Sue A. Keilbaugh

Linking Long-Term Dietary Patterns with Gut Microbial Enterotypes

The basic composition of the human gut microbiome is influenced by long-term diet: high fat and protein versus high fiber. Diet strongly affects human health, partly by modulating gut microbiome composition. We used diet inventories and 16S rDNA sequencing to characterize fecal samples from 98 individuals. Fecal communities clustered into enterotypes distinguished primarily by levels of Bacteroides and Prevotella. Enterotypes were strongly associated with long-term diets, particularly protein and animal fat (Bacteroides) versus carbohydrates (Prevotella). A controlled-feeding study of 10 subjects showed that microbiome composition changed detectably within 24 hours of initiating a high-fat/low-fiber or low-fat/high-fiber diet, but that enterotype identity remained stable during the 10-day study. Thus, alternative enterotype states are associated with long-term diet.

A novel therapy for colitis utilizing PPAR-gamma ligands to inhibit the epithelial inflammatory response.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily originally shown to play a critical role in adipocyte differentiation and glucose homeostasis, has recently been implicated as a regulator of cellular proliferation and inflammatory responses. Colonic epithelial cells, which express high levels of PPAR-gamma protein, have the ability to produce inflammatory cytokines that may play a role in inflammatory bowel disease (IBD). We report here that PPAR-gamma ligands dramatically attenuate cytokine gene expression in colon cancer cell lines by inhibiting the activation of nuclear factor-kappaB via an IkappaB-alpha-dependent mechanism. Moreover, thiazolidinedione ligands for PPAR-gamma markedly reduce colonic inflammation in a mouse model of IBD. These results suggest that colonic PPAR-gamma may be a therapeutic target in humans suffering from IBD.

High-fat diet determines the composition of the murine gut microbiome independently of obesity.

BACKGROUND & AIMS The composition of the gut microbiome is affected by host phenotype, genotype, immune function, and diet. Here, we used the phenotype of RELMbeta knockout (KO) mice to assess the influence of these factors. METHODS Both wild-type and RELMbeta KO mice were lean on a standard chow diet, but, upon switching to a high-fat diet, wild-type mice became obese, whereas RELMbeta KO mice remained comparatively lean. To investigate the influence of diet, genotype, and obesity on microbiome composition, we used deep sequencing to characterize 25,790 16S rDNA sequences from uncultured bacterial communities from both genotypes on both diets. RESULTS We found large alterations associated with switching to the high-fat diet, including a decrease in Bacteroidetes and an increase in both Firmicutes and Proteobacteria. This was seen for both genotypes (ie, in the presence and absence of obesity), indicating that the high-fat diet itself, and not the obese state, mainly accounted for the observed changes in the gut microbiota. The RELMbeta genotype also modestly influenced microbiome composition independently of diet. Metagenomic analysis of 537,604 sequence reads documented extensive changes in gene content because of a high-fat diet, including an increase in transporters and 2-component sensor responders as well as a general decrease in metabolic genes. Unexpectedly, we found a substantial amount of murine DNA in our samples that increased in proportion on a high-fat diet. CONCLUSIONS These results demonstrate the importance of diet as a determinant of gut microbiome composition and suggest the need to control for dietary variation when evaluating the composition of the human gut microbiome.

An open-label trial of the PPARγ ligand rosiglitazone for active ulcerative colitis

OBJECTIVES:Previous research has demonstrated that ligands for the γ subtype of peroxisome proliferator-activated receptors (PPARs) reduce inflammation in two different murine models of colitis. This study was designed to examine the potential efficacy of rosiglitazone, a ligand for the γ subtype of PPARs, as a therapy for active ulcerative colitis.METHODS:Fifteen patients with mild to moderately active ulcerative colitis despite therapy with 5-aminosalicylic acid compounds were enrolled in an open-label study of rosiglitazone (4 mg b.i.d. p.o.) for 12 wk. Thirteen of 15 patients were receiving concomitant therapy with corticosteroids and/or immunomodulator medications. Disease activity was measured with the Disease Activity Index.RESULTS:After 12 wk of therapy, four patients (27%) had achieved clinical remission, of whom three (20%) also had an endoscopic remission. Four additional patients (27%) had a clinical response without achieving remission. Two patients were hospitalized with worsened disease activity, and one patient was withdrawn for nephrotic syndrome.CONCLUSIONS:These data suggest that ligands for the γ subtype of PPARs may represent a novel therapy for ulcerative colitis. A double blind, placebo-controlled, randomized trial is warranted.

Absence of bacterially induced RELMβ reduces injury in the dextran sodium sulfate model of colitis

Although inflammatory bowel disease (IBD) is the result of a dysregulated immune response to commensal gut bacteria in genetically predisposed individuals, the mechanism(s) by which bacteria lead to the development of IBD are unknown. Interestingly, deletion of intestinal goblet cells protects against intestinal injury, suggesting that this epithelial cell lineage may produce molecules that exacerbate IBD. We previously reported that resistin-like molecule beta (RELMbeta; also known as FIZZ2) is an intestinal goblet cell-specific protein that is induced upon bacterial colonization whereupon it is expressed in the ileum and colon, regions of the gut most often involved in IBD. Herein, we show that disruption of this gene reduces the severity of colitis in the dextran sodium sulfate (DSS) model of murine colonic injury. Although RELMbeta does not alter colonic epithelial proliferation or barrier function, we show that recombinant protein activates macrophages to produce TNF-alpha both in vitro and in vivo. RELMbeta expression is also strongly induced in the terminal ileum of the SAMP1/Fc model of IBD. These results suggest a model whereby the loss of epithelial barrier function by DSS results in the activation of the innate mucosal response by RELMbeta located in the lumen, supporting the hypothesis that this protein is a link among goblet cells, commensal bacteria, and the pathogenesis of IBD.

High-level expression of I kappa B-beta in the surface epithelium of the colon: in vitro evidence for an immunomodulatory role.

The intestinal epithelium is spatially segregated into two compartments, one containing undifferentiated cells in a proliferative state and one with non‐proliferative differentiated cells. Although this epithelium can produce many immune‐modulating substances, emerging evidence suggests that the differentiated cell compartment is less immune responsive. Indeed, it is the differentiated cellular compartment that represents the interface between the highly antigenic luminal environment and the mucosal immune system. The NF‐κB/rel family of transcriptional activators play a critical role in regulating the inflammatory response by activating a wide variety of immune‐modulating genes. These transcription factors are maintained in an inactive state in the cytoplasmic compartment by interaction with inhibitory proteins of the IκB family. In this study we show by immunohistochemistry that IκB‐β is expressed at high levels specifically in the differentiated surface epithelium of the colonic mucosa. Using a naturally occurring compound found in the colon of vertebrates, butyrate, we provide evidence in an intestinal cell line that alteration of IκB‐β expression can modulate the transcriptional activation of the interleukin‐8 (IL‐8) gene by preventing the nuclear translocation of NF‐κB proteins. Therefore, the expression of IκB‐β in the differentiated surface epithelium of the colon may help these cells act as an immunological barrier to prevent activation of the mucosal immune system. J. Leukoc. Biol. 66: 1049–1056; 1999.

Immune-mediated signaling in intestinal goblet cells via PI3-kinase- and AKT-dependent pathways

In the intestinal epithelium, activation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT pathways, via growth factor-mediated signaling, has been shown to regulate cell proliferation and inhibit apoptosis. An immune-activated receptor critical for Th2 immune responses, IL-4Ralpha can also activate PI3-kinase via insulin receptor substrate (IRS)-dependent signaling. Here, using the intestinal goblet cell-specific gene RELMbeta, we investigated the effect of PI3-kinase activation via Th2 immune responses on the goblet cell phenotype. IL-13 stimulation activated PI3-kinase and AKT signal transduction in LS174T cells. Not only did pharmacological inhibition of PI3-kinase and AKT1/2 inhibit RELMbeta induction by IL-13, but AKT inhibition also significantly reduced constitutive basal expression of RELMbeta, a response reproduced by the simultaneous pharmacological inhibition of both epidermal growth factor receptor and IGF-I receptor signaling. In vivo, the disruption of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), an inhibitor of PI3-kinase activation, led to the activation of RELMbeta expression in the small intestine. Furthermore, induction of an intestinal Th2 immune response by infection with a small intestinal nematode parasite, Heligmosomoides polygyrus, led to enhanced epithelial cell proliferation, activation of AKT as demonstrated by the loss of Foxo1 nuclear localization, and robust induction of RELMbeta expression in wild-type, but not IL-4Ralpha knockout, mice. These results demonstrate that Th2 immune responses can regulate goblet cell responses by activation of PI3-kinase and AKT pathways via IL-4Ralpha.

W1832 Diversity in Human Gut Microbiome Composition Assessed by Pyrosequencing: Comparison of Sampling Methods

thy) and histologic (epithelial erosions, crypt hyperplasia, mononuclear cellular infiltrate) lesions were inversely associated with changes in composition of the intestinal microbiota of IL-10-/mice at all time points. Conclusions: Changes in the relative densities and spatial distribution of dominant bacterial groups occur and precede the development of colitis in IL-10-/mice. Archaea decreased in colitic IL-10-/mice and appear to associate with different bacterial species during active inflammation. These results indicate that the Archaea (methanogens) may serve as a biomarker for onset and progression of IBD. Further work on the diversity of methanogens as well as alterations in the concentration of methanogenic species is required to fully assess their biomarker potential.

Dynamic Response of the Human Virome to Dietary Intervention

regarding disease activity, adherence, side effects, andmeasurement of weight. An educational curriculum was delivered at the end of each session. Alerts and action plans were generated based on the results. The BAC arm underwent routine follow up, received written action plans and were given educational fact sheets. Seo Index and IBDQ scores were measured every 4 months for 1 year. Intention to treat analyses and analyses of trial completers using all available data were performed. Results: 25 patients were randomized to UC HAT and 22 to BAC. At baseline, 56% of UC HAT participants were on immune suppressants (IS) compared to 27% in BAC (p=0.05). BAC participants had higher QoL scores (191 vs. 172, p=0.02) and lower depression scores (16 vs. 21, p=0.01) than UC HAT at baseline. After 12 months, 11 (44%) participants withdrew in UC HAT compared to 5 (24%) in BAC. Disease activity, QoL, and adherence were not significantly different between groups at any time point post baseline in intention to treat analyses. An analysis of trial completers adjusting for baseline QoL showed that UC HAT participants had a decrease in Seo index scores of 11.9+/-6.6 points from baseline (p=0.08) compared to 1.2+/-6.0 in BAC (p=ns). Analysis of trial completers, adjusting for baseline disease knowledge, demonstrated improved QoL in UC HAT participants compared to BAC at 12 months (+12.5+/-5.9 vs. -3.8+/-5.3). The difference in IBDQ scores at 12 months from baseline between groups was 16.3+/-7.9 (p= 0.04). Conclusions: UC HAT did not improve disease activity or QoL compared to BAC after 1 year. However, UC HAT participants demonstrated improvements from baseline in disease activity and QoL in analyses of trial completers. Our results suggest a potential benefit of UC HAT. Further research is indicated to determine if telemedicine improves outcomes in patients with IBD.