Saccharomyces cerevisiae mannan induces sheep beta-defensin-1 expression via Dectin-2-Syk-p38 pathways in ovine ruminal epithelial cells.

The rumen epithelium of sheep serves as an immune interface with the environment and secretes antimicrobial peptides with bactericidal function against various pathogens. Sheep beta-defensin-1 (SBD-1), an antimicrobial peptide, is secreted from ovine ruminal epithelial cells (OREC) in response to microbial infections. Mannan, the main component of the Saccharomyces cerevisiae cell wall can stimulate innate and regulatory immune responses that could improve the gastrointestinal environment. We aimed at investigating the effects of mannan on SBD-1 expression and the downstream signaling pathways stimulated in OREC. We cultured OREC; assessed the effects of mannan on SBD-1 expression by qPCR and ELISA; and then investigated the underlying signaling pathways using qPCR, ELISA, Western blotting, immunohistochemistry, and immunohistofluorescence. Interestingly, mannan markedly upregulated SBD-1 expression in a concentration- and time-dependent manner. Dectin-2 Mouse mAb, Syk specific inhibitor R406, and specific inhibitors of the p38, ERK1/2, JNK, and NF-κB pathways attenuated mannan-induced SBD-1 expression to varying degrees. These results demonstrate that SBD-1 is upregulated by mannan via the Dectin-2-Syk axis, and this is regulated to a large extent through the mitogen-activated protein kinase (MAPK) p38 and less so through the ERK1/2 and JNK or the NF-κB pathway. Our findings highlight the immunomodulatory effects of mannan on OREC in terms of mannan-induced SBD-1 expression.

Modulation of ovine SBD-1 expression by Saccharomyces cerevisiae in ovine ruminal epithelial cells.

BACKGROUND: The ovine rumen is involved in host defense responses and acts as the immune interface with the environment. The ruminal mucosal epithelium plays an important role in innate immunity and secretes antimicrobial innate immune molecules that have bactericidal activity against a variety of pathogens. Defensins are cationic peptides that are produced by the mucosal epithelia and have broad-spectrum antimicrobial activity. Sheep ß-defensin-1 (SBD-1) is one of the most important antibacterial peptides in the rumen. The expression of SBD-1 is regulated by the probiotic, Saccharomyces cerevisiae (S.c); however, the regulatory mechanism has not yet been elucidated. In the current study, the effects of S.c on the expression and secretion of SBD-1 in ovine ruminal epithelial cells were investigated using quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). In addition, specific inhibitors were used to block the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), p38, JNK, and ERK1/2 signalling pathways separately or simultaneously, to determine the regulatory mechanism(s) governing S.c-induced SBD-1 upregulation. RESULTS: Incubation with S.c induced release of SBD-1 by ovine ruminal epithelial cells, with SBD-1 expression peaking after 12 h of incubation. The highest SBD-1 expression levels were achieved after treatment with 5.2 × 107 CFU∙mL- 1 S.c. Treatment with S.c resulted in significantly increased NF-κB, p38, JNK, ERK1/2, TLR2, and MyD88 mRNA expression. Whereas inhibition of mitogen-activated protein kinases (MAPKs) and NF-κB gene expression led to a decrease in SBD-1 expression. CONCLUSIONS: S.c was induced SBD-1 expression and the S.c-induced up-regulation of SBD-1 expression may be related to TLR2 and MyD88 in ovine ruminal epithelial cells. This is likely simultaneously regulated by the MAPKs and NF-κB pathways with the p38 axis of the MAPKs pathway acting as the primary regulator. Thus, the pathways regulating S.c-induced SBD-1 expression may be related to TLR2-MyD88-NF-κB/MAPKs, with the TLR2-MyD88-p38 component of the TLR2-MyD88-MAPKs signalling acting as the main pathway.

Enzyme kinetics and molecular modeling studies of G6PD(Mahidol) associated with acute hemolytic anemia.

G6PD(Mahidol) enzyme is the most common variant in the Achang Chinese ethnic group and clinically manifests as class II. In this study, G6PD(Mahidol) enzyme was characterized by molecular modeling to understand its kinetics. G6PD(Mahidol), G6PD(G487A) and G6PD(WT) proteins were heterologously expressed in the G6PD-deficient DF213 E. coli strain, purified and their steady-state kinetic parameters were determined. Compared with G6PD(WT), the Km, and Vmax of NADP+ with G6PD(G487A) were about 28-fold and 12-fold lower, respectively. The Ki values of dehydroepiandrosterone (DHEA), NADPH and ATP with G6PD(G487A) showed 29.5-fold, 2.36-fold reduction and 1.83-fold increase, respectively. A molecular modeling of G6PD(G487A) was performed based on the X-ray structure of human G6PD (PDB: 2BH9). It is suggested that Ser-163 might affect the stability of G6PD(G487A) alpha-helix d and beta-strand E, besides the conformation of beta-strand D. In conclusion, the biochemical and structural properties of G6PD(G487A) and G6PD(WT) enzymes are significantly different, which may be responsible for clinical diversity of G6PD deficiencies.

ß-glucan from Saccharomyces cerevisiae is involved in immunostimulation of ovine ruminal explants.

In this study, we investigated whether ß-glucan from Saccharomyces cerevisiae exerts beneficial effects on mucosal immunity in an ovine ruminal explant (ORE) model. Once the ORE model was established, viability was assessed through histological change, E-cadherin expression, CK-18 and Ki-67 distribution. Then, the OREs were co-cultured with ß-glucan, following which, gene and protein expression levels of sheep ß-defensin-1 (SBD-1), pro-inflammatory interleukin (IL)-6, and anti-inflammatory IL-10 were detected using quantitative real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). Hematoxylin & eosin staining, qPCR, and immunohistochemistry showed that the overall ORE structure was intact after 96 hours in culture, but explants cultured for more than 24 hours showed epithelial degradation. Therefore, we performed the follow-up test within 24 hours. qPCR and ELISA revealed that the gene and protein expression levels of SBD-1, IL-6, and IL-10 in the OREs significantly increased (P < 0.05) after treatment with ß-glucan compared with controls. This study identified the feasibility and optimal conditions of ORE culture and demonstrated that ß-glucan activates SBD-1, IL-6, and IL-10 secretion in OREs to promote mucosal immunity.

Dans la présente étude nous avons examiné si le ß-glucane de Saccharomyces cerevisiae amène des effets bénéfiques sur l'immunité mucosale dans un modèle d'explant ruminal ovin (ORE). Une fois que le modèle ORE fut établi, la viabilité fut évaluée via les changements histologiques, l'expression d'E-cadhérine et la distribution de CK-18 et Ki-67. Puis, les OREs furent co-cultivés avec du ß-glucane, après quoi, les degrés d'expression des gènes et des protéines ß-défensine-1 ovine (SBD-1), interleukine (IL)-6 pro-inflammatoire et IL-10 anti-inflammatoire furent détectés en utilisant une réaction d'amplification en chaîne par la polymérase quantitative en temps réel (qPCR) et une épreuve immuno-enzymatique (ELISA). Une coloration à l'hématoxyline et éosine, le qPCR et l'immunohistochimie ont montré que la structure globale d'ORE était intacte après 96 heures en culture, mais des explants cultivés pour plus de 24 heures présentaient une dégradation épithéliale. Par conséquent, nous avons effectué les tests de suivi en dedans de 24 heures. Les analyses par qPCR et ELISA ont révélé que les degrés d'expression des gènes et des protéines SBD-1, IL-6 et IL-10 dans les OREs augmentèrent de manière significative (P < 0,05) après un traitement avec du ß-glucane comparativement aux témoins. Cette étude a identifié la faisabilité et les conditions optimales pour la culture d'ORE et a démontré que le ß-glucane active la sécrétion de SBD-1, IL-6 et IL-10 dans les OREs afin de promouvoir l'immunité mucosale.(Traduit par Docteur Serge Messier).

Modulation of SBD-1 expression by Saccharomyces cerevisiae cell wall components in ovine ruminal epithelial cells.

The ovine rumen is an immune interface with the external environment, participating in host defence responses. Ovine ruminal epithelial cells (ORECs) not only have a physical barrier function, but also secrete sheep ß-defensin-1 (SBD-1), which plays a key role in innate and adaptive immunity. Prebiotics are potential alternatives to infeed antibiotics. Saccharomyces cerevisiae cell wall (S.c.CW) is rich in prebiotics, which play roles in improving the growth performance of animals and regulating immunity. Here, we investigated whether S.c.CW induces SBD-1 expression in ORECs, as well as the underlying mechanism. The regulatory mechanisms of S.c.CW-induced up-regulation of SBD-1 were determined using quantitative real-time PCR, enzyme-linked immunosorbent assay, and western blotting. S.c.CW significantly increased the expression of Toll-like receptor 2 (TLR2) and nuclear factor-kappa B (NF-κB), but had no effect on TLR4 expression. TLR2, MyD88, and NF-κB inhibition attenuated the induction of SBD-1 expression by S.c.CW. However, TLR4 inhibition only resulted in attenuated SBD-1 mRNA, having no effect on SBD-1 protein expression. Thus, we conclude that S.c.CW can induce SBD-1 expression and that this induction is regulated by the TLR2-MyD88-NF-κB pathway.

Saccharomyces cerevisiae ß-glucan-induced SBD-1 expression in ovine ruminal epithelial cells is mediated through the TLR-2-MyD88-NF-κB/MAPK pathway.

Ovine ruminal epithelial cells (ORECs) not only have a physical barrier function but also can secrete host defence peptides (HDPs), such as sheep ß-defensin-1 (SBD-1). As a feed additive, Saccharomyces cerevisiae can enhance the host's innate immunity. ß-glucan, a cell wall component of Saccharomyces cerevisiae, can stimulate innate immune responses and trigger the up-regulation of SBD-1 in ORECs. The signaling mechanisms involved in ß-glucan-induced SBD-1 expression are not completely understood. The aim of this study was to identify the receptors and intracellular pathways involved in the up-regulation of SBD-1 induced by ß-glucan. ORECs were cultured, and the regulatory mechanisms of ß-glucan-induced up-regulation of SBD-1 were detected using quantitative real-time PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and western blotting. TLR-2 and MyD88 knockdown or inhibition attenuated ß-glucan-induced SBD-1 expression. We also showed that inhibition of MAPK and NF-κB pathways significantly reduced ß-glucan-induced SBD-1 expression. These results demonstrate that ß-glucan-induced SBD-1 expression is TLR-2-MyD88-dependent and may be regulated by both MAPK and NF-κB pathways. Since NF-κB inhibition had a greater effect on the down-regulation of ß-glucan-induced SBD-1 expression, the NF-κB pathway may be the dominant signaling pathway involved in the regulation of defensin expression. Our studies demonstrate that ß-glucan-induced SBD-1 expression is mediated through the TLR-2-MyD88-NF-κB/MAPK pathway. Our results would contribute to the understanding of immunological modulations in the gastrointestinal tract triggered by probiotic yeast cell wall components.

ß-Glucan from Saccharomyces cerevisiae induces SBD-1 production in ovine ruminal epithelial cells via the Dectin-1-Syk-NF-κB signaling pathway.

The ruminal mucosal epithelium can secrete defensins, which play a key role in innate and adaptive immunity and are considered potential replacements for antibiotics. Of these, sheep ß-defensin-1 (SBD-1) is one of the most potent molecules produced by ovine ruminal epithelial cells (ORECs). ß-glucan, safe and effective immune activators, can stimulate innate and adaptive immune responses. Here we examined whether ß-glucan from Saccharomyces cerevisiae can induce SBD-1 expression in ORECs, as well as the underlying mechanism. First, ORECs were cultured, and quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to study the effects of different ß-glucan concentrations. Then western blotting, immunohistochemistry, and immunocytofluorescence were performed to investigate the regulatory mechanism of ß-glucan-induced SBD-1 upregulation. We show that ß-glucan can induce the release of SBD-1 from ORECs; the highest SBD-1 mRNA and protein expression was achieved after treatment with 10 µg/mL at 2 and 4 h. Moreover, ß-glucan-induced SBD-1 production was mediated by the activation of dendritic-cell-associated C-type lectin 1 (Dectin-1) receptors, Syk, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). These findings highlight the immunomodulatory effects of ß-glucan on ORECs.