IFN-γ Activates the TLR4-CCL5 Signaling Through Reducing Arginine Level, Leading to Enhanced Susceptibility of Bovine Mammary Epithelial Cells to Staphylococcus aureus.

Dairy cow mastitis is a common bacterial infectious disease which seriously threatens the development of the dairy cow industry. Previous studies have found that increased IFN-γ expression in dairy cows makes dairy cows more susceptible to mastitis, but the underlying mechanism is still not known. In this study, we utilized the in vitro bovine mammary epithelial cells (BMECs) model to explore the molecular mechanism via transcriptome sequencing technology, immunofluorescence, and Western blotting. It was found that IFN-γ promoted the adhesion and invasion of Staphylococcus aureus to BMECs through increasing the expression of TLR4-mediated CCL5 in BMECs. IFN-γ increased the activity of arginase II and reduced the level of arginine in cells, while the addition of arginine inhibited the expression of TLR4 and CCL5. An invasion experiment in mice further validated that IFN-γ treatment significantly increased the bacterial load in mammary glands and blood. However, the colonization and diffusion of S. aureus were interestingly decreased after Arg supplement. These data reveal that increased IFN-γ reduces arginine levels and activates TLR4-CCL5 signaling, leading to enhanced susceptibility of BMECs to S. aureus. Our findings are helpful to understand the pathogenesis of dairy cow mastitis and provide a theoretical basis for improvement of mastitis resistance in dairy cows.

TGF-ß1 promoted the infection of bovine mammary epithelial cells by Staphylococcus aureus through increasing expression of cells' fibronectin and integrin ß1.

Mastitis is a disease that affects dairy cattle and causes a decline in milk quality as well as economic loss worldwide. TGF-ß1 levels are usually increased during mastitis; however, it is unknown whether TGF-ß1 is involved in bovine mastitis. Therefore, this study evaluated the effects of TGF-ß1 on the susceptibility of bovine mammary epithelial cells (BMECs) to Staphylococcus aureus (S. aureus). The results revealed that S. aureus adhesion to and invasion of BMECs was significantly increased after cells were treated with TGF-ß1. Adhesion of S. aureus to BMECs was increased dramatically by upregulation of fibronectin (Fn) and integrin ß1 (ITGB1), while the increase in the susceptibility of BMECs to S. aureus was blocked by specific antibodies against either Fn or ITGB1. These results indicated that adhesion and invasion were increased by TGF-ß1-induced upregulation of both Fn and ITGB1. Furthermore, TGF-ß1 treatment prior to S. aureus infection significantly increased S. aureus colonization as well as Fn and ITGB1 expression in the mammary glands of mice. These results suggest that TGF-ß1 promoted the expression of Fn and ITGB1 on the surface of BMECs and contributed to mammary gland infection in vitro and in vivo. The results of this study imply that Fn and ITGB1 may be useful therapeutic targets for the treatment of mastitis in dairy cows.

Exosomes Derived from Bovine Mammary Epithelial Cells Treated with Transforming Growth Factor-ß1 Inhibit the Proliferation of Bovine Macrophages.

Transforming growth factor (TGF)-ß1 is a multifunctional cytokine that plays an important role in regulating immune cell proliferation. We speculate that high expression of TGF-ß1 may affect the immunity of dairy cows. In this study, untreated exosomes (un-exo) derived from an untreated bovine mammary epithelial cell line (MAC-T) and TGF-ß1-treated exosomes (t-exo) derived from TGF-ß1-treated MAC-T cells were isolated by ultracentrifugation and identified by electron microscopy and Western blotting. Then, un-exo and t-exo were used to treat a bovine macrophage cell line (BOSMAC), and the proliferative ability of BOSMAC cells was detected by an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and flow cytometry. The expression and phosphorylation levels of p38 were analyzed by q-PCR and Western blotting. The results showed that both exosome types exhibited the basic characteristics of exosomes. In BOSMAC cells treated with t-exo, significant inhibition of cell proliferation was observed, and the cell cycle progression was inhibited, while no difference was found between the un-exo and control groups. Only treatment with t-exo increased the expression and phosphorylation of p38, and the addition of the p38 inhibitor SB203580 abrogated the inhibition of BOSMAC cell proliferation by t-exo. Our results demonstrated that t-exo inhibited the proliferation of bovine macrophages by stimulating p38 MAPK and might interfere with immunity in dairy cattle. This finding may provide a new strategy for improving immunity and preventing breast-related diseases in dairy cows.

Interferon-γ regulates cell malignant growth via the c-Abl/HDAC2 signaling pathway in mammary epithelial cells.

Interferon-γ (IFN-γ) has been used to control cancers in clinical treatment. However, an increasing number of reports have suggested that in some cases effectiveness declines after a long treatment period, the reason being unclear. We have reported previously that long-term IFN-γ treatment induces malignant transformation of healthy lactating bovine mammary epithelial cells (BMECs) in vitro. In this study, we investigated the mechanisms underlying the malignant proliferation of BMECs under IFN-γ treatment. The primary BMECs used in this study were stimulated by IFN-γ (10 ng/mL) for a long term to promote malignancy. We observed that IFN-γ could promote malignant cell proliferation, increase the expression of cyclin D1/cyclin-dependent kinase 4 (CDK4), decrease the expression of p21, and upregulate the expression of cellular-abelsongene (c-Abl) and histone deacetylase 2 (HDAC2). The HDAC2 inhibitor, valproate (VPA) and the c-Abl inhibitor, imatinib, lowered the expression level of cyclin D1/CDK4, and increased the expression level of p21, leading to an inhibitory effect on IFN-γ-induced malignant cell growth. When c-Abl was downregulated, the HDAC2 level was also decreased by promoted proteasome degradation. These data suggest that IFN-γ promotes the growth of malignant BMECs through the c-Abl/HDAC2 signaling pathway. Our findings suggest that long-term application of IFN-γ may be closely associated with the promotion of cell growth and even the carcinogenesis of breast cancer.

A corn straw-based diet increases release of inflammatory cytokines in peripheral blood mononuclear cells of dairy cows.

Recent studies have shown that diet can affect the body's immunity. Roughage of dairy cows consists of a variety of plant materials which make different contributions to health. This study investigated the effect of different roughages on the immunity of dairy cows. Serum, peripheral blood mononuclear cells (PBMCs), and milk samples were collected from 20 multiparous mid-lactation cows fed mixed forage (MF)- or corn straw (CS)-based diets. Expression profile analysis was used to detect the differentially expressed genes (DEGs) from PBMCs. The results showed that milk protein in the MF group increased to 3.22 g/100 ml, while that of the CS group milk was 2.96 g/100 ml; by RNA sequencing, it was found that 1615 genes were differentially expressed between the CS group and the MF group among the 24 027 analyzed probes. Gene ontology (GO) and pathway analysis of DEGs suggested that these genes (especially genes coding cytokines, chemokine and its receptors) are involved in the immune response. Results were confirmed at the protein level via detecting the levels of interleukin-2 (IL-2), IL-6, IL-10, IL-12, leptin (LEP), interferon-γ (IFN-γ), transforming growth factor-ß1 (TGF-ß1), and tumor necrosis factor-α (TNF-α) in peripheral blood by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay analysis. Our data supported the conclusions that the protein content in milk of the MF group was higher than that of the CS group, the CS-based diets induced more release of cytokines than the MF-based diets in dairy cows' PBMCs, and milk protein content may be affected by cytokines.

Arginine inhibits the malignant transformation induced by interferon-gamma through the NF-κB-GCN2/eIF2α signaling pathway in mammary epithelial cells in vitro and in vivo.

Breast cancer is the most common female malignant tumors in the world. It seriously affects women's physical and mental health and the leading cause of cancer death among women. Our previous study demonstrated that diet-derived IFN-γ promoted the malignant transformation of primary bovine mammary epithelial cells by accelerating arginine depletion. The current study aimed to explore whether arginine addition could inhibit the degree of malignant transformation and its molecular mechanism. The results indicate that arginine addition could alleviate the malignant transformation of mammary epithelial cells induced by IFN-γ, including reducing cell proliferation, cell migration and colony formation, through the NF-κB-GCN2/eIF2α pathway. The in vivo experiments also consistently confirmed that arginine supplementation could significantly inhibit tumor growth in tumor-bearing mice. Furthermore, the investigation of the clinical data also revealed that the plasma or tissue from human breast cancer patients owned lower arginine level and higher IFN-γ level than that from patients with benign breast disease, showing IFN-γ may be a potential control target. Our findings demonstrate that arginine supplement could antagonize the malignant transformation of mammary epithelial cells induced by IFN-γ (nutritionally induced) both in vitro and in vivo, and IFN-γ was higher in breast cancer women. This might provide a novel strategy for the prevention and treatment of breast cancer regarding to nutrition.

TGF-ß1 promotes Staphylococcus aureus adhesion to and invasion into bovine mammary fibroblasts via the ERK pathway.

Fibroblasts are the structural base of mammary breast tissues. TGF-ß1 can regulate the fibrotic process; however, it remains unclear whether TGF-ß1 influences the susceptibility of fibroblasts to bacteria. Staphylococcus aureus (S. aureus) is a major bacterium in both chronic and subclinical mastitis in lactating cows that acts by invading host cells. To better understand the function of TGF-ß1 in bovine mammary fibroblasts' (BMFBs) susceptibility to bacteria as well as the mechanisms involved, a primary BMFB model was established by treating cells with TGF-ß1 followed by infection with S. aureus. The results revealed that the adhesion and invasion of S. aureus into BMFBs was significantly increased after cells were treated with 5 ng/ml TGF-ß1 for 12 h. Moreover, TGF-ß1 can increase Collagen I and α-SMA expression via activation of ERK signaling. However, the increased adhesion and invasion of S. aureus can be blocked by specific antibodies against either Collagen I or α-SMA, indicating that the increased adhesion and invasion are dependent on TGF-ß1-induced upregulation of both Collagen I and α-SMA. Using PD98059, an ERK inhibitor, could also decrease the adhesion and invasion of S. aureus. These results indicate that TGF-ß1 could promote S. aureus adhesion to and invasion into BMFBs by increasing Collagen I and α-SMA expression and may provide a novel target for controlling bovine mastitis.

TGF-ß1 promotes bovine mammary fibroblast proliferation through the ERK 1/2 signalling pathway.

The abnormal proliferation of bovine mammary fibroblasts (BMFBs) impairs mammary gland development and lactation. Severe manifestations develop into breast fibrosis, leading to the culling of cows and causing serious losses to the dairy industry. Transforming growth factor ß1 (TGF-ß1) is an important modulator of cell proliferation and extracellular matrix formation; however, limited information is available on BMFBs. In this study, a convenient and stable culture method for BMFBs was established. Treatment with 5 ng/mL of TGF-ß1 significantly promoted the proliferation of BMFBs and accelerated the cell cycle. TGF-ß1 stimulation for up to 12 h significantly increased the relative ERK1/2 mRNA expression and enhanced the protein expression of p-ERK1/2 and cyclin D1. Conversely, the ERK1/2 inhibitor PD98059 blocked these TGF-ß1 effects. Further exploration using a mouse model showed that TGF-ß1 significantly increased the proportion of fibroblasts and accelerating the cell transition from the G1 to G2/M phases. In addition, TGF-ß1 enhanced the expression of fibrosis markers, α-SMA and I Collagen, which could be blocked efficiently by the PD98059 in mouse mammary gland. Finally, immunofluorescence analysis confirmed that TGF-ß1 promoted fibroblast proliferation in healthy dairy cows after normal long-term dietary corn straw roughage supplementation. It is suggested that the diet may promote mammary fibroblast proliferation by raising the level of TGF-ß1. Our study provides new insights into how nutrition causes undesirable changes in mammary gland structure.

Adh enhances Actinobacillus pleuropneumoniae pathogenicity by binding to OR5M11 and activating p38 which induces apoptosis of PAMs and IL-8 release.

Members of the Trimeric Autotransporter Adhesin (TAA) family play a crucial role in the adhesion of Gram-negative pathogens to host cells, but the immunopathogenesis of TAAs remains unknown. Our previous studies demonstrated that Adh from Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is required for full bacterial pathogenicity. Alveolar macrophages are the first line of defense against respiratory infections. This study compared the interactions between porcine alveolar macrophages (PAMs) and wild-type A. pleuropneumoniae (5b WT) or an Adh-deletion strain (5b ΔAdh) via gene microarray, immunoprecipitation and other technologies. We found that Adh was shown to interact with the PAMs membrane protein OR5M11, an olfactory receptor, resulting in the high-level secretion of IL-8 by activation of p38 MAPK signaling pathway. Subsequently, PAMs apoptosis via the activation of the Fax and Bax signaling pathways was observed, followed by activation of caspases 8, 9, and 3. The immunological pathogenic roles of Adh were also confirmed in both murine and piglets infectious models in vivo. These results identify a novel immunological strategy for TAAs to boost the pathogenicity of A. pleuropneumoniae. Together, these datas reveal the high versatility of the Adh protein as a virulence factor and provide novel insight into the immunological pathogenic role of TAAs.

Arginine Supplementation Recovered the IFN-γ-Mediated Decrease in Milk Protein and Fat Synthesis by Inhibiting the GCN2/eIF2α Pathway, Which Induces Autophagy in Primary Bovine Mammary Epithelial Cells.

During the lactation cycle of the bovine mammary gland, autophagy is induced in bovine mammary epithelial cells (BMECs) as a cellular homeostasis and survival mechanism. Interferon gamma (IFN-γ) is an important antiproliferative and apoptogenic factor that has been shown to induce autophagy in multiple cell lines in vitro. However, it remains unclear whether IFN-γ can induce autophagy and whether autophagy affects milk synthesis in BMECs. To understand whether IFN-γ affects milk synthesis, we isolated and purified primary BMECs and investigated the effect of IFN-γ on milk synthesis in primary BMECs in vitro. The results showed that IFN-γ significantly inhibits milk synthesis and that autophagy was clearly induced in primary BMECs in vitro within 24 h. Interestingly, autophagy was observed following IFN-γ treatment, and the inhibition of autophagy can improve milk protein and milk fat synthesis. Conversely, upregulation of autophagy decreased milk synthesis. Furthermore, mechanistic analysis confirmed that IFN-γ mediated autophagy by depleting arginine and inhibiting the general control nonderepressible-2 kinase (GCN2)/eukaryotic initiation factor 2α (eIF2α) signaling pathway in BMECs. Then, it was found that arginine supplementation could attenuate IFN-γ-induced autophagy and recover milk synthesis to some extent. These findings may not only provide a novel measure for preventing the IFN-γ-induced decrease in milk quality but also a useful therapeutic approach for IFN-γ-associated breast diseases in other animals and humans.