Tolerance to Soluble Proteins: Oral Tolerance
The role of the intestinal microbiota on the OT process has been demonstrated by various experimental studies using GF mice. Results depend on the immune response considered, oral Ag, and experimental schedule used. In these experiments, immune responses to a specific Ag are compared in two groups of mice: the tolerant group where mice are fed with an Ag prior to the peripheral immunization with the same Ag, and the control group fed with only the buffer before the same peripheral immunization. Specific immune responses to the Ag used are then evaluated (Ab responses in serum or cellular response by delayed-type hypersensitivity) in both groups. The tolerant state is present when peripheral immune responses to the Ag are abolished or significantly decreased in the group Ag-fed as compared with the control group.
In an initial study, Wannemuehler and coworkers (81) showed that, in contrast to what is observed with the CV mice, gavage of GF mice with a particular antigen, sheep red blood cells (SRBC), does not enable suppression of immune responses to SRBC in serum. However, the OT process was re-established when LPS was administered orally prior to gavage. The authors concluded that Gram-negative bacteria play a fundamental role in the mechanisms responsible for OT. Subsequently, other experiments using adult GF mice fed with a soluble protein, OVA, in order to study the immune suppression of anti-OVA serum IgG response, demonstrated that it was possible to induce OT in GF mice. However, in contrast to what is observed with CV mice, the suppression was of very short duration, about 10-15 days, versus more than 5 months in CV mice (82). Similar results were obtained in human-microbiota-associated gnotobiotic mice (60). Colonization of the intestinal tract with E. coli alone prior to gavage was sufficient to restore lasting suppression (83), and the same results were obtained with another Gram-negative bacteria, Bacteroides (unpublished personal data), while in our experimental conditions, adult GF colonized with the strain of Bifidobacterium bifidum isolated from a baby's feces, had no effect on the serum IgG anti-OVA suppression (83).
Recently, in their experimental conditions, Sudo and coworkers (84) showed that in OVA-fed mice, the GF state does not allow suppression of the systemic anti-OVA IgE response in serum in contrast to what is observed with CV mice. Colonization of the intestinal tract by a strain of Bifidobacterium infantis restored the suppression but only when the strain colonized the intestinal tract of the mouse from birth. The importance of the presence of intestinal bacteria from birth in the optimization of the immune processes has also been suggested in a more recent study (60).
It is interesting to compare these experimental results to those described in human neonates by Lodinova-Zadnikova and coworkers (85). In their study, they colonized the digestive tract of babies just after birth with a given strain of E. coli. In these conditions E. coli is able to establish durably in the digestive tract of newborns as described previously (86). After 10 years (preterm infants) and 20 years (full-term infants), differences in occurrence of food allergies between colonized and control subjects were statistically significant; 21% versus 53%, and 36% versus 51% respectively. Furthermore, recent clinical trials using ingestion of a strain of probiotic, Lactobacillus rhamnosus GG, during the last month of pregnancy to women and after birth to babies during 6 months, reduced the incidence of atopic eczema in at-risk children during the first 4 years of life (87). However, in this case, IgE levels were not decreased in the treated group as compared with the placebo group. The protective mechanisms of these interventions are not elucidated.
All these experimental data show the importance that a single bacterial strain present in the intestinal digestive microbiota of infants may have with respect to the establishment of tolerance mechanisms. Are there E. coli, Bacteroides or some strains of Bifidobacterium which play this important role? First, as suggested by previous studies, it is not sure whether the mechanisms are the same for suppression of the various isotypes IgG and IgE (45,88), and consequently that the same bacteria are operating on them. Secondly, as described previously, all the strains belonging to the same bacterial genus have not the same immunoregulatory properties and it is conceivable that some Bifidobacterium strains may have regulatory properties on suppressive immune processes.
The cellular ways by which the bacteria are acting, and the exact bacterial components involved are not known. However, from an ecological point of view, it is important to note that some experimental data point out the importance of the neonatal period with respect to the ability to recognize bacterial messages.
An important question is why the intestinal microbiota does not mount an inflammatory response in the gut while this state is broken in pathologic conditions such as IBD?
The mechanisms by which commensal and non-pathogenic bacteria are tolerated by the IIS is beginning to be understood and may result from a cross-talk between bacteria, epithelium, and immune cells. In an interesting experimental study, Neish and co-workers (89) demonstrated, using an in vitro model of cultured human intestinal epithelial cells, that a non-pathogenic strain of Salmonella directly influenced the intestinal epithelium to limit inflammatory cytokine production. They showed that the immunosuppressive effect was due to the inhibition of the NFk-B activation pathway by blockage of IkB-a degradation. Another interesting conclusion from this study was that non-pathogenic bacteria, which do not belong to the commensal intestinal microbiota, are unable to induce inflammatory responses. Another study converges to an opposite conclusion (90). In several intestinal epithelial cell lines, the authors demonstrated that a commensal bacterial strain, Bacteroides vulgatus, was able to activate the NF-kB signaling pathway through IkB-a degradation and ReIA phosphorylation. However, the presence of TGF-P1 cytokine inhibits B. vulgatus--mediated NF-kB transcriptional activity showing that the responsiveness of intestinal epithelial cells to luminal enteric bacteria depends on a network of communication between immune and epithelial cells and their secreted mediators.
Recently, it was shown in vivo in mice, that the intestinal microbiota itself plays a regulatory role with respect to inhibition of the NFk-B activation pathway, by the way of another inhibitory factor, the peroxisome proliferator-activated receptor (PPARg) (61). The latter is highly expressed in the colon and its activation has anti-inflammatory effects, with protection against colitis. PPARg activators are able to limit inflammatory cytokine production through the inhibition of the NF-kB pathway. It has been suggested that PPARg could play an important role in homeostasis of the gut, especially in the colon. In patients with IBD, impaired expression of PPARg in colon epithelial cells was observed (61). In the same work, in vivo observations showed that the intestinal microbiota and TLR-4 regulates PPARg expression by epithelial cells of the colon. Indeed, it is highly expressed in CV mice while it is barely detectable in GF mice. When TLR-4 transfected CaCo-2 cells were incubated with LPS, an increase of PPARg expression was observed showing the involvement of TLR-4 in this process and suggesting that PPARg may be a regulatory factor able to shut down the TLR-4 signaling given by bacterial LPS abundant in the colon (61).
Taken together, these data provide evidence that the cross-talk existing between the IIS and intestinal microbiota pass through regulatory processes preventing inflammatory responses induced by activation of some nuclear factors, such as NF-kB, which could be different, or predominant, according to the intestinal site. They are mediated through the actions of commensal bacteria, but also through exogenous non-pathogenic bacteria action and this data is of importance in terms of nutrition. Indeed, we can ingest billions of exogenous bacteria in some foods such as fermented milks and some cheeses, without detrimental consequences. In terms of pathology, a lot of other questions concerning the mechanisms and origin of IBD have yet to be answered. Why is an activation of the NF-kB pathway observed in IBD? Is it due to some subsets of the intestinal microbiota, which are suddenly dominant in an unbalanced microbiota? Is it due to enteropathogens which can interact with the NF-kB pathway during infection? Or, is it due to a decrease and modification of mucus secretion allowing excessive adhesion of commensal bacteria? All these factors, and others, may be responsible.
It is interesting to give recent clinical results concerning oral administration of probiotics on the maintenance of the remission phase in IBD, either the use of a mixture of 8 strains of lactic-acid bacteria used as probiotics (VSL#3) in chronic pouchitis (91), or a yeast strain, Saccharomyces boulardii (92) or the E. coli Nissle 1917 (93) in ulcerative colitis. The mechanisms underlying such beneficial effects are still not known and they are multifactorial. From experimental data it has been suggested that a stimulation of the noninflammatory IL-10 cytokine production by ingestion of probiotics may be involved in such protective effect (94). Further experimental and clinical studies need to be conducted to further elucidate the mechanisms involved in the epithelium-bacterial cross talk.
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The first trimester is very important for the mother and the baby. For most women it is common to find out about their pregnancy after they have missed their menstrual cycle. Since, not all women note their menstrual cycle and dates of intercourse, it may cause slight confusion about the exact date of conception. That is why most women find out that they are pregnant only after one month of pregnancy.