MODULATION OF HOST INNATE IMMUNITY BY HEALTH-PROMOTING BACTERIA AND DIETARY COMPOUNDS

In its widest meaning, the probiotic approach consists in exogenous administration of microbial cells (or cell components) aimed at benefiting the host\u2019s health, both in terms of maintenance of homeostasis and also as alternative strategy for the prevention and/or treatment of infectious diseases. More recently, it has been demonstrated that an important way through which probiotics can exert their beneficial effects is the ability to interact with host\u2019s immune system, both at local and systemic level, thus having efficacy also in body niches different from the gut. Starting from these observations, in the first part of the PhD research activity we screened several bacterial strains for their potential use as probiotics for the pharyngeal mucosa. We tested the ability of bacteria employed in food industry and newly isolated from the pharynx of healthy volunteers to adhere to the human pharyngeal epithelium, and to antagonize the oro-pharyngeal pathogen S. pyogenes on FaDu cells and HaCat keratinocytes. Two oral bacterial strains, Streptococcus salivarius ST3, and the dairy starter Lactobacillus helveticus MIMLh5 were selected and compared with the oral commercial probiotic S. salivarius strain K12. These strains were sensitive to a variety of antibiotics routinely used for the control of upper respiratory tract infections. The in vitro immunological characterization performed on FaDu cells revealed that ST3 and MIMLh5 were all able to significantly reduce the activation of the nuclear-factor (NF)-\u3baB, both at baseline and in presence of the pro-inflammatory stimulus interleukin (IL)-1\u3b2, whereas showing different cytokine profile under the above mentioned conditions. We subsequently decided to characterized the effects of the combined use of strain ST3 and MIMLh5. We found that strains MIMLh5 and ST3 activated innate immunity by inducing in U937 human macrophages the expression of cyclooxygenase (COX)-2, a balanced IL10/Tumor-Necrosis Factor (TNF)-\u3b1 ratio, and we demonstrated that Toll-like receptor 2 (TLR-2) participates in the recognition of both strains. We also observed that these microorganisms grow efficiently when co-coltured in milk, suggesting that the preparation of a milk-based fermented product containing both strains can be a practical solution for the administration of these bacteria. Considered the ability of L. helveticus MIMLh5 to trigger immune responses also on murine bone marrow-derived dendritic cells, in the second part of the research we focused our attention on the possible molecular determinants involved in the immunostimulating activity of this strain. We studied MIMLh5 surface layer protein (SlpA) and we found that the bacterium and SlpA exerted anti-inflammatory effects on the intestinal epithelial Caco-2 cell line by reducing the activation of NF-\u3baB. On the contrary, MIMLh5 and SlpA acted as stimulators of the innate immune system by triggering the expression of pro-inflammatory factors TNF-\u3b1 and COX-2 in the human macrophage cell line U937 via recognition through TLR-2, whereas the protein had no effect on the anti-inflammatory IL10. When we tested MIMLh5 bacterial cells depleted from the protein, we observed a reduced pro-inflammatory activity, suggesting that SlpA plays a major role in mediating the immunostimulatory attitude of the bacterium, which could help to induce host\u2019s defenses against and responses towards infections. In the third part of the research we analyzed the effects on immune system of food compounds from vegetal origin. To this aim, we evaluated the immunomodulatory potential of different fractions extracted from wild blueberries (WB) powder. We observed that only the anthocyanin (ACN) fraction was effective in reducing the activation of NF-\u3baB on Caco-2 cells, whereas both the soluble and the phenolic fractions had no significant effects. Consequently, we used only the anthocyanin fraction for the subsequent characterization on U937 macrophages. We found that the presence of ACNs decreased the induction of TNF-\u3b1 triggered by lipopolysaccharide (LPS) from Escherichia coli on U937, particularly when the cells were pretretated with ACNs and afterwards treated with LPS. These data suggest that ACNs from WB might have a protective role towards inflammation and that, probably, the described anti-oxidant features of these compound might be partially mediated by direct effects on immune system. In conclusion, this PhD work evidenced the noticeable abilities of bacteria and dietary compounds to modulate host immune system responses. Particularly, this study suggests that the use of selected food-grade bacteria, bacterial components or dietary compounds has a promising potential for the maintenance of host health and the prevention of diseases