• colitis
• probiotics
• inflammatory bowel disease
• dendritic cells

There is considerable public, media, and scientific interest in “natural” products, including probiotics, in modulating intestinal inflammation and health.¹ Intestinal microflora are intimately involved in the generation of immunocompetent cells and tuning the balance between T helper 1 (Th₁) and Th₂ immunity during the development of the gut associated immune system. It is now generally accepted that the intestinal bacterial flora contributes significantly to the pathogenesis of inflammatory bowel disease (IBD) along with mucosal immune dysregulation and genetic susceptibility. Considerable research is focused on modifying the intestinal flora with probiotic bacteria to attenuate inflammatory activity and prevent relapses in ulcerative colitis, Crohn’s disease, and pouchitis. Although both Lactobacillus species and Bifidobacterium species are frequently used, the optimum use of probiotics in IBD requires greater understanding of their effects on the immune system.

A rationale for the use of probiotics in IBD stems from reports of dysbiosis in the intestinal flora in ulcerative colitis, Crohn’s disease, and pouchitis, either by conventional anaerobic culture or by analysis using molecular probes. It is however unclear whether such alterations in intestinal flora drives the inflammation or is a consequence of it. The practical application of probiotic strategy has been especially encouraged by the positive results of a trial in its use for the prevention and treatment of pouchitis.^2,3 The multispecies probiotics used pose special challenges in identifying precise mechanism of action, although alterations in faecal flora have been demonstrated.⁴ Despite some positive trials, generalisation from pouchitis to their use for all forms of IBD appears somewhat premature, however, as for example, a trial of administration of Lactobacillus GG after surgical resection for Crohn’s disease proved ineffective in preventing relapse.⁵ Further studies are therefore required in ulcerative colitis and Crohn’s disease before firm recommendations may be made.

Lactobacilli are a major constituent of the intestinal microflora and are frequently used as probiotics, often in the health food industry.⁶ Among the Lactobacillus genus, different species of bacteria induce distinct mucosal cytokine profiles in the gut immune system of BALB/c mice.⁷ For example, an increase in the Th₂ cytokines interleukin (IL)-10 and IL-4 was observed in mice fed Lactobacillus delbrueckii subspecies Bulgaricus and Lactobacillus casei whereas, in contrast, a significant induction of the Th₁ cytokines IL-2 and IL-12 was observed with Lactobacillus acidophilus. It is therefore important that notice is taken of which specific bacteria are being used.

Various knockout, transgenic, and adoptive transfer murine and rodent models of IBD have been generated and the requirement for bacterial colonisation to induce a IBD phenotype is virtually universal, despite the complexities of the immune network.⁸ In contrast, in the IL-10 knockout mice model of colitis, probiotic therapy with Lactobacillus species and Bifidobacterium species has been shown to be effective in reducing inflammation.^9,10 In animal models, probiotic therapy may prevent relapses of colitis, as shown by treatment with Lactobacillus GG in HLA-B27 transgenic rats after antibiotic treatment.¹¹ It is therefore clear that not all bacteria have the same actions on gut immune function. Separating them into “good” and “bad” bacteria, a marketing strategy often used in the commercial industry, is however a gross oversimplification, and takes no account of host differences as a contributory factor.

In this issue of the Gut, researchers from Cork, Ireland,¹² challenge the conventional hypothesis of mechanisms of probiotic efficacy by administering Lactobacillus salivarius subcutaneously to IL-10 knockout mice [see page 694]. The anti-inflammatory effect of subcutaneous administration was not specific as it was also seen in a murine model of arthritis. Non-viable bacteria could not be tested as the group receiving heat treated L salivarius had 100% mortality by week 10. No change in faecal microflora occurred as a result of this subcutaneous administration of L salivarius, suggesting a mechanism of action distinct from colonic floral modulation. Various indicators of altered immune function were seen with decreased tumour necrosis factor (TNF) and IL-12 levels being obtained from splenocytes that had been stimulated by Salmonella typhimurium. In contrast, transforming growth factor (TGF)-β levels were maintained. Such systemic anti-inflammatory activity is counterintuitive to a simplistic model of gut flora modulation, and leads to speculation about the molecules involved in driving immunomodulation.

The findings of the current study are not the first to suggest that probiotics have more than a local anti-inflammatory effect by modulating the flora. For example, Lactobacillus casei or Lactobacillus bulgaricus reduced the inflammatory response induced by coculture of bacteria with mucosal explants from Crohn’s disease affected intestinal tissue. In this study, a significant reduction of proinflammatory cytokines such as TNF was noted.¹³ Such anti-inflammatory effect might even be systemic, as shown by the bacteria CpG DNA experiments discussed later.

Probiotics may also influence mucosal cell-cell interactions and cellular “stability” by actions such as enhancement of intestinal barrier function by modulating cytoskeletal and tight junctional protein phosphorylation. For example, live probiotics such as Lactobacillus acidophilus or Streptococcus thermophilus protect in vitro intestinal epithelial cell lines (HT29, Caco-2) from pathogen invasion and adhesion by enteroinvasive Escherichia coli.¹⁴ Similarly, the non-pathogenic E coli strain Nissle 1917 inhibited adhesion and invasion of intestinal epithelial cell line (intestine 407) by adherent invasive E coli strains isolated from patients with Crohn’s disease.¹⁵

In “the age of the genome”, it is not surprising that much time and attention has been spent on studying the importance of the detailed bacterial DNA sequences in these effects. Bacterial DNA contains non-methylated CpG motifs which bind to toll-like receptor 9 (TLR-9). TLR-9 signalling is dependent on the adaptor protein MyD88. Such immunostimulatory DNA sequences (ISS-DNA or CpG DNA) of bacterial origin have been shown to reduce inflammation in rodent IBD models such as DSS induced colitis, hapten induced colitis in BALB/c mice, and the IL-10 knockout mice model of colitis. This reduction in inflammation was accompanied by inhibition of proinflammatory cytokine and chemokine production and suppression of induction of matrix metalloproteinases in the colon.¹⁶

Further evidence of the central role of bacterial DNA has come from novel experiments where both intragastric and subcutaneous administration of probiotic and E coli DNA attenuated the severity of DSS induced colitis.¹⁷ The form that this DNA takes appears crucial, as methylated probiotic DNA, calf thymus DNA, and DNAse treated probiotics were ineffective.

Given this complexity, do we need live bacteria, dead bacteria, or just the DNA? Unfortunately, the data are confusing and sometimes contradictory. TLR-9 and the adaptor protein MyD88 appear essential in signalling, and in their presence even non-viable bacteria can signal. In TLR-9 deficient mice, unlike TLR-2 or TLR-4 deficient mice, intragastric γ irradiated (that is, non-viable) probiotics had no effect on DSS induced colitis. Mice deficient in MyD88 did not respond to γ irradiated probiotics.¹⁷ The immune modulatory function of DNA has also been demonstrated in a study of peripheral blood mononuclear cells from healthy donors where Bifidobacterium genomic DNA caused induction of secretion of the anti-inflammatory IL-10.¹⁸ Given the high GC content of Bifidobacterium chromosomal DNA, it will be of interest to assess the effect of its subcutaneous administration in the IL-10 knockout model of colitis.

The immune modulatory properties of the various probiotic bacteria may differ, and this becomes problematic for the use of multispecies preparations. Furthermore, not all immunostimulatory oligonucleotides have the palindromic CpG motif. In one study, chromosomal DNA was purified from nine strains of Lactobacillus delbrueckii subspecies Bulgaricus and six strains of Streptococcus thermophilus derived from yoghurt starter cultures. Only DNA from L bulgaricus NIAI B6 induced significant proliferation of mice Peyer’s patch and splenic B cells although it did not contain a palindromic CpG motif.¹⁹ It is therefore clear that “the devil is in the detail” and extrapolation across DNA sequences and bacterial species may provide false impressions.

In addition to indirectly influencing gut flora and stimulating immune responses, the probiotic strains themselves produce antimicrobial peptides. Bacteriocin production is often associated with probiotic strains, and Lactobacillus salivarius cultures produce a broad spectrum bacteriocin that exhibits activity against a range of microorganisms such as Bacillus, Staphylococcus, Enterococcus, and Listeria species. Bacteriocins are synthesised in ribosomes as prepeptides before being released extracellularly, and their genetic locus in Lactobacillus salivarius has been identified.²⁰ Production of different classes of bacteriocins confers a competitive survival advantage in colonisation and therefore these molecules are most relevant within the intestinal flora, but their systemic effects require further study. Importantly, the production and activity of bacteriocin is not affected by spray drying which may facilitate commercial preparation.²¹

There has recently been much interest in the function of dendritic cells (DC) in controlling gut immune activity. DC act as the switch that determines the delicate balance between Th₁ and Th₂ immunity, as well as tolerance (Th₃). Therefore, it is likely that the DC phenotype and state of activation determine whether initiation of immunity or tolerance takes place and DC are likely to play a central role in mediating the effect of probiotic bacteria and determining the type of immune response that occurs. Different species of lactobacillus exert different DC activation patterns and the complexity of such interactions is exemplified by demonstration that Lactobacillus reuteri, a poor inducer of IL-12, is capable of inhibiting DC activation by other Lactobacillus species.²² The threshold of bacterial concentration necessary to induce cytokine production may be different for proinflammatory cytokines IL-12/TNF and anti-inflammatory/regulatory cytokine IL-10, permitting fine modulation of the immune response.²²

Evidence of probiotic strains affecting Th₁/Th₂ immune balance also comes from experiments in which stimulation of macrophages with Lactobacillus rhamnosus GG induced mRNA expression of the chemokines CCL2, CCL3, CCL5, CCL7, CCL19, CCL20, CXCL8, CXCL9, and CXCL10.²³ Such a Th₁ pattern of chemokine induction could explain the proposed antiallergenic properties of this probiotic strain and may benefit Th₂ oriented ulcerative colitis. Interestingly, studies of oral administration of these bacteria suggest that they may affect the systemic immune response. For example, oral administration of Lactobacillus rhamnosus GG to healthy volunteers for five weeks affected the systemic cellular immune response to intestinal microorganisms.²⁴

What about the host? Identification of NOD2 mutations associated with Crohn’s disease provides further support for the central role of bacteria in the pathogenesis.^25,26 Three NOD2 mutations located near or in the leucine rich repeats involve a frameshift mutation (Leu1007fsinsC) or two missense mutations (Gly908Arg and Arg702Trp). These alterations are associated with increased risk of development of Crohn’s disease and result in defective induction of nuclear factor κB (NFκB) activation by bacterial peptidoglycan and muramyl dipeptide (MDP). MDP induced activation of NFκB in mononuclear cells is absent in patients with Crohn’s disease homozygous for the Leu1007fsinsC mutation.^27,28 It is therefore interesting to speculate that NOD2 mutations associated with Crohn’s disease result in defective sensing of some bacteria which may precipitate inappropriate diffuse activation of NFκB and inflammation through non-NOD2 mechanisms. Repeating the experiments with Crohn’s mucosal explants¹³ from patients with homozygous, heterozygous, and double heterozygous NOD2 mutations, and appropriate controls cocultured with Lactobacilli, may provide interesting data using TNF readouts.

Our attempts to understand how bacteria modulate the immune system will undoubtedly yield novel therapeutic targets and therapeutic agents. Irrespective of whether or not dysbiosis is reproducibly confirmed in IBD, we need to consider probiotic therapy in terms of specific molecules modulating defined targets in the gut mucosal and systemic immune system, and move away from a simplistic concept of re-populating the intestinal flora with “friendly” bacteria. Bacteria are only “friendly” in the context of the desired immune modulation required for a specific disease. The environment to which the immune system is exposed to determines DC phenotype and state of activation and eventually drives the balance between effector and regulatory T cells (fig 1). Understanding probiotic action may permit modulation of the immune system, both locally and systemically. The article by Sheil and colleagues¹² is the right step towards stimulating further research where immunologists, microbiologists, and gastroenterologists can collaborate.

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Figure 1

Pathogen associated molecular patterns (PAMPs) derived from bacteria (including probiotics) are recognised by pattern recognition receptors (PRRs, such as Toll-like receptors). Initiation of dendritic cell (DC) maturation starts after ligation of PRRs, which also requires adaptor proteins such as MyD88 for signalling. Type of PAMPs determines the selective priming of DCs for production of T_H1, T_H2, and T_Reg lymphocyte polarising factors. Different PAMPs ligate to specific corresponding PRRs. IL, interleukin; TNF, tumour necrosis factor; TGF-β, transforming growth factor β.