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Probiotic Regulation of Vitamin D Receptor in Intestinal Inflammation
75
Values indicate mean ±SD, * p=<0.05 vs. control.
The vitamin D receptor (VDR) is a nuclear receptor that mediates most functions of vitamin D. VDR also has multiple critical functions in regulating bacterial colonization, tight junction structure, anti-inflammatory actions, and intestinal homeostasis. Low VDR expression and dysfunction of vitamin D/VDR signaling are reported in patients with inflammatory bowel disease (IBD). Hence, restoration of VDR function to control inflammation in IBD is desirable. Probiotics are used in the treatment of IBD. However, precisely how probiotics alter aberrant VDR signaling and effectively reduce inflammation is unknown. In the current study, we identified a novel role of probiotics in activating VDR activity, thus inhibiting intestinal inflammation, using cultured intestinal cell models, a probiotic mono-associated pig model, and VDR-deletion mice. We found that probiotics Lactobacillus rhamnosus strain GG (LGG) and Lactobacillus plantarum (LP) increased VDR protein expression in human epithelial colonic cells. Using the VDR Luciferase reporter vector, we detected increased transcriptional activity of VDR after probiotic treatment for only 1 hour. We also analyzed cyp24 (25hydroxyvitamin D3-24-hydroxylase), the enzyme that catabolizes the active form1, 25 vitamin D3, and the VDR target genes, antimicrobial peptide cathelicidin, and beta-defensin 4, at the transcriptional level. Our data indicate that probiotics increased the expression of the VDR target genes. To further understand the probiotics promotion of VDR protein expression, we investigated the half-life of VDR and its degradation. We found that VDR protein was degraded with a half-life of approximately 8 hours, and this rate of degradation was completely blocked by the proteasome inhibitor MG262. Some probiotics have an inhibitory role on proteasome activity. We speculate that VDR stabilization is via ubiquitination/proteasome inhibition by probiotics. Moreover, we used a probiotic mono-associated pig model to assess the probiotic effect on VDR expression In Vivo, we found intestinal VDR increased significantly after probiotic colonization compared to the ex-germ-free pig. Furthermore, the role of probiotic in regulating VDR signaling was tested In Vivo using a Salmonella-colitis model in VDR knockout and wild-type mice. Probiotic treatment conferred clinical and histologic protection from Salmonella infection in VDR+/+ mice, whereas probiotics had no effects in the VDR knockout mice. These data indicate that probiotics did not inhibit inflammation in mice lacking VDR. Taken together, we report here that probiotic treatment is able to enhance VDR expression and activity in the host. Understanding how probiotics enhance VDR signaling and inhibit inflammation will allow probiotics to be used effectively, resulting in innovative approaches to prevention and treatment of IBD.
73 Effects of Probiotics on Host Immune Function and Behaviour Are Dependent Upon Genotype and Diet Lisa Kish, Haley Bell, Nic Moore, Naomi Hotte, Karen Madsen Diet in western societies is characterized by a high intake of fat and refined carbohydrates which can alter gut microflora. Gut bacteria can modulate immune function as well as behaviour through the gut-brain axis. Lactobacillus helveticus and Bifidobacterium infantis are probiotics used clinically with immunomodulatory effects. The aim of this study was to examine if host diet alters the ability of probiotics to modulate immune function and behaviour under normal and inflammatory conditions. Methods: At weaning, wild type (WT) and IL-10 deficient (IL-10-/-) 129/SvEv mice were placed on a standard mouse chow or a western style chow (fat 40%, carb 40%) ± Lactobacillus helveticus ROO52 (109 cfu/d) or Bifidobacterium infantis ROO33 (109 cfu/d) for 21 days. Animal weight and food eaten were monitored weekly. To examine mucosal immune function, large and small intestine were homogenized and analyzed for cytokine expression by MesoScale discovery platform. Spatial memory and anxiety-like behavior was assessed in a Barnes maze. Terminal Restriction Fragment Length Polymorphism (TRFLP) was used to analyze the faecal microflora in stool samples. Results: WT and IL-10-/- mice on the western diet had a significantly higher weight gain per mass of food eaten. L. helveticus, but not B. infantis, reduced the western-diet induced weight gain. Mice on the western diet also had decreased microbial diversity and alterations in composition within the phyla Firmicutes. IL-10-/- mice on normal chow developed colitis with high expression of pro-inflammatory cytokines. A western diet further increased expression of IL-1β and IL-12 in the small intestine, but had an immunosuppressive effect in the colon, with decreased expression of IFNγ, IL-1β, IL-8, and TNFα. L. helveticus prevented the western-diet induced alterations in cytokine expression in the small intestine, but not in the colon. L. Helveticus induced IL-1β, IL-2, IL-4, and IL-5 in WT mice on a chow, but not a western, diet. A western diet suppressed IFNγ, IL-1β, and IL-8 expression in the colon of WT mice, and this was not prevented by L. helveticus. IL-10-/- mice had greater anxiety and reduced exploratory behavior in the Barnes maze as compared with WT mice. Mice on the western diet had decreased learning ability and enhanced anxiety. L. helveticus enhanced learning ability and increased exploratory behavior in IL-10-/- mice on both diets. In WT mice, L. helveticus increased exploratory behaviour only in mice on the western diet. Conclusion: Both WT and IL-10-/- mice on a western diet had increased weight gain, altered gut microflora, an impairment of learning behavior, and altered gut cytokine secretion. The ability of probiotic strains to modulate these factors was genotype, strain- and diet-dependent. These findings have direct relevance to the clinical use of probiotics.
76 Effect of Oligofructose Enriched Inulin (OF-IN) on Bacterial Composition and Disease Activity of Crohn's Disease Patients: Results From a Double-Blind Randomized Controlled Trial Marie Joossens, Vicky De Preter, Vera Ballet, Kristin Verbeke, Kathleen Machiels, Paul J. Rutgeerts, Severine Vermeire Background and Aim The presence of intestinal dysbiosis is well-established in Crohn's disease (CD) patients. Hence improving the disease course by modulation of the intestinal microbiota is an appealing therapeutic approach. Prebiotics are food ingredients that can improve health by modulating the colonic microbiota. Although some small open-label studies with prebiotics in CD already showed promising results, we aimed to study the effect of oligofructose enriched inulin (OF-IN) on the microbiota composition in a double blind placebo-controlled trial. Methods We performed a single centre, randomized controlled trial in inactive and mild to moderately active CD patients to study the impact of a 4-week supplementation with OF-IN on the predominant microbiota using denaturing gradient gel electrophoresis (DGGE) and real time PCR. The disease activity was measured with the Harvey-Bradshaw index (HBI) and statistical analyses were performed with SPSS 17.0. In total 67 patients were randomized, 34 to OF-IN treatment, 33 to placebo. Results In patients receiving OF-IN, the median number of B. longum increased from 8.3 to 8.7 log10/g (ITT: p=0.03) and the median number of R. gnavus decreased after treatment from 8.8 to 8.1 log10/g (PP: p=0.03). Furthermore a positive correlation between improvement in diseaseactivity and increase in number of B. longum was found in patients with active disease receiving OF-IN (p=0.02). (Figure 1) In patients randomized to placebo, no significant changes in microbiota composition or clinical activity were found after 4-weeks intervention. Conclusion In this first double-blind randomized controlled trial with OF-IN in inactive and mild to moderately active CD patients we demonstrated that OF-IN intake increased the number of B. longum and decreased the number of R. gnavus. The positive correlation between improvement in disease activity and increase of number of B. longum is encouraging for longer follow-up studies in CD patients with this prebiotic.
74 Probiotic Conditioned Media Induces an Anti-Inflammatory Macrophage Phenotype In Vivo and In Vitro Michelle Taylor, Vandana Gambhir, Curtis Noordhof, Oliver Jones, Shu-Mei He, Erika C. Claud, Jun Sun, Sam Basta, Elaine O. Petrof Background: Probiotic bacteria, such as Lactobacillus plantarum (Lp), have been shown to reduce the inflammation in animal models of colitis. Some probiotics can decrease proinflammatory cytokine release from macrophages In Vitro. In mouse models of colitis, pro-inflammatory cytokine levels are elevated along with increased numbers of M1 (“proinflammatory”), macrophages. When M2 (“anti-inflammatory”), macrophages predominate, inflammation is reduced. Although some protective effects of probiotics require direct bacterial-epithelial cell-to-cell contact, Lp confers its anti-inflammatory effects via bioactive factors secreted into conditioned media (Lp-CM). Hence, we sought to determine whether Lp-CM exerts systemic effects In Vivo by affecting macrophages, key mediators of the innate immune response. Hypothesis: L. plantarum produces bioactive factors (Lp-CM) that block inflammation by modulating macrophage phenotype. Methods: (1) Mice were infected with Salmonella after streptomycin pretreatment, then gavaged with Lp-CM 4 hours prior to infection and every 24 hours until they were sacrificed 48 hours post-infection. Macrophages were harvested from peritoneum, spleen and bone marrow. (2) Mice were given 3% DSS for 5 days followed by 2 days of water, and gavaged daily with Lp-CM. Macrophages were harvested and cultured at the time of sacrifice. (3) RAW 264.7 murine monocytes were pretreated with Lp-CM or vehicle for 4 hours prior to LPS stimulation. Macrophage cytokine release was measured in all 3 models by ELISA (e.g. TNFα, IL-6, IL-12, and IL-10), or by immunocytochemical detection, following LPS stimulation (1μg/mL). Results: Macrophages from the peritoneal cavity, spleen, and bone marrow of Salmonella-infected mice treated with Lp-CM had reduced pro-inflammatory cytokine release (TNFα, IL-6, and IL-12) and increased anti-inflammatory cytokine release (IL-10) compared to macrophages from Salmonella-infected mice treated with vehicle. The cytokine profile of macrophages from Salmonellainfected mice treated with Lp-CM resembled the profile of macrophages from uninfected mice. Immunocytochemistry supported the presence of increased IL-10 in macrophages from Salmonella-infected mice treated with Lp-CM versus vehicle. Macrophages isolated from the peritoneal cavity and spleen of DSS mice treated with Lp-CM possessed an antiinflammatory cytokine profile with significantly decreased TNFα and increased IL-10 compared to DSS mice treated with vehicle. Lp-CM pretreated RAW cells released significantly more IL-10 and less TNFα than cells pretreated with vehicle. Conclusion: Lp-CM treatment induces an anti-inflammatory macrophage cytokine profile both In Vivo, in two mouse models of colitis, and In Vitro, in a macrophage cell line.
S-19
AGA Abstracts
AGA Abstracts
Probiotic Regulation of Vitamin D Receptor in Intestinal Inflammation Sonia Yoon, Shaoping Wu, Yong-guo Zhang, Rong Lu, Elaine O. Petrof, Lijuan Yuan, Erika C. Claud, Jun Sun
Values indicate mean ±SD, * p=<0.05 vs. control.
The vitamin D receptor (VDR) is a nuclear receptor that mediates most functions of vitamin D. VDR also has multiple critical functions in regulating bacterial colonization, tight junction structure, anti-inflammatory actions, and intestinal homeostasis. Low VDR expression and dysfunction of vitamin D/VDR signaling are reported in patients with inflammatory bowel disease (IBD). Hence, restoration of VDR function to control inflammation in IBD is desirable. Probiotics are used in the treatment of IBD. However, precisely how probiotics alter aberrant VDR signaling and effectively reduce inflammation is unknown. In the current study, we identified a novel role of probiotics in activating VDR activity, thus inhibiting intestinal inflammation, using cultured intestinal cell models, a probiotic mono-associated pig model, and VDR-deletion mice. We found that probiotics Lactobacillus rhamnosus strain GG (LGG) and Lactobacillus plantarum (LP) increased VDR protein expression in human epithelial colonic cells. Using the VDR Luciferase reporter vector, we detected increased transcriptional activity of VDR after probiotic treatment for only 1 hour. We also analyzed cyp24 (25hydroxyvitamin D3-24-hydroxylase), the enzyme that catabolizes the active form1, 25 vitamin D3, and the VDR target genes, antimicrobial peptide cathelicidin, and beta-defensin 4, at the transcriptional level. Our data indicate that probiotics increased the expression of the VDR target genes. To further understand the probiotics promotion of VDR protein expression, we investigated the half-life of VDR and its degradation. We found that VDR protein was degraded with a half-life of approximately 8 hours, and this rate of degradation was completely blocked by the proteasome inhibitor MG262. Some probiotics have an inhibitory role on proteasome activity. We speculate that VDR stabilization is via ubiquitination/proteasome inhibition by probiotics. Moreover, we used a probiotic mono-associated pig model to assess the probiotic effect on VDR expression In Vivo, we found intestinal VDR increased significantly after probiotic colonization compared to the ex-germ-free pig. Furthermore, the role of probiotic in regulating VDR signaling was tested In Vivo using a Salmonella-colitis model in VDR knockout and wild-type mice. Probiotic treatment conferred clinical and histologic protection from Salmonella infection in VDR+/+ mice, whereas probiotics had no effects in the VDR knockout mice. These data indicate that probiotics did not inhibit inflammation in mice lacking VDR. Taken together, we report here that probiotic treatment is able to enhance VDR expression and activity in the host. Understanding how probiotics enhance VDR signaling and inhibit inflammation will allow probiotics to be used effectively, resulting in innovative approaches to prevention and treatment of IBD.
73 Effects of Probiotics on Host Immune Function and Behaviour Are Dependent Upon Genotype and Diet Lisa Kish, Haley Bell, Nic Moore, Naomi Hotte, Karen Madsen Diet in western societies is characterized by a high intake of fat and refined carbohydrates which can alter gut microflora. Gut bacteria can modulate immune function as well as behaviour through the gut-brain axis. Lactobacillus helveticus and Bifidobacterium infantis are probiotics used clinically with immunomodulatory effects. The aim of this study was to examine if host diet alters the ability of probiotics to modulate immune function and behaviour under normal and inflammatory conditions. Methods: At weaning, wild type (WT) and IL-10 deficient (IL-10-/-) 129/SvEv mice were placed on a standard mouse chow or a western style chow (fat 40%, carb 40%) ± Lactobacillus helveticus ROO52 (109 cfu/d) or Bifidobacterium infantis ROO33 (109 cfu/d) for 21 days. Animal weight and food eaten were monitored weekly. To examine mucosal immune function, large and small intestine were homogenized and analyzed for cytokine expression by MesoScale discovery platform. Spatial memory and anxiety-like behavior was assessed in a Barnes maze. Terminal Restriction Fragment Length Polymorphism (TRFLP) was used to analyze the faecal microflora in stool samples. Results: WT and IL-10-/- mice on the western diet had a significantly higher weight gain per mass of food eaten. L. helveticus, but not B. infantis, reduced the western-diet induced weight gain. Mice on the western diet also had decreased microbial diversity and alterations in composition within the phyla Firmicutes. IL-10-/- mice on normal chow developed colitis with high expression of pro-inflammatory cytokines. A western diet further increased expression of IL-1β and IL-12 in the small intestine, but had an immunosuppressive effect in the colon, with decreased expression of IFNγ, IL-1β, IL-8, and TNFα. L. helveticus prevented the western-diet induced alterations in cytokine expression in the small intestine, but not in the colon. L. Helveticus induced IL-1β, IL-2, IL-4, and IL-5 in WT mice on a chow, but not a western, diet. A western diet suppressed IFNγ, IL-1β, and IL-8 expression in the colon of WT mice, and this was not prevented by L. helveticus. IL-10-/- mice had greater anxiety and reduced exploratory behavior in the Barnes maze as compared with WT mice. Mice on the western diet had decreased learning ability and enhanced anxiety. L. helveticus enhanced learning ability and increased exploratory behavior in IL-10-/- mice on both diets. In WT mice, L. helveticus increased exploratory behaviour only in mice on the western diet. Conclusion: Both WT and IL-10-/- mice on a western diet had increased weight gain, altered gut microflora, an impairment of learning behavior, and altered gut cytokine secretion. The ability of probiotic strains to modulate these factors was genotype, strain- and diet-dependent. These findings have direct relevance to the clinical use of probiotics.
76 Effect of Oligofructose Enriched Inulin (OF-IN) on Bacterial Composition and Disease Activity of Crohn's Disease Patients: Results From a Double-Blind Randomized Controlled Trial Marie Joossens, Vicky De Preter, Vera Ballet, Kristin Verbeke, Kathleen Machiels, Paul J. Rutgeerts, Severine Vermeire Background and Aim The presence of intestinal dysbiosis is well-established in Crohn's disease (CD) patients. Hence improving the disease course by modulation of the intestinal microbiota is an appealing therapeutic approach. Prebiotics are food ingredients that can improve health by modulating the colonic microbiota. Although some small open-label studies with prebiotics in CD already showed promising results, we aimed to study the effect of oligofructose enriched inulin (OF-IN) on the microbiota composition in a double blind placebo-controlled trial. Methods We performed a single centre, randomized controlled trial in inactive and mild to moderately active CD patients to study the impact of a 4-week supplementation with OF-IN on the predominant microbiota using denaturing gradient gel electrophoresis (DGGE) and real time PCR. The disease activity was measured with the Harvey-Bradshaw index (HBI) and statistical analyses were performed with SPSS 17.0. In total 67 patients were randomized, 34 to OF-IN treatment, 33 to placebo. Results In patients receiving OF-IN, the median number of B. longum increased from 8.3 to 8.7 log10/g (ITT: p=0.03) and the median number of R. gnavus decreased after treatment from 8.8 to 8.1 log10/g (PP: p=0.03). Furthermore a positive correlation between improvement in diseaseactivity and increase in number of B. longum was found in patients with active disease receiving OF-IN (p=0.02). (Figure 1) In patients randomized to placebo, no significant changes in microbiota composition or clinical activity were found after 4-weeks intervention. Conclusion In this first double-blind randomized controlled trial with OF-IN in inactive and mild to moderately active CD patients we demonstrated that OF-IN intake increased the number of B. longum and decreased the number of R. gnavus. The positive correlation between improvement in disease activity and increase of number of B. longum is encouraging for longer follow-up studies in CD patients with this prebiotic.
74 Probiotic Conditioned Media Induces an Anti-Inflammatory Macrophage Phenotype In Vivo and In Vitro Michelle Taylor, Vandana Gambhir, Curtis Noordhof, Oliver Jones, Shu-Mei He, Erika C. Claud, Jun Sun, Sam Basta, Elaine O. Petrof Background: Probiotic bacteria, such as Lactobacillus plantarum (Lp), have been shown to reduce the inflammation in animal models of colitis. Some probiotics can decrease proinflammatory cytokine release from macrophages In Vitro. In mouse models of colitis, pro-inflammatory cytokine levels are elevated along with increased numbers of M1 (“proinflammatory”), macrophages. When M2 (“anti-inflammatory”), macrophages predominate, inflammation is reduced. Although some protective effects of probiotics require direct bacterial-epithelial cell-to-cell contact, Lp confers its anti-inflammatory effects via bioactive factors secreted into conditioned media (Lp-CM). Hence, we sought to determine whether Lp-CM exerts systemic effects In Vivo by affecting macrophages, key mediators of the innate immune response. Hypothesis: L. plantarum produces bioactive factors (Lp-CM) that block inflammation by modulating macrophage phenotype. Methods: (1) Mice were infected with Salmonella after streptomycin pretreatment, then gavaged with Lp-CM 4 hours prior to infection and every 24 hours until they were sacrificed 48 hours post-infection. Macrophages were harvested from peritoneum, spleen and bone marrow. (2) Mice were given 3% DSS for 5 days followed by 2 days of water, and gavaged daily with Lp-CM. Macrophages were harvested and cultured at the time of sacrifice. (3) RAW 264.7 murine monocytes were pretreated with Lp-CM or vehicle for 4 hours prior to LPS stimulation. Macrophage cytokine release was measured in all 3 models by ELISA (e.g. TNFα, IL-6, IL-12, and IL-10), or by immunocytochemical detection, following LPS stimulation (1μg/mL). Results: Macrophages from the peritoneal cavity, spleen, and bone marrow of Salmonella-infected mice treated with Lp-CM had reduced pro-inflammatory cytokine release (TNFα, IL-6, and IL-12) and increased anti-inflammatory cytokine release (IL-10) compared to macrophages from Salmonella-infected mice treated with vehicle. The cytokine profile of macrophages from Salmonellainfected mice treated with Lp-CM resembled the profile of macrophages from uninfected mice. Immunocytochemistry supported the presence of increased IL-10 in macrophages from Salmonella-infected mice treated with Lp-CM versus vehicle. Macrophages isolated from the peritoneal cavity and spleen of DSS mice treated with Lp-CM possessed an antiinflammatory cytokine profile with significantly decreased TNFα and increased IL-10 compared to DSS mice treated with vehicle. Lp-CM pretreated RAW cells released significantly more IL-10 and less TNFα than cells pretreated with vehicle. Conclusion: Lp-CM treatment induces an anti-inflammatory macrophage cytokine profile both In Vivo, in two mouse models of colitis, and In Vitro, in a macrophage cell line.
S-19
AGA Abstracts
AGA Abstracts
Probiotic Regulation of Vitamin D Receptor in Intestinal Inflammation Sonia Yoon, Shaoping Wu, Yong-guo Zhang, Rong Lu, Elaine O. Petrof, Lijuan Yuan, Erika C. Claud, Jun Sun