Probiotics in inflammatory bowel disease: is it all gut flora modulation?

T here is considerable public, media, and scientific interest in ‘‘natural’’ products, including probiotics, in modulating intestinal inflammation and health. 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. 3 The multispecies probiotics used pose special challenges in identifying precise mechanism of action, although alterations in faecal flora have been demonstrated. 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. 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. Among the Lactobacillus genus, different species of bacteria induce distinct mucosal cytokine profiles in the gut immune system of BALB/c mice. 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. 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. 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. 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, 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)-b 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. 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. Similarly, the nonpathogenic 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. 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 620 COMMENTARY