Exploring probiotics-host interactions : intestinal immune and defence responses to Lactobacillus reuteri in health and disease

Abstract: Lactobacillus reuteri, a ubiquitous inhabitant of the mammalian gastrointestinal (GI) tract has known health-promoting effects and various strains are commercially available as probiotics. Several probiosis mechanisms have been suggested in L. reuteri’s mode of action, but the mediators and factors involved are not well understood. This thesis examined the function of probiotics, particularly L. reuteri, in the GI tract by equipping L. reuteri ATCC PTA 6475 and R2LC with reporter genes (luminescence and fluorescence) and a mutant of strain 6475 was generated by inactivation of chaperon dnaK. Different in vitro and in vivo applications of fluorescent and luminescent strains were evaluated, and it was demonstrated that flow cytometry can be a powerful method for determination of plasmid persistence. Biophotonic imaging (BPI) enabled low doses (~1x10^5) of luminescent bacteria to be monitored in the GI tract and revealed retention of large numbers of bacteria in the stomach up to 3 hours post-gavage. The effect of four strains of L. reuteri (6475, R2LC, DSM 17938, 1563F) was examined in an epithelial infection model using IPEC-J2 cells induced by enterotoxigenic E. coli. By analysing transepithelial electrical resistance (TEER) and leakage of FITC-dextran, it was shown that L. reuteri pre-treatment prevented damage by ETEC to epithelial monolayer integrity. The strains also reduced expression of pro-inflammatory cytokines (TNF-alfa and IL-6) and maintained expression of adherens junction(E-cadherin) and upregulated tight junction (ZO-1) proteins. To further explore the L. reuteri mode of action, five mutants were evaluated in DSS-induced acute colitis and IPEC-J2 models. It was found that dnaK-, pduC-, cmbA-, amidase- and srtA- may not play major roles in the mechanisms by which 6475 maintains mucosal integrity and counteracts inflammation. However, mutants 6475 pduC- and 6475 cmbA- had a tendency to weaken the protective effect of 6475 in the colitis model. Studies on the effects of L. reuteri strains on mast cell activation and inflammatory response revealed no inhibition of degranulation mediated by IgE-antigen activation, but down-regulated expression of the pro-inflammatory cytokines IL-6 and IL-13, irrespective of degranulation. Thus pre-treatment with L. reuteri strains can protect against intestinal barrier dysfunction and mucosal inflammation, partly through altering junctional complex proteins, mediators of immunity and mast cells.

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