Primary Bovine Mammary Epithelial Cells Research Articles

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.

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.

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.

Enhancement of PPARα-Inhibited Leucine Metabolism-Stimulated β-Casein Synthesis and Fatty Acid Synthesis in Primary Bovine Mammary Epithelial Cells.

Leucine, a kind of branched-chain amino acid, plays a regulatory role in the milk production of mammalian mammary glands, but its regulatory functions and underlying molecular mechanisms remain unknown. This work showed that a leucine-enriched mixture (LEUem) supplementation increased the levels of milk protein and milk fat synthesis in primary bovine mammary epithelial cells (BMECs). RNA-seq of leucine-treated BMECs indicated alterations in lipid metabolism, translation, ribosomal structure and biogenesis, and inflammatory response signaling pathways. Meanwhile, the supplementation of leucine resulted in mTOR activation and increased the expression of BCKDHA, FASN, ACC, and SCD1. Interestingly, the expression of PPARα was independently correlated with the leucine-supplemented dose. PPARα activated by WY-14643 caused significant suppression of lipogenic genes expression. Furthermore, WY-14643 attenuated leucine-induced β-casein synthesis and enhanced the level of BCKDHA expression. Moreover, promoter analysis revealed a peroxisome-proliferator-response element (PPRE) site in the bovine BCKDHA promoter, and WY-14643 promoted the recruitment of PPARα onto the BCKDHA promoter. Together, the present data indicate that leucine promotes the synthesis of β-casein and fatty acid and that PPARα-involved leucine catabolism is the key target.

Successful editing and maintenance of lactogenic gene expression in primary bovine mammary epithelial cells

In vitro investigation of bovine lactation processes is limited by a lack of physiologically representative cell models. This deficiency is most evident through the minimal or absent expression of lactation-specific genes in cultured bovine mammary tissues. Primary bovine mammary epithelial cells (pbMECs) extracted from lactating mammary tissue and grown in culture initially express milk protein transcripts at relatively representative levels. However, expression drops dramatically after only three or four passages, which greatly reduces the utility of primary cells to model and further examine lactogenesis. To investigate the effects of alternate alleles in pbMECs including effects on transcription, we have developed methods to deliver CRISPR-Cas9 gene editing reagents to primary mammary cells, resulting in very high editing efficiencies. We have also found that culturing the cells on an imitation basement membrane composed of Matrigel, results in the restoration of a more representative lactogenic gene expression profile and the cells forming three-dimensional structures in vitro. Here, we present data from four pbMEC lines recovered from pregnant cows and detail the expression profile of five key milk synthesis genes in these MECs grown on Matrigel. Additionally, we describe an optimised method for preferentially selecting CRISPR-Cas9-edited cells conferring a knock-out of DGAT1, using fluorescence-activated cell sorting (FACS). The combination of these techniques facilitates the use of pbMECs as a model to investigate the effects of gene introgressions and genetic variation in lactating mammary tissue.

Investigating Genetic Characteristics of Chinese Holstein Cow’s Milk Somatic Cell Score by Genetic Parameter Estimation and Genome-Wide Association

The quality and safety of milk is challenged by cow mastitis, and the value of somatic cell score (SCS) in milk is closely related to the occurrence of mastitis. This study aimed to analyze the genetic characteristics of SCS across the first three parities in Chinese Holstein cattle, as well as to investigate potential candidate genes and biological processes that may play a potential role in the progress of cow mastitis. In this respect, we evaluated genetic parameters and conducted a genome-wide association study based on the test-day records of SCS for Chinese Holstein cows; we also validated key candidate genes using a quantitative reverse transcription PCR (RT-qPCR) experiment in primary bovine mammary epithelial cells (bMECs). The heritability of the SCS 305-day performance in milk varied between 0.07 and 0.24, and decreased with increasing parity. As the time interval grew larger, the genetic and permanent environmental correlations with the number of days in milk (DIM) weakened. Six significant single-nucleotide polymorphisms (SNPs) were identified in the association analysis, one of which was located within the exonic region of CD44. This exon-associated SNP may modify the activity of the protein encoded by the CD44. A total of 32 genes within the two hundred kilobase (kb) range of significant SNPs were detected, and these genes were markedly enriched in eight Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 22 biological processes, mainly participating in the progress of transmembrane transport, inflammatory factor regulation, cellular responses, the Toll-like receptor signaling pathway, and the MAPK signaling pathway. Nine genes, including the PKD2, KCNAB1, SLC35A4, SPP1, IBSP, CD14, CD44, MAPK10, and ABCG2 genes, were selected as candidate genes that could have critical functions in cow mastitis. These findings can serve as a foundation for molecular breeding and as valuable data for reducing the incidence of mastitis of Chinese Holstein cattle at the molecular level.

Lysine promotes proliferation and β-casein synthesis through the SLC6A14-ERK1/2-CDK1-mTOR signaling pathway in bovine primary mammary epithelial cells

Lysine, as the first limiting amino acid in dairy cows, has been shown to play an important role in milk synthesis and cell proliferation. However, the underlying mechanism remains unclear. In this study, we isolated bovine primary mammary epithelial cells (BMECs) and studied the mechanism in which lysine promotes cell proliferation and β-casein synthesis through overexpression and knockdown of CDK1 and supplements BCH, U0126, and rapamycin in BMECs. Results show that 0.7 mM lysine can significantly promote cell proliferation and the synthesis of β-casein in BMECs. In addition, lysine activates the ERK signaling pathway to promote the expression of CDK1. Further studies have shown that CDK1 can promote cell proliferation and the synthesis of β-casein through the mTOR signaling pathway in BMECs. Lastly, lysine can promote cell proliferation and the synthesis of β-casein through SLC6A14 in BMECs. The above results indicate that lysine promotes cell proliferation and the synthesis of β-casein through the SLC6A14-ERK-CDK1-mTOR signaling pathway in BMECs.

Investigation of the pathophysiology of bacterial mastitis using precision-cut bovine udder slices

Mastitis in cattle is a major health problem as well as incurring high costs for the dairy industry. To assess the suitability of precision-cut bovine udder slices (PCBUS) for bovine mastitis studies, we infected PCBUS with 2 different Staphylococcus aureus strains. Accordingly, we investigated both the tissue response to infection based on immune mediators at the mRNA and protein levels and the invasion of bacteria within the tissue. The studied proteins represent immune mediators of early inflammation [IL-1β, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2)] and showed a time-dependent increase in concentration. Infection of PCBUS with S. aureus resulted in increased expression of proinflammatory cytokines and chemokines such as TNF-α, C-C motif chemokine ligand 20 (CCL20), IL-1β, IL-6, and IL-10, but not C-X-C motif chemokine ligand 8 (CXCL8), lingual antimicrobial peptide (LAP), or S100 calcium binding protein A9 (S100A9) at the mRNA level. To compare the data acquired with this model, we carried out investigations on primary bovine mammary epithelial cells. Our results showed that the immune responses of both models-PCBUS and primary bovine mammary epithelial cells-were similar. In addition, investigations using PCBUS enabled us to demonstrate adherence of bacteria in the physiological cell network. These findings support the use of PCBUS in studies designed to further understand the complex pathophysiological processes of infection and inflammation in bovine mastitis and to investigate alternative therapies for mastitis.

GPRC6A is a key mediator of palmitic acid regulation of lipid synthesis in bovine mammary epithelial cells.

Fatty acids (FAs) can promote lipid synthesis in the mammary gland via stimulating lipogenic gene expression, but the underlying molecular mechanism is still not fully understood. Here, we showed the dose-dependent effects of palmitic acid (PA) on lipid synthesis in primary bovine mammary epithelial cells (BMECs) and explored the corresponding molecular mechanism. BMECs were treated with PA (0, 50, 100, 150, and 200 μM), and the 100 μM treatment had the best stimulatory effect on lipid synthesis and expression and maturation of sterol regulatory element-binding protein 1c (SREBP-1c) in cells. Inhibition of phosphatidylinositol 3-kinase (PI3K) almost totally blocked the stimulation of PA on SREBP-1c expression, whereas protein kinase Cα (PKCα) knockdown only partially decreased the stimulation of PA on SREBP-1c expression but abolished the stimulation of PA on its maturation. Knockdown of GPR120 did not change the stimulation of PA on the SREBP-1c signaling. G protein-coupled receptor family C group 6 member A (GPRC6A) knockdown almost totally blocked the stimulation of FA on PI3K and PKCα phosphorylation as well as SREBP-1c expression and maturation. Furthermore, PA dose-dependently promoted GPRC6A expression and plasma membrane localization. Together, these above results reveal that GPRC6A is a key mediator of PA signaling to lipid synthesis in BMECs via the PI3K/PKCα-SREBP-1c pathways.

SV40T/E6E7-Induced Proliferation Is Involved in the Activity of E2F3 in Bovine Mammary Epithelial Cells.

Simple SummaryA previous study demonstrated that SV40T does not require E2F1, E2F2, and E2F3 activators to induce proliferation in mouse embryo fibroblasts (MEFs). Our results showed that, at an early stage, primary bovine mammary epithelial cells (BMECs) lacking the E2F1 expression have the capacity to proliferate and show E2F2 and E2F3 slight protein levels. At a late stage, primary BMECs deficient for E2F3 completely abolish any proliferative ability and exhibit a severe cell senescence signal, although the E2F2 can be expressed at a late stage of primary BMECs. Compared with the late stage of primary BMECs, the BMECs immortalized by SV40T and E6E7 restored the protein level of E2F3. In conclusion, this study revealed a molecular mechanism where E2F3 controls the BMECs’ proliferation and senescence.The E2F family of transcription factor is divided into activators and repressors that control cell proliferation. Bovine mammary epithelial cells (BMECs) can be immortalized using human papillomavirus 16 E6E7 (HPV16 E6E7) and simian vacuolating virus 40 large T antigen (SV40T). In addition, SV40T does not require E2F1, E2F2, and E2F3 activators to induce proliferation in mouse embryo fibroblasts (MEFs). However, we report that E2F3 activator is required to induce the proliferation of BMECs. Our results showed that, at an early stage, primary BMECs lacking the E2F1 expression have the capacity to proliferate and show E2F2 and E2F3 slight protein levels. At a late stage, primary BMECs deficient for E2F3 completely abolish any proliferative ability and exhibit a severe cell senescence signal, although the E2F2 can be expressed at a late stage of primary BMECs. Compared with the late stage of primary BMECs, the BMECs immortalized by SV40T and E6E7 restored the protein level of E2F3 and enhanced the CDK4, CDK6, cyclin D3, and CDK2 protein level, leading to proliferating robustly. Surprisingly, it was found that p53, p21Cip1, and p27Kip1 were upregulated in SV40T and E6E7-immortalized BMECs, relatively to primary BMECs. Notably, Cdc2 was almost expressed in primary BMECs. However, Cdc2 was elevated in BMECs immortalized by SV40T and E6E7. In conclusion, this study revealed a molecular mechanism where E2F3 controls the BMECs’ proliferation and senescence.

Lycium barbarum Polysaccharide Inhibits E. coli-Induced Inflammation and Oxidative Stress in Mammary Epithelial Cells of Dairy Cows via SOCS3 Activation and MAPK Suppression

Escherichia coli (E. coli) is one of the main causative agents of mastitis in dairy cows. Lycium barbarum polysaccharide (LBP) has a variety of physiological effects as it has antioxidants, it is hypoglycemic, it has anti-aging properties, it is neuroprotective, immune boosting, and it has anti-inflammatory effects in vivo and in vitro. In this study, we examined whether LBP affects the expression of pro-inflammatory factors, and the mitogen-activated protein kinase (MAPK) signaling pathway via activation of the suppressor of cytokine signaling-3 (SOCS3) in E. coli-induced primary bovine mammary epithelial cell (pbMEC) inflammatory responses. The experiment was designed with the control group (NC), cells were treated with E. coli for 6 h as the E. coli group (E. coli), and cells were pretreated with 100 μg/mL or 300 μg/mL of LBP for 24 h, followed by the addition of E. coli for 6 h as the E. coli + low level (E + LL) or E. coli + high level (E + HL) groups. The addition of LBP did not alter the cell viability of pbMEC in a dose-dependent assay. Pretreatment with LBP significantly decreased the expression of pro-inflammatory genes (IL1B, MAPK14, COX-2, iNOS) and proteins (COX-2, IL-1β, TNF-α) in the cells challenged by E. coli as compared with the control group (p &lt; 0.05). E. coli stimulation significantly increased the production of reactive oxygen species (ROS) and malondialdehyde (MDA) in pbMEC, and decreased the antioxidants’ capacity with regard to decreased superoxide dismutase (SOD) and total antioxidant capacity (T-AOC); however, pretreatment with LBP reversed the oxidative stress and inhibition of antioxidants in cells challenged by E. coli. Moreover, LBP reversed the upregulated expression of the components of the MAPK pathway (increased phosphorylation level of p38, JNK, and ERK), followed by E. coli stimulation. Consistently, cells exposed to E. coli strengthened the staining of p38, whereas pretreatment of LBP weakened the staining of p38 in cells challenged by E. coli. Notably, the expression of SOCS3 was increased by LBP added to the cells in a dose-dependent manner. Additionally, the level of decreased expression of proinflammatory factors (IL-1β, TNF-α, and COX-2) was higher in the E + LL group than in the E + HL group. These results indicate that LBP pretreatment is effective in the alleviation of E. coli-induced inflammatory and oxidative responses in pbMEC through activation of SOCS3 and depression of MAPK signaling. As such, this might help us to develop molecular strategies for mitigating the detrimental effects of clinical bovine mastitis.

Stearic Acid Activates the PI3K-mTOR-4EBP1/S6K and mTOR-SREBP-1 Signaling Axes through FATP4-CDK1 To Promote Milk Synthesis in Primary Bovine Mammary Epithelial Cells.

Stearic acid (SA), an 18-carbon long-chain saturated fatty acid, has great potential for promoting lactation. Therefore, this study investigates the effects and mechanism of SA on milk synthesis in primary bovine mammary epithelial cells (BMECs). In our study, we found that SA significantly increased β-casein and triglycerides, and the effect was most significant at 100 μM. Signaling pathway studies have found that SA affects milk synthesis by upregulating cyclin-dependent kinase 1 (CDK1) to activate PI3K-mTOR-4EBP1/S6K and mTOR-SREBP-1 pathways. Furthermore, we detected fatty acid transport proteins (FATPs) when BMECs were treated with SA; the mRNA levels of FATP3 (3.713 ± 0.583) and FATP4 (40.815 ± 8.959) were significantly upregulated at 100 μM. Subsequently, we constructed FATP4-siRNA and found that SA was transported by FATP4 into BMECs, promoting milk synthesis. Collectively, these results revealed that SA activated PI3K-mTOR-4EBP1/S6K and mTOR-SREBP-1 signaling axes through FATP4-CDK1 to promote milk synthesis in BMECs.

In vitro effects of 5-Hydroxy-L-tryptophan supplementation on primary bovine mammary epithelial cell gene expression under thermoneutral or heat shock conditions

Serotonin (5-HT) is an autocrine-paracrine molecule within the mammary gland regulating homeostasis during lactation and triggering involution after milk stasis. Exposure of dairy cows to hyperthermia during the dry period alters mammary gland involution processes leading to reduced subsequent yields. Herein, primary bovine mammary epithelial cells (pBMEC) under thermoneutral (TN, 37 °C) or heat shock (HS, 41.5 °C) conditions were cultured with either 0, 50, 200, or 500 μM 5-Hydroxy-L-tryptophan (5-HTP; 5-HT precursor) for 8-, 12- or 24-h. Expression of 95 genes involved in 5-HT signaling, involution and tight junction regulation were evaluated using a Multiplex RT-qPCR BioMark Dynamic Array Circuit. Different sets of genes were impacted by 5-HTP or temperature, or by their interaction. All 5-HT signaling genes were downregulated after 8-h of HS and then upregulated after 12-h, relative to TN. After 24-h, apoptosis related gene, FASLG, was upregulated by all doses except TN-200 μM 5-HTP, and cell survival gene, FOXO3, was upregulated by HS-50, 200 and 500 μM 5-HTP, suggesting 5-HTP involvement in cell turnover under HS. Supplementing 5-HTP at various concentrations in vitro to pBMEC modulates the expression of genes that might aid in promoting epithelial cell turn-over during involution in dairy cattle under hyperthermia.

Effects of Essential Amino Acid Deficiency on General Control Nonderepressible 2/Eukaryotic Initiation Factor 2 Signaling and Proteomic Changes in Primary Bovine Mammary Epithelial Cells.

We hypothesized that the general control nonderepressible 2 (GCN2)/eukaryotic initiation factor 2 (eIF2) signaling pathway and intracellular protein synthesis (PS) are regulated to maintain milk PS in primary bovine mammary epithelial cells (MECs) under essential amino acid (EAA) starvation conditions. We cultured MECs with 0%, 2% (depletion), and 100% (control) EAA for two exposure times (8 and 24 h), followed by three refeeding (RF) times with 100% EAA (0, 8, and 24 h). Subsequently, we measured cell viability, total protein concentration, and proliferation. Western blotting was used to quantify the levels of casein and the expression of total GCN2 and eIF2, as well as phosphorylated GCN2 (GCN2P) and eIF2 (eIF2P). The ISOQuant method was used to assess MEC proteomes, and the resultant data were analyzed using the Kruskal–Wallis test, nonpaired Wilcoxon rank post-hoc test, and ANOVA–Tukey test, as well as principal component analyses and multiple regressions models. Differences in cell viability were observed between the control versus the depleted and repleted MECs, respectively, where 97.2–99.8% viability indicated low cell death rates. Proliferation (range, 1.02–1.55 arbitrary units (AU)) was affected by starvation for 12 and 24 h and repletion for 24 h, but it was not increased compared with the control. Total protein expression was unaffected by both depletion and repletion treatments (median 3158 µg/mL). eIF2P expression was significantly increased (p < 0.05) after treatment with 2% EAA for 8 and 24 h compared with 2% EAA with 8 h + 24 h RF and 2% EAA with 24 h + 8 h RF. GCN2P also showed significantly increased expression (p < 0.05) after treatment with 2% EAA for 24 h compared with the control and 2% EAA with 24 h + 8 h RF. Intracellular casein/α-tubulin expression was unaffected by 2% EAA compared with control (0.073 ± 0.01 AU versus 0.086 ± 0.02 AU, respectively). We studied 30 of the detected 1180 proteins, 16 of which were differentially expressed in starved and refed MECs. Cells faced with EAA deficiency activated the GCN2P/eIF2P pathway, and the lack of change in the levels of casein and other milk proteins suggested that the EAA deficit was mitigated by metabolic flexibility to maintain homeostasis.

Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein.

Mastitis is one of the most serious diseases that causes losses in the dairy industry, seriously impairing milk production and milk quality, and even affecting human health. Menthol is a cyclic monoterpene compound obtained from the stem and leaves of peppermint, which has a variety of biological activities, including anti-inflammatory and antioxidant activity. The purpose of this study was to investigate the preventive effect of menthol on the lipopolysaccharide-induced inflammatory response in primary bovine mammary gland epithelial cells (BMECs) and its anti-inflammatory mechanism. First, BMECs were isolated and amplified from the udders of Holstein cows by enzymatic hydrolysis. BMECs were treated with menthol (10, 50, 100, 200 μM) for 1h, followed by lipopolysaccharide (5μg/ml) for 12 h. Lipopolysaccharide treatment upregulated the protein levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (INOS) and the mRNA abundance of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), while menthol was able to inhibit this effect. The inhibitory effect of menthol on proinflammatory factors was significantly reduced when autophagy was blocked using 3-Methyladenine (5μg/ml), an inhibitor of autophagy. Furthermore, lipopolysaccharide treatment reduced the expression levels of milk lipids and milk proteins, which were inhibited by menthol. In addition, menthol (200 μM) treatment was able to significantly upregulate the expression level of autophagy-related protein LC3B, downregulate the expression level of P62, promote the expression abundance of autophagy-related gene mRNA, and enhance significantly enhance autophagic flux. Interestingly, treatment of BMECs with menthol (200 μM) promoted the phosphorylation of AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1) and increased the nuclear localization of nuclear factor-E2 associated factor 2 (Nrf-2). When the AMPK pathway was blocked using compound C (10μg/ml), an inhibitor of AMPK, autophagy was significantly inhibited. Autophagy levels were significantly decreased after blocking the Nrf-2 pathway using ML385 (5μg/ml), an inhibitor of Nrf-2. Overall, the data suggest that menthol activates the AMPK-ULK1 pathway to initiate the onset of autophagy and maintains the level of autophagy through the AMPK-Nrf-2 pathway. In conclusion, the findings suggest that menthol may alleviate the inflammatory response in BMECs via the AMPK/ULK1/Nrf-2/autophagy pathway.

Escherichia coli and Staphylococcus aureus Differentially Regulate Nrf2 Pathway in Bovine Mammary Epithelial Cells: Relation to Distinct Innate Immune Response.

Escherichia coli and Staphylococcus aureus are major mastitis causing pathogens in dairy cattle but elicit distinct immune and an inflammatory response in the udder. However, the host determinants responsible for this difference remains largely unknown. Our initial studies focused on the global transcriptomic response of primary bovine mammary epithelial cells (pbMECs) to heat-killed E. coli and S. aureus. RNA-sequencing transcriptome analysis demonstrates a significant difference in expression profiles induced by E. coli compared with S. aureus. A major differential response was the activation of innate immune response by E. coli, but not by S. aureus. Interestingly, E. coli stimulation increased transcript abundance of several genes downstream of Nrf2 (nuclear factor erythroid 2-related factor 2) that were enriched in gene sets with a focus on metabolism and immune system. However, none of these genes was dysregulated by S. aureus. Western blot analysis confirms that S. aureus impairs Nrf2 activation as compared to E. coli. Using Nrf2-knockdown cells we demonstrate that Nrf2 is necessary for bpMECs to mount an effective innate defensive response. In support of this notion, nuclear Nrf2 overexpression augmented S. aureus-stimulated inflammatory response. We also show that, unlike E. coli, S. aureus disrupts the non-canonical p62/SQSTM1-Keap1 pathway responsible for Nrf2 activation through inhibiting p62/SQSTM1 phosphorylation at S349. Collectively, our findings provide important insights into the contribution of the Nrf2 pathway to the pathogen-species specific immune response in bovine mammary epithelial cells and raise a possibility that impairment of Nrf2 activation contributes to, at least in part, the weak inflammatory response in S. aureus mastitis.

Circadian Gene PER2 Silencing Downregulates PPARG and SREBF1 and Suppresses Lipid Synthesis in Bovine Mammary Epithelial Cells.

Simple SummaryThe present study was constructed to determine the effects of the core circadian clock gene, Period 2 (PER2), on lipid synthesis in bovine mammary epithelial cells (BMECs). Data revealed that PER2-regulated genes were involved in fatty acid de novo synthesis, desaturation, TAG accumulation, and lipid droplet secretion in primary BMECs, partly by inhibiting PPARG and SREBF1. Our overall data suggests that PER2 in bovine mammary cells plays a role in regulating milk fat synthesis directly, or via the activation of the transcription regulators PPARG and SREBF1. This study provides molecular evidence underscoring a link between the circadian clock and lipid metabolism in bovines.PER2, a circadian clock gene, is associated with mammary gland development and lipid synthesis in rodents, partly via regulating peroxisome proliferator-activated receptor gamma (PPARG). Whether such a type of molecular link existed in bovines was unclear. We hypothesized that PER2 was associated with lipid metabolism and regulated cell cycles and apoptosis in bovine mammary epithelial cells (BMECs). To test this hypothesis, BMECs isolated from three mid-lactation (average 110 d postpartum) cows were used. The transient transfection of small interfering RNA (siRNA) was used to inhibit PER2 transcription in primary BMECs. The silencing of PER2 led to lower concentrations of cellular lipid droplets and triacylglycerol along with the downregulation of lipogenic-related genes such as ACACA, FASN, LPIN1, and SCD, suggesting an overall inhibition of lipogenesis and desaturation. The downregulation of PPARG and SREBF1 in response to PER2 silencing underscored the importance of circadian clock signaling and the transcriptional regulation of lipogenesis. Although the proliferation of BMECs was not influenced by PER2 silencing, the number of cells in the G2/GM phase was upregulated. PER2 silencing did not affect cell apoptosis. Overall, the data provided evidence that PER2 participated in the coordination of mammary lipid metabolism and was potentially a component of the control of lipid droplets and TAG synthesis in ruminant mammary cells. The present data suggested that such an effect could occur through direct effects on transcriptional regulators.

Lycium barbarum polysaccharides alleviate LPS-induced inflammatory responses through PPARγ/MAPK/NF-κB pathway in bovine mammary epithelial cells.

Current research examined Lycium barbarum polysaccharides (LBP) for combating LPS-induced inflammatory responses in primary bovine mammary epithelial cells. We uncovered a preventive role of LBP in reducing detrimental effects induced by LPS including inhibition of NF-κB and MAPK along with peroxisome proliferator-activated receptor-γ (PPARγ) activation. The decrease in cell proliferation due to LPS was curtailed by pretreatment with LBP. Moreover, the effect of LBP on regulation of inflammatory responses in bovine mammary epithelial cell was PPARγ dependent. Collectively, data suggest that LBP reverses LPS-induced inflammatory response via MAPK/NF-κB signaling in a PPARγ-activation-dependent manner. Thus, the study provides new insights into therapeutic strategies for combating mastitis using LBP and highlighted the link between PPARγ and regulation of mammary cell inflammation.

Directed Expression of Tracheal Antimicrobial Peptide as a Treatment for Bovine-Associated Staphylococcus Aureus-Induced Mastitis in Mice.

Bovine mastitis is perplexing the dairy industry since the initiation of intensive dairy farming, which has caused a reduction in the productivity of cows and an escalation in costs. The use of antibiotics causes a series of problems, especially the formation of bacterial antimicrobial resistance. However, there are limited antibiotic-free therapeutic strategies that can effectively relieve bacterial infection of bovine mammary glands. Hence, in this study, we constructed a mammary gland tissue-specific expression vector carrying the antimicrobial peptide of bovine-derived tracheal antimicrobial peptide (TAP) and evaluated it in both primary bovine mammary epithelial cells (pBMECs) and mice. The results showed that the vector driven by the β-lactoglobulin gene (BLG) promoter could efficiently direct the expression of TAP in pBMECs and the mammary gland tissue of mice. In addition, significant antibacterial effects were observed in both in vitro and in vivo experiments when introducing this vector to bovine-associated Staphylococcus aureus-treated pBMECs and mice, respectively. This study demonstrated that the mammary gland tissue-specific expression vector could be used to introduce antimicrobial peptide both in in vitro and in vivo and will provide a new therapeutic strategy in the treatment of bovine mastitis.

Sirtuin 3 inhibits nuclear factor-κB signaling activated by a fatty acid challenge in bovine mammary epithelial cells

Susceptibility to mastitis is highest during the peripartal (transition) period and is often concomitant with other comorbidities such as ketosis. Although infection with pathogenic microorganisms and immune-dysfunction around calving clearly play key roles in mastitis development, other metabolic factors also contribute. Sirtuin 3 (SIRT3), a mitochondrial deacetylase regulating energy and redox homeostasis, antagonizes the lipotoxic effects of nonesterified fatty acids (NEFA). Thus, we hypothesized that increases in circulating NEFA concentrations, as observed in the transition period, provokes inflammatory responses that can be reversed via activation of SIRT3. Here we aimed to study (1) proinflammatory NF-κB signaling and SIRT3 abundance in mammary tissue of ketotic cows and healthy controls, and (2) the effect of SIRT3 on NF-κB activation in bovine mammary epithelial cells (BMEC) treated with high levels of NEFA. The mammary gland biopsy samples were from a previous study, which included 15 healthy cows and 15 ketotic cows. Primary BMEC were isolated from 3 healthy Holstein cows with collagenase III digestion. Purified BMEC were incubated with or without SIRT3 overexpression adenovirus for 48 h, then treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 24 h. Mammary tissue of ketotic cows was associated with lower protein abundance of SIRT3 along with greater NF-κB P65 phosphorylation levels (p-NF-κB P65), p-NF-κB P65:NF-κB P65 ratio, and mRNA abundance of IL1B and IL6. In BMEC, exogenous NEFA dose-dependently reduced protein abundance of SIRT3, but increased p-NF-κB P65, p-NF-κB P65:NF-κB P65 ratio, and mRNA abundance of IL1B and IL6. Compared with green fluorescent protein adenovirus vector + NEFA, overexpression of SIRT3 in NEFA-treated BMEC downregulated p-NF-κB P65 and mRNA abundance of IL1B and IL6. Immunofluorescence indicated that overexpression of SIRT3 inhibited nuclear translocation of NF-κB P65. Overall, our data demonstrated that ketosis is associated with a reduction in SIRT3 abundance and activation of NF-κB signaling in the mammary gland. In vitro data provided evidence that high NEFA concentrations inhibit SIRT3, which contributes to enhanced NF-κB signaling including nuclear translocation and a pro-inflammatory response. The data suggest a promising role of SIRT3 as a target for helping alleviate localized inflammation of the mammary gland resulting from exposure to high concentrations of NEFA.