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PDF file - 226K, Relative Abundance of Indicator Bacterial Phylotypes in Mice Fecal bacteria of age- and litter-matched SPF-N and SPF-S mice (n = 4-5 for each type) were subjected to an indicator analysis.

PDF file - 197K, Phyla Legend.

PDF file - 94K, Effect of Oral Administration of L. johnsonii on Genotoxicity and Inflammation.

PDF file - 551K, Relative Abundance of OTU of CM and RM Mice by Genotype and Intestinal Region.

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PDF file - 61K, Relative Abundance of Phyla of CM and RM Mice by Genotype and Intestinal Region.

PDF file - 369K, Reverse PCR primers used in the Illumina-based high throughput sequence analysis of bacterial rRNA genes.

Background: Host–microbe interactions at the intestinal mucosal–luminal interface (MLI) are critical factors in the biology of inflammatory bowel disease (IBD). Methods: To address this issue, we performed a series of investigations integrating analysis of the bacteria and metaproteome at the MLI of Crohn's disease, ulcerative colitis, and healthy human subjects. After quantifying these variables in mucosal specimens from a first sample set, we searched for bacteria exhibiting strong correlations with host proteins. This assessment identified a small subset of bacterial phylotypes possessing this host interaction property. Using a second and independent sample set, we tested the association of disease state with levels of these 14 “host interaction” bacterial phylotypes. Results: A high frequency of these bacteria (35%) significantly differentiated human subjects by disease type. Analysis of the MLI metaproteomes also yielded disease classification with exceptional confidence levels. Examination of the relationships between the bacteria and proteins, using regularized canonical correlation analysis (RCCA), sorted most subjects by disease type, supporting the concept that host–microbe interactions are involved in the biology underlying IBD. Moreover, this correlation analysis identified bacteria and proteins that were undetected by standard means-based methods such as analysis of variance, and identified associations of specific bacterial phylotypes with particular protein features of the innate immune response, some of which have been documented in model systems. Conclusions: These findings suggest that computational mining of mucosa-associated bacteria for host interaction provides an unsupervised strategy to uncover networks of bacterial taxa and host processes relevant to normal and disease states. (Inflamm Bowel Dis 2012;)

This study examined bacteria-immune interactions in a mouse model possessing microbiota-dependent immune regulatory features similar to those occurring in human atopy, colitis, and immune regulation. Associations between the abundance of several bacterial phylotypes and immunoregulatory target cell types were identified, suggesting that they may play a role in these phenotypes.

Microorganisms appear to play important yet ill-defined roles in the etiologies of Crohn's disease (CD) and ulcerative colitis (UC).1–3 CD and UC occur in regions of the intestine where enteric bacteria are found in the highest concentrations.4 Contact with intestinal contents triggers mucosal inflammation in CD,5 while diversion of the fecal stream promotes intestinal healing.6 The most direct evidence for the importance of microorganisms in disease etiology comes from investigations with animals. In numerous rodent models, colitis is absent when the animals are kept in a “germ-free” state, but it rapidly develops when standard intestinal microorganisms are introduced.7 Evidence also indicates that disease etiology involves specific bacteria, and or an aberrant immunological response to specific intestinal microorganisms. In animal studies, monoassociation experiments have shown that the type of colitis is dependent on the bacterial species.8,9 In addition, various antibiotics, possessing different taxonomic targets, have been shown to exhibit varying abilities to prevent and treat colitis in HLA-B27 rats10 and IL-10–/– mice.11 In human studies, evidence for the involvement of specific bacteria includes the differing abilities of various antibiotic therapies to induce disease remission.12–14 Evidence that the immunological responses to these bacteria are involved in disease etiology include the ability of CBir1-selective T cells to cause colitis when transferred to immunodeficient mice15 and the ability to utilize seroreactivity to specific microbial antigens for disease stratification.16 Prior investigations in our laboratories have demonstrated the utility of population-based approaches for identifying microorganisms involved in specific in situ processes such as plant-pathogen suppression in soil.17,18 The first step in this approach is to create or identify a series of samples/subjects with various levels of a specific functional parameter/phenotype. Extensive microbial community analyses are then performed on these samples or subjects. Finally, analyses are performed to identify associations between the abundance of specific taxa and the levels of the functional parameter/phenotype. In this study we used this experimental approach to identify bacteria and bacterial rRNA genes associated with the development of colitis in IL-10–/– mice. Various levels of colitis were examined by monitoring bacterial populations in 2 different environments, where the disease progression rates were different, and over a 6-month period, throughout the development of colitis. Bacteria and bacterial rRNA genes with both positive and negative associations with colitis were identified. Although such trends can be interpreted in several ways, we suggest that negative associations may represent particularly important trends in inflammatory bowel disease (IBD), because they may facilitate the identification of resident microbiota that are being targeted by aberrant immunological responses.

The composition of the intestinal microbial community is a distinctive individual trait that may divergently influence host biology. Because dendritic cells (DC) regulate the quality of the host response to microbiota, we evaluated DC in mice bearing distinct enteric microbial communities divergent for colitis susceptibility. Surprisingly, a selective, systemic reduction of plasmacytoid dendritic cells (pDC) was observed in isogenic mice with different microbiota: restricted flora (RF) vs specific pathogen free (SPF). This reduction was not observed in germfree mice, suggesting that the pDC deficiency was not simply due to a lack of intestinal microbial products. The microbial action was linked to cytotoxic CD8+ T cells, since pDC in RF mice were preserved in the CD8−/− and perforin−/− genotypes, partially restored by anti-CD8β Ab, and augmented in SPF mice bearing the TAP−/− genotype. Direct evidence for pDC cytolysis was obtained by rapid and selective pDC depletion in SPF mice transferred with RF CD8+ T cells. These data indicate that commensal microbiota, via CTL activation, functionally shape systemic immune regulation that may modify risk of inflammatory disease.

This chapter examines experimental design considerations for a population-based approach for identifying microorganisms involved in specific in situ functions. Although this chapter focuses on a particular population-based approach, many of the experimental design considerations discussed here apply to a wide range of rRNA gene-based population studies and sequence selective PCR assays. This chapter examines an experimental approach that uses the population-based strategy. The approach has the following three phases: (i) identifying populations of rRNA genes whose abundances correlate with the functional parameter, (ii) validating the rRNA gene correlates identified in phase I by using an independent quantitative assay, and (iii) isolating the microorganisms identified by the rRNA gene correlates and reintroducing them into the environment to assess their functions in situ. This approach was recently used to identify microorganisms that suppress the population development of the plant parasitic nematode Heterodera schachtii in southern California soil. Functional gradients are created by manipulating the microbial community with methods such as differential heat treatments, targeted antimicrobial agents, and nutritional amendments. Microbial community composition is examined by rRNA gene analysis. Nucleotide sequence analysis of rRNA gene clone libraries can be used to generate detailed depictions of microbial community composition. PCR assays can be validated by using them to amplify DNAs extracted from different environmental samples and then cloning and sequencing several randomly selected clones from each sample. The assays can be considered selective if they exclusively amplify the target sequence.

Ataxia-telangiectasia is a genetic disorder associated with high incidence of B-cell lymphoma. Using an ataxia-telangiectasia mouse model, we compared lymphoma incidence in several isogenic mouse colonies harboring different bacterial communities, finding that intestinal microbiota are a major contributor to disease penetrance and latency, lifespan, molecular oxidative stress, and systemic leukocyte genotoxicity. High-throughput sequence analysis of rRNA genes identified mucosa-associated bacterial phylotypes that were colony-specific. Lactobacillus johnsonii, which was deficient in the more cancer-prone mouse colony, was causally tested for its capacity to confer reduced genotoxicity when restored by short-term oral transfer. This intervention decreased systemic genotoxicity, a response associated with reduced basal leukocytes and the cytokine-mediated inflammatory state, and mechanistically linked to the host cell biology of systemic genotoxicity. Our results suggest that intestinal microbiota are a potentially modifiable trait for translational intervention in individuals at risk for B-cell lymphoma, or for other diseases that are driven by genotoxicity or the molecular response to oxidative stress. Cancer Res; 73(14); 4222–32. ©2013 AACR.

Aberrant interactions between the host and the intestinal bacteria are thought to contribute to the pathogenesis of many digestive diseases. However, studying the complex ecosystem at the human mucosal-luminal interface (MLI) is challenging and requires an integrative systems biology approach. Therefore, we developed a novel method integrating lavage sampling of the human mucosal surface, high-throughput proteomics, and a unique suite of bioinformatic and statistical analyses. Shotgun proteomic analysis of secreted proteins recovered from the MLI confirmed the presence of both human and bacterial components. To profile the MLI metaproteome, we collected 205 mucosal lavage samples from 38 healthy subjects, and subjected them to high-throughput proteomics. The spectral data were subjected to a rigorous data processing pipeline to optimize suitability for quantitation and analysis, and then were evaluated using a set of biostatistical tools. Compared to the mucosal transcriptome, the MLI metaproteome was enriched for extracellular proteins involved in response to stimulus and immune system processes. Analysis of the metaproteome revealed significant individual-related as well as anatomic region-related (biogeographic) features. Quantitative shotgun proteomics established the identity and confirmed the biogeographic association of 49 proteins (including 3 functional protein networks) demarcating the proximal and distal colon. This robust and integrated proteomic approach is thus effective for identifying functional features of the human mucosal ecosystem, and a fresh understanding of the basic biology and disease processes at the MLI.

Enteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla:Ascomycota,Basidiomycota,Chytridiomycota, andZygomycota. The largest assemblages of fungal rRNA sequences were related to the generaAcremonium,Monilinia,Fusarium,Cryptococcus/Filobasidium,Scleroderma,Catenomyces,Spizellomyces,Neocallimastix,Powellomyces,Entophlyctis,Mortierella, andSmittiumand the orderMucorales. The majority of bacterial rRNA gene clones were affiliated with the taxaBacteroidetes,Firmicutes,Acinetobacter, andLactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.