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Effects of lipoteichoic and arachidonic acids on the immune-regulatory mechanism of bovine mammary epithelial cells using multi-omics analysis.

ABSTRACT: Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows. It mainly utilizes the properties of its pathogenic factor, lipoteichoic acid (LTA), to elicit a host-cell inflammatory response and evade the host-cell immune response. Arachidonic acid (AA) has a regulatory role in the inflammatory response, cell metabolism, and apoptosis. The study aimed to establish a cell model by determining the optimal concentration of LTA and AA for cell induction using the Cell Counting Kit-8 assay and the quantitative polymerase chain reaction of interleukin (IL)-1β, IL-2, and IL-6. MAC-T cells were planted in 36 10-cm² culture dishes at a density of 1 × 10⁷ cells per dish. They were treated with LTA for 24 h to constitute the LTA group and with AA for 12 h to constitute the AA group. The cells were pretreated with LTA for 24 h followed by treatment with AA for 12 h to constitute the LTA + AA group. Using proteomic, transcriptomic, and metabolomic analyses, this study determined that LTA can regulate the expression of Actin Related protein 2/3 complex (ARPC)3, ARPC4, Charged Multivesicular Body Protein 3, protein kinase cGMP-dependent, NF-κB Inhibitor Alpha,and other genes to affect cellular metabolism, immune regulation and promote apoptosis. In contrast, AA was observed to regulate the expression of genes such as ARPC3, ARPC4, Charged Multivesicular Body Protein 3, Laminin Gamma 1, Insulin Receptor, Filamin B, and Casein Kinase 1 Epsilon to inhibit cellular apoptosis and promote immune regulation, which provides a theoretical basis for future studies.

SUBMITTER: Dong W

PROVIDER: S-EPMC9450935 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Effects of lipoteichoic and arachidonic acids on the immune-regulatory mechanism of bovine mammary epithelial cells using multi-omics analysis.

Dong Weitao W Chen Yan Y Zhang Quanwei Q Zhao Xiaoxuan X Liu Peiwen P He Haijian H Lu Ting T He Yuxuan Y Du Xianghong X Hu Junjie J Zhao Xingxu X Zhang Yong Y

Frontiers in veterinary science 20220824

<i>Staphylococcus aureus</i> is one of the most important pathogens causing mastitis in dairy cows. It mainly utilizes the properties of its pathogenic factor, lipoteichoic acid (LTA), to elicit a host-cell inflammatory response and evade the host-cell immune response. Arachidonic acid (AA) has a regulatory role in the inflammatory response, cell metabolism, and apoptosis. The study aimed to establish a cell model by determining the optimal concentration of LTA and AA for cell induction using th ...[more]

PMID: 36090174

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Project description:The mammary gland of the cow is particularly susceptible to infections of a wide range of pathogenic bacteria, including both Gram-positive and Gram-negative bacteria. The endotoxins of these pathogenic bacteria include peptidoglycan (PGN), lipoteichoic acid (LTA) and lipopolysaccharide (LPS), and they are the pathogen-associated molecular patterns (PAMPs) to induce mastitis. LPS can directly inhibit proliferation and milk fat synthesis of bovine mammary epithelial cells (BMECs) while inducing mastitis, but it is unclear whether PGN and LTA also have such effects. Furthermore, since the three PAMPs usually appear simultaneously in the udder of cows with mastitis, their synergistic effects on proliferation and milk fat synthesis of BMECs are worth investigating. The immortalized BMECs (MAC-T cells) were stimulated for 24 h using various concentrations of PGN, LTA and LPS, respectively, to determine the doses that could effectively cause inflammatory responses. Next, the cells were stimulated for 24 h with no endotoxins (CON), PGN, LTA, LPS, PGN + LTA, and PGN + LTA + LPS, respectively, with the predetermined doses to analyze their effects on proliferation and milk fat synthesis of BMECs. PGN, LTA and LPS successfully induced inflammatory responses of BMECs with doses of 30, 30 and 0.1 μg/mL, respectively. Although the proliferation of BMECs was significantly inhibited in the following order: LTA < PGN + LTA < PGN + LTA + LPS, there was no change in cell morphology and cell death. LTA significantly promoted the expression of fatty acid synthesis-related genes but did not change the content of intracellular triglyceride (TG), compared with the CON group. The mRNA expression of fatty acid synthesis-related genes in the LPS group was the lowest among all the groups. Meanwhile, LPS significantly decreased the content of intracellular non-esterified fatty acids (NEFAs) and TG, compared with the CON group. PGN had no effects on milk fat synthesis. Co-stimulation with PGN, LTA and LPS significantly increased the expression of fat acid synthesis-related genes and the intracellular NEFAs, but decreased intracellular TG, compared with sole LPS stimulation. Collectively, PGN, LTA and LPS showed an additive effect on inhibiting proliferation of BMECs. The promoting role of LTA in fatty acid synthesis might offset the negative effects of LPS in this regard, but co-stimulation with PGN, LTA and LPS significantly decreased intracellular TG content.

Project description:Staphylococcus aureus, a major pathogen for the mammary gland of dairy ruminants, elicits the recruitment of neutrophils into milk during mastitis, but the mechanisms are incompletely understood. We investigated the response of the bovine mammary gland to muramyl dipeptide (MDP), an elementary constituent of the bacterial peptidoglycan, alone or in combination with lipoteichoic acid (LTA), another staphylococcal microbial-associated molecular pattern (MAMP). MDP induced a prompt and marked influx of neutrophils in milk, and its combination with LTA elicited a more intense and prolonged influx than the responses to either stimulus alone. The concentrations of several chemoattractants for neutrophils (CXCL1, CXCL2, CXCL3, CXCL8, and C5a) increased in milk after challenge, and the highest increases followed challenge with the combination of MDP and LTA. MDP and LTA were also synergistic in inducing in vitro chemokine production by bovine mammary epithelial cells (bMEpC). Nucleotide-binding oligomerization domain 2 (NOD2), a major sensor of MDP, was expressed (mRNA) in bovine mammary tissue and by bMEpC in culture. The production of interleukin-8 (IL-8) following the stimulation of bMEpC by LTA and MDP was dependent on the activation of NF-κB. LTA-induced IL-8 production did not depend on platelet-activating factor receptor (PAFR), as the PAFR antagonist WEB2086 was without effect. In contrast, bMEpC and mammary tissue are known to express Toll-like receptor 2 (TLR2) and to respond to TLR2 agonists. Although the levels of expression of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β were increased by LTA and MDP at the mRNA level, no protein could be detected in the bMEpC culture supernatant. The level of induction of IL-6 was low at both the mRNA and protein levels. These results indicate that MDP and LTA exert synergistic effects to induce neutrophilic inflammation in the mammary gland. These results also show that bMEpC could contribute to the inflammatory response by recognizing LTA and MDP and secreting chemokines but not proinflammatory cytokines. Overall, this study indicates that the TLR2 and NOD2 pathways could cooperate to trigger an innate immune response to S. aureus mastitis.

Project description: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.

Dataset Information

Effects of lipoteichoic and arachidonic acids on the immune-regulatory mechanism of bovine mammary epithelial cells using multi-omics analysis.

Publications

Effects of lipoteichoic and arachidonic acids on the immune-regulatory mechanism of bovine mammary epithelial cells using multi-omics analysis.

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