Recent molecular-based investigations have confirmed the species diversity and metabolic complexity of human microbiota. It is also increasingly clear that human gut microbiota have a crucial impact on host health, both as a source of infection and environmental insult and, conversely, as protection against disease by maintaining gut function. Thus, investigators are beginning to develop microbiota management employing by probiotics and prebiotics, including mannoprotein . Nevertheless, little information is available in the literature to analyze the effects in the gut of the molecular components of pro-prebiotics during a bacterial infection. This manuscript evaluates the effects of mannoprotein, a component of the yeast cell wall, in a rat model of Salmonella infection. Microbiological analysis revealed a protective effect by these compounds that decreased the number of animals infected and the number of organs (spleen, liver and gut) invaded by the infection. Furthermore, at a molecular level, the infected animals treated with mannoprotein showed a lower pro-inflammatory response than the untreated infected control animals, which did not receive mannoprotein.
It is generally assumed that an intestinal proinflammatory mechanism is a characteristic response to pathogenic intestinal bacteria, whereas the commensal bacteria should not induce this response, which interrupt the symbiosis between the bacteria and their mammalian host. Epithelial cells lining the gut have recently been identified as key players in the regulation of the initial steps of host proinflammatory responses to intraluminal bacteria via their controlled expression of PAMP receptors. One attractive hypothesis is that intestinal epithelial cells are hyporesponsive to commensal intraluminal bacteria because of the low PAMP receptor number on the apical surface on the epithelial cells , whereas invasive bacteria have access to, and can activate, PAMP receptors in the intracellular space or on the basolateral membrane. In support of the hypothesis, our study shows that mannoproteins apparently modulate the proinflammatory response either because they form a physical barrier to the Salmonella infection or they disrupt the interaction required for activation of NF-κB at a given molecular level, thus blocking further signalling cascades beyond that level. Induction of the NF-κB pathway relies on the interaction of flagellin, which is common to all flagellated bacteria, with the TLR5 expressed in situ in the ileum, in both the basal and apical compartments of the epithelial cells and enterocytes. In fact, mannoproteins inhibited campylobacter adherence and invasion of Caco-2 cells in a recent in vitro study . Thus, mannoproteins would seem good candidates to protect against infection through an intestinal route.
In our study, an inflammatory cytokine response was induced in the gut of the rats after oral infection with small amounts of a Salmonella typhimurium innoculus. In our experiments the control group showed higher TNF-α and IL-6 mRNA expression than the mannoprotein-treated rats. This phenomenon was observed in the three tissues analyzed: jejunum, ileum and colon. IL-1β mRNA expression was only affected in the Ileum of control rats, which showed higher expression levels than treated animals. This response agrees with previous studies in murine small intestine, in which both IL-6 and TNF-α were induced after exposure to S. typhimurium [35–38]. IL-1β mRNA synthesis is increased in intestinal epithelial cells after Salmonella infection . In contrast to the other cytokines studied, IL-1β enhances the innate response by activating NF-κB [39, 40]. We observed a clear action by mannoprotein on proinflammatory cytokine mRNA expression since these levels were lower in the treated animals than the controls. This suggests an anti-inflammatory effect whereby mannoprotein could be involved in some protective mechanisms against oral infection by Samonella typhimurium.
Our study also measured the expression of TLR5, the specific Salmonella typhimurium flagellin receptor, at both mRNA and protein levels. Our results demonstrated decreased levels of gene and protein expression in the mannoprotein treated group. Thus, mannoprotein itself is able to regulate TLR5 expression after infection by Salmonella. No data are available in the literature about this regulation, but there are recent studies on TLR5/flagellin interaction and the activation cascades involved in this process. These signaling cascades activate the NF-κB pathway and induce a proinflammatory response that mediates up-regulation of cytokines, chemokines and adhesion molecules, etc. Protein modifiers of cellular apoptotic pathways are integral components of this response. This observation strongly supports the notion that apoptotic activation proceeds in parallel with proinflammatory activation [41–43]. Concerning the inflammatory response in our results, the decreased TLR5 levels of in the mannoprotein group could explain the lower levels of IL-1β, TNF-α and IL-6 mRNA expression found in treated rats compared to their controls.
The crypt proliferative cells and apical and central apoptotic cells of the villi were immunohistochemically analysed in order to establish a possible effect of mannoprotein in proliferative and apoptotic response in Ileal tissue. Interestingly, the number of apoptotic cells in the control group was significantly higher than in the mannoprotein group. Nevertheless, when we analyzed the proliferative response in tissue these were no differences between the groups. This could be explained by the fact that the highest levels of TLR5 were found in the control group where they possibly activated both the pro-inflammatory and the apoptotic pathways as suggested in a recent study where both pathways were activated the inappropriate location of flagellin . We cannot confirm this possibility, although it seems likely, because we did not determine flagellin levels or position in our study. Furthermore, other in vitro studies have indicated that Salmonella provokes apoptosis in macrophages [44, 45] and in HT-29 epithelial cells after 24 hours of co-culture . On a different level a recent study with chickens, reports that mannoprotein diet enrichment increased jejunal villus height and thymal weight, changes that could point to an increased immune response capacity and improved gut function . Summarising, the administration of mannoprotein could exert a protective effect by inhibiting the apoptosis induced by oral infection with Salmonella.
In conclusion, Mannoprotein administration in a liquid diet seems to protect the intestinal tissue against the effects of Salmonella triphymorium. This protection seems to be effected by down regulation of the proinflammatory response and of TLR5 expression, in addition to the inhibition of apoptosis. Nevertheless, the molecular mechanism by which mannoprotein is able to regulate this response remains unclear and is still under investigation. These results could open up new avenues in the therapeutic strategy for treatment of inflammatory gut processes induced by microbia.