Recent data suggest that gut commensal microbiota is a dynamic factor with an active relationship with the host, through the interaction with specific bacterial recognition systems, and directly involved in the regulation of gastrointestinal sensory, motor and secretory responses. In particular, gut commensal microbiota has been implicated in the pathophysiology of inflammatory bowel disease (IBD). This work aims to demonstrate how environmental variations lead to spontaneous adaptive changes of gut commensal microbiota in rats, and how these changes affect the expression of bacterial recognition systems and modulate gut inflammation and the expression of endogenous sensory systems (in particular the cannabinoid system). First, we determined changes in cecal commensal microbiota and expression of Toll Like Receptors (TLR), (TLR-2) and (TLR-4) in rats bred under microbiologically-controlled conditions (barrier), under standard conditions (conventional) and in barrier animals adapted to standard conditions (barrier/conventional). In these groups, cecal microbiota was analyzed by fluorescence in situ hybridization (FISH) and microbial profiles were assessed by terminal restriction fragment length polymorphism (t-RFLP). As to correlate microbiota and bacterial recognition systems within the gut, cecal expression of TLR-2 and TLR-4 was also determined by RT-PCR. Total number of cecal bacteria was similar in the three groups. However, the barrier group showed a higher number of strict anaerobic bacteria, namely Bacteroides spp and Clostridium spp, while Bifidobacterium spp and Lactobacillus spp were scarce. Re-housing the barrier-bred rats into conventional conditions led to a microbiota with intermediate characteristics between the barrier and the conventional groups. Richness of the cecal microbial ecosystem was similar in the three groups. Although a relative time-dependent variation was observed in microbiota composition, the barrier group showed high consistency over time. Expression levels of TLR-2 and TLR-4 were relatively low, without clear correlation with the microbiota. Only TLR-4 showed a tendency to be over-expressed in the barrier group. Thereafter, a model of indomethacin-induced acute enteritis (ileitis) in rats was used to assess inflammation-induced changes in gut commensal microbiota as well as differences in the susceptibility to inflammation between rats bred under high hygienic conditions (SPF) adapted to conventional conditions and rats bred and maintained under conventional conditions. Results obtained show specific, inflammation-induced, qualitative and quantitative changes of ileal and cecal comensal microbiota. Moreover, gut bacterial recognition systems are also affected by acute inflammation, with an over-expression of both TLR-2 and TLR-4. As it relates to inflammation per se, rats breds under conventional conditions showed, in general, higher susceptibility to inflammation than SPF animals adapted to the conventional environment; as demonstrated using clinical histological and biochemical parameters. Finally, we assessed how gut comensal microbiota affects other intestinal sensory mechanisms, in particular cannabinoid receptor-dependent mechanisms. For this, we determined the cecal expression of cannabinoid receptors 1 (CB1) and 2 (CB2) in the same groups of rats described above: 1) maintained under barrier conditions (SPF); 2) maintained under conventional conditions; and 3) bred under barrier conditions but adapted to a conventional environment. In general, both receptors were expressed in cecal tissues, although levels of expression of CB2 were higher than those of CB1. Moreover, independently of the receptor subtype considered expression levels diminished with the change from barrier to conventional conditions. These variations correlated positively with spontaneous changes in the counts of Bacteroides spp and Clostridium spp and negatively with the counts of Bifidobacterium spp. As a whole, these results show that gut commensal microbiota is a dynamic system, which varies and adapts spontaneously to the environmental conditions of maintenance of laboratory animals. These changes involve variations in gut homeostasis and affect the expression of bacterial recognition receptors, namely TLR-2 and TLR-4, and cannabinoid receptors (CB1 and CB2) and the susceptibility to acute intestinal inflammation. These observations suggest that gut commensal microbiota is a variable factor that can affect the experimental output and that, therefore, must be considered in the interpretation of studies, particularly in the field of gastrointestinal.
Influencia del ambiente sobre la microbiota comensal del intestino de la rata: Implicaciones en la inflamación y en mecanismos sensoriales
Teran Ventura, E. (Author). 21 Dec 2009
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis