Host-probiotic interaction in inflammatory models (relación huésped-probiótico sobre modelos de inflamación)

Resumen

ABSTRACT In the last decade much attention has been drawn to probiotic bacteria, since the potential of certain strains to modulate the immune system and attenuate intestinal inflammation could be demonstrated in animal experimentation as well as in clinical studies. The intestinal mucosa is furnished with biochemical systems that discriminate commensal bacteria from pathogens. Both, intestinal epithelial cells (IECs) and dendritic cells are involved in the recognition of bacterial signals and in the maintenance of the fine homeostatic balance between tolerance and immunity, including the recovery after inflammation. However, the understanding of the molecular mechanism, the knowledge of genes regulated by probiotic bacteria as well as the identification of the bacterial components responsible for this anti-inflammatory effect is just starting to be unraveled. In an attempt to gain insights on such a complex system, transcriptomic approaches were used to study the interaction of probiotic bacteria with the host under inflammatory conditions and genetic manipulation procedures were implemented to characterize Lactobacillus proteins with likely functionality. The anti-inflammatory properties of several probiotic strains were studied in a human ex vivo colon organ culture system and in HT-29 cells as model for IECs. Gene expression profiles obtained with whole genome cDNA arrays revealed that L. casei BL23, L. plantarum 299v, as well as a non-adherent mutant strain, were able to globally counteract the pro-inflammatory gene expression induced by phorbol 12-myristate 13-acetate/ionomycin (PMA/IO) and reduce significantly expression levels of important cytokines (IL-2, IFN-¿, IL-17A) and chemokines (CXCL9, CXCL11). In IECs (HT 29), TNF-¿ (TNF) provoked a proinflammatory status and DNA microarrays showed that a small number of TNF-induced genes were repressed upon BL23 treatment, some of them related to ubiquitination/proteasome systems. Then, expression of chemokines and NF-kappaB related genes was assessed in HT-29 with a variety of probiotic and commensal strains. None of these strains were able to counteract the TNF-induced inflammatory events. However, a clearly distinct expression pattern could be found between the Gram positive lactic acid bacteria and the Gram negative E. coli strains, which was hallmarked by a strong induction of IL-8, GCP-2, CCL20, A20, BCL3 by E. coli. This led to propose a model for the control of NF-¿B in IEC by commensal bacteria: non-pathogenic E. coli induce A20, a natural feed-back inhibitor of the pathway, while L. casei BL23 and lactobacilli may interfere with the TNF-induced degradation of I-kappa B-alpha. This study also tried to investigate likely bacterial elements interacting with mucosa receptors. Two proteins, p40 and p75, from L. rhamnosus GG had been related to anti-apoptotic and cell protective effects on IEC. Homologous proteins were found in the genome of the L. casei BL23 and were also present in different strains of the Lactobacillus casei/paracasei and Lactobacillus rhamnosus group. In BL23, inactivation of the gene encoding p40 did not result in a significant difference in phenotype whereas a mutation in the gene encoding p75 produced cells that formed very long chains. Purified glutathione-S-transferase (GST)-p40 and -p75 fusion proteins were able to hydrolyze the muropeptides from L. casei cell walls. Both fusions bound to mucin, collagen and to intestinal epithelial cells and stimulated epidermal growth factor receptor (EGFR) phosphorylation (external collaboration), indicating that these proteins related to the cell-wall metabolism are likely involved in the probiotic effects described for this group of bacteria. This study investigated the host/probiotic interaction with advanced techniques. The results obtained reinforced the role of probiotics in the maintenance of homeostasis in the intestinal mucosa and rendered further clues on the regulatory mechanisms involved and on the function of probiotic proteins.