The exact etiology of chronic IBD is still unknown but seems complex and multifactorial. From human studies of and animal models for experimental colitis, increasing evidence is generated that the resident intestinal flora plays a critical role in the development of the intestinal inflammation on a given genetic background. IL-2- and IL-10-knockout mice as well as HLA-B27 transgenic rats developed colitis when raised in specific-pathogen-free conditions, but they remained almost disease free under germfree conditions (32
). Furthermore, clinical observations demonstrate beneficial effects of probiotic microorganisms in the treatment of IBD. EcN especially was shown to be as effective as mesalazine in the maintenance of remission in patients with ulcerative colitis (17
). So far, however, no animal model of experimental colitis has been tested for further investigation of the mechanisms by which EcN may exert its beneficial effects.
Using a preventive setup in the present study, we demonstrated that EcN has immunomodulatory properties in acute and chronic experimental colitis, possibly mediated by translocation into MLN and alteration of the proinflammatory cytokine secretion profile—but EcN has no short-term effect on acute toxic mucosal damage.
We have chosen two different models of colitis to test the efficacy of EcN in acute and chronic experimental intestinal inflammation. The modified transfer model (26
), based on the CD4+
T-cell model, first described by Powrie et al. (29
), is characterized by a massive influx of mononuclear cells into the colonic mucosa, elevated levels of proinflammatory cytokines, and a chronic, severe wasting syndrome. The degree of colitis in this model correlates with the extent of luminal bacterial growth (1
) and therefore makes it suitable for experimental probiotic therapy. On the other hand, administration of DSS in drinking water induces acute colitis in mice (10
) with patchy mucosal damage, including focal crypt loss, followed by acute transmural infiltration with inflammatory cells. It has been shown that neither T nor B cells are required for the induction of acute DSS-induced colitis, as this model can also be used for SCID mice (13
). However, DSS colitis also seems to be dependent on the intestinal microflora (2
The use of the probiotic microorganism EcN in SCID mice following transfer of CD4+
T cells ameliorated the development of intestinal inflammation, as was demonstrated by a healthier clinical appearance of the animals, lower histological scores, and changes in cytokine secretion profiles of mesenteric lymphocytes. A marked reduction in the secretion of the proinflammatory cytokine IFN-γ was observed, which is thought to contribute predominantly to the perpetuation of intestinal inflammation, as seen in many previous murine models of colitis (14
). Additionally, MLN cells from EcN-treated colitic SCID mice secreted less IL-5 and IL-6. IL-6 seems to play a major role in intestinal inflammation, since blockage of the IL-6 receptor in a murine model of colitis successfully prevented both wasting disease and the development of macroscopic and histologic lesions (16
). On the other hand, there was no effect on the secretion of IL-10, an antiinflammatory cytokine secreted predominantly by regulatory T cells.
Both the CD4+
and the CD4+
models are characterized by chronic intestinal inflammation and a severe wasting syndrome. While the intestinal inflammation is significantly ameliorated, treatment with EcN had no effect on the weight loss in this model. It can be hypothesized that probiotic therapy influences the intestinal inflammation but does not have an impact on the multiorgan pathology, thus contributing to the wasting syndrome as described previously by Powrie and Mason (30
). Whether a longer observation period would be needed to record recovery of the weight loss in EcN treated mice remains unclear.
Different results were seen when EcN was administered in a model of acute colitis. In contrast to the effects of the probiotics on clinical and histopathological scores in chronic intestinal inflammation, application of EcN did not ameliorate the histological appearance of the acute DSS-induced colitis in mice. Histology in the acute DSS colitis model reveals a characteristic patchy pattern with noninflamed mucosa appearing next to necrotic tissue. However, the dominant parameter for the histology score in the acute phase is destruction of the mucosal architecture rather than the influx of inflammatory cells, which is most likely due to a primarily toxic effect of DSS. The beneficial effect of probiotic administration, however, is obviously limited to the immunologic modification of colitis, as demonstrated by a pronounced reduction of proinflammatory cytokine secretion, which is more relevant in the chronic phase of intestinal inflammation (13
The microbial analysis of cecal contents of mice from the transfer model revealed that after 8 weeks of oral administration, EcN did not replace the resident E. coli population but rather led to an increase in the total E. coli concentration. Furthermore, subcultures of homogenized MLN showed a translocation of viable members of Enterobacteriaceae. Via REP-PCR, these colonies were identified as resident Enterobacteriaceae from the intestinal flora and as colonies from the orally administered EcN. This finding is even more interesting in context with our observation that bacteria could not be isolated from MLN derived from healthy BALB/c mice following administration of EcN for 8 weeks.
Intestinal inflammation leading to a breakdown of the intestinal epithelial barrier function that would subsequently allow microorganisms from within the gut lumen to translocate to mucosal lymphatic tissue was always suggested (4
). There are previous reports that bacterial translocation occurs also in healthy mice but is limited to 10 to 20% of mice analyzed (5
). In as yet unpublished observations (for which healthy BALB/c mice were used), we were able to detect a peak concentration of translocated microorganisms within MLN and Peyer's patches at 6 to 12 h post-oral administration, followed by a steady decline until no bacteria could be isolated from lymphoid tissue at 48 h post-oral administration (unpublished data).
The fact that neither translocated EcN nor translocated resident E. coli
bacteria could be isolated from MLN of healthy BALB/c mice after 8 weeks of treatment confirms the hypothesis that under healthy conditions only a minor translocation of luminal intestinal bacteria to mesenterical lymphatic tissue takes place. These are then cleared rapidly by phagocytosis (45
). However, under inflammatory conditions, the epithelial barrier is disrupted, and luminal bacteria, including the orally administered EcN, are able to continuously translocate in very large quantities to Peyer's patches and MLN, as seen in the chronic model of colitis. A Th1-mediated T-cell response is induced, and proinflammatory cytokines secreted by antigen-presenting cells and effector T lymphocytes perpetuate the inflammation (9
). The oral administration and subsequent translocation of EcN might positively influence this systemic reaction by an induction of regulatory T-cell responses. It has been shown in vitro that probiotic bacteria, in contrast to other bacterial strains, are able to reduce antigen-presenting cell activation after phagocytosis (7
). This could be an explanation for the reduction of proinflammatory cytokine secretion seen in both of our in vivo models after oral EcN administration. In this context, it is likely that this mechanism of action would have no immediate effect on acute colitis but would rather change the severity of inflammation in chronic colitis.
In summary, we have shown that EcN is effective in modifying intestinal immunologic responses which result in the amelioration of chronic experimental murine colitis but have no clinical effect on the severity of acute mucosal damage. These findings are in accordance with clinical observations in which probiotic therapy was beneficial only in the maintenance of remission of chronic pouchitis and ulcerative colitis but had no effect for acute IBD. However, despite the successful use of probiotic therapy in cases of human pouchitis and ulcerative colitis, our suggestions of possible underlying antiinflammatory mechanisms, deployed by probiotic bacteria, cannot be fully extrapolated to the human situation since these experiments were performed under standardized conditions that are not feasible for human trials.
The analysis of intestinal microflora in this experimental setting was limited to Enterobacteriaceae
, and the impact on the composition of the microflora by probiotic therapy therefore remains elusive. Note that no final conclusion can be drawn regarding a direct impact of EcN on cytokine production that reduces inflammatory activity as demonstrated previously in other studies (35
). On the other hand, an alteration in cytokine levels because of reduced inflammatory activity that is mediated by a modulation of the gut flora can also be anticipated (42
). However, as demonstrated in this report, EcN is able to translocate into MLN and to modulate the proinflammatory cytokine secretion profile of mesenterial lymphocytes in both models of murine colitis, without significant amelioration of the acute DSS colitis. It can therefore be speculated that EcN directly modulates the immune response.