NUTRITION AND IMMUNITY: MOLECULAR APPROACHES

Nutrition plays an important role in modulating livestock species immunity. Therefore, this thesis aimed at evaluating the effects of different dietary molecules used in animal nutrition on mammalian and avian immunity. Both, in vitro functional analyses and OMIC technologies (proteomics and miRNAomics) were implemented herein for an integral characterization of the molecules’ impact on the animals’ immune response. Specifically, in this thesis the in vitro impact of the n-6 conjugated linoleic acid (CLA), citrus pectin (CP), and porcine milk exosomes and n-3 polyunsaturated fatty acids (PUFA) on bovine, chicken and porcine mononuclear cells immune response was evaluated, respectively. In the first study, the in vitro activity of CLA on bovine monocytes apoptosis and immune activities, including chemotaxis, phagocytosis, killing capability, and extracellular reactive oxygen species (ROS) production was assessed. Anti-apoptotic effects and an increase in extracellular ROS production during experimental pro-inflammatory conditions were observed, only when using the mixture of the two main isomers of CLA in equal proportions (50:50). The present results demonstrated for the first time that CLA does have immunomodulatory effects on some functions of bovine monocytes in vitro and that the CLA (50:50) mixture is more effective than the CLA isomers individually. The proteomics analysis performed on bovine peripheral blood mononuclear cells (PBMC) revealed that CLA (50:50) mixture does modulate bovine PBMC proteome, supporting the antiapoptotic and immunomodulatory effects observed in the previous in vitro study on bovine monocytes, and propose a potential cytoprotective role of CLA (50:50) mixture against oxidative stress. In the second study, the in vitro activity of CP on chicken monocytes viability, apoptosis, chemotaxis and phagocytosis was assessed. The study demonstrated for the first time that CP inhibits monocytes’ chemotaxis and phagocytosis in vitro, suggesting a potential anti-inflammatory activity. The proteomics analysis carried out on chicken PBMC provided a proteomics background to the anti-inflammatory activity of CP, demonstrating that the in vitro reduction of phagocytosis and chemotaxis is associated with changes in proteins related to the actin cytoskeleton. In the third study, the in vitro activity of porcine milk exosomes on porcine monocytes viability, apoptosis, chemotaxis, phagocytosis, killing capability and extracellular ROS production was assessed. Milk exosomes were successfully purified from sows’ milk and characterized using their size, concentration, morphology, and exosome protein markers. This study reported for the first time that porcine milk exosomes can be internalized by porcine monocytes in vitro and that they can modulate the cell's immune response, by decreasing their chemotaxis and phagocytosis; and increasing their ROS production under resting and pro-inflammatory conditions. The proteomics analysis performed on porcine PBMC demonstrated for the first time that porcine milk exosomes can modulate porcine PBMC proteome in vitro. Moreover, the gene ontology (GO) functional analyses revealed that porcine milk exosomes enrich biological processes related to innate immune-related processes and exosome uptake processes, supporting the immunomodulatory effects and the exosome internalization observed in the previous in vitro study. In the last study, the in vitro activity of the n-3 PUFA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on porcine monocytes viability, apoptosis, chemotaxis, phagocytosis, and intracellular, extracellular and total ROS production was assessed. The results of the study showed that DHA and EPA at the highest concentration (200 µM) decreased porcine monocytes' viability. In addition, it was reported for the first time that DHA and EPA can exert differential in vitro immunomodulatory effects in pigs, by dampening monocytes' chemotaxis and potentiating their intracellular oxidative burst, respectively. The proteomics and miRNAomics analyses were not performed for this study. Instead, a first glance on the results from the bioinformatic analyses of the miRNAomics data of all the rest of the studies is presented herein. In conclusion, this thesis provides both, a phenotypical and molecular characterization of the in vitro impact of these dietary molecules on bovine, porcine and chicken immune responses.