Metagenomic approaches for defining the pathogenesis of inflammatory bowel diseases

Abstract

The human gastrointestinal tract is home to immense and complex populations of microorganisms. Using recent technical innovations, the diversity present in this human body habitat is now being analyzed in detail. This review focuses on the microbial ecology of the gut in inflammatory bowel diseases and on how recent studies provide an impetus for using carefully designed, comparative metagenomic approaches to delve into the structure and activities of the gut microbial community and its interrelationship with the immune system.

Figure 1. Bacterial SSU rRNA gene-based surveys of the gut microbiota

(A) Cartoon of the general structure of the bacterial 16S rRNA gene, showing conserved and variable regions (based on http://www.rna.ccbb.utexas.edu/). Targets for PCR primers are shown. The numbers associated with the primers are referenced to the E. coli 16S rDNA gene. The targeted sites for PCR vary depending upon whether the goal is to sequence the entire gene or subdomains containing variable regions. The read length achievable with the current generation of massively parallel sequencers limit the portion of the gene that can be characterized: currently favored regions are V2-V3 (Hamady et al., 2008) and V6 (Huber et al., 2007). (B) Analysis of diversity in the human gut microbial community. Collector's curves of observed and estimated richness are shown. Richness is estimated to be 18,000 (genus-level OTUs with ≥95 %ID) and 36,000 at the species-level (≥99 %ID) using the Chao1 estimator.

Figure 2. Bacterial phyla identified in the human gut microbiota

Distribution of predominant bacterial phylotypes in the human intestinal tract. The graphs depict relative abundance as a function of location along the cephalocaudal axis of the distal gut. DNA sequences were compiled from the studies of Eckburg et al. (2005) and Frank et al. (2007). Phylogenetic classifications were made by parsimony insertion of aligned sequences into an 16S rRNA tree provided by the Greengenes database (DeSantis et al., 2006), using ARB (Ludwig et al, 2004). Abbreviations: IBD, samples from patients with either Crohn's disease or ulcerative colitis (Frank et al., 2007); Normal, data from healthy young adults from Eckburg et al (2005) plus the non-IBD controls reported in Frank et al. (2007); n.d., not done. A total of 18,405 16S rRNA sequences were analyzed (5,405 IBD, 13,000 normal).

Figure 3. Host-microbial interactions affected by IgA are modulated as result of stimulation of the immune system at different sites

Secretory IgA, antimicrobial peptides, and mucus are among the factors that comprise the mucosal barrier. The ability of different microorganisms to develop resistance to these factors, or to degrade mucus, will contribute to greater microbial pattern recognition receptor-mediated signaling in epithelial cells, dendritic cells (DCs), macrophages, and B cells. Where these organisms prime the adaptive immune system will depend on how far they can penetrate the barrier, and where the host traffics microbial cells and their antigens. Numbers in the figure refer to the following processes. (1) The simplest response loop may be the innate immune system's induction of IgA production in the lamina propria: NK, DC, epithelial cell-derived cytokines (APRIL, IL-10, TGF, TNF among others) and membrane-bound receptors (e.g. CD40-CD40 ligand) induce IgA production from recruited B cells. (2) Bacteria, bound or unbound by IgA in the lumen, are actively transported to Peyer's patches and associated lymphoid organs where T cell priming and help are more likely. (3) DCs transport whole bacteria and microbial antigens to mesenteric lymph nodes where priming of T cells and B cells programs them to home to the lamina propria, and where IgA induction is favored. (4) Commensal microbes (and invasive pathogens) are transported and trapped by the innate immune system in the spleen, where a systemic immune response is induced (lymphocytes primed in the spleen are less likely to home to the gut, and more likely to switch to non-IgA subclasses).