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Simple Summary: In addition to their function as energy sources, monosaccharides are used to build complex structured oligo- and polysaccharides, which play numerous essential roles as functional biomolecules. Such bioactive sugars are also key components of milk, since they have positive impacts on intestinal development, the gut microbiome, and an effective immune system, along with the learning and memory ability of offspring. Moreover, milk oligo- and polysaccharides have anti-adhesive properties against pathogenic microorganisms and viruses and are, therefore, important for the health of the mammary gland and the offspring. One key monosaccharide of such oligo- and polysaccharides is the sialic acid N-acetylneuraminic acid (Neu5Ac). Since bovine milk is not only important for calf health but also, in the case of colostrum, as a functional food for humans, it is of particular interest, at which time of lactation the highest amounts of these bioactive molecules are found in bovine milk. Our results demonstrate that on the day of calving, the highest amounts of Neu5Ac and its polymers are present in bovine milk and, thus, the sialic acid-dependent benefits of bovine milk are also highest at this time. Sialylated milk oligosaccharides and glycoconjugates have several positive effects on the mucosal barrier, the gut microbiome, and an effective immune system. For this reason, they are important biomolecules for mammary gland health and optimal development of offspring. In milk, the major sialic acid, N-acetylneuraminic acid (Neu5Ac), can be attached as monosialyl-residues or as polymers. To investigate the sialylation processes during lactation of German Holstein cows, we analyzed udder tissue in addition to milk at different time points of lactation. The analysis of the milk samples revealed that both the levels of Neu5Ac and its polymer, polysialic acid (polySia), rapidly decreased during the first three days of lactation, and a high interindividual variance was observed. In mature milk, however, the sialylation status remains relatively constant. The results indicate that mammary gland epithelial cells are one source for milk polySia, since immunohistochemistry of udder tissue exhibited strong polySia staining in these cells. Furthermore, both polysialyltransferases, ST8SiaII and ST8SiaIV, are expressed. Based on known functions of monosialyl residues and polySia, we discuss the potential impact of these biomolecules and the consequences of the heterogeneous sialylation status of milk in relation to udder health and offspring health. [ABSTRACT FROM AUTHOR]

Abstract Background Udder infections with environmental pathogens like Escherichia coli are a serious problem for the dairy industry. Reduction of incidence and severity of mastitis is desirable and mild priming of the immune system either through vaccination or with low doses of immune stimulants such as lipopolysaccharide LPS was previously found to dampen detrimental effects of a subsequent infection. Monocytes/macrophages are known to develop tolerance towards the endotoxin LPS (endotoxin tolerance, ET) as adaptation strategy to prevent exuberant inflammation. We have recently observed that infusion of 1 μg of LPS into the quarter of an udder effectively protected for several days against an experimentally elicited mastitis. We have modelled this process in primary cultures of mammary epithelial cells (MEC) from the cow. MEC are by far the most abundant cells in the healthy udder coming into contact with invading pathogens and little is known about their role in establishing ET. Results We primed primary MEC cultures for 12 h with LPS (100 ng/ml) and stimulated three cultures either 12 h or 42 h later with 107/ml particles of heat inactivated E. coli bacteria for six hours. Priming-related alterations in the global transcriptome of those cells were quantified with Affymetrix microarrays. LPS priming alone caused differential expression of 40 genes and mediated significantly different response to a subsequent E. coli challenge of 226 genes. Expression of 38 genes was enhanced while that of 188 was decreased. Higher expressed were anti-microbial factors (β-defensin LAP, SLPI), cell and tissue protecting factors (DAF, MUC1, TGM1, TGM3) as well as mediators of the sentinel function of MEC (CCL5, CXCL8). Dampened was the expression of potentially harmful pro-inflammatory master cytokines (IL1B, IL6, TNF-α) and immune effectors (NOS2, matrix metalloproteases). Functional network analysis highlighted the reduced expression of IL1B and of IRF7 as key to this modulation. Conclusion LPS-primed MEC are fitter to repel pathogens and better protected against misguided attacks of the immune response. Attenuated is the exuberant expression of factors potentially promoting immunopathological processes. MEC therefore recapitulate many aspects of ET known so far from professional immune cells.

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Excessive stimulation of the TLR4 axis through LPS reduces the expression of some cytokine genes in immune cells, while stimulating the expression of immune defense genes during a subsequent bacterial infection. This endotoxin tolerance (ET) is mediated via epigenetic mechanisms. Priming the udder of cows with LPS was shown to induce ET in mammary epithelial cells (MEC), thereby protecting the udder against reinfection for some time. Seeking alternatives to LPS priming we tried to elicit ET by priming MEC with either lipopeptide (Pam2CSK4) via the TLR2/6 axis or inhibitors of histone-modifying enzymes. Pre-incubation of MEC with Pam2CSK4 enhanced baseline and induced expression of bactericidal (β-defensin; SLPI) and membrane protecting factors (SAA3, TGM3), while reducing the expression of cytokine- and chemokine-encoding genes (TNF, IL1β) after a subsequent pathogen challenge, the latter, however, not as efficiently as after LPS priming. Pre-treating MEC with various inhibitors of histone H3 modifiers (for demethylation, acetylation or deacetylation) all failed to induce any of the protective factors and only resulted in some dampening of cytokine gene expression after the re-challenge. Hence, triggering immune functions via the TLR axis, but not through those histone modifiers, induced the beneficial phenomenon of ET in MEC. [ABSTRACT FROM AUTHOR]