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1097 Butyrate Enhances LPS-Stimulated Conventional Dendritic Cell TH17 Induction
mutation that inactivates PI3Kγ enzymatic activity (‘PI3Kγ-kinase-dead mice') and a control group, consisting of 10 wild-type mice. We induced a mild colitis through administration of 1.5% Dextran Sodium Sulphate (DSS) in their drinking water for 7 days. On day 8 we sacrificed the mice and scored blood loss and stool consistency. The average of the scores for weight loss, blood loss and stool consistency was defined as the disease activity index. A pathologist examined the degree of inflammation by scoring influx of inflammatory cells, crypt loss, ulceration, fibrosis, edema and the area involved. Neutrophils, macrophages and T-cells were stained using standard immunohistochemical techniques and counted. Results: The PI3Kγ-kinase-dead mice developed a significantly milder colitis than the control group. On day 8 they had gained 1.1% ± 0.9% weight, whereas the wild-type mice had lost 1.4% ± 0.6% weight, P=0.027. Their disease activity index was lower than control mice (0.63 ± 0.14, versus 1.63 ± 0.24, P=0.002). They also scored better on the histology-parameters (0.8 ± 0.1, versus 1.1 ± 0.1, P=0.02). Influx of T-cells and macrophages was significantly reduced in the PI3Kγ-kinase-dead mice (7.9 ± 0.6 versus 10.5 ± 0.9 positive cells per intercrypt area, P=0.02, and 8.7 ± 0.9, versus 11.1 ± 0.8 positive cells per intercrypt area, P=0.03, respectively). Conclusions: These data show that mice lacking functional PI3Kγ develop less severe colitis upon induction with DSS. These results suggest that the reduced influx of inflammatory cells could play an important role in this outcome and that inhibition of PI3Kγ might be a valuable target in the treatment of IBD.
is also significantly attenuated. Bacterial persistence is well-known to elicit granulomatous inflammation,2 and therefore likely to be of primary relevance to the pathogenesis of CD. References 1. J Pathol 2008; 214: 260 2. J Clin Pathol 1983; 36: 723
Antigen Presenting Cells (APC) from Crohn's Patients Are Less Susceptible to Yeast Mannan-Induced Immunomodulation Compared to Healthy APC Thomas Schaffer, Stefan Mueller, Frank Seibold Background and Aims: Yeast mannan inhibits T cell proliferation in mixed leukocytes reactions from healthy individuals, but not in those from Crohn's disease (CD) patients characterized by anti-S. cerevisiae antibodies (ASCA) and deficiency for mannose-binding lectin (MBL). We wondered whether it is the T cells, antigen presenting cells (APC), or both, which behave differently in these patients. Furthermore, it has been shown that yeast mannan interferes with intracellular killing of bacteria by adherent monocytes from healthy individuals. Thus, it was of interest, whether bactericidal activity of monocytes from CD patients would be differently affected by mannan. Methods: Alloreactive T cell proliferation was measured with or without mannan using APC from CD patients and T cells from healthy controls, and vice-versa. A T cell receptor transgenic mouse model was used to study effects of mannan on APC as compared to responder T cells in the setting of antigen-specific proliferation. Adherent monocytes were co-cultured with an adherent-invasive E. coli (LF82) or with S. aureus in the presence or absence of mannan, and survival of bacteria was assessed. Results: At 4 μg/ml, mannan inhibited allo-reactive T cell proliferation by 40% when APC were derived from healthy controls. When APC were derived from ASCA+/MBL- CD patients, comparable inhibition was achieved only at more than 5-times higher mannan concentrations. Mannan had no effect on anti-CD3/CD28-mediated proliferation of purified human or mouse CD4 T cells. On the other hand, exposure of bone-marrow-derived APC to mannan led to about 50% reduced ovalbumin peptide-specific T cell proliferation. In agreement with published results, monocytes from healthy controls allowed more than 5-fold increased bacterial survival at high (1 mg/ml) mannan concentration. However, this effect was not observed with monocytes from CD patients. Furthermore, if extracellular bacteria were eradicated with gentamicin, bacterial survival was no longer enhanced, indicating that at high mannan concentration bacteria were more efficiently retained at the surface of healthy control monocytes while intracellular killing remained unchanged. Conclusion: Yeast mannan has immunomodulatory effect on APC but not on T cells. This immunomodulation is more likely exerted on the cell surface rather than intracellularly. ASCA+/MBL- CD patients' APC are less susceptible to mannan-mediated immunomodulation. Reduced immunomodulatory effects of mannan on CD patients' APC may support the generation of a mannan-specific adaptive immune response.
1094 The Impact of HDAC Inhibition On CD4+ T Cells in Chronic Intestinal Inflammation Rainer Glauben, Elena Sonnenberg, Thorsten Stroh, Inka Fedke, Paolo Mascagni, Martin Zeitz, Britta Siegmund The impact of epigenetic modifications, especially the modification of the histone acetylation patterns and resulting modified gene expression profile represents a promising new therapeutic approach for various diseases. Several histone deacetylase (HDAC) inhibitors are currently in clinical studies for a variety of solid and hematological cancers. Recently, an anti-inflammatory potency was demonstrated by our group for HDAC inhibitors In Vitro for lamina propria mononuclear cells and CD4+ cells and In Vivo by using different models of experimental colitis in mice. To further evaluate the relevance of acetylation/deactetylation during the process of T cell development and activation, naïve T helper cells were polarized to Th1, Th2, Th17 or T reg cells in the presence or absence of ITF2357. While the generation of Th1 and Th2 cells was not affected, the In Vitro generation of FoxP3+cells could be enhanced in the presence of ITF2357. In contrast, the differentiation to Th17 cells was suppressed under the same conditions. To define the role of single HDAC in intestinal inflammation, the expression pattern of the HDAC 1-11 in naïve or activated CD4+ T cells, compared to In Vitro generated Th1 or Th2 cells as well as FoxP3+ and Th17 cells was analyzed via real-time PCR. Here, a significant down-regulation of HDAC mRNA during the polarization process occurred. The expression pattern of the T cell subsets varied between Th1 and Th2 cells on the one hand and Th17 and FoxP3+ T cells on the other hand. Namely, HDAC 5 and 9 expression in Th17 and FoxP3+ cells was significantly increased compared to the other cell types. Additionally, we analyzed the expression pattern of CD4+ effector cells sorted from the colon, spleen or peripheral lymph nodes from healthy or colitis mice. Again, HDAC 5 and 9 were significantly increased at the site of inflammation. To characterize the significance of single HDAC in T cell responses, CD4+ T cells were transfected with small interfering RNA against the HDAC 1-11 before activation. This specific gene knock-down mimicked the effect of HDAC inhibitors regarding their anti-inflammatory potency. Here again, HDAC 5, 7,and 9 could be identified as crucial factors in CD4+ T cell activation. The present study indicates, that unspecific HDAC inhibition at the site of inflammation exerts an anti-inflammatory potency and modulates T cell differentiation and development. Furthermore, we are now able to target single HDAC, thus allowing for a further specification of the anti-inflammatory effects observed. In conclusion, specific histone modifications might represent a novel therapeutic target for chronic intestinal inflammation.
1097 Butyrate Enhances LPS-Stimulated Conventional Dendritic Cell TH17 Induction Bradford E. Berndt, Teresa W. Wang, Stephanie Y. Owyang, Min Zhang, John Y. Kao Background: Gut microbiota play an important role in the maintenance of the intestinal immune homeostasis. Gut microbes are responsible for breaking down otherwise indigestible dietary fibers into short-chain fatty acids, of which butyrate is the most abundantly produced fatty acid with metabolic activity in the gut. Butyrate is also a known histone deacetylase inhibitor, a class of drug known to have immunosuppressive properties. We recently reported that conventional dendritic cells (DCs) were required for the development of DSS colitis, and thus speculate butyrate may alter DC function and suppress chronic DSS colitis. Aim: To study the effect of butyrate on LPS-stimulated DCs and on chronic DSS colitis. Methods: Day-6 bone marrow-derived DCs from B6 mice were treated with H2O, or LPS (1μg/ml) with of without butyrate (1mM) for 18 hrs. DC activation markers (CD40, CD80, and CD86) were measured by FACS and cytokine release (IL-12, IL-23) was measured by ELISA. Expression of IL-12p40, IL-12p35, and IL-23p19 mRNA were measured by qRT-PCR. B6 mice receiving three 1-week cycles of 1.5%-3% DSS in drinking water with or without i.p. butyrate (1g/kg in H2O daily) were analyzed on day 34. Results: Butyrate reduced LPSinduced DC maturation by downregulating CD40 (↓44%), CD80 ↓37%) and CD86 (↓11.3%) surface expressions. Butyrate significantly inhibited DC IL-12 but increased IL-23 cytokine productions (LPS vs LPS+butyrate: IL-12=9227+1911 vs 2018+497 pg/ml, p<0.05; IL-23= 1567+261 vs 2892+415 pg/ml, p<0.05). Butyrate downregulated LPS-induced DC mRNA expressions of IL-12p35 but not IL-12p40 or IL-23p19. Coculture of LPS-stimulated DC with splenocytes in the presence or absence of butyrate showed that butyrate increased IL17 production but not IFN-γ by lymphocytes. In Vivo, mice with chronic DSS colitis treated with daily butyrate injections showed a trend towards lower rectal bleeding score compared to no butyrate controls (0.5 vs 1.25, p=0.11), and 54% reduction of IL-1β and 69% reduction in MIP-2α mRNA expression in the mouse colon. Conclusion: Butyrate inhibits LPS-induced DC maturation and IL-12 release but upregulated IL-23 release. Butyrate did not inhibit Th1 induction but did increase Th17 induction by LPS-stimulated DCs. Daily injection of butyrate showed a trend towards less severe chronic DSS colitis. Given that neutralization of IL-17 aggravates DSS colitis (Ogawa et al. Clin Immunol. 2004:110:55-62), we speculate that increased induction of Th17 cells by butyrate-conditioned DCs may be protective against experimental colitis by the influence of Th17 cells on specific colitogenic microbiota.
1095 Neutrophil Accumulation and Bacterial Clearance Is Delayed in Patients with Crohn's Disease Bu'Hussain Hayee, Farooq Rahman, Andrew M. Smith, Daniel J. Marks, Trevor J. Sweeting, Stuart L. Bloom, Anthony Segal Introduction. Recent work provides evidence for a failure of acute inflammation in Crohn's disease (CD), in which attenuated neutrophil accumulation at sites of bacterial infection is thought to result in inadequate bacterial clearance,1 providing a stimulus for granulomatous inflammation.2 This study was conducted to determine whether the related phenomena of neutrophil accumulation and bacterial clearance could be characterised in patients with CD. Methods. Local and national ethical and Radiation Safety permission was obtained. An antibiotic-sensitive clinical isolate of E. coli was labelled in culture with 32P and killed by UV-irradiation. Labelled bacteria (1 kBq activity) were mixed with identical, unlabelled UVirradiated E. coli (3x107 bacteria per inoculum). The mixture was injected subcutaneously into both forearms of study subjects, and its clearance determined by measuring radioactivity at the injection sites over the subsequent 72 hours. Clearance kinetics were modeled using a nested mixed effects model with non-linear dynamics. An injection of autologous, indium111-oxine labelled neutrophils has also been employed to investigate neutrophil accumulation at sites of bacterial injection. Results. 14 healthy controls (HC), 16 patients with quiescent CD and 3 patients with ulcerative colitis (UC) were studied. All subjects displayed twophase clearance kinetics with an initial rapid phase lasting ~4 hours. Clearance was significantly delayed in CD compared to HC (p<10-5) and normal in UC. Extrapolating clearance curves to a point when 99% of injected bacteria should have been removed gave total clearance times of 10.2 days (95% CI: 8.3-13.0) for HC, 7.1 days (5.4-10.4) for UC, and 44.3 days (21.8 - ∞) in CD. Neutrophil accumulation to sites of bacterial injection was grossly delayed in 5 patients with CD compared to HC (p<0.0001). Conclusions. This is the first study to use radiolabelled bacteria in human subjects to examine their clearance kinetics. It clearly demonstrates a gross defect in bacterial clearance in patients with CD, and prolonged persistence in their tissues. As expected, neutrophil accumulation to these sites
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is also significantly attenuated. Bacterial persistence is well-known to elicit granulomatous inflammation,2 and therefore likely to be of primary relevance to the pathogenesis of CD. References 1. J Pathol 2008; 214: 260 2. J Clin Pathol 1983; 36: 723
Antigen Presenting Cells (APC) from Crohn's Patients Are Less Susceptible to Yeast Mannan-Induced Immunomodulation Compared to Healthy APC Thomas Schaffer, Stefan Mueller, Frank Seibold Background and Aims: Yeast mannan inhibits T cell proliferation in mixed leukocytes reactions from healthy individuals, but not in those from Crohn's disease (CD) patients characterized by anti-S. cerevisiae antibodies (ASCA) and deficiency for mannose-binding lectin (MBL). We wondered whether it is the T cells, antigen presenting cells (APC), or both, which behave differently in these patients. Furthermore, it has been shown that yeast mannan interferes with intracellular killing of bacteria by adherent monocytes from healthy individuals. Thus, it was of interest, whether bactericidal activity of monocytes from CD patients would be differently affected by mannan. Methods: Alloreactive T cell proliferation was measured with or without mannan using APC from CD patients and T cells from healthy controls, and vice-versa. A T cell receptor transgenic mouse model was used to study effects of mannan on APC as compared to responder T cells in the setting of antigen-specific proliferation. Adherent monocytes were co-cultured with an adherent-invasive E. coli (LF82) or with S. aureus in the presence or absence of mannan, and survival of bacteria was assessed. Results: At 4 μg/ml, mannan inhibited allo-reactive T cell proliferation by 40% when APC were derived from healthy controls. When APC were derived from ASCA+/MBL- CD patients, comparable inhibition was achieved only at more than 5-times higher mannan concentrations. Mannan had no effect on anti-CD3/CD28-mediated proliferation of purified human or mouse CD4 T cells. On the other hand, exposure of bone-marrow-derived APC to mannan led to about 50% reduced ovalbumin peptide-specific T cell proliferation. In agreement with published results, monocytes from healthy controls allowed more than 5-fold increased bacterial survival at high (1 mg/ml) mannan concentration. However, this effect was not observed with monocytes from CD patients. Furthermore, if extracellular bacteria were eradicated with gentamicin, bacterial survival was no longer enhanced, indicating that at high mannan concentration bacteria were more efficiently retained at the surface of healthy control monocytes while intracellular killing remained unchanged. Conclusion: Yeast mannan has immunomodulatory effect on APC but not on T cells. This immunomodulation is more likely exerted on the cell surface rather than intracellularly. ASCA+/MBL- CD patients' APC are less susceptible to mannan-mediated immunomodulation. Reduced immunomodulatory effects of mannan on CD patients' APC may support the generation of a mannan-specific adaptive immune response.
1094 The Impact of HDAC Inhibition On CD4+ T Cells in Chronic Intestinal Inflammation Rainer Glauben, Elena Sonnenberg, Thorsten Stroh, Inka Fedke, Paolo Mascagni, Martin Zeitz, Britta Siegmund The impact of epigenetic modifications, especially the modification of the histone acetylation patterns and resulting modified gene expression profile represents a promising new therapeutic approach for various diseases. Several histone deacetylase (HDAC) inhibitors are currently in clinical studies for a variety of solid and hematological cancers. Recently, an anti-inflammatory potency was demonstrated by our group for HDAC inhibitors In Vitro for lamina propria mononuclear cells and CD4+ cells and In Vivo by using different models of experimental colitis in mice. To further evaluate the relevance of acetylation/deactetylation during the process of T cell development and activation, naïve T helper cells were polarized to Th1, Th2, Th17 or T reg cells in the presence or absence of ITF2357. While the generation of Th1 and Th2 cells was not affected, the In Vitro generation of FoxP3+cells could be enhanced in the presence of ITF2357. In contrast, the differentiation to Th17 cells was suppressed under the same conditions. To define the role of single HDAC in intestinal inflammation, the expression pattern of the HDAC 1-11 in naïve or activated CD4+ T cells, compared to In Vitro generated Th1 or Th2 cells as well as FoxP3+ and Th17 cells was analyzed via real-time PCR. Here, a significant down-regulation of HDAC mRNA during the polarization process occurred. The expression pattern of the T cell subsets varied between Th1 and Th2 cells on the one hand and Th17 and FoxP3+ T cells on the other hand. Namely, HDAC 5 and 9 expression in Th17 and FoxP3+ cells was significantly increased compared to the other cell types. Additionally, we analyzed the expression pattern of CD4+ effector cells sorted from the colon, spleen or peripheral lymph nodes from healthy or colitis mice. Again, HDAC 5 and 9 were significantly increased at the site of inflammation. To characterize the significance of single HDAC in T cell responses, CD4+ T cells were transfected with small interfering RNA against the HDAC 1-11 before activation. This specific gene knock-down mimicked the effect of HDAC inhibitors regarding their anti-inflammatory potency. Here again, HDAC 5, 7,and 9 could be identified as crucial factors in CD4+ T cell activation. The present study indicates, that unspecific HDAC inhibition at the site of inflammation exerts an anti-inflammatory potency and modulates T cell differentiation and development. Furthermore, we are now able to target single HDAC, thus allowing for a further specification of the anti-inflammatory effects observed. In conclusion, specific histone modifications might represent a novel therapeutic target for chronic intestinal inflammation.
1097 Butyrate Enhances LPS-Stimulated Conventional Dendritic Cell TH17 Induction Bradford E. Berndt, Teresa W. Wang, Stephanie Y. Owyang, Min Zhang, John Y. Kao Background: Gut microbiota play an important role in the maintenance of the intestinal immune homeostasis. Gut microbes are responsible for breaking down otherwise indigestible dietary fibers into short-chain fatty acids, of which butyrate is the most abundantly produced fatty acid with metabolic activity in the gut. Butyrate is also a known histone deacetylase inhibitor, a class of drug known to have immunosuppressive properties. We recently reported that conventional dendritic cells (DCs) were required for the development of DSS colitis, and thus speculate butyrate may alter DC function and suppress chronic DSS colitis. Aim: To study the effect of butyrate on LPS-stimulated DCs and on chronic DSS colitis. Methods: Day-6 bone marrow-derived DCs from B6 mice were treated with H2O, or LPS (1μg/ml) with of without butyrate (1mM) for 18 hrs. DC activation markers (CD40, CD80, and CD86) were measured by FACS and cytokine release (IL-12, IL-23) was measured by ELISA. Expression of IL-12p40, IL-12p35, and IL-23p19 mRNA were measured by qRT-PCR. B6 mice receiving three 1-week cycles of 1.5%-3% DSS in drinking water with or without i.p. butyrate (1g/kg in H2O daily) were analyzed on day 34. Results: Butyrate reduced LPSinduced DC maturation by downregulating CD40 (↓44%), CD80 ↓37%) and CD86 (↓11.3%) surface expressions. Butyrate significantly inhibited DC IL-12 but increased IL-23 cytokine productions (LPS vs LPS+butyrate: IL-12=9227+1911 vs 2018+497 pg/ml, p<0.05; IL-23= 1567+261 vs 2892+415 pg/ml, p<0.05). Butyrate downregulated LPS-induced DC mRNA expressions of IL-12p35 but not IL-12p40 or IL-23p19. Coculture of LPS-stimulated DC with splenocytes in the presence or absence of butyrate showed that butyrate increased IL17 production but not IFN-γ by lymphocytes. In Vivo, mice with chronic DSS colitis treated with daily butyrate injections showed a trend towards lower rectal bleeding score compared to no butyrate controls (0.5 vs 1.25, p=0.11), and 54% reduction of IL-1β and 69% reduction in MIP-2α mRNA expression in the mouse colon. Conclusion: Butyrate inhibits LPS-induced DC maturation and IL-12 release but upregulated IL-23 release. Butyrate did not inhibit Th1 induction but did increase Th17 induction by LPS-stimulated DCs. Daily injection of butyrate showed a trend towards less severe chronic DSS colitis. Given that neutralization of IL-17 aggravates DSS colitis (Ogawa et al. Clin Immunol. 2004:110:55-62), we speculate that increased induction of Th17 cells by butyrate-conditioned DCs may be protective against experimental colitis by the influence of Th17 cells on specific colitogenic microbiota.
1095 Neutrophil Accumulation and Bacterial Clearance Is Delayed in Patients with Crohn's Disease Bu'Hussain Hayee, Farooq Rahman, Andrew M. Smith, Daniel J. Marks, Trevor J. Sweeting, Stuart L. Bloom, Anthony Segal Introduction. Recent work provides evidence for a failure of acute inflammation in Crohn's disease (CD), in which attenuated neutrophil accumulation at sites of bacterial infection is thought to result in inadequate bacterial clearance,1 providing a stimulus for granulomatous inflammation.2 This study was conducted to determine whether the related phenomena of neutrophil accumulation and bacterial clearance could be characterised in patients with CD. Methods. Local and national ethical and Radiation Safety permission was obtained. An antibiotic-sensitive clinical isolate of E. coli was labelled in culture with 32P and killed by UV-irradiation. Labelled bacteria (1 kBq activity) were mixed with identical, unlabelled UVirradiated E. coli (3x107 bacteria per inoculum). The mixture was injected subcutaneously into both forearms of study subjects, and its clearance determined by measuring radioactivity at the injection sites over the subsequent 72 hours. Clearance kinetics were modeled using a nested mixed effects model with non-linear dynamics. An injection of autologous, indium111-oxine labelled neutrophils has also been employed to investigate neutrophil accumulation at sites of bacterial injection. Results. 14 healthy controls (HC), 16 patients with quiescent CD and 3 patients with ulcerative colitis (UC) were studied. All subjects displayed twophase clearance kinetics with an initial rapid phase lasting ~4 hours. Clearance was significantly delayed in CD compared to HC (p<10-5) and normal in UC. Extrapolating clearance curves to a point when 99% of injected bacteria should have been removed gave total clearance times of 10.2 days (95% CI: 8.3-13.0) for HC, 7.1 days (5.4-10.4) for UC, and 44.3 days (21.8 - ∞) in CD. Neutrophil accumulation to sites of bacterial injection was grossly delayed in 5 patients with CD compared to HC (p<0.0001). Conclusions. This is the first study to use radiolabelled bacteria in human subjects to examine their clearance kinetics. It clearly demonstrates a gross defect in bacterial clearance in patients with CD, and prolonged persistence in their tissues. As expected, neutrophil accumulation to these sites
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