MAC-T Cells Research Articles

Effect of hyperthermia on cell viability, amino acid transfer, and milk protein synthesis in bovine mammary epithelial cells.

The reduction of milk yield caused by heat stress in summer is the main condition restricting the economic benefits of dairy farms. To examine the impact of hyperthermia on bovine mammary epithelial (MAC-T) cells, we incubated the MAC-T cells at thermal-neutral (37°C, CON group) and hyperthermic (42°C, HS group) temperatures for 6 h. Subsequently, the cell viability and apoptotic rate of MAC-T cells, apoptosis-related genes expression, casein and amino acid transporter genes, and the expression of the apoptosis-related proteins were examined. Compared with the CON group, hyperthermia significantly decreased the cell viability (p < 0.05) and elevated the apoptotic rate (p < 0.05) of MAC-T cells. Moreover, the expression of heat shock protein (HSP)70, HSP90B1, Bcl-2-associated X protein (BAX), Caspase-9, and Caspase-3 genes was upregulated (p < 0.05). The expression of HSP70 and BAX (pro-apoptotic) proteins was upregulated (p < 0.05) while that of B-cell lymphoma (BCL)2 (antiapoptotic) protein was downregulated (p < 0.05) by hyperthermia. Decreased mRNA expression of mechanistic target of rapamycin (mTOR) signaling pathway-related genes, amino acid transporter genes (SLC7A5, SLC38A3, SLC38A2, and SLC38A9), and casein genes (CSNS1, CSN2, and CSN3) was found in the heat stress (HS) group (p < 0.05) in contrast with the CON group. These findings illustrated that hyperthermia promoted cell apoptosis and reduced the transport of amino acids into cells, which inhibited the milk proteins synthesis in MAC-T cells.

Single-Chain Fragment Variables Targeting Leukocidin ED Can Alleviate the Inflammation of Staphylococcus aureus-Induced Mastitis in Mice.

Staphylococcus aureus is a vital bovine mastitis pathogen causing huge economic losses to the dairy industry worldwide. In our previous studies, leukotoxin ED (LukED) was detected in most S. aureus strains isolated from bovine mastitis. Here, four single-chain fragment variables (scFvs) (ZL8 and ZL42 targeting LukE, ZL22 and ZL23 targeting LukD) were obtained using purified LukE and LukD proteins as the antigens after five rounds of bio-panning. The complementarity-determining region 3 (CDR3) of the VH domain of these scFvs exhibited significant diversities. In vitro, the scFvs significantly decreased LukED-induced cell killing by inhibiting the binding of LukED to chemokine receptors (CCR5 and CXCR2) and reduced the death rates of bovine neutrophils and MAC-T cells caused by LukED and S. aureus (p < 0.05). In an S. aureus-induced mouse mastitis model, histopathology and MPO results revealed that scFvs ameliorated the histopathological damages and reduced the infiltration of inflammatory cells (p < 0.05). The ELISA and qPCR assays showed that scFvs reduced the transcription and expression levels of Tumor Necrosis Factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8 and IL-18 (p < 0.05). The overall results demonstrated the protective anti-inflammatory effect of scFvs in vitro and in vivo, enlightening the potential role of scFvs in the prevention and treatment of S. aureus-induced mastitis.

Aclonifen induces bovine mammary gland epithelial cell death by disrupting calcium homeostasis and inducing ROS production

Herbicides play key roles in agriculture. Aclonifen is a diphenyl ether herbicide that is widely used for sunflower, potato, corn, and wheat crops. Since it has a long half-life, it is considered persistent and can easily accumulate in the environment. Therefore, livestock and humans are at risk of exposure to aclonifen. Importantly, aclonifen is toxic to several mammals such as rats, mice, and dogs. However, the toxicity of aclonifen in cattle remains unclear. Therefore, we sought to investigate its toxicity in cattle using bovine mammary gland epithelial cells (MAC-T). We found that aclonifen induces sub-G1 phase arrest and represses MAC-T proliferation. In addition, aclonifen caused mitochondrial dysfunction, as evidenced by excessive ROS production and loss of mitochondrial membrane potential. Furthermore, cytosolic and mitochondrial calcium homeostases were disrupted after aclonifen treatment. Moreover, aclonifen treatment caused alterations in the PI3K/AKT and MAPK signaling pathways, which are involved in the regulation of cell survival and death. In conclusion, aclonifen causes MAC-T cell death through mitochondrial dysfunction and the collapse of calcium homeostasis.

Fluroxypyr-1-methylheptyl ester causes apoptosis of bovine mammary gland epithelial cells by regulating PI3K and MAPK signaling pathways and endoplasmic reticulum stress

Fluroxypyr-1-methylheptyl ester (FPMH) is an auxin herbicide that is widely applied to crops and pastures to block growth of post-emergence weeds. Several studies have reported the toxicity of FPMH in aquatic vertebrates. However, the adverse impacts of FPMH on mammals, including domestic animals, have not been reported. The purpose of our current study is to assess the impact of FPMH on the bovine mammary system and milk production. To evaluate the toxicity of FPMH on the mammary glands of lactating cows, the bovine mammary gland epithelial cell line, MAC-T, was exposed to various concentrations (0, 5, 7.5, 10, 15, and 20 μM) of FPMH for 24 h, and then various assessments were performed. The results showed that FPMH dose-dependently reduced MAC-T cell viability following exposure to FPMH and induced mitochondrial depolarization and apoptosis. FPMH also modulated signaling through the PI3K and MAPK pathways. In addition, the expression levels of proteins related to endoplasmic reticulum (ER) stress were upregulated, indicating induction of ER stress, and calcium homeostasis was disrupted following FPMH treatment. In conclusion, our investigation suggests that FPMH may be toxic to the bovine mammary system and may decrease dairy production.

Evaluation of the relative expression of genes associated with adherence after different hours of co-culture between Streptococcus uberis and MAC-T cells

Streptococcus uberis is an environmental pathogen associated with subclinical and clinical IMI in both lactating and non-lactating cows. RC19 strain was isolated from a cow with subclinical mastitis, qualitatively classified as moderate biofilm producer in Todd Hewitt medium (THB), and it showed a high value of the adhered bacteria (CFU/ml). Hence, the aims of this study were (a) to determine ability to adhere to and internalize into epithelial cells MAC-T for 1, 2 and 3 h, (b) to evaluate the relative expression of adherence-associated genes from co-cultures of S. uberis with MAC-T cells at 1, 2 and 3 h. We hypothesized that upon contact with bovine mammary epithelial cells, S. uberis upregulates adherence-associated genes encoding adhesins, which enable it a higher adherence to and/or internalization into host cells. Four to six genes increased their R with regard to the control after initial contact with MAC-T cells (group 1) at 1, 2 and 3 h. The highest value of R was observed at 2 h after co-culture between RC19 and MAC-T cells.

Transcriptome sequencing analysis for the identification of stable lncRNAs associated with bovine Staphylococcus aureus mastitis

BackgroundStaphylococcus aureus (S. aureus) mastitis is one of the most difficult diseases to treat in lactating dairy cows worldwide. S. aureus with different lineages leads to different host immune responses. Long non-coding RNAs (lncRNAs) are reported to be widely involved in the progress of inflammation. However, no research has identified stable lncRNAs among different S. aureus strain infections. In addition, folic acid (FA) can effectively reduce inflammation, and whether the inflammatory response caused by S. aureus can be reduced by FA remains to be explored.MethodslncRNA transcripts were identified from Holstein mammary gland tissues infected with different concentrations of S. aureus (in vivo) and mammary alveolar cells (Mac-T cells, in vitro) challenged with different S. aureus strains. Differentially expressed (DE) lncRNAs were evaluated, and stable DE lncRNAs were identified in vivo and in vitro. On the basis of the gene sequence conservation and function conservation across species, key lncRNAs with the function of potentially immune regulation were retained for further analysis. The function of FA on inflammation induced by S. aureus challenge was also investigated. Then, the association analysis between these keys lncRNA transcripts and hematological parameters (HPs) was carried out. Lastly, the knockdown and overexpression of the important lncRNA were performed to validate the gene function on the regulation of cell immune response.ResultsLinear regression analysis showed a significant correlation between the expression levels of lncRNA shared by mammary tissue and Mac-T cells (P < 0.001, R2 = 0.3517). lncRNAs PRANCR and TNK2–AS1 could be regarded as stable markers associated with bovine S. aureus mastitis. Several HPs could be influenced by SNPs around lncRNAs PRANCR and TNK2–AS1. The results of gene function validation showed PRANCR regulates the mRNA expression of SELPLG and ITGB2 within the S. aureus infection pathway and the Mac-T cells apoptosis. In addition, FA regulated the expression change of DE lncRNA involved in toxin metabolism and inflammation to fight against S. aureus infection.ConclusionsThe remarkable association between SNPs around these two lncRNAs and partial HP indicates the potentially important role of PRANCR and TNK2–AS1 in immune regulation. Stable DE lncRNAs PRANCR and TNK2–AS1 can be regarded as potential targets for the prevention of bovine S. aureus mastitis. FA supplementation can reduce the negative effect of S. aureus challenge by regulating the expression of lncRNAs.

Beta-Sitosterol Promotes Milk Protein and Fat Syntheses-Related Genes in Bovine Mammary Epithelial Cells.

Simple SummaryThe levels of milk fats and proteins are important indexes used to evaluate milk quality. Generally, feed additives are used to improve milk quality. This study aimed to investigate the effect of β-sitosterol on milk fat and protein gene expression in bovine mammary epithelial cells. β-sitosterol increased the β-casein levels in bovine mammary epithelial cells and promoted the expression of milk fat and protein synthesis-related genes, suggesting the use of β-sitosterol as a potential feed additive to improve milk quality in dairy cows.β-sitosterol, a phytosterol with multiple biological activities, has been used in the pharmaceutical industry. However, there are only a few reports on the use of β-sitosterol in improving milk synthesis in dairy cows. This study aimed to investigate the effects of β-sitosterol on milk fat and protein syntheses in bovine mammary epithelial cells (MAC-T) and its regulatory mechanism. MAC-T cells were treated with different concentrations (0.01, 0.1, 1, 5, 10, 20, 30, or 40 μM) of β-sitosterol, and the expression levels of milk protein and fat synthesis-related genes and proteins were analyzed. β-sitosterol at 0.1, 1, and 10 μM concentrations promoted the mRNA and protein expression of β-casein. β-sitosterol (0.1, 1, 10 μM) increased the mRNA and protein expression levels of signal transducer activator of transcription 5 (STAT5), mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase beta-1 (S6K1) of the JAK2/STAT5 and mTOR signaling pathways. It also stimulated the milk fat synthesis-related factors, including sterol regulatory element-binding protein 1 (SREBP1), peroxisome proliferator-activated receptor-gamma (PPARγ), acetyl-CoA carboxylase (ACC), lipoprotein lipase (LPL), and stearyl CoA desaturase (SCD). β-sitosterol (0.1, 1, 10 μM) also significantly increased the expression of growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and hypoxia-inducible factor-1α (HIF-1α)-related genes. Notably, the compound inhibited the expression of the negative regulator, the suppressor of cytokine signaling 2 (SOCS2) at the two lower concentrations (0.1, 1 μM), but significantly promoted the expression at the highest concentration (30 μM). These results highlight the role of β-sitosterol at concentrations ranging from 0.1 to 10 μM in improving milk protein and fat syntheses, regulating milk quality. Therefore, β-sitosterol can be used as a potential feed additive to improve milk quality in dairy cows.

Protective Roles of Folic Acid in the Responses of Bovine Mammary Epithelial Cells to Different Virulent Staphylococcus aureus Strains.

Simple SummaryMastitis caused by zoonotic Staphylococcus aureus (S. aureus) is hard to cure. The cross-talk between host, pathogen, and environment influences bovine resistance to S. aureus infection. Our previous study observed that the inflammation of bovine mammary gland could be reduced by folic acid, i.e., a kind of beneficial micronutrient. However, the heterogeneity of inflammatory responses in bovine mammary epithelial cells challenged with different virulent S. aureus strains and the protective effects of folic acid on improving host defense against S. aureus infection are still unclear. RNA-seq was performed to investigate the effects of different S. aureus lineages and folic acid on the global transcriptome of bovine mammary epithelial cells (Mac-T cell line). Functional enrichment analysis indicated different S. aureus strains share few differentially expressed and spliced genes and activate host differentially inflammatory responses, and folic acid plays the protective role in host defense against the S. aureus challenge partially through activating cytoplasmic DNA sensing and tight junction pathway. This study provides novel insights into the improvement of S. aureus mastitis defense.Mastitis caused by Staphylococcus aureus (S. aureus) infection is one of the most difficult diseases to treat in dairy cattle. Exploring the biological progression of S. aureus mastitis via the interaction between host, pathogen, and environment is the key to an effective and sustainable improvement of animal health. Here, two strains of S. aureus and a strain of MRSA (Methicillin-resistant Staphylococcus aureus) isolated from cows with different inflammation phenotypes were used to challenge Mac-T cells and to investigate their effects on the global transcriptome of the cells, then to explore the potential regulatory mechanisms of folic acid on S. aureus mastitis prevention. Differential gene expression or splicing analysis showed that different strains of S. aureus led to distinct transcriptional responses from the host immune system. Folic acid could protect host defense against the challenge of S. aureus and MRSA partially through activating cytoplasmic DNA sensing and tight junction pathway. ZBP1 at the upstream of cytoplasmic DNA sensing pathway was verified and related to anti-pathogen through RNA interference. Further enrichment analysis using these transcriptome data with cattle large-scale genome-wide association study (GWAS) data confirmed that ZBP1 gene is highly associated with bovine somatic cell score (SCS) trait. Our data shed light on the potential effect of FA through regulating key gene and then protect host cells’ defense against S. aureus and MRSA.

Anti-Staphylococcus aureus Single-Chain Fragment Variables Play a Protective Anti-Inflammatory Role In Vitro and In Vivo.

Staphylococcus aureus is a causative agent of bovine mastitis, capable of causing significant economic losses to the dairy industry worldwide. This study focuses on obtaining single-chain fragment variables (scFvs) against the virulence factors of S. aureus and evaluates the protective effect of scFvs on bovine mammary epithelial (MAC-T) cells and mice mammary gland tissues infected by S. aureus. After five rounds of bio-panning, four scFvs targeting four virulence factors of S. aureus were obtained. The complementarity-determining regions (CDRs) of these scFvs exhibited significant diversities, especially CDR3 of the VH domain. In vitro, each of scFvs was capable of inhibiting S. aureus growth and reducing the damage of MAC-T cells infected by S. aureus. Preincubation of MAC-T cells with scFvs could significantly attenuate the effect of apoptosis and necrosis compared with the negative control group. In vivo, the qPCR and ELISA results demonstrated that scFvs reduced the transcription and expression of Tumor Necrosis Factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8, and IL-18. Histopathology and myeloperoxidase (MPO) results showed that scFvs ameliorated the histopathological damages and reduced the inflammatory cells infiltration. The overall results demonstrated the positive anti-inflammatory effect of scFvs, revealing the potential role of scFvs in the prevention and treatment of S. aureus infections.

Bovine mammary alveolar MAC-T cells afford a tool for studies of bovine milk exosomes in drug delivery

Bovine milk exosomes (BMEs) have attracted attention as vehicles for delivering RNA therapeutics. BMEs originate in mammary alveolar cells. Here, we determined whether bovine mammary alveolar MAC-T cells afford a tool to assess RNA delivery by BMEs. MAC-T cells exosomes (MAC-T BMEs) and BMEs were harvested by differential ultracentrifugation. Exosome size, morphology, microRNA content and marker proteins were assessed using nanoparticle tracking analysis, transmission electron microscopy, real-time PCR and immunoblot analysis, respectively. MAC-T cells were genetically engineered to secrete MAC-T BMEs endogenously labeled with a near-infrared fluorescent protein and tissue distribution was compared to fluorophore-labeled BMEs following intravenous injection in C57BL/6 mice. Morphology and size were similar in MAC-T BMEs and BMEs (94 ± 5.8 nm and 101 ± 4.2 nm, p > 0.05). Both preparations expressed miR-320a, miR-200c and let-7a-5p (positive controls) but not miR-1 (negative control). Exosome marker proteins, CD9, CD63, CD81 and Tsg101, were detected in both MAC-T BMEs and BMEs. Distribution in mouse tissues was similar for both preparations, with liver being the primary accumulation site. Collectively, MAC-T BMEs afford a tool for BMEs-based RNA delivery studies.

Effect of Heat Stress on Bovine Mammary Cellular Metabolites and Gene Transcription Related to Amino Acid Metabolism, Amino Acid Transportation and Mammalian Target of Rapamycin (mTOR) Signaling.

Simple SummaryThis study mainly employed metabolomics technology to determine changes of intracellular metabolite concentrations related to milk protein synthesis induced by heat stress (HS) in bovine mammary epithelial cells. HS was associated with significant differences in intracellular amino acid metabolism resulting in an increase in the intracellular amino acid concentrations. Moreover, HS promoted amino acid transportation and the activity of the mammalian target of rapamycin (mTOR) signaling pathway, which plays an important role as a central regulator of cell metabolism, growth, proliferation and survival. Greater expression of the alpha-S2-casein gene (CSN1S2) was also observed during HS. Overall, our study indicated that bovine mammary epithelial cells may have the ability to resist HS damage and continue milk protein synthesis partly through enhanced intracellular amino acid absorption and metabolism and by activating the mTOR signaling pathway during HS.Heat stress (HS) is one of the most serious factors to negatively affect the lactation performance of dairy cows. Bovine mammary epithelial cells are important for lactation. It was demonstrated that HS decreases the lactation performance of dairy cows, partly through altering gene expression within bovine mammary epithelial tissue. However, the cellular metabolism mechanisms under HS remains largely unknown. The objective of this study was to determine whether HS induced changes in intracellular metabolites and gene transcription related to amino acid metabolism, amino acid transportation and the mTOR signaling pathway. Immortalized bovine mammary epithelial cell lines (MAC-T cells, n = 5 replicates/treatment) were incubated for 12 h at 37 °C (Control group) and 42 °C (HS group). Relative to the control group, HS led to a greater mRNA expression of heat shock protein genes HSF1, HSPB8, HSPA5, HSP90AB1 and HSPA1A. Compared with the control group, metabolomics using liquid chromatography tandem–mass spectrometry identified 417 differential metabolites with p < 0.05 and a variable importance in projection (VIP) score >1.0 in the HS group. HS resulted in significant changes to the intracellular amino acid metabolism of glutathione, phenylalanine, tyrosine, tryptophan, valine, leucine, isoleucine, arginine, proline, cysteine, methionine, alanine, aspartate and glutamate. HS led to a greater mRNA expression of the amino acid transporter genes SLC43A1, SLC38A9, SLC36A1, and SLC3A2 but a lower mRNA expression of SLC7A5 and SLC38A2. Additionally, HS influenced the expression of genes associated with the mTOR signaling pathway and significantly upregulated the mRNA expression of mTOR, AKT, RHEB, eIF4E and eEF2K but decreased the mRNA expression of TSC1, TSC2 and eEF2 relative to the control group. Compared with the control group, HS also led to greater mRNA expression of the CSN1S2 gene. Overall, our study indicates that bovine mammary epithelial cells may have the ability to resist HS damage and continue milk protein synthesis partly through enhanced intracellular amino acid absorption and metabolism and by activating the mTOR signaling pathway during HS.

Study on the Regulation Mechanism of Lipopolysaccharide on Oxidative Stress and Lipid Metabolism of Bovine Mammary Epithelial Cells

The long-term feeding of a high-concentrate diet (the concentrate ratio is greater than 60 %) leads to mammary gland inflammatory response in ruminants and decreased quality in dairy cows and affects the robust development of the dairy industry. The main reason is closely related to elevated lipopolysaccharide (LPS) in the body. In this experiment, a bovine mammary epithelial cell line (MAC-T) was used as a model, and LPS at different concentrations (0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, 10000 ng/ml) was added to the cells. The cell survival rate, oxidative stress indicators, total lipid droplet area, triglyceride content and key genes regulating lipid metabolism were detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), assay kit, microscope observation and RT-PCR methods to explore the regulatory mechanism of mammary health and milk fat synthesis. The results showed that compared with those of the control group, the survival rates of cells were significantly decreased after 9 h of stimulation with 1000 ng/ml and 10000 ng/ml LPS (P&lt;0.01). The contents of superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) in cells were significantly decreased (P&lt;0.05). Compared with that of the control group, the content of malondialdehyde (MDA) in cells was significantly increased (P&lt;0.05) after stimulation with 10000 ng/ml LPS for 9 h. After 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS, the total lipid drop area and triglyceride (TG) content of MAC-T cells were significantly decreased (P&lt;0.05). The expression levels of fatty acid synthesis-related genes Acetyl-CoA carboxylase (ACC) and Stearoyl-CoA desaturase 1 (SCD-1) were significantly decreased after 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS (P&lt;0.05), while the expression levels of Fatty Acid synthetase (FAS) were significantly decreased after stimulation with 1000 ng/ml and 10000 ng/ml LPS (P&lt;0.05). TG synthesis by the related gene Diacylglycerol acyltransferase-1 (DGAT1) was significantly lower than that of the control group after stimulation with 1000 ng/ml and 10000 ng/ml LPS for 9 h (P&lt;0.05), and Diacylglycerol acyltransferase-2 (DGAT2) also showed a significant decrease after 10000 ng/ml LPS stimulation (P&lt;0.05). In conclusion, adding different concentrations of LPS to MAC-T cells not only led to a decrease in cell activity, resulting in oxidative damage, but also affected fatty acid and TG synthesis, which may ultimately be closely related to the decrease in milk fat synthesis.

Study on the regulation mechanism of lipopolysaccharide on oxidative stress and lipid metabolism of bovine mammary epithelial cells.

The long-term feeding of a high-concentrate diet (the concentrate ratio is greater than 60 %) leads to mammary gland inflammatory response in ruminants and decreased quality in dairy cows and affects the robust development of the dairy industry. The main reason is closely related to elevated lipopolysaccharide (LPS) in the body. In this experiment, a bovine mammary epithelial cell line (MAC-T) was used as a model, and LPS at different concentrations (0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, 10000 ng/ml) was added to the cells. The cell survival rate, oxidative stress indicators, total lipid droplet area, triglyceride content and key genes regulating lipid metabolism were detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), assay kit, microscope observation and RT-PCR methods to explore the regulatory mechanism of mammary health and milk fat synthesis. The results showed that compared with those of the control group, the survival rates of cells were significantly decreased after 9 h of stimulation with 1000 ng/ml and 10000 ng/ml LPS (P<0.01). The contents of superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) in cells were significantly decreased (P<0.05). Compared with that of the control group, the content of malondialdehyde (MDA) in cells was significantly increased (P<0.05) after stimulation with 10000 ng/ml LPS for 9 h. After 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS, the total lipid drop area and triglyceride (TG) content of MAC-T cells were significantly decreased (P<0.05). The expression levels of fatty acid synthesis-related genes Acetyl-CoA carboxylase (ACC) and Stearoyl-CoA desaturase 1 (SCD-1) were significantly decreased after 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS (P<0.05), while the expression levels of Fatty Acid synthetase (FAS) were significantly decreased after stimulation with 1000 ng/ml and 10000 ng/ml LPS (P<0.05). TG synthesis by the related gene Diacylglycerol acyltransferase-1 (DGAT1) was significantly lower than that of the control group after stimulation with 1000 ng/ml and 10000 ng/ml LPS for 9 h (P<0.05), and Diacylglycerol acyltransferase-2 (DGAT2) also showed a significant decrease after 10000 ng/ml LPS stimulation (P<0.05). In conclusion, adding different concentrations of LPS to MAC-T cells not only led to a decrease in cell activity, resulting in oxidative damage, but also affected fatty acid and TG synthesis, which may ultimately be closely related to the decrease in milk fat synthesis.

On the Biocompatibility and Teat Retention of In Situ Gelling Intramammary Formulations: Cattle Mastitis Prevention and Treatment.

Treatment and prevention of cattle mastitis remains a formidable challenge due to the anatomical and physiological constraints of the cow udder. In this study, we investigated polymeric excipients and solvents that can form, (when combined) novel, non-toxic and biocompatible in situ gelling formulations in the mammary gland of bovine cattle. We also report on a new approach to screen intramammary formulations using fresh excised cow teats. Fourteen hydrophilic polymers and six solvents were evaluated for in vitro cytotoxicity and biocompatibility towards cultured bovine mammary epithelial cells (MAC-T), microscopic and macroscopic examination upon contact with excised cow teats. No significant cytotoxicity (p > 0.05) was observed with polyethylene oxides, hydroxypropyl methylcellulose, carboxymethyl cellulose, sodium alginate and xanthan gum. Polycarbophil and carbopol polymers showed significantly higher cytotoxicity (p < 0.05). Concentration-dependent cytotoxicity was observed for glycerin, propylene glycol, polyethylene glycol 400, ethanol, N-methyl-2-pyrrolidone and 2-pyrrolidone, with the 2-pyrrolidone solvents showing higher cytotoxic effects (p < 0.05). In situ gelling formulations comprising hydroxypropyl methylcellulose or carboxymethyl cellulose and solvents in specific ratios were biocompatible at higher concentrations with MAC-T cells compared to alginates. All investigated formulations could undergo in situ sol-to-gel phase transformation, forming non-toxic gels with good biocompatibility in excised cow teats hence, showing potential for use as intramammary carriers for sustained drug delivery.

MiR-142-5p regulates lipopolysaccharide-induced bovine epithelial cell proliferation and apoptosis via targeting BAG5.

Bovine mastitis is a threat to the health of the dairy cow. MicroRNAs (miRs) serve an important role in the progression of bovine mastitis, regulating immune and defense responses. The present study aimed to investigate the possible effects and mechanisms of bovine mastitis underlying miR-142-5p and Bcl-2 associated athanogene 5 (BAG5) in in vitro lipopolysaccharide (LPS)-induced models. Reverse transcription-quantitative PCR and western blotting were performed to determine mRNA and protein expression levels, respectively. ELISAs were conducted to assess the levels of cytokines and an immunofluorescence assay was performed to determine the expression of BAG5. Cell Counting Kit-8, clone formation and 5-ethynyl-2'-deoxyuridine assays were conducted to determine cell viability and proliferation of bovine mammary epithelial MAC-T cells, respectively. Flow cytometry was performed to measure MAC-T cell cycle distribution and apoptosis, and a luciferase assay was conducted to verify whether BAG5 was a target of miR-142-5p. The results indicated that miR-142-5p was upregulated in MAC-T cells treated with LPS compared with the control group. miR-142-5p mimics transfection significantly activated the cytokines TNF-α, IL-1β, IL-6 and IL-8, and significantly increased the expression levels of NF-κB signaling pathway-related proteins in LPS-treated cells. The luciferase activity of MAC-T cells treated with miR-142-5p mimics and BAG5 3'untranslated region wild type decreased, compared with mutant type. By contrast, BAG5 overexpression significantly downregulated the levels of cytokines, including TNF-α, IL-1β, IL-6 and IL-8, in LPS-treated cells. BAG5 overexpression significantly promoted cell proliferation and viability, decreased apoptosis, and regulated Caspase-3, Caspase-9, Bcl-2 and Bax expression in LPS-treated MAC-T cells, which was significantly reversed by transfection with miR-142-5p mimics. In conclusion, the results of the present study suggested that miR-142-5p may promote the progression of bovine mastitis via targeting BAG5. Therefore, the present study provided the foundations for future investigations.

Anti-inflammation of epicatechin mediated by TMEM35A and TMPO in bovine mammary epithelial cell line cells and mouse mammary gland

Epicatechin (EC) has significant antiinflammation, antioxidation, and anticancer activities. It also provides a new alternative treatment for mastitis, which can result in great economic losses in the dairy industry if left untreated. The purpose of this study was to investigate the anti-inflammatory effects of EC on mastitis and the underlying mechanism using in vivo and in vitro systems. The use of ELISA and immunohistochemistry assays showed that EC treatment at 1.5, 7.5, 15, and 30 mg/mL decreased protein expression of inflammatory mediators, including cyclooxygenase-2 and inducible nitric oxide synthase; inflammatory cytokines, which were composed of IL-1β, TNF-α, and IL-6 in lipopolysaccharide (LPS)-stimulated bovine mammary epithelial cell line (MAC-T); and mouse mammary gland, together with reduced filtration of T lymphocytes in the mouse mammary gland. Furthermore, EC treatment reduced LPS-induced phosphorylation levels of p65 and inhibitor of NF-κB, and blocked nuclear translocation of p65 as revealed by western blot and immunofluorescence test in MAC-T cells and the mouse mammary gland. Epicatechin also attenuated LPS-induced phosphorylation levels of mitogen-activated protein kinase members (i.e., p38, c-Jun N-terminal kinase 1/2 and extracellular regulated protein kinases 1/2). Using RNA-seq and tandem mass tag analyses, upregulation of TMEM35A and TMPO proteins was disclosed in MAC-T cells cotreated with LPS and EC. Although clustered regularly interspaced short palindromic repeats/Cas9-based knockdown of TMEM35A and TMPO attenuated abundance of phosphorylated (p)-p65, p-p38, TNF-α, and iNOS, overexpression of TMEM35A reversed EC-mediated effects in TMPO knockdown cells. Moreover, interaction between TMEM35A and TMPO was detected using the co-immunoprecipitation method. In conclusion, our data demonstrated that EC inhibited LPS-induced inflammatory response in MAC-T cells and the mouse mammary gland. Importantly, TMEM35A mediated the transmembrane transport of EC, and the interaction between TMEM35A and TMPO inhibited MAPK and NF-κB pathways.

Antibacterial Activity of Ikarugamycin against Intracellular Staphylococcus aureus in Bovine Mammary Epithelial Cells In Vitro Infection Model.

Simple SummaryAntibiotics are widely used for the treatment and control of bovine mastitis. However, the treatment has only been partially effective, as the cure percentage only ranging from 10–30%. Infection by Staphylococcus aureus (S. aureus) is particularly difficult to treat due to the bacteria’s ability to enter and resides inside the host cells. Most antibiotics are ineffective against intracellular bacterial due to the poor penetration into host cells to achieve optimal intracellular bactericidal bioavailability levels. There is therefore, an increasing need to evaluate candidate active substances and develop novel antibiotics effective against intracellular persistence infection. In this study, we examine the potential antibacterial properties of ikarugamycin compound as an alternative drug candidate to be explored for treating persistent bovine mastitis caused by intracellular S. aureus using bovine mammary cell line as an in vitro infection model. We also assessed the potential cytotoxicity effect of ikarugamycin in the infection model. We found that, the ikarugamycin possessed intracellular killing activity against S. aureus within the mammary epithelial cell. This finding highlights the potential application of ikarugamycin as a novel antimicrobial for the treatment of S. aureus mastitis.Staphylococcus aureus is an ubiquitous and versatile pathogen associated with a wide range of diseases. In animals, this bacterium is one of the causative agents of bovine mastitis, responsible for huge economic losses in the dairy industry. Besides the development of antibiotic resistance, the intracellular survival of S. aureus within udder cells has rendered many antibiotics ineffective, leading to therapeutic failure. Our study therefore aims to investigate the in vitro bactericidal activity of ikarugamycin (IKA) against intracellular S. aureus using a bovine mammary epithelial cells (Mac-T cells) infection model and determine the cytotoxic effect. Minimum inhibitory concentration (MIC) was used to determine the antibacterial activity of IKA, and Mac-T cells were infected with S. aureus using gentamicin protection assay. IKA intracellular antibacterial activity assays were used to determine the bactericidal activity of IKA against intracellular S. aureus. The cytotoxicity of IKA against Mac-T cells was evaluated using the resazurin assay. We showed that, S. aureus is susceptible to IKA with a MIC value of 0.6 μg/mL. IKA at 4 × MIC and 8 × MIC have bactericidal activity by reducing 3 and 5 logs10 CFU/mL of S. aureus in the first six-hour of treatment respectively. In addition, IKA demonstrated intracellular killing activity by killing 90% of intracellular S. aureus at 5 μg/mL. This level is comparatively lower than 9.2 μg/mL determined as the half-maximal inhibitory concentration (IC50) of IKA required to kill 50% of Mac-T cells, highlighting a lower concentration required for bactericidal effect compared to the cytotoxic effect. The study highlighted that importance of IKA as a potential antibiotic candidate to be explored for the in vivo efficacy in treating S. aureus mastitis.

MiR-125b regulates inflammation in bovine mammary epithelial cells by targeting the NKIRAS2 gene

Mastitis is a complex inflammatory disease caused by pathogenic infection of mammary tissue in dairy cows. The molecular mechanism behind its occurrence, development, and regulation consists of a multi-gene network including microRNA (miRNA). Until now, there is no report on the role of miR-125b in regulating mastitis in dairy cows. This study found that miR-125b expression is significantly decreased in lipopolysaccharide (LPS)-induced MAC-T bovine mammary epithelial cells. Also, its expression is negatively correlated with the expression of NF-κB inhibitor interacting Ras-like 2 (NKIRAS2) gene. MiR-125b target genes were identified using a double luciferase reporter gene assay, which showed that miR-125b can bind to the 3′ untranslated region (3′ UTR) of the NKIRAS2, but not the 3′UTR of the TNF-α induced protein 3 (TNFAIP3). In addition, miR-125b overexpression and silencing were used to investigate the role of miR-125b on inflammation in LPS-induced MAC-T. The results demonstrate that a reduction in miR-125b expression in LPS-induced MAC-T cells increases NKIRAS2 expression, which then reduces NF-κB activity, leading to low expression of the inflammatory factors IL-6 and TNF-α. Ultimately, this reduces the inflammatory response in MAC-T cells. These results indicate that miR-125b is a pro-inflammatory regulator and that its silencing can alleviate bovine mastitis. These findings lay a foundation for elucidating the molecular regulation mechanism of cow mastitis.

Lactobacillus rhamnosus GR-1 Prevents Escherichia coli-Induced Apoptosis Through PINK1/Parkin-Mediated Mitophagy in Bovine Mastitis

Escherichia coli is one of the most important pathogens that cause clinical mastitis in dairy cattle worldwide and lead to severe economic losses. Antibiotics are often used to treat this inflammatory disease; however, antimicrobial resistance and environmental pollution cannot be ignored. Probiotic is the best alternative; however, its mechanisms of action to prevent mastitis remain unclear. Moreover, the role of probiotics in regulating mitophagy, a selective autophagy that maintains mitochondrial quality, needs to be explored. E. coli infection induced NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3) inflammasome assembly, Caspase-1 activation, and apoptosis in MAC-T cells. Infection also resulted in mitochondrial damage and subsequent increase in reactive oxygen species (ROS) production. Moreover, inhibition of ROS release by scavenger N-acetyl-L-cysteine (NAC) abrogated the importance of ROS in NLRP3 assembly and apoptosis in MAC-T cells. Pretreatment with Lactobacillus rhamnosus GR-1 (LGR-1), a probiotic, alleviated E. coli-induced NLRP3 inflammasome activation and apoptosis via ROS inhibition. Besides, E. coli infection inhibited mitophagy while LGR-1 pretreatment augmented PINK1/Parkin–mediated mitophagy activation, which further blocked ROS generation. To explore the effect of LGR-1 in vivo, a mouse mastitis model was established. The results showed that LGR-1 pretreatment had preventive and protective effects on E. coli induced mastitis, and could reduce cytokines levels such as IL-1β and TNF-α. In accordance with the results in vitro, E. coli can inhibit mitophagy and activate NLRP3 inflammasome and apoptosis, while LGR-1 can weaken the effect of E. coli. Taken together, our data indicated that LGR-1 pretreatment induced PINK1/Parkin-mediated mitophagy that eliminated damaged mitochondria and reduced ROS production and NLRP3 inflammasome activation, which subsequently decreased E. coli-induced apoptosis. To conclude, our study suggests that therapeutic strategies aiming at the upregulation of mitophagy under E. coli-induced mastitis may preserve mitochondrial function and provide theoretical support for the application of probiotics in bovine mastitis.

Relative virulence of Staphylococcus aureus bovine mastitis strains representing the main Canadian spa types and clonal complexes as determined using in vitro and in vivo mastitis models

Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of β-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap-), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1β and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus.