Response Of Mammary Epithelial Cells Research Articles

Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway.

Mastitis is one of three bovine diseases recognized as a cause of substantial economic losses every year throughout the world. Niacin is an important feed additive that is used extensively for dairy cow nutrition. However, the mechanism by which niacin acts on mastitis is not clear. The aim of this study is to investigate the mechanism of niacin in alleviating the inflammatory response of mammary epithelial cells and in anti-mastitis. Mammary glands, milk, and blood samples were collected from mastitis cows not treated with niacin (n = 3) and treated with niacin (30 g/d, n = 3) and healthy cows (n = 3). The expression of GPR109A, IL-6, IL-1β, and TNF-α in the mammary glands of the dairy cows with mastitis was significantly higher than it was in the glands of the healthy dairy cows. We also conducted animal experiments in vivo by feeding rumen-bypassed niacin. Compared with those in the untreated mastitis group, the somatic cell counts (SCCs) and the expression of IL-6, IL-1β, and TNF-α in the blood and milk were lower. In vitro, we isolated the primary bovine mammary epithelial cells (BMECs) from the mammary glands of the healthy cows. The mRNA levels of IL-6, IL-1β, TNF-α, and autophagy-related genes were detected after adding niacin, shRNA, compound C, trans retinoic acid, 3-methyladenine to BMECs. Then GPR109A, AMPK, NRF-2, and autophagy-related proteins were detected by Western blot. We found that niacin can activate GPR109A and phosphorylate AMPK, and promote NRF-2 nuclear import and autophagy to alleviate LPS-induced inflammatory response in BMECs. In summary, we found that niacin can reduce the inflammatory response of BMECs through GPR109A/AMPK/NRF-2/autophagy. We also preliminarily explored the alleviative effect of niacin on mastitis in dairy cows.

Inhibition of histone deacetylases is the major pathway mediated by astaxanthin to antagonize LPS-induced inflammatory responses in mammary epithelial cells.

Mastitis is amajor inflammatory response of the mammary gland due to various pathogenic invasions and is a serious disease that affects the production yield and health status of cows. Astaxanthin (AST), a xanthophyll carotenoid, is a secondary metabolite synthesized by microalgaeand yeasts that has been reported to suppress various inflammatory responses. However, the protective effect of AST on lipopolysaccharide (LPS)-induced mammary epithelial cells has not yet been reported. The present study results indicated that AST treatment markedly attenuated the oxidative stress markers and nitric oxide (NO) while improving the anti-oxidant enzymes in LPS exposed cells. On the other hand, LPS-exposed cells showed nuclear translocation of nuclear factor-κB (NF-κB) with the activation of inflammatory cytokines such as monocyte chemoattractantprotein-1, tumor necrosis factor-α, interferon-γ, and interleukin-6 (IL-6). In addition, mRNA expression analysis revealed that the histone deacetylase (HDAC) -1, -2, -3, -6, -7 and pentraxin 3 (PTX3) expressions were increased in the LPS group. Furthermore, the activity of HDAC was increased to 2-fold with a significant reduction in the histone acetyltransferase activity in cells exposed to LPS. However, AST was able to inhibit the nuclear translocation of NF-κB with attenuated HDAC activity. Intriguingly, HDAC inhibition studies demonstrated that the cytokines such as IL-4, IL-8, granulocyte-mcrophage colony stimulating factor, C-reactive protein, IL-17A, and IL-22 were significantly suppressedwhichwere upregulated in LPS treatment; while AST was found acting byimproving the anti-inflammatory cytokineIL-10, and thioredoxin reductase levels. Collectively, these findings provide novel insights into the role of HDACs in regulating cellular processes involved in the pathogenesis of LPS-induced mastitis as well as the potential use of AST as a therapeutic in treatment for controlling disease progression.

Protective Effects of Lixisenatide against Lipopolysaccharide-Induced Inflammation Response in MAC-T Bovine Mammary Epithelial Cells: A Therapeutic Implication in Mastitis.

Mastitis is acute inflammation caused by microbial infections in the mammary glands. This disease is extremely harmful to lactating mothers. The preferred clinical strategy is antibiotic treatment, but this method results in resistance and side effects. Lixisenatide, a kind of glucagon-like peptide-1 (GLP-1) receptor agonist, is typically used for the treatment of type II diabetes. It is unknown whether lixisenatide possesses a beneficial role in mastitis. In the current study, we assessed the protective effects of lixisenatide against lipopolysaccharide (LPS) stimulation in MAC-T bovine mammary epithelial cells (MECs). Our findings show that lixisenatide attenuated LPS-induced oxidative stress by reducing reactive oxygen species (ROS) production and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases-1 (NOX-1) expression in MAC-T MECs. Additionally, lixisenatide inhibited LPS-induced expression and secretion of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and interleukin 1β (IL-1β). We also found that lixisenatide suppressed LPS-induced expression of matrix metalloproteinase 2 (MMP-2) and metalloproteinase 9 (MMP-9), and reduced the expression of toll-like receptor 4 (TLR4) (a typical receptor of LPS), its downstream molecule myeloid differentiation factor 88 (MyD88), and the phosphorylation of TGF β-activated kinase 1 (TAK1). Notably, lixisenatide decreased the nuclear levels of nuclear factor-κB (NF-κB) and its transcriptional activity. These findings suggest that lixisenatide might become a possible therapeutic agent for the treatment of mastitis by weakening oxidative stress and the inflammatory response in MECs.

1,25-hydroxyvitamin D3 decreases endoplasmic reticulum stress-induced inflammatory response in mammary epithelial cells.

Recent studies indicated that intramammary administration of active vitamin D3 hormone (1,25D3) inhibits the inflammatory process associated with mastitis. We hypothesized that attenuation of endoplasmic reticulum (ER) stress by 1,25D3 in mammary epithelial cells (MECs) is an important cellular mechanism contributing to this beneficial effect of intramammary treatment with 1,25D3. To test this hypothesis, the effect of 1,25D3 was studied on induction of ER stress in a transformed human MEC line, MCF-7 cells. Treatment with two different ER stress inducers, thapsigargin (TG) and tunicamycin (TM), caused a dose-dependent induction of ER stress as evident from up-regulation of protein kinase RNA-like ER kinase (PERK), heat shock protein family A (Hsp70) member 5 (HSPA5), activating transcription factor (ATF4), ATF6, DNA damage inducible transcript 3 (DDIT3) and spliced X-box binding protein 1 (sXBP1) and impaired cell viability and decreased expression of vitamin D receptor (VDR) in MCF-7 cells (P < 0.05). Treatment with 1,25D3 (100 nM) inhibited TG (10 nM)- and TM (1 μg/mL)-induced mRNA and/or protein levels of ATF4, ATF6, DDIT3 and HSPA5 in MCF-7 cells (P < 0.05). In addition, 1,25D3 (100 nM) antagonized the effect of TG (10 nM) and TM (1 μg/mL) on mRNA and protein levels of VDR and mRNA levels of genes involved in production and degradation of 1,25D3 in MCF-7 cells (P < 0.05). Moreover, 1,25D3 (100 nM) inhibited nuclear factor-κB (NF-κB) activation in response to TM (10 nM) and TG (1 μg/mL) in MCF-7 cells. In conclusion, the present findings show that 1,25D3 is effective in attenuating ER stress and the NF-κB-driven inflammatory response in MCF-7 cells. This indicates that attenuation of ER stress by 1,25D3 in MECs may contribute to the recently observed inhibitory effect of intramammary treatment of dairy cows with 1,25D3 on the inflammatory process associated with mastitis.

A Primary Cell and Organoid Platform for Evaluating Pharmacological Responses in Mammary Epithelial Cells.

An essential process in predicting the in vivo pharmacological activity of a candidate molecule involves the evaluation of target responses using established model systems. While these models largely comprise immortalized cells, which are often serially passaged as monolayers on uniformly stiff substrates and are modified to overexpress one or more components of the pathway-of-interest, the importance of cell identity, heterogeneity, and three-dimensional (3D) context to target response is gaining increasing attention. Here, we assess intracellular calcium responses in mouse mammary epithelial cells in three distinct model systems: 3D primary organoids, 2D primary epithelial cells, and 2D immortalized cells. Specifically, we assess intracellular calcium responses to a number of extracellular signals implicated in the regulation of basal (or myoepithelial) cell function. These findings provide further insights into cell type and context-specific pharmacological responses in mammary epithelial cells and highlight the opportunities and challenges in the adoption of architecturally complex and heterogeneous in vitro assays in pharmacological research.

Meloxicam affects the inflammatory responses of bovine mammary epithelial cells

Nonsteroidal anti-inflammatory drugs are used as supportive therapy with antimicrobial treatments for mastitis in cows to alleviate pain of the inflamed mammary gland. They act mainly by inhibition of cyclooxygenases. Meloxicam (MEL) is a drug designed for cyclooxygenase-2 selectivity, which is upregulated upon inflammation, acting as a key enzyme for the conversion of arachidonic acid to prostaglandins. Although some studies in dairy cows showed positive results in recovery from mastitis when MEL was added to the treatments, direct effects of MEL on the immune system of mastitic cows are unknown. The aim of this study was to investigate effects of MEL on the immune response of bovine mammary epithelial cells (MEC) with or without simultaneous immune stimulation by pathogen-associated molecular patterns of common mastitis pathogens. Mammary epithelial cells from 4 cows were isolated and cultured. To evaluate dose effects of MEL, MEC were challenged with or without 0.2 µg/mL lipopolysaccharide (LPS; serotype O26:B6 from Escherichia coli) with addition of increasing concentrations of MEL (0, 0.25, 0.5, 1.0, 1.5, or 2.0 mg/mL). The addition of MEL prevented the increase of mRNA expression of key inflammatory factors in LPS-challenged MEC in a dose-dependent manner. To investigate the effects of MEL on pathogen-specific immune responses of MEC, treatments included challenges with LPS from E. coli and lipoteichoic acid from Staphylococcus aureus with or without 1.5 mg/mL MEL for 3, 6, and 24 h. Meloxicam prevented the increase of mRNA abundance of key inflammatory mediators in response to LPS and lipoteichoic acid, such as tumor necrosis factor, serum amyloid A, inducible nitric oxide synthase, and the chemokines IL-8 and CXC chemokine ligands 3 and 5. The prostaglandin E2 synthesis in challenged and nonchallenged cells was reduced by MEL within 24 h. Furthermore, MEL reduced the viability and consequently the total RNA yield of the cells. However, mRNA abundance of apoptosis-related enzymes was not affected by any treatment. Meloxicam had clear dose-dependent effects on the immune response of MEC to pathogen-associated molecular patterns of common mastitis pathogens by preventing increased expression of important factors involved in inflammation. This nonsteroidal anti-inflammatory drug also has detrimental effects on cell viability. How these effects would influence the elimination of pathogens from an infected mammary gland during mastitis therapy with meloxicam needs to be further investigated.

Abstract 1028: Extracellular matrix dimensionality reduces cellular cortical tension to stimulate pro-survival signaling in mammary epithelial cells

Abstract Tumor dormancy is a clinical phenomenon in which disseminated tumor cells remain asymptomatic and undetectable over a long period of time. Dormant cells are able to maintain quiescence in hostile microenvironments, escape frontline cancer therapies and evade the immune system, as well as their propensity to reactivate from latency and cause metastatic relapse. Although tumor dormancy is an important problem in the treatment of cancer, the molecular mechanisms underlying this complex process remain unclear. Because dormant cells are frequently found surrounded by a laminin-rich ECM, we hypothesized that ECM dimensionality intrinsically affects cell behaviors that predispose to dormancy. Accordingly, we assessed the biophysical and biochemical response of mammary epithelial cells (MECs) to compliant polyacrylamide (PA) gels and micropatterned surfaces in which ligand presentation and ECM dimensionality were modulated to recapitulate different ECM landscapes. Using traction force microscopy, atomic force microscopy indentation, and laser ablation studies, we found that a 3D ECM led to a drop in cortical tension of MECs. Computational modeling predicted that reduced cortical tension should lead to an increase in the number and/or residence time of actin protrusions as well as a net increase in negative membrane curvature. To test the prediction, we ectopically expressed F-actin and plasma membrane markers in MECs to examine the plasma membrane topography and actin protrusion dynamics. Indeed, MECs in 3D ECM had longer and more stable actin protrusions and more negative membrane curvature inducing proteins, including Exo70, at the plasma membrane. The enrichment of Exo70 at the plasma membrane accompanied the activation of Arf6, which led to an increase in Rac/p38 pro-survival signaling pathway. Consistently, we found that the non-spread cells were able to survive in 3D ECM but the non-spread MECs plated on either a soft 2D PA gels or rigid micropatterned adhesive islands died. We next examined if reduced cortical tension is sufficient to activate pro-survival pathways in cells in 2D. Importantly, pharmacological inhibition of myosin in non-spread MECs on a 2D ECM, which causes loss of cortical tension, increased negative membrane curvature and cell viability. Conversely, genetic knockdown of the negative curvature-inducing protein Exo70 compromised MEC survival in a 3D ECM. Our results provide the first evidence demonstrating that ECM dimensionality alters the biophysical properties of cells to modulate plasma membrane curvature and activate pro-survival signaling pathways. Our findings also offer a unique perspective for why Arf6 and Rac GTPases have been implicated in cancer aggression and suggest that targeting the tissue ECM or cellular cortical tension may provide a novel therapeutic approach to target dormant cells. Citation Format: FuiBoon Kai, Guanqing Ou, Alexandra Long, Wei Guo, Richard Tourdot, Ravi Radhakrishnan, Christopher Chen, Sophie Dumont, Valerie M. Weaver. Extracellular matrix dimensionality reduces cellular cortical tension to stimulate pro-survival signaling in mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1028.

Variational Autoencoding Tissue Response to Microenvironment Perturbation.

This work applies deep variational autoencoder learning architecture to study multi-cellular growth characteristics of human mammary epithelial cells in response to diverse microenvironment perturbations. Our approach introduces a novel method of visualizing learned feature spaces of trained variational autoencoding models that enables visualization of principal features in two dimensions. We find that unsupervised learned features more closely associate with expert annotation of cell colony organization than biologically-inspired hand-crafted features, demonstrating the utility of deep learning systems to meaningfully characterize features of multi-cellular growth characteristics in a fully unsupervised and data-driven manner.

Molecular Actions of Thyroid Hormone on Breast Cancer Cell Migration and Invasion via Cortactin/N-WASP

The thyroid hormone triiodothyronine (T3) plays a fundamental role in growth regulation, differentiation, metabolism and cellular movement. These processes are particularly important considering that deregulation of T3 levels could promote abnormal responsiveness of mammary epithelial cells, which may lead to the development and progression of breast cancer (BC). Once cells migrate and invade different tissues, BC metastasis is the main cause of cancer-related death because it is particularly difficult to revert this multistep process. Cell migration integrates several steps that induce changes in cell structure and morphology to promote BC cell invasion. These sequential steps include actin cytoskeleton remodeling, focal adhesion complex formation and, finally, the turnover of branched actin filament networks. In this article, we demonstrate that T3 has the ability to modify the Epithelial-Mesenchymal Transition process. In addition, we show that T3 induces actin cytoskeleton reorganization, triggers focal adhesion formation and, as a consequence, promotes actin nucleation via non-genomic pathway. These events are specifically modulated by T3 via integrin αvβ3 to FAK/paxillin/cortactin/N-WASP/Arp2/3 complex signaling pathway, increasing cell adhesion, migration and invasion of T-47D BC cells. We suggest that T3 influences the progression of tumor metastasis by controlling signaling pathways that converge in cell motility. This knowledge is crucial for the development of novel therapeutic strategies for BC treatment.

Ketoprofen affects the mammary immune response in dairy cows in vivo and in vitro

Nonsteroidal anti-inflammatory drugs are commonly administered parenterally in addition to antimicrobial mastitis therapy to increase the well-being of the diseased animal. As mastitis is usually a localized infection of mammary tissue, we tested the hypothesis that a local administration of nonsteroidal anti-inflammatory drugs through the teat canal could have anti-inflammatory effects on the affected area. We investigated the effects of intramammarily administered ketoprofen (KET) during an LPS-induced immune response on somatic cell count (SCC) and blood-milk barrier integrity. In addition, we investigated the effects of KET on the mRNA abundance of immune factors and their prostaglandin E2 secretion in primary bovine mammary epithelial cells in vitro. Six cows received 0.2 µg of LPS (serotype O26:B6) together with 50 mg of KET into one quarter and LPS only in the opposing quarter. The increase of SCC and of serum albumin (SA) and IgG concentrations and the increase of lactate dehydrogenase (LDH) activity in milk induced by LPS were lower in quarters that received KET in addition. In 3 cows, intramammary KET (50 mg) without additional LPS did not affect SCC, SA, IgG, and LDH in milk. Effects of KET on the immune response of mammary epithelial cells in vitro were investigated in cells from 3 cows challenged with or without LPS (0.2 µg/mL) and with or without additional KET in 2 concentrations (1.25 or 2.5 mg/mL). Ketoprofen reduced the LPS-induced increase of mRNA abundance of tumor necrosis factor α, IL-8, serum amyloid A, and cyclooxygenase-2. The mRNA abundance of cyclooxygenase-1 and prostaglandin E synthase was reduced in cells without LPS challenge by addition of KET at 2.5 mg/mL. Furthermore, the LPS-induced secretion of prostaglandin E2 of mammary epithelial cells into the supernatant could not be detected if KET was added. The results demonstrate that intramammary KET diminishes the increase of SCC and reduces the impairment of the blood-milk barrier (based on SA and LDH in milk), leading to a reduced IgG concentration in milk during LPS-induced mastitis. In mammary epithelial cells, KET limits the expression of several immune factors that are increased during an immune response. In summary, intramammary administration of KET reduces the inflammatory response in the mammary gland. However, it remains unclear whether the inhibited transfer of immune cells and IgG from blood into milk after KET administration would reduce the success of the immune defense in infectious mastitis.

HDAC1/2‐mediated regulation of JNK and ERK phosphorylation in bovine mammary epithelial cells in response to TNF‐α

Bovine mammary epithelial cells (MAC-Ts) are a common cell line for the study of mammary epithelial inflammation; these cells are used to mechanistically elucidate molecular underpinnings that contribute to bovine mastitis. Bovine mastitis is the most prevalent form of disease in dairy cattle that culminates in annual losses of two billion dollars for the US dairy industry. Thus, there is an urgent need for improved therapeutic strategies. Histone deacetylase (HDAC) inhibitors are efficacious in rodent models of inflammation, yet their role in bovine mammary cells remain unclear. HDACs have traditionally been studied in the regulation of nucleosomal DNA, in which deacetylation of histones impact chromatin accessibility and gene expression. Using MAC-T cells stimulated with tumor necrosis factor α (TNF-α) as a model for mammary cell inflammation, we report that inhibition of HDACs1 and 2 (HDAC1/2) attenuated TNF-α-mediated inflammatory gene expression. Of note, we report that HDAC1/2-mediated inflammatory gene expression was partly regulated by c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) phosphorylation. Here, we report that HDAC1/2 inhibition attenuated JNK and ERK activation and thus inflammatory gene expression. These data suggest that HDACs1 and 2 regulate inflammatory gene expression via canonical (i.e., gene expression) and noncanonical (e.g., signaling dependent) mechanisms. Whereas, further studies using primary cell lines and animal models are needed. Our combined data suggest that HDAC1/2-specific inhibitors may prove efficacious for the treatment of bovine mastitis.

Sensing of Escherichia coli and LPS by mammary epithelial cells is modulated by O-antigen chain and CD14.

Escherichia coli is one of the major pathogens causing mastitis in dairy cattle. Yet, the factors which mediate the ability for E. coli to develop in the bovine mammary gland remain poorly elucidated. In a mouse model, infections induced by the reference mastitis E. coli P4 showed a strong colonisation of the mammary gland, while this strain had a low stimulating power on cells of the PS bovine mammary epithelial cell line. In order to understand if such a reduced response contributes to the severity of infection, a library of random mutants of P4 strain was screened to identify mutants inducing stronger response of PS cells. Among hyper-stimulating mutants, six were altered in genes involved in biosynthesis of lipopolysaccharide (LPS) and had lost their O-polysaccharide region, suggesting that the presence of O-antigen impairs the response of PS cells to LPS. Using purified smooth (S) and rough (R) fractions of LPS, we showed that the R-LPS fraction induced a stronger response from PS cells than the smooth LPS fraction. Biological activity of the S-LPS fraction could be restored by the addition of recombinant bovine CD14 (rbCD14), indicating a crucial role of CD14 in the recognition of S-LPS by Mammary Epithelial Cells (MEC). When S-LPS and R-LPS were injected in udder quarters of healthy lactating cows, an inflammation developed in all infused quarters, but the S-LPS induced a more intense pro-inflammatory response, possibly in relation to sizeable concentrations of CD14 in milk. Altogether, our results demonstrate that the O-antigen modulates the pro-inflammatory response of MEC to LPS, that S-LPS and R-LPS trigger different responses of MEC and that these responses depend on the presence of CD14.

Blimp-1/PRDM1 is a critical regulator of Type III Interferon responses in mammary epithelial cells

The transcriptional repressor Blimp-1 originally cloned as a silencer of type I interferon (IFN)-β gene expression controls cell fate decisions in multiple tissue contexts. Conditional inactivation in the mammary gland was recently shown to disrupt epithelial cell architecture. Here we report that Blimp-1 regulates expression of viral defense, IFN signaling and MHC class I pathways, and directly targets the transcriptional activator Stat1. Blimp-1 functional loss in 3D cultures of mammary epithelial cells (MECs) results in accumulation of dsRNA and expression of type III IFN-λ. Cultures treated with IFN lambda similarly display defective lumen formation. These results demonstrate that type III IFN-λ profoundly influences the behavior of MECs and identify Blimp-1 as a critical regulator of IFN signaling cascades.

Identification and characterization of bovine mammary peptide transporters in response to tripeptide and lactogenic hormone treatment

Oligopeptide transportation is mediated by the peptide transporter (PepT), which consists of two isoforms, PepT1 and PepT2. Because PepT play essential roles in amino acid metabolism and cell growth, the aim of the present study was to identify these transporters in bovine mammary glands and to analyze the potential functions of these transporters in mammary epithelial cells. Abundance of PepT1 and PepT2 mRNA was successfully measured in both mammary glands and cultured mammary epithelial cells. In addition, the two proteins were examined using immunohistochemistry, immunocytochemistry, and Western blots. The response of mammary epithelial cells to tripeptide and lactogenic hormone treatment was assayed. The PepT mRNA abundance of cultured epithelial cells and secreted protein in the culture medium were increased after tri-peptide substitution and addition of hormones such as insulin, hydrocortisone, and prolactin. The response of mammary epithelial cells to tripeptide and hormone treatments suggests that PepT affects the mammary gland function and increases bovine milk production.

SDF-1 in Mammary Fibroblasts of Bovine with Mastitis Induces EMT and Inflammatory Response of Epithelial Cells.

Fibroblasts constitute the majority of the stromal cells within bovine mammary gland, yet the functional contributions of these cells to mastitis and fibrosis and the mechanism are poorly understood. In this study, we demonstrate that inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix (ECM) proteins, chemokines and cytokines compared to normal fibroblasts (NFs) from dairy cows during lactation. The INFs induced epithelial-mesenchymal transition (EMT) and inflammatory responses of mammary epithelial cells in a vitro co-culture model. These functional contributions of INFs to normal epithelial cells were mediated through their ability to secrete stromal cell-derived factor 1 (SDF-1). SDF-1 was highly secreted/expressed by INFs, lipopolysaccharide (LPS) -treated NFs, lipoteichoic acid (LTA) -treated NFs, as well as mastitic tissue compared to their counterparts. Exogenous SDF-1 promoted EMT on epithelial cells through activating NF-κB pathway, induced inflammation response and inhibited proliferation of epithelial cells. In addition, SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland, which was attenuated by a specific inhibitor of the receptor of SDF-1. Our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1, which could result in the inflammation spread and fibrosis within mammary gland.

Potassium carbonate as a cation source for early-lactation dairy cows fed high-concentrate diets

Previous studies reported that addition of K2CO3 to high-concentrate diets improved milk fat synthesis, although the mechanism is yet to be established. The objective of the current experiment was to investigate the effects of dietary cation-anion difference (DCAD), cation source, and buffering ability of the mineral supplement on rumen biohydrogenation of fatty acids and production performance in dairy cows fed a high-concentrate diet. Thirty-five early-lactation Holstein cows (25 multiparous ruminally fistulated and 10 primiparous nonfistulated) were used in a randomized complete block design (7 blocks) with 33-d periods, including a 5-d pre-treatment collection period used as a covariate. Diets were (1) control, a basal diet [47% nonfibrous carbohydrates, DCAD (Na + K - Cl - S) = 65 mEq/kg of dry matter (DM)] containing 40% forage (including 60% corn silage) and 60% concentrate, (2) K2CO3 (control + K2CO3, 1.8% of DM, DCAD = 326 mEq/kg of DM), (3) KHCO3 (control + KHCO3, 2.6% of DM, DCAD = 324 mEq/kg of DM), (4) KCl (control + KCl, 2.0% of DM, DCAD = 64 mEq/kg of DM), and (5) Na2CO3 (control + Na2CO3, 1.4% of DM, DCAD = 322 mEq/kg of DM). Pre-planned orthogonal contrasts were used to assess the effects of K2CO3 (control vs. K2CO3), buffering ability (K2CO3 vs. KHCO3), DCAD (K2CO3 vs. KCl), and cation type (K2CO3 vs. Na2CO3). Supplementing K2CO3 in a high-concentrate diet did not improve milk fat yield or 4% fat-corrected milk yield. Milk fat concentration was greater in cows fed K2CO3 compared with control (4.03 vs. 3.26%). Milk yield tended to decrease (34.5 vs. 38.8 kg/d) and lactose yield decreased in cows fed K2CO3 as compared with KCl (1.64 vs. 1.87 kg/d). Milk fat concentration of trans-10 18:1 was increased when cows were fed Na2CO3 as compared with K2CO3. A positive relationship was observed between concentrations of anteiso 15:0 and trans-10,cis-12 18:2 in milk fat from cows receiving K2CO3. Milk Na concentration was increased, whereas milk Cl was decreased with K2CO3 as compared with KHCO3 or KCl. A positive relationship was established between milk Cl concentration and milk yield (R2 = 0.34) across all dietary treatments. Cation-anion difference (Na + K - Cl - S) in ruminal fluid was increased with K2CO3 as compared with control or KCl. Blood pH tended to decrease in cows fed KCl compared with K2CO3. Our results suggest that mineral supplementation tends to affect milk and milk fat synthesis and that factors other than DCAD, potassium ion, or buffer ability may be implicated. The variations observed in mineral composition of milk suggest an allostatic process to maintain an ionic equilibrium in mammary epithelial cells in response to mineral composition of the diet.

Variant innate immune responses of mammary epithelial cells to challenge by Staphylococcus aureus, Escherichia coli and the regulating effect of taurine on these bioprocesses

Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are important pathogens causing subclinical and clinical bovine mastitis, respectively. Taurine, an organic acid found in animal tissues, has been used for the treatment of various superficial infections and chronic inflammations. We challenged a bovine mammary epithelial cell (MEC) line (MAC-T) or a mouse mammary epithelial cell line (EpH4-Ev) with either E. coli or S. aureus and compared the responses of MECs to these 2 pathogens. We also examined the regulatory effects of taurine on these responses. Receptor analyses showed that both TLR2 and TLR4 are upregulated upon exposure to either E. coli or S. aureus. Taurine pre-treatment dampened upregulation to some extent. E. coli and S. aureus stimulated comparable levels of ROS, which could be inhibited by taurine pre-treatment. E. coli infection elicited a dramatic change in iNOS expression. Taurine significantly decreased iNOS expression in the S. aureus challenged group. Protein microarray demonstrated that 32/40 and 8/40 inflammatory molecules/mediators were increased after E. coli or S. aureus challenge, respectively. The fold changes of most molecules were higher in the E. coli infection group than that in the S. aureus infection group. Taurine negatively regulated the inflammatory profile in both bacterial infections. Pro-inflammatory cytokines (such as TNF-α) connected with TLR activation were down-regulated by taurine pre-treatment. The influence of TAK-242 and OxPAPC on cytokine/molecule expression profiles to E. coli challenge are different than to S. aureus. Some important factors (MyD88, TNF-α, IL-1β, iNOS and IL-6) mediated by TLR activation were suppressed either in protein microarray or special assay (PCR/kits) or both. TAK-242 restrained ROS production and NAGase activity similar to the effect of taurine in E. coli challenge groups. The detection of 3 indices (T-AOC, SOD and MDA) reflecting oxidative stress in vivo, showed that taurine improved the antioxidant ability of cells. We conclude that taurine can regulate the inflammatory response during infection with E. coli and prevent cell damage by affecting the signaling pathways mediated by TLRs and by improving the antioxidant ability of cells. In S. aureus infections, taurine's antioxidant ability may be the primary means of resistance to inflammation. This study provides a better understanding of the inflammatory mechanisms of E. coli and S. aureus mastitis, and it provides a putative strategy for the prevention of this disease.

Extracellular milieu grossly alters pathogen-specific immune response of mammary epithelial cells.

BackgroundConsiderably divergent data have been published from attempts to model the E. coli vs. S. aureus specific immune reaction of the udder using primary cultures of bovine mammary epithelial cells from cows (pbMEC). Some groups reported a swift, strong and transient inflammatory response against challenges with E. coli and only a weak and retarded response against S. aureus, in agreement with the respective reaction of the udder. Others found almost the reverse. Presence or absence of fetal calf serum distinguished the experimental setting between both groups. We examined here if this causes the divergent reaction of the pbMEC towards both pathogen species. We challenged pbMEC with proteins from heat killed E. coli or S. aureus pathogens or purified TLR2 and TLR4 ligands. The stimuli were applied in normal growth medium with (SM10) or without (SM0) 10 % fetal calf serum, or in the basal medium supplemented with 10 mg/ml milk proteins (SM Milk).ResultsWithdrawal of FCS slowed down and decreased the extent by which E. coli or LPS enhanced the expression of cyto- and chemokine encoding genes through impaired TLR4 signalling but enforced their expression during stimulation with S. aureus. SM Milk strongly quenched the induction of those genes. S. aureus strain specific differences in the reaction of the pbMEC could only be recorded in SM0. NF-κB factors were activated by E. coli in all stimulation media, but only to a small extent by S. aureus, solely in SM0. Purified ligands for TLR2 stimulated expression of those genes and activated NF-κB equally well in SM10 and SM0. The mRNA destabilizing factor tristetraproline was only induced by E. coli in SM10 and by purified PAMPs.ConclusionsOur data cross validate the correctness of previously published divergent data on the pathogen-specific induction of key immune genes in pbMEC. The differences are due to the presence of FCS, modulating signalling through TLR4 and TLR-unrelated pathogen receptors. S. aureus does not substantially activate any TLR signalling in MEC. Rather, receptors distinct from TLRs perceive the presence of S. aureus and control the immune response against this pathogen in MEC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0489-3) contains supplementary material, which is available to authorized users.

Advances in bovine immunology - new tools and new insights to tackle old foes.

Advances in bovine immunology - new tools and new insights to tackle old foes.

Collagen density regulates xenobiotic and hypoxic response of mammary epithelial cells

Breast density, where collagen I is the dominant component, is a significant breast cancer risk factor. Cell surface integrins interact with collagen, activate focal adhesion kinase (FAK), and downstream cell signals associated with xenobiotics (AhR, ARNT) and hypoxia (HIF-1α, ARNT). We examined if mammary cells cultured in high density (HD) or low density (LD) collagen gels affected xenobiotic or hypoxic responses. ARNT production was significantly reduced by HD culture and in response to a FAK inhibitor. Consistent with a decrease in ARNT, AhR and HIF-1α reporter activation and VEGF production was lower in HD compared to LD. However, P450 production was enhanced in HD and induced by AhR and HIF-1α agonists, possibly in response to increased NF-κB activaton. Thus, collagen density differentially regulates downstream cell signals of AhR and HIF-1α by modulating the activity of FAK, the release of NF-κB transcriptional factors, and the levels of ARNT.