Invited critical review
The immunophysiological impact of bacterial CpG DNA on the gut

https://doi.org/10.1016/j.cca.2005.05.017Get rights and content

Abstract

Both the endogenous commensal flora and a dysregulated mucosal immune response have been implicated as contributing to the pathogenesis of human intestinal disease. Unmethylated cytosine–guanine (CpG)-containing DNA, the ligand for Toll-like receptor 9 (TLR9), is a recently recognized microbial product with immunostimulatory and immunoregulatory effects. TLR9 is expressed by many cell types located in the intestine, including epithelial cells and classical immune cells. The physiological impact of the juxtaposition of these factors (bacterial DNA and responsive cells) in the gut therefore bears consideration. Here we discuss studies that examine the interaction between CpG DNA and the intestine, focusing on activation of epithelial cells, administration of CpG-containing oligonucleotides as therapy for experimental inflammatory enteropathies, and the role of CpG DNA in mediating the beneficial effects of bacterial probiotics.

Introduction

Colonization of the sterile neonatal intestine begins during passage through the birth canal and upon exposure to the ex utero environment, and within hours postpartum bacteria can be detected in infant fecal material. By adulthood, the human gut (primarily the proximal colon) has a resident population of ∼1014 bacteria composed of an estimated 500 different species [1]. Recent models purport that this complex microbiota exists within and adhered to the intestinal mucus as a biofilm-like community of symbiont and commensal organisms [2], [3]. While the relationship of the host with its normal microbiota is in general poorly understood, it is becoming apparent that the microbes confer both health benefits and, under certain circumstances, will contribute to disease. For example, the short-chain fatty acid butyrate is generated by bacterial metabolism of indigestible carbohydrates and acts as a growth factor for colonic epithelial cells [4]. Furthermore, it is generally accepted that the presence and activity of the microbiota limit colonization of the colon by potential pathogens through competitive exclusion, a process that can be indirect (i.e., consumption of available nutrients) or direct (elaboration of antimicrobial products, e.g., bacteriocins). Contrasted against these beneficial effects, a substantial amount of evidence supports the hypothesis that in concert with a dysregulated immune response the normal gut microbiota participates in driving the pathogenesis of idiopathic disorders such as the inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC) [5].

A voluminous literature exists pertaining to the interaction of host immune and non-immune cells with bacterial pathogens, bacterial products (e.g., fMLP, superantigen) and to a lesser extent the normal commensal microflora, in the context of the gastrointestinal tract (GIT). Pathogens and commensal bacteria are sources of a variety of molecules that are structurally very similar, if not identical (e.g., nucleic acids, lipoproteins); this realization led to a resurgence in interest for considering a role for commensal bacteria, and their products, in disorders of unknown etiology, of which IBD is a classic example. Unmethylated cytosine–guanine (CpG)-rich DNA is a relatively new addition to the assemblage of microbial products that are recognized by the vertebrate innate immune system. The wealth of information on CpG DNA precludes a comprehensive discussion, so instead we concentrate our comments on the impact of bacterial CpG DNA on the gut.

Section snippets

The gastrointestinal tract

The human intestine houses a microbial community that outnumbers host eukaryotic cells by an order of magnitude. There are two fundamental strategies for managing this immense antigenic load-restriction within the gut lumen 1) effectively separate them from the body proper, and 2) neutralization of organisms that penetrate into the mucosa. The former is accomplished primarily by the barrier function of the epithelium and its secreted products, and the latter is the responsibility of the mucosal

Innate immunity and recognition of bacterial components

Immune processes are generally classified into one of two broad categories. Innate immunity depends on a variety of classical (e.g., macrophages) and non-classical (e.g., epithelial) cells employing germ-line encoded (i.e., genetically inherited) receptors to recognize foreign antigen and organisms. In contrast, adaptive immunity is the domain of T and B lymphocytes that, through genome recombination events, express clonally unique antigen receptors (T cell receptor and immunoglobulin), the

CpG DNA

Krieg has recently reviewed the discovery of CpG-containing DNA and the initial experiments that began to define its immunostimulatory activity [17]. Recognition of CpG DNA as an immunostimulant resulted from analysis of synthetic single-stranded antisense oligodeoxynucleotides (AS-ODN) [17], which, ironically, were being employed to suppress cellular responses — not activate them. AS-ODN are designed as short cDNA-complementary for the target mRNA transcript. When transduced into cells

The CpG DNA receptor: Toll-like receptor 9

An important advance in our understanding of innate immunity has been the identification and continuing characterization of the Toll-like receptors (TLR), a family of conserved PRRs that specifically recognize a small number of relatively static microbial structures or products (MAMPs). The immunological role of Toll receptors was originally identified in Drosophila with the finding that Toll mutant flies quickly succumbed to overwhelming fungal infection [25]. This was followed by

CpG and the gastrointestinal tract

TLR9 mRNA expression has been demonstrated in several immune cell-types including human B cells, plasmacytoid DC, NK cells, and monocyte/macrophages [17], [19], [47], [48], [49]. More novel is the finding that TLR9 is expressed in numerous non-immune cell types, including epithelia. We preface the following discussion with the opinion that demonstration of TLR9 protein expression, at least as determined with currently available commercial antibodies, must be considered critically. In our hands,

CpG DNA and the modulation of gut disease

Studies utilizing various models of atopic disease, including experimental asthma, have demonstrated that administration of CpG DNA reduces the severity of pathology associated with these models [70]. Allergy and atopy are generally accepted as being initiated, propagated and/or exacerbated by ongoing T helper cell type-2 (Th2) responses. In accordance with the Th1/Th2 balance paradigm, delivery of Th1-stimulatory CpG DNA would neutralize a Th2-shift by promoting the production of Th1-type

Conclusion

The study of CpG-containing bacterial DNA and its immunophysiological impact is a burgeoning area of innate immunity; continuing this research is necessary to further define the important and sometimes deleterious relationship shared between host and indigenous microbiota. Of interest also will be determining what association, if any, exists between the virulence of microbial pathogens and the nature of their DNA, e.g., hyper/hypo-methylation, and CpG motif content and context. Widespread

Acknowledgements

We would like to acknowledge funding support through grants from the Canadian Institutes of Health Research (CIHR) and the Crohn's and Colitis Foundation of Canada (CCFC).

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