Charged Amino Acids in the Transmembrane Helix Strongly Affect the Enzyme Activity of Aromatase.

Estrogens play critical roles in embryonic development, gonadal sex differentiation, behavior, and reproduction in vertebrates and in several human cancers. Estrogens are synthesized from testosterone and androstenedione by the endoplasmic reticulum membrane-bound P450 aromatase/cytochrome P450 oxidoreductase complex (CYP19/CPR). Here, we report the characterization of novel mammalian CYP19 isoforms encoded by CYP19 gene copies. These CYP19 isoforms are all defined by a combination of mutations in the N-terminal transmembrane helix (E42K, D43N) and in helix C of the catalytic domain (P146T, F147Y). The mutant CYP19 isoforms show increased androgen conversion due to the KN transmembrane helix. In addition, the TY substitutions in helix C result in a substrate preference for androstenedione. Our structural models suggest that CYP19 mutants may interact differently with the membrane (affecting substrate uptake) and with CPR (affecting electron transfer), providing structural clues for the catalytic differences.

A brief history of galectin evolution.

Galectins are a family of carbohydrate-binding proteins found in vertebrates in great abundance and diversity in terms of both structure and ligand-binding properties as well as physiological function. Proteins with clear relationships to vertebrate galectins are already found in primitive Bilateria. The increasing amount of accessible well-annotated bilaterian genomes has allowed us to reveal, through synteny analyses, a new hypothesis about the phylogenetic history of the galectin family in this animal group. Thus, we can trace the genomic localization of the putative ancestral Bilateria galectin back to the scallops as a still very primitive slow-evolving bilaterian lineage. Intriguingly, our analyses show that the primordial galectin of the Deuterostomata most likely exhibited galectin-8-like characteristics. This basal standing galectin is characterized by a tandem-repeat type with two carbohydrate recognition domains as well as by a sialic acid binding property of the N-terminal domain, which is typical for galectin-8. With the help of synteny, the amplification of this potential primordial galectin to the broad galectin cosmos of modern jawed vertebrates can be reconstructed. Therefore, it is possible to distinguish between the paralogs resulting from small-scale duplication and the ohnologues generated by whole-genome duplication. Our findings support a substantially new hypothesis about the origin of the various members of the galectin family in vertebrates. This allows us to reveal new theories on the kinship relationships of the galectins of Gnatostomata. In addition, we focus for the first time on the galectines of the Cyclostomata, which as a sister group of jawed vertebrates providing important insights into the evolutionary history of the entire subphylum. Our studies also highlight a previously neglected member of the galectin family, galectin-related protein 2. This protein appears to be a widespread ohnologue of the original tandem-repeat ancestor within Gnathostomata that has not been the focus of galectin research due to its nonclassical galactose binding sequence motif and the fact that it was lost during mammalian evolution.

Body-Related Attitudes, Personality, and Identity in Female Adolescents with Anorexia Nervosa or Other Mental Disorders.

The psychological integration of body-related attitudes (BodyRA) is a critical developmental task in adolescence. Adolescents must adapt to their changing body image and body satisfaction. For young people, BodyRA (body dissatisfaction, bulimia, and drive for thinness) are connected to insecurities, which can disturb identity integration and personality development. Our goal was to evaluate the importance of BodyRA also for other mental disorders other than anorexia nervosa (AN), and the association between BodyRA with temperament and personality traits and identity diffusion. Data for the period of 2012 to 2019 were retrospectively analyzed from a convenience sample of patients in a child and adolescent psychiatric hospital (n = 114). The patients were 13 to 17 years of age and had a BMI of 11.9−36.1 kg/m2. As expected, BodyRA were found to be more pronounced in AN, as well as in borderline personality disorder (BPD), depression (DD), and attention deficit hyperactivity disorder (ADHD). BodyRA correlated significantly with internalizing problems in patients with DD (r = 0.428−0.565, p < 0.01) and BPD (r = 0.680, p < 0.01) as well as with BMI (r = 0.404, p < 0.01) in patients with DD. Moreover, we detected significant correlations with impaired identity development in patients with DD (r = 0.482−0.565, p < 0.01) and BPD (r = 0.681−0.703, p < 0.01). BodyRA also correlated significantly with the personality traits of harm avoidance (r = 0.377−0.541, p < 0.01) and self-directedness (r = −0.537−−0.635, p < 0.01) in DD. These personality traits and bulimia were used as predictors for identity diffusion in the investigated disorders of this study. We conclude that BodyRA, harm avoidance and self-directedness are associated with identity development in adolescent females with mental disorders.

Evaluation of blood cell viability rate, gene expression, and O-GlcNAcylation profiles as indicative signatures for fungal stimulation of salmonid cell models.

Fungal diseases of fish are a significant economic problem in aquaculture. Using high-throughput expression analysis, we identified potential transcript markers in primary head kidney and secondary embryonic cells from salmonid fish after stimulation with the inactivated fungi Mucor hiemalis and Fusarium aveneacium and with purified fungal molecular patterns. The transcript levels of most of the 45 selected genes were altered in head-kidney cells after 24 h of stimulation with fungal antigens. Stimulation with the inactivated fungus M. hiemalis induced the most pronounced transcriptional changes, including the pathogen receptor-encoding genes CLEC18A and TLR22, the cytokine-encoding genes IL6 and TNF, and the gene encoding the antimicrobial peptide LEAP2. In parallel, we analyzed the total GlcNAcylation status of embryonic salmonid cells with or without stimulation with inactivated fungi. O-GlcNAcylation modulates gene expression, intracellular protein, and signal activity, but we detected no significant differences after a 3-h stimulation. A pathway analysis tool identified the "apoptosis of leukocytes" based on the expression profile 24 h after fungal stimulation. Fluorescence microscopy combined with flow cytometry revealed apoptosis in 50 % of head-kidney leukocytes after 3 h stimulation with M. hiemalis, but this level decreased by > 5% after 24 h of stimulation. The number of apoptotic cells significantly increased in all blood cells after a 3-h stimulation with fungal molecular patterns compared to unstimulated controls. This in vitro approach identified transcript-based parameters that were strongly modulated by fungal infections of salmonid fish.

Milk Polysialic Acid Levels Rapidly Decrease in Line with the N-Acetylneuraminic Acid Concentrations during Early Lactation in Dairy Cows.

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.

Cellular detection of the chemokine receptor CXCR4 in bovine mammary glands and its distribution and regulation on bovine leukocytes.

Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.

The Loss of Polysialic Acid Impairs the Contractile Phenotype of Peritubular Smooth Muscle Cells in the Postnatal Testis.

In the testis, the germinal epithelium of seminiferous tubules is surrounded by contractile peritubular cells, which are involved in sperm transport. Interestingly, in postnatal testis, polysialic acid (polySia), which is also an essential player for the development of the brain, was observed around the tubules. Western blotting revealed a massive decrease of polySia from postnatal day 1 towards puberty, together with a fundamental reduction of the net-like intertubular polySia. Using polysialyltransferase knockout mice, we investigated the consequences of the loss of polySia in the postnatal testis. Compared to postnatal wild-type animals, polySia knockouts showed slightly reduced smooth muscle actin (SMA) immunostaining of peritubular smooth muscle cells (SMCs), while calponin, marking more differentiated SMCs, dramatically decreased. In contrast, testicular SMA and calponin immunostaining remained unchanged in vascular SMCs in all genotypes. In addition, the cGMP-dependent protein kinase PKG I, a key enzyme of SMC relaxation, was nearly undetectable in the peritubular SMCs. Cell proliferation in the peritubular layer increased significantly in the knockouts, as shown by proliferating cell nuclear anti (PCNA) staining. Taken together, in postnatal testis, the absence of polySia resulted in an impaired differentiation of peritubular, but not vascular, SMCs to a more synthetic phenotype. Thus, polySia might influence the maintenance of a differentiated phenotype of non-vascular SMCs.

Indication of Premelanosome Protein (PMEL) Expression Outside of Pigmented Bovine Skin Suggests Functions Beyond Eumelanogenesis.

The premelanosome protein (PMEL) is important for fibril formation within melanosomes during vertebrate melanogenesis. Fibrils form a matrix for pigment deposition within pigmented tissues such as skin and hair. PMEL mutations are known to modulate eumelanic pigmentation in vertebrates. However, in bovines, PMEL mutations were also found to alter pheomelanic pigmentation resulting in coat color dilution. Furthermore, epistatic effects of a mutated PMEL allele were detected in the phenotypic expression of the bovine hair defect "rat-tail syndrome" (RTS) characterized by charcoal coat color and hair deformation. Reports about PMEL gene expression in non-pigmented tissues raised the hypothesis that there may be unknown functions of the PMEL protein beyond eumelanin deposition to PMEL fibrils. In our study, we analysed the PMEL protein expression in pigmented skin and non-pigmented bovine tissues (non-pigmented skin, thyroid gland, rumen, liver, kidney, and adrenal gland cortex). We found that a processed form of the bovine PMEL protein is expressed in pigmented as well as in non-pigmented tissues, which is in line with gene expression data from targeted RT-PCR and whole transcriptome RNAseq analysis. The PMEL protein is located in membranes and within the cytosol of epithelial cells. Based on our data from bovine tissues, we concluded that at least in cattle PMEL potentially has additional, yet unexplored functions, which might contribute to effects of PMEL mutations on pheomelanin coat color dilution and charcoal coat color in RTS animals. However, indication of PMEL protein in unpigmented cells and tissues will require further confirmation in the future, because there have been no confirmed reports before, which had detected bovine PMEL protein with specific antibodies either in pigmented or unpigmented tissue.

Gene expression profiles of bovine uninucleate trophoblast cells and trophoblast giant cells: a data note.

OBJECTIVES: In the bovine placenta, intimate fetomaternal contact is restricted to placentomes. Within the placentomes fetal chorionic villi interdigitate with corresponding maternal caruncular crypts. The trophoblast epithelium covering the chorionic villi consists of 80% uninucleate trophoblast cells (UTCs) and 20% trophoblast giant cells (TGCs). TGCs migrate toward the endometrium and fuse with endometrial cells to form short-lived fetomaternal hybrid cells. Thereby the TGCs transport molecules of fetal origin across the placental barrier into the maternal compartment. The UTC/TGC ratio is constant during pregnancy because UTCs can differentiate into new TGCs to replace spent TGCs. However, our understanding of this differentiation process was sparse. Therefore, we collected the data to study the gene expression profiles in UTCs and TGCs and to identify differently expressed genes between the two trophoblast cell populations. Using Gene Ontology analysis, we wanted to identify biological processes and pathways that play an important role in the differentiation of UTCs into TGCs. DATA DESCRIPTION: Bovine placentas were from days 118 to 130 of gestation. We obtained virtually pure UTCs and TGCs using a fluorescence-activated cell sorting (FACS) method. Total RNA was extracted from the UTC and TGC isolates, labeled and hybridized to Affymetrix Bovine Gene 1.0 ST Arrays.

Trophoblast cell differentiation in the bovine placenta: differentially expressed genes between uninucleate trophoblast cells and trophoblast giant cells are involved in the composition and remodeling of the extracellular matrix and O-glycan biosynthesis.

BACKGROUND: In the bovine placenta, intimate fetomaternal contacts are restricted to discrete placentomes. Here, widely branched fetal chorionic villi interdigitate with corresponding maternal caruncular crypts. The fetal trophoblast epithelium covering the chorionic villi consists of approximately 80% uninucleate trophoblast cells (UTCs) and 20% binuclear trophoblast giant cells (TGCs). The weakly invasive TGCs migrate toward the caruncle epithelium and eventually fuse with individual epithelial cells to form short-lived fetomaternal hybrid cells. In this way, molecules of fetal origin are transported across the placental barrier and released into the maternal compartment. The UTC/TGC ratio in the trophoblast remains almost constant because approximately as many new TGCs are produced from UTCs as are consumed by the fusions. The process of developing TGCs from UTCs was insufficiently understood. Therefore, we aimed to detect differentially expressed genes (DEGs) between UTCs and TGCs and identify molecular functions and biological processes regulated by DEGs. RESULTS: We analyzed gene expression patterns in virtually pure UTC and TGC isolates using gene arrays and detected 3193 DEGs (p < 0.05; fold change values < - 1.5 or > 1.5). Of these DEGs, 1711 (53.6%) were upregulated in TGCs and 1482 (46.4%) downregulated. Gene Ontology (GO) analyses revealed that molecular functions and biological processes regulated by DEGs are related to the extracellular matrix (ECM) and its interactions with cellular receptors, cell migration and signal transduction. Furthermore, there was some evidence that O-glycan biosynthesis in TGCs may produce sialylated short-chain O-glycans (Tn antigen, core 1 O-glycans), while the synthesis of other O-glycan core structures required for the formation of complex (i.e., branched and long-chain) O-glycans appears to be decreased in TGCs. CONCLUSION: The differentiation of UTCs into TGCs particularly regulates genes that enable trophoblast cells to interact with their environment. Significant differences between UTCs and TGCs in ECM composition indicate reduced anchoring of TGCs in the surrounding matrix, which might contribute to their migration and their weakly invasive interaction with the maternal endometrium. Furthermore, increased expression of sialylated short chain O-glycans by TGCs could facilitate the modulation of maternal immune tolerance.

Limitations and Off-Target Effects of Tryptophan-Related IDO Inhibitors in Cancer Treatment.

Immunooncology is still a growing area in cancer therapy. Drugs within this therapeutic approach do not directly target/attack the tumor but interfere with immune checkpoints and target or reprogram key metabolic pathways critical for anti-cancer immune defense. Indolamine 2,3-dioxygenase 1 (IDO1) and the tryptophan (TRP)-kynurenine pathway were identified as critical mechanisms in cancer immune escape and their inhibition as an approach with promising therapeutic potential. Particularly, a multitude of IDO1 inhibiting tryptophan analogs are widely applied in several clinical trials. However, this therapy results in a variety of implications for the patient's physiology. This is not only due to the inhibition of an enzyme important in almost every organ and tissue in the body but also because of the general nature of the inhibitor as an analog of a proteinogenic amino acid as well as the initiation of cellular detoxification known to affect inflammatory pathways. In this review we provide a deeper insight into the physiological consequences of an IDO1 inhibiting therapy based on TRP related molecules. We discuss potential side and off-target effects that contribute to the interpretation of unexpected positive as well as negative results of ongoing or discontinued clinical studies while we also highlight the potential of these inhibitors independent of the IDO1 signaling pathway.

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue.

BACKGROUND: In the mammary gland transcriptome of lactating dairy cows genes encoding milk proteins are highly abundant, which can impair the detection of lowly expressed transcripts and can bias the outcome in global transcriptome analyses. Therefore, the aim of this study was to develop and evaluate a method to deplete extremely highly expressed transcripts in mRNA from lactating mammary gland tissue. RESULTS: Selective RNA depletion was performed by hybridization of antisense oligonucleotides targeting genes encoding the caseins (CSN1S1, CSN1S2, CSN2 and CSN3) and whey proteins (LALBA and PAEP) within total RNA followed by RNase H-mediated elimination of the respective transcripts. The effect of the RNA depletion procedure was monitored by RNA sequencing analysis comparing depleted and non-depleted RNA samples from Escherichia coli (E. coli) challenged and non-challenged udder tissue of lactating cows in a proof of principle experiment. Using RNase H-mediated RNA depletion, the ratio of highly abundant milk protein gene transcripts was reduced in all depleted samples by an average of more than 50% compared to the non-depleted samples. Furthermore, the sensitivity for discovering transcripts with marginal expression levels and transcripts not yet annotated was improved. Finally, the sensitivity to detect significantly differentially expressed transcripts between non-challenged and challenged udder tissue was increased without leading to an inadvertent bias in the pathogen challenge-associated biological signaling pathway patterns. CONCLUSIONS: The implementation of selective RNase H-mediated RNA depletion of milk protein gene transcripts from the mammary gland transcriptome of lactating cows will be highly beneficial to establish comprehensive transcript catalogues of the tissue that better reflects its transcriptome complexity.

Effects of 1-Methyltryptophan on Immune Responses and the Kynurenine Pathway after Lipopolysaccharide Challenge in Pigs.

An enhanced indoleamine 2,3-dioxygenase 1 (IDO1) activity is associated with an increased mortality risk in sepsis patients. Thus, the preventive inhibition of IDO1 activity may be a promising strategy to attenuate the severity of septic shock. 1-methyltryptophan (1-MT) is currently in the interest of research due to its potential inhibitory effects on IDO1 and immunomodulatory properties. The present study aims to investigate the protective and immunomodulatory effects of 1-methyltryptophan against endotoxin-induced shock in a porcine in vivo model. Effects of 1-MT were determined on lipopolysaccharide (LPS)-induced tryptophan (TRP) degradation, immune response and sickness behaviour. 1-MT increased TRP and its metabolite kynurenic acid (KYNA) in plasma and tissues, suppressed the LPS-induced maturation of neutrophils and increased inactivity of the animals. 1-MT did not inhibit the LPS-induced degradation of TRP to kynurenine (KYN)-a marker for IDO1 activity-although the increase in KYNA indicates that degradation to one branch of the KYN pathway is facilitated. In conclusion, our findings provide no evidence for IDO1 inhibition but reveal the side effects of 1-MT that may result from the proven interference of KYNA and 1-MT with aryl hydrocarbon receptor signalling. These effects should be considered for therapeutic applications of 1-MT.

The first line of defence: insights into mechanisms and relevance of phagocytosis in epithelial cells.

Epithelial tissues cover most of the external and internal surfaces of the body and its organs. Inevitably, these tissues serve as first line of defence against inorganic, organic, and microbial intruders. Epithelial cells are the main cell type of these tissues. Besides their function as cellular barrier, there is growing evidence that epithelial cells are of particular relevance as initial sensors of danger and also as executers of adequate defence responses. These cells feature various essential functions to maintain tissue integrity in health and disease. In this review, we survey some of the different innate immune functions of epithelial cells in mucosal tissues being constantly exposed to a plethora of harmless contaminants but also of pathogens. We discuss how epithelial cells avoid inadequate immune responses in such conditions. In particular, we will focus on the diverse types and mechanisms of phagocytosis used by epithelial cells to not only maintain homeostasis but to also harness the host response against invading pathogens.

Pathogen-specific responses in the bovine udder. Models and immunoprophylactic concepts.

Bovine mastitis is a disease of major economic effects on the dairy industry worldwide. Experimental in vivo infection models have been widely proven as an effective tool for the investigation of pathogen-specific host immune responses. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are two common mastitis pathogens with an opposite clinical outcome of the disease. E. coli and S. aureus have proven to be valid surrogates to model clinical and subclinical mastitis respectively. Contemporary transcriptome profiling studies demonstrated that the transcriptomic response in the teat reflects the course of pathogen-specific mastitis, being ultimately determined by the immune response of the mammary epithelial cells. After an experimental in vivo challenge, E. coli induces a vigorous early transcriptional response in udder tissue being quantitatively and - notably - qualitatively distinct from the much weaker response against an S. aureus infection. E. coli mastitis models proved that the local response in the infected udder quarters is accompanied by a response in non-infected neighbouring udder quarters modulating systemically their immune responsiveness. Immunomodulation of the udder was investigated in animal models. Pathophysiological consequences were studied after intramammary administration of cytokines, chemokines, growth factors, steroidal anti-inflammatory drugs, or priming of tissue resident cells with pathogen-derived molecules. The latter approaches resulted only in a temporal protection of the udder, reducing transiently the risk of infection but sustained lowering of the severity of an eventually occurring mastitis. They offer an alternative to vaccination trials, which over decades also did not yield protection against new infections.

Differentiating Staphylococcus aureus from Escherichia coli mastitis: S. aureus triggers unbalanced immune-dampening and host cell invasion immediately after udder infection.

The etiology determines quality and extent of the immune response after udder infection (mastitis). Infections with Gram negative bacteria (e.g. Escherichia coli) will quickly elicit strong inflammation of the udder, fully activate its immune defence via pathogen receptor driven activation of IκB/NF-κB signaling. This often eradicates the pathogen. In contrast, Gram-positive bacteria (e.g. Staphylococcus aureus) will slowly elicit a much weaker inflammation and immune response, frequently resulting in chronic infections. However, it was unclear which immune regulatory pathways are specifically triggered by S. aureus causing this partial immune subversion. We therefore compared in first lactating cows the earliest (1-3 h) udder responses against infection with mastitis causing pathogens of either species. Global transcriptome profiling, bioinformatics analysis and experimental validation of key aspects revealed as S. aureus infection specific features the (i) failure to activating IκB/NF-κB signaling; (ii) activation of the wnt/ß-catenin cascade resulting in active suppression of NF-κB signaling and (iii) rearrangement of the actin-cytoskeleton through modulating Rho GTPase regulated pathways. This facilitates invasion of pathogens into host cells. Hence, S. aureus mastitis is characterized by eliciting unbalanced immune suppression rather than inflammation and invasion of S. aureus into the epithelial cells of the host causing sustained infection.

TLR ligands, but not modulators of histone modifiers, can induce the complex immune response pattern of endotoxin tolerance in mammary epithelial cells.

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 (ß-defensin; SLPI) and membrane protecting factors ( SAA3, TGM3), while reducing the expression of cytokine- and chemokine-encoding genes ( TNF, IL1ß) 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.

Kynurenic Acid: The Janus-Faced Role of an Immunomodulatory Tryptophan Metabolite and Its Link to Pathological Conditions.

Tryptophan metabolites are known to participate in the regulation of many cells of the immune system and are involved in various immune-mediated diseases and disorders. Kynurenic acid (KYNA) is a product of one branch of the kynurenine pathway of tryptophan metabolism. The influence of KYNA on important neurophysiological and neuropathological processes has been comprehensively documented. In recent years, the link of KYNA to the immune system, inflammation, and cancer has become more apparent. Given this connection, the anti-inflammatory and immunosuppressive functions of KYNA are of particular interest. These characteristics might allow KYNA to act as a "double-edged sword." The metabolite contributes to both the resolution of inflammation and the establishment of an immunosuppressive environment, which, for instance, allows for tumor immune escape. Our review provides a comprehensive update of the significant biological functions of KYNA and focuses on its immunomodulatory properties by signaling via G-protein-coupled receptor 35 (GPR35)- and aryl hydrocarbon receptor-mediated pathways. Furthermore, we discuss the role of KYNA-GPR35 interaction and microbiota associated KYNA metabolism for gut homeostasis.

Early transcriptional events in the udder and teat after intra-mammary Escherichia coli and Staphylococcus aureus challenge.

Intra-mammary bacterial infections can result in harmful clinical mastitis or subclinical mastitis with persistent infections. Research during the last decades closely examined the pathophysiology of inflamed udders. Initial events after pathogen perception but before the onset of mastitis have not been examined in vivo The objective of this study was to develop a mastitis model in cows by monitoring initial transcriptional pathogen-specific host response before clinical signs occur. We applied a short-term infection model to analyse transcripts encoding chemokines, cytokines and antimicrobial molecules in the teat cistern (TC) and lobulo-alveolar parenchyma (LP) up to 3 h after challenge with E and Staphylococcus aureus Both pathogens elicited an immune reaction by 1 h after challenge. Escherichia coli induced all analysed factors (CCL20, CXCL8, TNF, IL6, IL12B, IL10, LAP, S100A9); however, S. aureus failed to induce IL12B, IL10, LAP and S100A9 expression. The E. coli-induced up-regulation was 25-105 times greater than that after S. aureus challenge. Almost all the responses were restricted to the TC. The short-term mastitis model demonstrates that a divergent pathogen-specific response is generated during the first h. It confirms that the first transcripts are generated in the TC prior to a response in the LP.