Impact of Yeast-Derived β-Glucans on the Porcine Gut Microbiota and Immune System in Early Life.

Piglets are susceptible to infections in early life and around weaning due to rapid environmental and dietary changes. A compelling target to improve pig health in early life is diet, as it constitutes a pivotal determinant of gut microbial colonization and maturation of the host's immune system. In the present study, we investigated how supplementation of yeast-derived β-glucans affects the gut microbiota and immune function pre- and post-weaning, and how these complex systems develop over time. From day two after birth until two weeks after weaning, piglets received yeast-derived β-glucans or a control treatment orally and were subsequently vaccinated against Salmonella Typhimurium. Faeces, digesta, blood, and tissue samples were collected to study gut microbiota composition and immune function. Overall, yeast-derived β-glucans did not affect the vaccination response, and only modest effects on faecal microbiota composition and immune parameters were observed, primarily before weaning. This study demonstrates that the pre-weaning period offers a 'window of opportunity' to alter the gut microbiota and immune system through diet. However, the observed changes were modest, and any long-lasting effects of yeast-derived β-glucans remain to be elucidated

A window of opportunity : Modulation of the porcine gut microbiota and immune system by feed additives in early life

In early life and around weaning piglets are susceptible to infection due to abrupt dietary, social and environmental changes that may directly or indirectly impact gut health and animal performance. In the past, antibiotic growth promoters (AGPs) were intensively used in the livestock industry to prevent the development of gastro-intestinal disorders and to optimise animal performance. However, because of the worldwide concern on the emergence of multi-drug resistant bacteria, the use of in-feed AGPs has been banned in the European Union since 2006. As a result, sustainable alternatives to in-feed AGPs that promote animal health and performance received great interest. In the last few decades, researchers have been studying the effects of several promising microbial and non-microbial nutritional factors for pigs. Pre- and probiotics, for example, have been shown to influence the porcine gut microbiota, improve gut epithelial barrier function and modulate the immune system. It is evident that these complex systems are inherently linked to each other and are important determinants of health and disease. However, dietary interventions are mostly administered post-weaning. During this period, feed additives are less likely to exert strong effects because of the with age increasing resilience of the gut microbiota to (dietary) perturbations. In order to induce transient and/or long-lasting changes, we hypothesized that early life serves as a ‘window of opportunity’ to modulate the gut microbiota, the gut epithelial barrier, and the immune system with the aim to make pigs more resilient to infections during the weaning period and later in life.Dendritic cells (DCs) are important immune cells that link the innate and the adaptive immune system and are the first cells to encounter (dietary) antigens that pass the gut epithelial barrier. In this thesis we assessed the immunostimulating potential of feed additives in vitro, using fresh and cryopreserved bone marrow-derived dendritic cells (BMDCs). Prior to the in vitro study, we selected two promising feed additives that are commercially available; yeast-derived β-glucans (MacroGard®) and the Gram-negative probiotic strain E. coli Nissle 1917 (Mutaflor®/Ponsocol®). We showed that E. coli Nissle 1917 (EcN), but not yeast-derived β-glucans, induces a dose-dependent upregulation of the DC maturation marker CD80/86. In addition, we demonstrated that both feed additives promote a dose-dependent production of several cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-10. Overall, EcN more significantly enhanced upregulation of CD80/86 and induced significantly higher levels of cytokines than yeast-derived β-glucans. These immunomodulatory effects were observed in fresh as well as cryopreserved in vitro cultured porcine BMDCs. Taken together, these results illustrated that both yeast-derived β-glucans and EcN are promising candidates to modulate the immune system, but they do so in a differential manner. A more mature DC phenotype may contribute to a more efficient response to infections. Moreover, both fresh and cryopreserved BMDCs can be used as in vitro pre-screening tools that enable an evidence-based prediction of the potential immunomodulatory effects of feed additives.We also investigated the presence, abundance, and location of EcN in the porcine gastrointestinal tract after administration of different concentrations of EcN. In a pilot study, we sampled faeces at different time points, and at dissection we collected digesta from different gut segments. In addition, the pH of digesta were measured and the mesenteric lymph node (MLN) was removed to investigate if EcN is able to translocate to porcine gut-associated lymphoid tissues (GALT). Overall, no adverse health effects were observed that were attributed to EcN. EcN was detected by qPCR in faeces of all piglets that received either high, medium, or low concentrations of EcN (109, 108, or 107 CFU/mL, respectively). In addition, no major differences with regards to the relative abundance of EcN were observed between these piglets. Although at a low abundance, we also detected EcN in one of the control piglets, indicating cross-contamination. This underlines the importance of implementing adequate hygiene measures when designing in vivo studies that involve microbial products such as probiotics. Another interesting observation was the detection of EcN in the MLN of a single animal that received the highest concentration of EcN, which suggests that EcN is able to (spontaneously) translocate to GALT. Taken together, this pilot study shows that after peroral administration of EcN in early life, EcN can be detected in feaces, digesta, and MLN of pre-weaning piglets.After investigating the effects of feed additives in vitro study, we analyzed if oral supplementation of yeast-derived β-glucans (MacroGard®) and EcN in early life affects the porcine gut microbiota and immune system in vivo. In addition, this study also provided valuable insights into the temporal development of these complex systems. From day 2 after birth until day 44 post-weaning, piglets perorally received yeast-derived β-glucans, EcN or a control treatment (water) every other day. On days 21 and 45, all pigs were vaccinated against Salmonella enterica serovar Typhimurium (Salmoporc ®) to determine the effect of yeast-derived β-glucans and EcN on the vaccination response. To study the gut microbiota composition and immune function, faeces, digesta, blood, and tissue samples were collected at different time points during the study. Overall, yeast-derived β-glucans and EcN did not affect the vaccination response. In addition, (modest) effects were observed on faecal microbiota composition (e.g., reduced α-diversity) and immune parameters (e.g., enhanced IL-10 production by stimulated MLN cells). Furthermore, most effects were transient and only observed in the pre-weaning period, and no long-lasting effects were detected.Based on the in vitro and in vivo results presented in this thesis, I conclude that yeast-derived β-glucans and EcN are able to modulate the porcine gut microbiota and the immune system, but in a differential manner. In addition, the majority of these effects were observed pre-weaning, which is in line with our hypothesis that early life serves as a ‘window of opportunity’ to modulate the porcine gut microbiota and the immune system by feed additives. Effects observed post-weaning were modest and any (long-lasting) health effects induced by yeast-derived β-glucans and EcN remain to be elucidated

Data from: Differential immunomodulation of porcine bone marrow derived dendritic cells by E. coli Nissle 1917 and β-glucans

This in vitro study assessed the immunomodulatory properties of yeast-derived β-glucans (MacroGard®) and the Gram-negative probiotic E. coli Nissle 1917 (EcN) using fresh and cryopreserved porcine BMDCs

Data from: Differential immunomodulation of porcine bone marrow derived dendritic cells by E. coli Nissle 1917 and β-glucans

This in vitro study assessed the immunomodulatory properties of yeast-derived β-glucans (MacroGard®) and the Gram-negative probiotic E. coli Nissle 1917 (EcN) using fresh and cryopreserved porcine BMDCs

Differential immunomodulation of porcine bone marrow derived dendritic cells by E. coli Nissle 1917 and β-glucans.

In early life and around weaning, pigs are at risk of developing infectious diseases which compromise animal welfare and have major economic consequences for the pig industry. A promising strategy to enhance resistance against infectious diseases is immunomodulation by feed additives. To assess the immune stimulating potential of feed additives in vitro, bone marrow-derived dendritic cells were used. These cells play a central role in the innate and adaptive immune system and are the first cells encountered by antigens that pass the epithelial barrier. Two different feed additives were tested on dendritic cells cultured from fresh and cryopreserved bone marrow cells; a widely used commercial feed additive based on yeast-derived β-glucans and the gram-negative probiotic strain E. coli Nissle 1917. E. coli Nissle 1917, but not β-glucans, induced a dose-dependent upregulation of the cell maturation marker CD80/86, whereas both feed additives induced a dose-dependent production of pro- and anti-inflammatory cytokines, including TNFα, IL-1β, IL-6 and IL-10. Furthermore, E. coli Nissle 1917 consistently induced higher levels of cytokine production than β-glucans. These immunomodulatory responses could be assessed by fresh as well as cryopreserved in vitro cultured porcine bone marrow-derived dendritic cells. Taken together, these results demonstrate that both β-glucans and E. coli Nissle 1917 are able to enhance dendritic cell maturation, but in a differential manner. A more mature dendritic cell phenotype could contribute to a more efficient response to infections. Moreover, both fresh and cryopreserved bone marrow-derived dendritic cells can be used as in vitro pre-screening tools which enable an evidence based prediction of the potential immune stimulating effects of different feed additives

Impact of yeast-derived β-glucans on the porcine gut microbiota and immune system in early life

Piglets are susceptible to infections in early life and around weaning due to rapid environmental and dietary changes. A compelling target to improve pig health in early life is diet, as it constitutes a pivotal determinant of gut microbial colonization and maturation of the host’s immune system. In the present study, we investigated how supplementation of yeast-derived β-glucans affects the gut microbiota and immune function pre-and post-weaning, and how these complex systems develop over time. From day two after birth until two weeks after weaning, piglets received yeast-derived β-glucans or a control treatment orally and were subsequently vaccinated against Salmonella Typhimurium. Faeces, digesta, blood, and tissue samples were collected to study gut microbiota composition and immune function. Overall, yeast-derived β-glucans did not affect the vaccination response, and only modest effects on faecal microbiota composition and immune parameters were observed, primarily before weaning. This study demonstrates that the pre-weaning period offers a ‘window of opportunity’ to alter the gut microbiota and immune system through diet. However, the observed changes were modest, and any long-lasting effects of yeast-derived β-glucans remain to be elucidated.</p

Data from: Impact of Yeast-Derived β-Glucans on the Porcine Gut Microbiota and Immune System in Early Life

This dataset contains data collected during an in vivo experiment with piglets as part of the PhD Thesis Projects of Hugo de Vries and Mirelle Geervliet (first authors of the manuscript)

Data underlying the publication: ‘Effects of E. coli Nissle 1917 on the Porcine Gut Microbiota and Immune System in Early Life’

This dataset contains data collected during an in vivo experiment with pigs at the Wageningen University as part of the PhD Thesis Projects of Mirelle Geervliet and Hugo de Vries (first authors of the manuscript). This research project was made possible by The Netherlands Organisation for Scientific Research and Vereniging Diervoeders Nederland (VDN)

Agaricus subrufescens fermented rye affects the development of intestinal microbiota, local intestinal and innate immunity in suckling-to-nursery pigs

Background: Agaricus subrufescens is considered as one of the most important culinary-medicinal mushrooms around the world. It has been widely suggested to be used for the development of functional food ingredients to promote human health ascribed to the various properties (e.g., anti-inflammatory, antioxidant, and immunomodulatory activities). In this context, the interest in A. subrufescens based feed ingredients as alternatives for antibiotics has also been fuelled during an era of reduced/banned antibiotics use. This study aimed to investigate the effects of a fermented feed additive -rye overgrown with mycelium (ROM) of A. subrufescens—on pig intestinal microbiota, mucosal gene expression and local and systemic immunity during early life. Piglets received ROM or a tap water placebo (Ctrl) perorally every other day from day 2 after birth until 2 weeks post-weaning. Eight animals per treatment were euthanized and dissected on days 27, 44 and 70. Results: The results showed ROM piglets had a lower inter-individual variation of faecal microbiota composition before weaning and a lower relative abundance of proteobacterial genera in jejunum (Undibacterium and Solobacterium) and caecum (Intestinibacter and Succinivibrionaceae_UCG_001) on day 70, as compared to Ctrl piglets. ROM supplementation also influenced gut mucosal gene expression in both ileum and caecum on day 44. In ileum, ROM pigs showed increased expression of TJP1/ZO1 but decreased expression of CLDN3, CLDN5 and MUC2 than Ctrl pigs. Genes involved in TLR signalling (e.g., TICAM2, IRAK4 and LY96) were more expressed but MYD88 and TOLLIP were less expressed in ROM pigs than Ctrl animals. NOS2 and HIF1A involved in redox signalling were either decreased or increased in ROM pigs, respectively. In caecum, differentially expressed genes between two groups were mainly shown as increased expression (e.g., MUC2, PDGFRB, TOLLIP, TNFAIP3 and MYD88) in ROM pigs. Moreover, ROM animals showed higher NK cell activation in blood and enhanced IL-10 production in ex vivo stimulated MLN cells before weaning. Conclusions: Collectively, these results suggest that ROM supplementation in early life modulates gut microbiota and (local) immune system development. Consequently, ROM supplementation may contribute to improving health of pigs during the weaning transition period and reducing antibiotics use

Effects of Escherichia coli Nissle 1917 on the Porcine Gut Microbiota, Intestinal Epithelium and Immune System in Early Life

Early in life and particularly around weaning, piglets are susceptible to infections because of abrupt social, environmental, and dietary changes. Dietary interventions with probiotic bacteria have gained popularity because of the increased awareness of the direct link between diet and health. In this study, piglets received the probiotic strain Escherichia coli Nissle 1917 (EcN) or a control treatment perorally from day 2 after birth until 2 weeks post-weaning. To investigate spatio-temporal effects of EcN on the gut microbiota composition, intestinal epithelial gene expression and immune system, feces, digesta, blood, scraping material and mesenteric lymph node tissue were collected at different time points. In addition, oral vaccinations against Salmonella enterica serovar Typhimurium were administered on days 21 and 45 of the study to assess the immunocompetence. EcN-treated pigs showed a reduced diversity of taxa within the phylum Proteobacteria and a lower relative abundance of taxa within the genus Treponema during the pre-weaning period. Moreover, EcN induced T cell proliferation and Natural Killer cell activation in blood and enhanced IL-10 production in ex vivo stimulated mesenteric lymph node cells, the latter pointing toward a more regulatory or anti-inflammatory state of the local gut-associated immune system. These outcomes were primarily observed pre-weaning. No significant differences were observed between the treatment groups with regards to body weight, epithelial gene expression, and immune response upon vaccination. Differences observed during the post-weaning period between the treatment groups were modest. Overall, this study demonstrates that the pre-weaning period offers a ‘window of opportunity’ to modulate the porcine gut microbiota and immune system through dietary interventions such as EcN supplementation