the proinflammatory phenotype of both murine and human intestinal immune cells from chronic inflamed tissue. In vivo blockade of GSK3‐&bgr; resulted in a shift from NF‐&kgr;B activity toward CREB activity in murine MLC and LPMC. Conclusions: Blockade of GSK3‐&bgr; attenuates excessive proinflammatory TLR‐mediated immune responses. GSK3‐&bgr; inhibition therefore constitutes a promising therapeutic option for selectively reducing exaggerated intestinal immune reactions toward the luminal flora in inflammatory bowel disease. (Inflamm Bowel Dis 2010)

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.

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‐γ 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.