Eva Sonck

The effect of β-glucans on porcine leukocytes.

beta-Glucans are conserved glucose polymers found in the cell walls of plants, fungi, yeasts and bacteria. They have the capacity to activate innate immunity, thereby enhancing defence barriers. Besides differences in type of linkage and branching, beta-glucans can vary in solubility, molecular mass, tertiary structure, polymer charge and solution conformation. All these characteristics may influence their immunomodulating effects. In this study, the effect of seven beta-glucans that differed in origin (fungi, yeast, seaweed, bacteria or algae) and structure (linear or branched; soluble, gel or particulate) were tested on peripheral blood mononuclear cells (PBMC) and neutrophils of the pig. We looked at lymphocyte proliferation, reactive oxygen species (ROS), production by neutrophils and monocytes and cytokine production. The soluble beta-glucans Laminarin and Sleroglucan did not activate ROS-production of monocytes and neutrophils while the particulate beta-glucans (beta-glucan from algae (Euglena gracilis)) and glucan preparations from bakers yeast (Macrogard, Saccharomyces cerevisiae and Zymosan) had a stimulating effect. The highest stimulation of lymphocyte proliferation occurred by Curdlan (bacteria), Zymosan and the beta-glucan of E. gracilis, especially at high concentrations (200 microg/ml and 800 microg/ml). TNF-alpha was particularly stimulated by Macrogard and S. cerevisiae, while all beta-glucans (except Laminarin) induced IL-1beta. Furthermore, it was interesting that all beta-glucans and in particular Curdlan, gave rise to IL-10 secretion, whereas any beta-glucan induced the release of IL-8, IL-4, IL-12, IL-6 or IFN-gamma.

Identification of the porcine C-type lectin dectin-1

Beta-glucans are conserved glucose polymers found in the cell walls of plants, fungi, yeasts and bacteria. They have a backbone of beta-(1-3)-linked glucose units with beta-(1-6)-glucan-linkages. Although a number of receptors are thought to play a role in mediating the biological response to beta-glucans, dectin-1, a C-type lectin, was described as the most important receptor. Dectin-1 belongs to the large family of pattern recognition receptors (PRRs) which recognize conserved pathogen-associated molecular patterns (PAMPs). Here, we report the identification and characterization of dectin-1 in the pig. We identified two major isoforms (GenBank acc. no. FJ386383 and FJ386384), which differ by the presence of a stalk region separating the carbohydrate recognition domain (CRD) from the transmembrane region. Furthermore, a third minor isoform (acc. no. FJ386385) was identified which was a variation of the primary isoform with a deletion in the transmembrane and stalk region. At the nucleotide level, the full length porcine dectin-1 comprises 744bp and is 88% identical to the bovine dectin-1 and 82% identical to the human dectin-1. Messenger RNA transcripts for porcine dectin-1 were detected in the stomach, the small intestine, colon and rectum, the spleen, the mesenterial lymph nodes and the lungs. The transcript was not expressed in the liver, kidneys, the bladder, the heart, the brains and the skin.

Optimized FaeG Expression and a Thermolabile Enterotoxin DNA Adjuvant Enhance Priming of an Intestinal Immune Response by an FaeG DNA Vaccine in Pigs

ABSTRACT One of the problems hindering the development of DNA vaccines is the relatively low immunogenicity often seen in humans and large animals compared to that in mice. In the present study, we tried to enhance the immunogenicity of a pcDNA1/faeG19 DNA vaccine in pigs by optimizing the FaeG expression plasmid and by coadministration of the plasmid vectors encoding the A and B subunits of the Escherichia coli thermolabile enterotoxin (LT). The insertion of a Kozak sequence and optimization of vector (cellular localization and expression) and both vector and codon usage were all shown to enhance in vitro FaeG expression compared to that of pcDNA1/faeG19. Subsequently, pcDNA1/faeG19 and the vector-optimized and the vector-codon-optimized construct were tested for their immunogenicity in pigs. In line with the in vitro results, antibody responses were better induced with increasing expression. The LT vectors additionally enhanced the antibody response, although not significantly, and were necessary to induce an F4-specific cellular response. These vectors were also added because LT has been described to direct the systemic response towards a mucosal immunoglobulin A (IgA) response in mice. Here, however, the intradermal FaeG DNA prime-oral F4 boost immunization resulted in a mainly systemic IgG response, with only a marginal but significant reduction in F4+E. coli fecal excretion when the piglets were primed with pWRGFaeGopt and pWRGFaeGopt with the LT vectors.

Cell type-specific differences in β-glucan recognition and signalling in porcine innate immune cells.

β-glucans exert receptor-mediated immunomodulating activities, including oxidative burst activity and cytokine secretion. The role of the β-glucan receptors dectin-1 and complement receptor 3 (CR3) in the response of immune cells towards β-glucans is still unresolved. Dectin-1 is considered as the main β-glucan receptor in mice, while recent studies in man show that CR3 is more important in β-glucan-mediated responses. This incited us to elucidate which receptor contributes to the response of innate immune cells towards particulate β-glucans in pigs as the latter might serve as a better model for man. Our results show an important role of CR3 in β-glucan recognition, as blocking this receptor strongly reduced the phagocytosis of β-glucans and the β-glucan-induced ROS production by porcine neutrophils. Conversely, dectin-1 does not seem to play a major role in β-glucan recognition in neutrophils. However, recognition of β-glucans appeared cell type-specific as both dectin-1 and CR3 are involved in the β-glucan-mediated responses in pig macrophages. Moreover, CR3 signalling through focal adhesion kinase (FAK) was indispensable for β-glucan-mediated ROS production and cytokine production in neutrophils and macrophages, while the Syk-dependent pathway was only partly involved in these responses. We may conclude that CR3 plays a cardinal role in β-glucan signalling in porcine neutrophils, while macrophages use a more diverse receptor array to detect and respond towards β-glucans. Nonetheless, FAK acts as a master switch that regulates β-glucan-mediated responses in neutrophils as well as macrophages.

Varying Effects of Different β-Glucans on the Maturation of Porcine Monocyte-Derived Dendritic Cells

ABSTRACT β-Glucans are well known for their immunomodulatory capacities in humans and mice. For this reason, together with the European ban on growth-promoting antibiotics, β-glucans are intensively used in pig feed. However, as shown in the present study, there is much variation in the stimulatory capacities of β-glucans from different sources. Since dendritic cells (DCs) are the first cells that are encountered after an antigen is taken up by the intestinal epithelial cell barrier, we decided to investigate the effect of two concentrations (5 and 10 μg/ml) of five commercial β-glucan preparations, differing in structure and source, on porcine monocyte-derived dendritic cells (MoDCs). Although all β-glucans gave rise to a significant reduction of the phagocytic activity of DCs, only Macrogard induced a significant phenotypic maturation. In addition to Macrogard, zymosan, another β-glucan derived from Saccharomyces cerevisiae, and curdlan also significantly improved the T-cell-stimulatory capacity of MoDCs. Most interesting, however, is the cytokine secretion profile of curdlan-stimulated MoDCs, since only curdlan induced significant higher expression levels of interleukin-1β (IL-1β), IL-6, IL-10, and IL-12/IL-23p40. Since the cytokine profile of DCs influences the outcome of the ensuing immune response and thus may prove valuable in intestinal immunity, a careful choice is necessary when β-glucans are used as dietary supplement.

Immunomodulation of porcine leukocytes and dendritic cells by β-(1,3) glucans

Infecties die kort na het spenen van biggen optreden vormen een van de grootste problemen in de varkenshouderij en hebben hoge sterftecijfers en significante economische verliezen tot gevolg. Om deze problemen te bestrijden, werden in het verleden verschillende soorten antibiotica toegevoegd aan het varkensvoeder. Overvloedig en langdurig gebruik van antibiotica werken echter het ontstaan van resistentie in de hand. Daarom werd het gebruik van antibiotica als groeibevorderaar in veevoeder volledig verboden in de EU vanaf 2006. Alternatieven voor antibiotica zijn onder meer gericht op het verhogen van de natuurlijke afweer van het dier. Door hun potentieel immunomodulerend vermogen zijn -(1,3)-(1,6)-glucanen mogelijks interessant als voedersupplement na het spenen. Gecontroleerde studies zijn echter nodig om de effectiviteit van deze preparaten na te gaan. Deze thesis bestudeert het effect van een aantal structureel verschillende -glucaanpreparaten op leukocyten en dendritische cellen (DCs) van het varken. Verder werd in mijn doctoraat ook nagegaan of dectine-1, dat in mens en muis beschreven wordt als belangrijkste -glucaanreceptor, ook aanwezig is in het varken, en welke rol deze receptor speelt in -glucaan-geinduceerde immuuneffecten. In een eerste luik werd nagegaan of dectine-1 in het varken geexpresseerd werd. Er werden drie dectine-1 isovormen geidentificeerd. Dectine-1 mRNA werd gedetecteerd in de maag, de dunne darm, het colon en rectum, de milt, de mesenteriale lymfeknopen en de longen. In een tweede luik werd de dosisrespons van zeven verschillende -glucaanpreparaten op leukocyten van het varken bestudeerd. Oplosbare -glucanen (laminarine, scleroglucaan) vertoonden bijna geen activiteit in tegenstelling tot partikelvormige -glucanen, waarbij zowel vertakte (Macrogard, het glucaan van Saccharomyces cerevisiae en zymosan) als onvertakte -glucanen (curdlan en het glucaan van Euglena gracilis) leukocyten van het varken stimuleren. Deze stimulerende activiteit is dosis-, en vooral voor de onvertakte -glucanen, celtype afhankelijk. Ook contaminatie met grote hoeveelheden lipopolysaccharide (LPS) speelt waarschijnlijk een rol in de waargenomen immuuneffecten. In een derde luik werd nagegaan of de -glucaan-geinduceerde immuunrespons door varkensleukocyten, gemedieerd wordt via dectine-1. Hiertoe blokkeerden we -glucaanbinding aan dectine-1 met laminarin, een specifieke dectine-1 inhibitor. Dectine-1 is waarschijnlijk niet betrokken in -glucaanherkenning en –signalisatie in zowel monocyten als neutrofielen van het varken. De rol van dectine-1 in -glucaan-geinduceerde lymfocytenproliferatie is minder duidelijk. Verdere studies moeten uitwijzen welke -glucaanreceptoren wel een belangrijke rol spelen bij het varken. In een vierde luik werd onderzocht of -glucanen maturatie van monocyt-afgeleide dendritische cellen (MoDCs) kunnen veroorzaken. Zoals bij leukocyten, is het DC maturerend vermogen sterk afhankelijk van het -glucaanpreparaat en van LPS contaminatie. Omdat het cytokineprofiel van DCs de uitkomst van de immuunrespons kan bepalen wat uitermate belangrijk is voor de immuniteit, toont dit hoofdstuk aan dat een voorzichtige keuze van -glucanen noodzakelijk is bij hun gebruik als voedersupplement. In een vijfde luik werd onderzocht of -glucaanrijke gistomhulsels kunnen fungeren als een potentiele carrier om antigeen selectief te richten naar DCs. Als modelantigeen werd F4-fimbriae van F4 enterotoxigene Escherichia coli (ETEC) gebruikt. De resultaten tonen aan dat F4-geladen gist omhulsels efficient worden opgenomen door DCs en hun maturatie kunnen veroorzaken. Verdere onderzoek moet echter uitwijzen of gistomhulsels wel degelijk kunnen gebruikt worden in DC targeting. Dit werk levert een bijdrage tot het beter begrijpen van het werkingsmechanisme van structureel verschillende -glucanen ter hoogte van het immuunsysteem van varkens. De resultaten uit deze studie leggen de basis voor verder onderzoek naar de specifieke rol van dectine-1 in -glucaan-geinduceerde immuunresponsen