Numerous cell types are involved in maintenance of the intestinal tissue. However, the main players are cells of the epithelial lining and the immune system. Human peripheral blood mononuclear cells (PBMCs) are used to investigate the effect of food bioactives on various immune cells. These cells are easily isolated from blood of healthy donors or buffy coats (leukocyte concentrates, a by-product from hospital Blood Banks in the manufacturing of red blood cell and thrombocyte concentrates from anti-coagulated whole blood). PBMCs have a different composition, phenotype and activation status than cells found in intestinal tissue. However, this is a useful test system for investigation of immune modulatory effects of food bioactive compounds. Methods for the isolation of PBMCs and how they are used to investigate effects of bioactive components are discussed in this chapter.

BackgroundOmega-3 PUFAs are known to have anti-inflammatory properties, and different mechanisms are involved. GPR120 is a G-protein coupled receptor that has recently received attention because of its anti-inflammatory signalling properties after binding omega-3 PUFAs. However, both omega-3 and omega-6 PUFAs are natural GPR120 ligands. The aim of this study was to study possible differences in GPR120-mediated signalling events after treatment with different long-chain PUFAs in intestinal epithelial cells. We also investigated possible GPR120-mediated anti-inflammatory effects of different long-chain PUFAs that may be relevant in the understanding of how dietary PUFAs influence inflammatory responses in inflammatory diseases such as IBD.MethodsWe used Caco-2 cells as a model system to study GPR120-mediated signalling events because we found this cell line to express GPR120, but not GPR40, another plasma membrane receptor for medium- and long chain fatty acids. Increase in cytosolic Ca2+concentration, activation of MAP kinase ERK1/2 and the inhibition of IL-1β induced NF-κB activity were studied to reveal potential differences in the activation of GPR120 by the omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the omega-6 PUFA arachidonic acid (AA).ResultsWe found that EPA, DHA and AA enhanced the cytosolic concentration of the second messenger Ca2+ with the same efficiency, but with different kinetics. Both omega-3 and omega-6 PUFAs activated MAP kinase ERK1/2, but differences regarding kinetics and intensity were also observed in this pathway. ERK1/2 activation was shown to be dependent upon EGFR and Raf-1. We further investigated the ability of EPA, DHA and AA to inhibit NF-κB activity in Caco-2 cells. All PUFAs tested were able to inhibit IL-1β induced breakdown of IκBα after binding to GPR120, but with different potency.ConclusionsOur results show that EPA, DHA and AA elicit the same signalling events, but with different kinetics and efficiency through GPR120 in Caco-2 cells. We show, for the first time, that both omega-3 and omega-6 PUFAs inhibit NF-κB activation in intestinal epithelial cells. Our results may be important for understanding how dietary PUFAs influence inflammatory processes relevant in delineating effects of PUFAs in the treatment of IBD.

ABSTRACTThe probiotic lactic acid bacteriumLactobacillus plantarumis a potential delivery vehicle for mucosal vaccines because of its generally regarded as safe (GRAS) status and ability to persist at the mucosal surfaces of the human intestine. However, the inherent immunogenicity of vaccine antigens is in many cases insufficient to elicit an efficient immune response, implying that additional adjuvants are needed to enhance the antigen immunogenicity. The goal of the present study was to increase the proinflammatory properties ofL. plantarumby expressing a long (D1 to D5 [D1-D5]) and a short (D4-D5) version of the extracellular domain of invasin from the human pathogenYersinia pseudotuberculosis. To display these proteins on the bacterial surface, four different N-terminal anchoring motifs fromL. plantarumwere used, comprising two different lipoprotein anchors, a transmembrane signal peptide anchor, and a LysM-type anchor. All these anchors mediated surface display of invasin, and several of the engineered strains were potent activators of NF-κB when interacting with monocytes in cell culture. The most distinct NF-κB responses were obtained with constructs in which the complete invasin extracellular domain was fused to a lipoanchor. The proinflammatoryL. plantarumstrains constructed here represent promising mucosal delivery vehicles for vaccine antigens.

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