Bovine Mammary Epithelial Cells Research Articles

Bioinsecticide PA1b alleviates lipopolysaccharide-induced inflammation and impairment of bovine mammary epithelial cells

Pea Albumin 1, subunit b (PA1b) is a 37 amino acid peptide. It was extracted from pea seeds and showed significant insecticidal activity against certain insects, such as the mosquitoes Culex pipiens and Aedes aegyptii, cereal weevils (genus Sitophilus), and certain species of aphids. Considering that pea seeds are regularly consumed by humans and mammals, PA1b is assumed to be a promising bioinsecticide with no allergenicity or toxicity to hosts. To clarify this aspect, PA1b was applied to bovine mammary epithelial cells challenged with lipopolysaccharide (LPS). The results revealed that LPS induced inflammatory cytokine tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL6) and monocyte chemoattractant protein 1 (MCP-1) secretion, while PA1b depressed these cytokines release via inhibiting NF-κB signaling activation. In addition, PA1b protected mammary epithelial cells from impairment caused by LPS, because it reduced cell membrane permeability and subsequently reconstructed mammary epithelial cell viability. Moreover, it inhibited cell apoptosis accompanied with alleviated oxidative stress. Furthermore, PA1b prevented opening of mitochondrial permeability transition pores, in turn up-regulated mitochondrial membrane potential and ATP production. Therefore, PA1b improved mitochondrial function, which contributed to re-construction of mammary epithelial cell viability. In conclusion, PA1b alleviates LPS-induced inflammation of bovine mammary epithelial cells via inhibiting NF-κB signaling activation and protects bovine mammary epithelial cells by improving mitochondrial function. PA1b is a good therapeutic survival factor for mammary epithelial cells.

Multi-omics dataset of bovine mammary epithelial cells stimulated by ten different essential amino acids

Application of high-throughput sequencing and screening help to detect the transcriptional and metabolic discrepancies in organs provided with various levels of nutrients. The influences of individual essential amino acid (EAA) administration on transcriptomic and metabolomic profilings of bovine mammary epithelial cells (BMECs) were systematically investigated. A RNA sequencing and liquid chromatography-tandem mass spectrometry generated a comprehensive comparison of transcriptomics, non-targeted metabolomics and targeted amino acids profilings of BMECs with individual EAA stimulation by turn. The sequencing data and raw LC-MS/MS data of samples were presented in the databases of Gene Expression Omnibus, MetaboLights and Figshare for efficient reuse, including exploring the divergences in metabolisms between different EAAs and screening valuable genes and metabolites regulating casein synthesis.

Evaluation of the Antimicrobial, Cytotoxic, and Physical Properties of Selected Nano-Complexes in Bovine Udder Inflammatory Pathogen Control.

Mastitis in dairy cows is a worldwide problem faced by dairy producers. Treatment mainly involves antibiotic therapy, however, due to widespread antibiotic resistance among bacteria, such treatments are no longer effective. For this reason, scientists are searching for new solutions to combat mastitis, which is caused by bacteria, fungi, and algae. One of the most promising solutions, nanotechnology, is attracting research due to its biocidal properties. The purpose of this research was to determine the biocidal properties of nanocomposites as a potential alternative to antibiotics in the control of mastitis, as well as to determine whether the use of nanoparticles and what concentration is safe for the breeder and the animal. In this study, the effects of Ag, Au, Cu, Fe, and Pt nanoparticles and their complexes were evaluated in relation to the survival of bacteria and fungi isolated from cattle diagnosed with mastitis, their physicochemical properties, and their toxicity to bovine and human mammary epithelial cells BME-UV1 and HMEC (human microvascular endothelial cells). Moreover, E. coli, S. aureus, C. albicans, and Prototheca sp. invasion was assessed using the alginate bead (bioprinted) model. The NPs were tested at concentrations of 25, 12.5, 6.25, 3.125, 1.56 mg/l for Au, Ag, Cu and Fe NPs, and 10, 5, 2.5, 1.25, 0.625 mg/l for Pt. With the exception of Fe and Pt, all exhibited biocidal properties against isolates, while the AgCu complex had the best effect. In addition, nanoparticles showed synergistic effects, while the low concentrations had no toxic effect on BME-UV1 and HMEC cells. Synergistic effects of nanoparticles and no toxicity to bovine and human cells might, in the future, be an effective alternative in the fight against microorganisms responsible for mastitis, and the implementation of research results in practice would reduce the percentage of dairy cows suffering from mastitis. The problem of increasing antibiotic resistance is posing a global threat to human's and animal's health, and requires comprehensive research to evaluate the potential use of nanoparticles - especially their complexes - as well as to determine whether nanoparticles are safe for the breeders and the animals. The conducted series of studies allows further consideration of the use of the obtained results in practice, creating a potentially new alternative to antibiotics in the treatment and prevention of mastitis in dairy cattle.

The mechanistic role of natural antimicrobials in preventing Staphylococcus aureus invasion of MAC-T cells using an in vitro mastitis model.

Starting primarily as an inflammation of the mammary gland, mastitis is frequently driven by infectious agents such as Staphylococcus aureus. Mastitis has a large economic impact globally, which includes diagnostic, treatment, and the production costs not to mention the potential milk contamination with antimicrobial residues. Currently, mastitis prevention and cure depends on intramammary infusion of antimicrobials, yet, their overuse risks engendering resistant pathogens, posing further threats to livestock. In our study we aimed to investigate, in vitro, using bovine mammary epithelial cells (MAC-T), the efficacy of the AuraShield an antimicrobial mixture (As) in preventing S. aureus attachment, internalisation, and inflammation. The antimicrobial mixture (As) included: 5% maltodextrin, 1% sodium chloride, 42% citric acid, 18% sodium citrate, 10% silica, 12% malic acid, 9% citrus extract and 3% olive extract (w/w). Herein we show that As can significantly reduce both adherence and invasion of MAC-T cells by S. aureus, with no impact on cell viability at all concentrations tested (0.1, 0.2, 0.5, 1%) compared with untreated controls. The anti-apoptotic effect of As was achieved by significantly reducing cellular caspase 1, 3 and 8 activities in the infected MAC-T cells. All As concentrations were proven to be subinhibitory, suggesting that Ac can reduce S. aureus virulence without bacterial killing and that the effect could be dual including a host modulation effect. In this context, we show that As can reduce the expression of S. aureus clumping factor (ClfB) and block its interaction with the host Annexin A2 (AnxA2), resulting in decreased bacterial adherence in infection of MAC-T cells. Moreover, the ability of As to block AnxA2 had a significant decreasing effect on the levels of pro inflammatory cytokine released upon S. aureus interaction with MAC-T cells. The results presented in this study indicate that mixtures of natural antimicrobials could potentially be considered an efficient alternative to antibiotics in treating S. aureus induced mastitis.

Cross-Oxygen Gradients Transcriptomic Comparison Revealed the Central Role of MAPK and Hippo in Hypoxia-Mediated Mammary Proliferation Inhibition.

The role of hypoxia in terms of affecting mammary epithelial cells (MECs) proliferation is closely associated with the milk synthesis of lactating mammals. Primary bovine MECs were cultured at 1, 6, 11, 16, and 21% O2 for 24 h. The results showed that cell proliferation decreased linearly, and hypoxic inducible factor (HIF)-1α expression increased linearly along with the declining O2. The linear increase in oxidative stress resulted in the accumulation of malondialdehyde and reactive oxygen species and decreased antioxidant enzyme activities following the reduced O2. Concerning mitochondria, the dynamin-related protein 1 showed improved expression, and optin atrophy protein 1 decreased along with the decreasing O2 gradient, which led to decreased mitochondrial mass and mitophagy emerging under 1% O2. Oxygen concentration-trend RNA-seq analysis was conducted. Specifically, HIF-1-MAPK (1% O2), PI3K-Akt-MAPK (6% O2), and p53-Hippo (11 and 16% O2) were found to primarily regulate cell proliferation in response to hypoxia compared with normoxia (21%), respectively. In conclusion, our study suggests that bMEC proliferation is suppressed in low-oxygen conditions, and is exacerbated following the reduced oxygen supply. The cross-oxygen gradient comparisons suggest that MAPK and Hippo, which are core pathways of mammary cell proliferation, are repressed by hypoxia via oxidative-stress-dependent signals.

SNAT2-mediated regulation of estrogen and progesterone in the proliferation of goat mammary epithelial cells

The development of the goat mammary gland is mainly under the control of ovarian hormones particularly estrogen and progesterone (P4). Amino acids play an essential role in mammary gland development and milk production, and sodium-coupled neutral amino acid transporter 2 (SNAT2) was reported to be expressed in the mammary gland of rats and bovine mammary epithelial cells, which may affect the synthesis of milk proteins or mammary cell proliferation by mediating prolactin, 17β-estradiol (E2) or methionine function. However, whether SNAT2 mediates the regulatory effects of E2 and P4 on the development of the ruminant mammary gland is still unclear. In this study, we show that E2 and P4 could increase the proliferation of goat mammary epithelial cells (GMECs) and regulate SNAT2 mRNA and protein expression in a dose-dependent manner. Further investigation revealed that SNAT2 is abundantly expressed in the mammary gland during late pregnancy and early lactation, while knockdown and overexpression of SNAT2 in GMECs could inhibit or enhance E2- and P4-induced cell proliferation as well as mammalian target of rapamycin (mTOR) signaling. We also found that the accelerated proliferation induced by SNAT2 overexpression in GMECs was suppressed by the mTOR signaling pathway inhibitor rapamycin. This indicates that the regulation of GMECs proliferation mediated by SNAT2 in response to E2 and P4 is dependent on the mTOR signaling pathway. Finally, we found that the total content of the amino acids in GMECs changed after knocking-down and overexpressing SNAT2. In summary, the results demonstrate that the regulatory effects of E2 and P4 on GMECs proliferation may be mediated by the SNAT2-transported amino acid pathway. These results may offer a novel nutritional target for improving the development of the ruminant mammary gland and milk production.

The preventive effects of Lactobacillus casei 03 on Escherichia coli-induced mastitis in vitro and in vivo

BackgroundLactobacillus casei possesses many kinds of bioactivities, such as anti-inflammation and anti-oxidant, and has been applied to treating multiple inflammatory diseases. However, its role in mastitis prevention has remained ambiguous.MethodsThis study aimed to examine the mechanisms underlying the preventive effects of L. casei 03 against E. coli- mastitis utilizing bovine mammary epithelial cells (BMECs) and a mouse model.ResultsIn vitro assays revealed pretreatment with L. casei 03 reduced the apoptotic ratio and the mRNA expression levels of IL1β, IL6 and TNFα and suppressed phosphorylation of p65, IκBα, p38, JNK and ERK in the NF-κB signaling pathway and MAPK signaling pathway. Furthermore, in vivo tests indicated that intramammary infusion of L. casei 03 relieved pathological changes, reduced the secretion of IL1β, IL6 and TNFα and MPO activity in the mouse mastitis model.ConclusionsThese data suggest that L. casei 03 exerts protective effects against E. coli-induced mastitis in vitro and in vivo and may hold promise as a novel agent for the prevention and treatment of mastitis.

The Complement Component 4 Binding Protein α Gene: A Versatile Immune Gene That Influences Lipid Metabolism in Bovine Mammary Epithelial Cell Lines.

Complement component 4 binding protein α (C4BPA) is an immune gene which is responsible for the complement regulation function of C4BP by binding and inactivating the Complement component C4b (C4b) component of the classical Complement 3 (C3) invertase pathway. Our previous findings revealed that C4BPA was differentially expressed by comparing the transcriptome in high-fat and low-fat bovine mammary epithelial cell lines (BMECs) from Chinese Holstein dairy cows. In this study, a C4BPA gene knockout BMECs line model was constructed via using a CRISPR/Cas9 system to investigate the function of C4BPA in lipid metabolism. The results showed that levels of triglyceride (TG) were increased, while levels of cholesterol (CHOL) and free fatty acid (FFA) were decreased (p < 0.05) after knocking out C4BPA in BMECs. Additionally, most kinds of fatty acids were found to be mainly enriched in the pathway of the biosynthesis of unsaturated fatty acids, linoleic acid metabolism, fatty acid biosynthesis, and regulation of lipolysis in adipocyte. Meanwhile, the RNA-seq showed that most of the differentially expressed genes (DEGs) are related to PI3K-Akt signaling pathway. The expressions of 3-Hydroxy-3-Methylglutaryl-CoA Synthase 1 (HMGCS1), Carnitine Palmitoyltransferase 1A (CPT1A), Fatty Acid Desaturase 1 (FADS1), and Stearoyl-Coenzyme A desaturase 1 (SCD1) significantly changed when the C4BPA gene was knocked out. Collectively, C4BPA gene, which is an immune gene, played an important role in lipid metabolism in BMECs. These findings provide a new avenue for animal breeders: this gene, with multiple functions, should be reasonably utilized.

Genes and pathways revealed by whole transcriptome analysis of milk derived bovine mammary epithelial cells after Escherichia coli challenge

Mastitis, inflammation of the mammary gland, is the costliest disease in dairy cattle and a major animal welfare concern. Mastitis is usually caused by bacteria, of which staphylococci, streptococci and Escherichia coli are most frequently isolated from bovine mastitis. Bacteria activate the mammary immune system in variable ways, thereby influencing the severity of the disease. Escherichia coli is a common cause of mastitis in cattle causing both subclinical and clinical mastitis. Understanding of the molecular mechanisms that activate and regulate the host response would be central to effective prevention of mastitis and breeding of cows more resistant to mastitis. We used primary bovine mammary epithelial cell cultures extracted noninvasively from bovine milk samples to monitor the cellular responses to Escherichia coli challenge. Differences in gene expression between control and challenged cells were studied by total RNA-sequencing at two time points post-challenge. In total, 150 and 440 (Padj < 0.05) differentially expressed genes were identified at 3 h and 24 h post-challenge, respectively. The differentially expressed genes were mostly upregulated at 3 h (141/150) and 24 h (424/440) post-challenge. Our results are in line with known effects of E. coli infection, with a strong early inflammatory response mediated by pathogen receptor families. Among the most significantly enriched early KEGG pathways were the TNF signalling pathway, the cytokine-cytokine receptor interaction, and the NF-kappa B signalling pathway. At 24 h post-challenge, most significantly enriched were the Influenza A, the NOD-like receptor signalling, and the IL-17 signaling pathway.

MiR-425-5p Regulates Proliferation of Bovine Mammary Epithelial Cells by Targeting TOB2.

In recent years, rising temperatures have caused heat stress (HS), which has had a significant impact on livestock production and growth, presenting considerable challenges to the agricultural industry. Research has shown that miR-425-5p regulates cellular proliferation in organisms. However, the specific role of miR-425-5p in bovine mammary epithelial cells (BMECs) remains to be determined. The aim of this study was to investigate the potential of miR-425-5p in alleviating the HS-induced proliferation stagnation in BMECs. The results showed that the expression of miR-425-5p significantly decreased when BMEC were exposed to HS. However, the overexpression of miR-425-5p effectively alleviated the inhibitory effect of HS on BMEC proliferation. Furthermore, RNA sequencing analysis revealed 753 differentially expressed genes (DEGs), comprising 361 upregulated and 392 downregulated genes. Some of these genes were associated with proliferation and thermogenesis through enrichment analyses. Further experimentation revealed that TOB2, which acts as a target gene of miR-425-5p, is involved in the regulatory mechanism of BMEC proliferation. In summary, this study suggests that miR-425-5p can promote the proliferation of BMECs by regulating TOB2. The miR-425-5p/TOB2 axis may represent a potential pathway through which miR-425-5p ameliorates the proliferation stagnation of BMECs induced by HS.

CGA alleviates LPS-induced inflammation and milk fat reduction in BMECs through the NF-κB signaling pathway

Mastitis is an easy clinical disease in dairy cows, which seriously affects the milk yield and quality of dairy cows. Chlorogenic acid (CGA), a polyphenolic substance, is abundant in Eucommia ulmoides leaves and has anti-inflammatory and anti-oxidative stress effects. Here, we explore whether CGA attenuated lipopolysaccharide (LPS)-induced inflammation and decreased milk fat in bovine mammary epithelial cells (BMECs). 10 μg/mL LPS was used to induce mastitis in BMECs. QRT-PCR, Western blotting, oil red O staining, and triglyceride (TG) assay were used to examine the effects of CGA on BMECs, including inflammatory response, oxidative stress response, and milk fat synthesis. The results showed that CGA repaired LPS-induced inflammation in BMECs. The expression of IL-6, IL-8, TNF-α, IL-1β, and iNOS was decreased, and the expression levels of CHOP, XCT, NRF2, and HO-1 were increased, which reduced the oxidative stress level of cells and alleviated the reduction of milk fat synthesis. In addition, the regulation of P65 phosphorylation by CGA suggests that CGA may exert its anti-inflammatory and anti-oxidative effects through the NF-κB signaling pathway. Our study showed that CGA attenuated LPS-induced inflammation and oxidative stress, and restored the decrease in milk fat content in BMECs by regulating the NF-κB signaling pathway.

The serine protease 2 gene regulates lipid metabolism through the LEP/ampkα1/SREBP1 pathway in bovine mammary epithelial cells

Molecular breeding has brought about significant transformations in the milk market and production system during the twenty-first century. The primary economic characteristic of dairy production pertains to milk fat content. Our previous transcriptome analyses revealed that serine protease 2 (PRSS2) is a candidate gene that could impact milk fat synthesis in bovine mammary epithelial cells (BMECs) of Chinese Holstein dairy cows. To elucidate the function of the PRSS2 gene in milk fat synthesis, we constructed vectors for PRSS2 overexpression and interference and assessed intracellular triglycerides (TGs), cholesterol (CHOL), and nonesterified fatty acid (NEFA) contents in BMECs. Fatty acid varieties and components were also quantified using gas chromatography‒mass spectrometry (GC‒MS) technology. The regulatory pathway mediated by PRSS2 was validated through qPCR, ELISA, and WB techniques. Based on our research findings, PRSS2 emerges as a pivotal gene that regulates the expression of associated genes, thereby making a substantial contribution to lipid metabolism via the leptin (LEP)/Adenylate-activated protein kinase, alpha 1 catalytic subunit (AMPKα1)/sterol regulatory element binding protein 1(SREBP1) pathway by inhibiting TGs and CHOL accumulation while potentially promoting NEFA synthesis in BMECs. Furthermore, the PRSS2 gene enhances intracellular medium- and long-chain fatty acid metabolism by modulating genes related to the LEP/AMPKα1/SREBP1 pathway, leading to increased contents of unsaturated fatty acids C17:1N7 and C22:4N6. This study provides a robust theoretical framework for further investigation into the underlying molecular mechanisms through which PRSS2 influences lipid metabolism in dairy cows.

Alfalfa xenomiR-162 targets G protein subunit gamma 11 to regulate milk protein synthesis in bovine mammary epithelial cells.

It was shown that microRNAs (miRNAs) play an important role in milk protein synthesis. However, the post-transcriptional regulation of casein expression by exogenous miRNA (xeno-miRNAs) in ruminants remains unclear. This study explores the regulatory roles of alfalfa xeno-miR162 on casein synthesis in bovine mammary epithelial cells (bMECs). The effects of alfalfa xenomiR-162 and G protein subunit gamma 11 (GNG11) on proliferation and milk protein metabolism of bMECs were detected by 5-Ethynyl-2'-Deoxyuridine (EdU) staining, flow cytometry, cell counting kit-8 (CCK-8), enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Dual-luciferase reporter assay was used to verify the targeting relationship between GNG11 and xenomiR-162. Results showed that over-expression of xenomiR-162 inhibited cell proliferation but promoted apoptosis, which also up-regulated the expression of several casein coding genes, including CSN1S1, CSN1S2, and CSN3, while decreasing the expression of CSN2. Furthermore, the targeting relationship between GNG11 and xenomiR-162 was determined, and it was confirmed that GNG11 silencing also inhibited cell proliferation but promoted apoptosis and reduced the expression of casein coding genes and genes related to the mammalian target of rapamycin (mTOR) pathway. Alfalfa xenomiR-162 appears to regulate bMECs proliferation and milk protein synthesis via GNG11 in the mTOR pathway, suggesting that this xeno-miRNA could be harnessed to modulate CSN3 expression in dairy cows, and increase κ-casein contents in milk.

Zearalenone triggers programmed cell death and impairs milk fat synthesis via the AKT-mTOR-PPARγ-ACSL4 pathway in bovine mammary epithelial cells.

Zearalenone (ZEN), a mycotoxin from Fusarium fungi, impairs fertility and milk production in female animals; however, the mechanisms remain poorly understood. Using the bovine mammary epithelial cells (MAC-T) as the model, this study investigated the impacts of ZEN on programmed cell death (PCD) and milk fat synthesis and explored the underlying mechanism. We found that 10ng/mL prolactin (PRL) notably enhanced the differentiation of MAC-T cells, promoting the expression of genes related to the synthesis of milk fat, protein, and lactose. Next, the toxic effects of different doses of ZEN on the differentiated MAC-T with PRL treatment were determined. 10 and 20 μM ZEN significantly reduced cell viability, induced oxidative stress, and triggered PCD (e.g., apoptosis and necrosis). Notably, ZEN exposure downregulated the mRNA/protein levels of critical factors involved in milk fat synthesis by disrupting the AKT-mTOR-PPARγ-ACSL4 pathway. Interestingly, melatonin (MT), known for its antioxidant properties, protected against the above ZEN-induced effects by enhancing the binding of PPARγ to the promoter regions of ACSL4, which led to the upregulated expression of the ACSL4 gene. These results underscored the potential of MT to mitigate the adverse effects of ZEN on mammary cells, highlighting a way for potential therapeutic intervention.

Bovine mammary epithelial cells can grow and express milk protein synthesis genes at reduced fetal bovine serum concentration.

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions.

DNAJA1 positively regulates amino acid-stimulated milk protein and fat synthesis in bovine mammary epithelial cells.

Several cellular processes, including the recovery of misfolded proteins, the folding of polypeptide chains, transit of polypeptides across the membrane, construction and disassembly of protein complexes, and modulation of protein control, are carried out by DnaJ homologsubfamily A member 1(DNAJA1), which belongs to the DnaJ heat-shock protein family. It is unknown if DNAJA1 regulates the production of milk in bovine mammary epithelium cells (BMECs). Methionine and leucine increased DNAJA1 expression and nuclear location, as seen by us. In contrast to DNAJA1 knockdown, overexpression of DNAJA1 boosted the production of milk proteins and fats as well as mammalian target of rapamycin (mTOR) and sterol regulatory element binding protein-1c(SREBP-1c). As a result of amino acids, mTOR and SREBP-1c gene expression are stimulated, and DNAJA1 is a positive regulator of BMECs'amino acid-induced controlled milk protein and fat production.

Bovine neutrophils stimulated with Streptococcus uberis induce neutrophil extracellular traps, and cause cytotoxicity and transcriptional upregulation of inflammatory cytokine genes in bovine mammary epithelial cells.

This study aimed to understand the response of neutrophils stimulated by Streptococcus uberis, a major cause of mastitis. It was found that the production of neutrophil extracellular traps (NETs) was induced in milk clots from mastitic milk produced by S. uberis-infected bovine udders. The release of NETs from neutrophils stimulated by S. uberis was investigated. Bovine neutrophils cocultured with S. uberis in vitro released the components of NETs, which contained extracellular DNA and elastase. Bovine mammary epithelial cells (BMECs) incubated in coculture supernatants containing components of NETs, caused cytotoxicity and transcriptional upregulation of inflammatory cytokines, including of interleukin (IL) -1β, tumor necrosis factor (TNF)-α, IL-6, and IL-8, in BMECs. These findings suggest that bovine neutrophils stimulated by S. uberis induce responses that cause exacerbated inflammation, such as NET formation, cytotoxicity against BMECs, and increased production of inflammatory cytokines. Bovine neutrophil responses stimulated by S. uberis could be involved in the progression of S. uberis-induced mastitis.

Genes associated with fitness and disease severity in the pan-genome of mastitis-associated Escherichia coli.

Bovine mastitis caused by Escherichia coli compromises animal health and inflicts substantial product losses in dairy farming. It may manifest as subclinical through severe acute disease and can be transient or persistent in nature. Little is known about bacterial factors that impact clinical outcomes or allow some strains to outcompete others in the mammary gland (MG) environment. Mastitis-associated E. coli (MAEC) may have distinctive characteristics which may contribute to the varied nature of the disease. Given their high levels of intraspecies genetic variability, virulence factors of commonly used MAEC model strains may not be relevant to all members of this group. In this study, we sequenced the genomes of 96 MAEC strains isolated from cattle with clinical mastitis (CM). We utilized clinical severity data to perform genome-wide association studies to identify accessory genes associated with strains isolated from mild or severe CM, or with high or low competitive fitness during in vivo competition assays. Genes associated with mastitis pathogens or commensal strains isolated from bovine sources were also identified. A type-2 secretion system (T2SS) and a chitinase (ChiA) exported by this system were strongly associated with pathogenic isolates compared with commensal strains. Deletion of chiA from MAEC isolates decreased their adherence to cultured bovine mammary epithelial cells. The increased fitness associated with strains possessing this gene may be due to better attachment in the MG. Overall, these results provide a much richer understanding of MAEC and suggest bacterial processes that may underlie the clinical diversity associated with mastitis and their adaptation to this unique environment.

Innate immune response of bovine mammary epithelial cells in Mycoplasma bovis mastitis using an in vitro model of bovine mammary gland infection.

Mycoplasma bovis mastitisis highly contagious and disrupts lactation, posing a significant threat to the dairy industry. While the mammary gland's defence mechanism involves epithelial cells and mononuclear cells (MNC), their interaction with M. bovis remains incompletely understood. In this study, we assessed the immunological reactivity of bovine mammary epithelial cells (bMEC) to M. bovis through co-culture with MNC. Upon co-culture with MNC, the mRNA expression levels of interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α in bMEC stimulated by M. bovis showed a significant increase compared to monoculture. Additionally, when stimulated with M. bovis, the culture supernatant exhibited significantly higher concentrations of IL-6 and interferon (IFN)-γ, while IL-1β concentration tended to be higher in co-culture with MNC than in monoculture. Furthermore, the mRNA expression levels of toll-like receptor (TLR) 2 in bMEC stimulated with M. bovis tended to increase, and TLR4 significantly increased when co-cultured with MNC compared to monocultures. However, the surface expression levels in bMEC did not exhibit significant changes between co-culture and monoculture. Overall, our research indicates that the inflammatory response of bMEC is increased during co-culture with MNC, suggesting that the interaction between bMEC and MNC in the mammary gland amplifies the immune response to M. bovis in cows affected by M. bovis mastitis.

Acylated Ghrelin Activates PI3K/mTOR Signaling Pathway by Promoting ThPOK Acetylation to Promote Milk Fat Synthesis in Bovine Mammary Epithelial Cells.

Ghrelin regulates diverse physiological activities. However, the effects of this hormone on the milk fat synthesis remain unknown. This study aimed to investigate the effect of acylated ghrelin (AG) on milk fat synthesis by modifying the expression (knockdown or overexpression) of growth hormone secretagogue receptor 1a (GHSR1a) and Th-inducing POK (ThPOK) in primary bovine mammary epithelial cells (BMECs). The results showed that AG significantly increased the triglyceride relative content from 260.83 ± 9.87 to 541.67 ± 8.38 in BMECs via GHSR1a. ThPOK functions as a key regulatory target downstream of AG, activating the PI3K and mTOR signaling pathways to promote milk fat synthesis in BMECs. Moreover, AG-regulated ThPOK by increasing the EP300 activity, which promoted ThPOK acetylation to protect it from proteasomal degradation. In conclusion, AG increases ThPOK acetylation and stabilizes ThPOK through GHSR1a, thereby activating the PI3K/mTOR signaling pathway and ultimately promoting the milk fat synthesis in BMECs.