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Klebsiella pneumoniae (K. pneumoniae) is one of the main environmental pathogens causing bovine mastitis. The incidence of bovine mastitis caused by K. pneumoniae is increasing worldwide. Selenium is an essential trace element that has multiple physiological functions, such as antioxidant and anti-inflammatory activities. Therefore, this study aimed to verify whether selenomethionine (SeMet) could contribute to alleviating the inflammatory injury and oxidative damage induced by K. pneumoniae. Bovine mammary epithelial cells were cultured in vitro and pretreated with 4 μM SeMet before being infected with K. pneumoniae. Western blot analysis was used to detect the expression of the related proteins in the NF-κB and Nrf2 signaling pathways. The gene expression levels of IL-1β, IL-6, IL-8, TNF-α, Nrf2, Keap1, NQO-1 and HO-1 were detected using RT-qPCR. The levels of MDA, GSH-PX, SOD, CAT and T-AOC were detected by commercial assay kits. Flow cytometry was used to determine the level of intracellular ROS, and immunofluorescence was used to detect the nuclear localization of Nrf2 protein. Briefly, SeMet downregulated the phosphorylation levels of IκBα and p65 proteins and the gene expression levels of IL-1β, IL-6, IL-8 and TNF-α were also decreased. Moreover, the protein and gene expression levels of Nrf2, NQO-1 and HO-1 were upregulated, and the nuclear expression of Nrf2 protein was also promoted, which enhanced the activity of antioxidant enzymes. In conclusion, SeMet protected BMECs from inflammatory injury and oxidative stress induced by K. pneumoniae by inhibiting the NF-κB and activating the Nrf2 signaling pathway.

Staphylococcus aureus is a pathogen that is the causative agent of several human and veterinary infections and plays a critical role in the clinical and subclinical mastitis of cattle. Autophagy is a conserved pathogen defence mechanism in eukaryotes. Studies have reported that S aureus can subvert autophagy and survive in cells. Staphylococcus aureus survival in cells is an important cause of chronic persistent mastitis infection. However, it is unclear whether S aureus can escape autophagy in innate immune cells. In this study, initiation of autophagy due to the presence of S aureus was detected in bovine macrophages. We observed autophagic vacuoles increased after S aureus infection of bovine macrophages by transmission electron microscopy (TEM). It was also found that S aureus‐infected bovine macrophages increased the expression of LC3 at different times(0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 hours). Data also showed the accumulation of p62 induced by S aureus infection. Application of autophagy regulatory agents showed that the degradation of p62 was blocked in S aureus induced bovine macrophages. In addition, we also found that the accumulation of autophagosomes promotes S aureus to survive in macrophage cells. In conclusion, this study indicates that autophagy occurs in S aureus‐infected bovine macrophages but is blocked at a later stage of autophagy. The accumulation of autophagosomes facilitates the survival of S aureus in bovine macrophages. These findings provide new insights into the interaction of S aureus with autophagy in bovine macrophages.

Background Bacterial infections are common in postpartum dairy cows. Cortisol level has been observed to increase in dairy cows during peripartum period, and is associated with the endometrial innate immunity against pathogens like E.coli. However, the mechanism underlying how cortisol regulates E.coli-induced inflammatory response in bovine endometrial epithelial cells (BEEC) remains elusive. Results Cortisol decreased the expressions of IL1β, IL6, TNF-α, IL8, and TLR4 mRNA in BEEC treated with LPS or heat-killed E.coli, but up-regulated these gene expressions in BEEC stimulated by live E.coli. Conclusion Cortisol exerted the anti-inflammatory action on LPS- or heat-killed E.coli-stimulated BEEC, but the pro-inflammatory action on live E.coli-induced BEEC.

Staphylococcus aureus (S. aureus) is known to induce chronic and persistent bovine mammary infection, which affects milk quality and leads to premature culling. The ability of S. aureus to invade mammalian cells protects it from clearance by the immune system. Mitophagy is important in cell homeostasis, and can be utilized by pathogens for immune escape. However, mitophagy's role in S. aureus-associated bovine mastitis remains unclear. Here, S. aureus infection induced mitophagy and enhanced mitochondrial translocation of parkin in MAC-T cells. After mitophagy inhibition by Mdivi-1 treatment or PTEN-induced putative kinase 1 (PINK1) silencing in MAC-T cells infected with S. aureus, NOD-like receptor protein 3 (NLRP3) inflammasome activation and p65 and IκBα phosphorylation were increased. Meanwhile, PINK1 overexpression had the opposite effects. In addition, NLRP3 inflammasome overactivation and enhanced p65 and IκBα phosphorylation caused by PINK1 silencing were reversed by MitoTEMPO. Furthermore, PINK1/parkin-mediated mitophagy promoted S. aureus survival and contributed to persistent S. aureus infection. These findings provide new insights into S. aureus invasion in bovine mastitis.

Background Bovine endometrial epithelial cells (BEECs) undergo regular regeneration after calving. Elevated cortisol concentrations have been reported in postpartum cattle due to various stresses. However, the effects of the physiological level of cortisol on proliferation in BEECs have not been reported. The aim of this study was to investigate whether cortisol can influence the proliferation properties of BEECs and to clarify the possible underlying mechanism. Methods BEECs were treated with different concentrations of cortisol (5, 15 and 30 ng/mL). The mRNA expression of various growth factors was detected by quantitative reverse transcription-polymerase chain reaction (qPCR), progression of the cell cycle in BEECs was measured using flow cytometric analysis, and the activation of the Wnt/β-catenin and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways was detected with Western blot and immunofluorescence. Results Cortisol treatment resulted in upregulated mRNA levels of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF); however, it had no influence on transforming growth factor-beta1 (TGF-β1). Cortisol (15 ng/mL) accelerated the cell cycle transition from the G0/G1 to the S phase. Cortisol upregulated the expression of β-catenin, c-Myc, and cyclinD1 and promoted the phosphorylation of PI3K and AKT. Conclusions These results demonstrated that cortisol may promote proliferation in BEECs by increasing the expression of some growth factors and activating the Wnt/β-catenin and PI3K/AKT signaling pathways.

Staphylococcus aureus is an important pathogen causing chronic and subclinical mastitis of cows. Autophagy is an important regulatory mechanism that participates in the elimination of invading pathogenic organisms. Here, we hypothesize that autophagy is involved in the process of Staph. aureus survival in bovine mammary epithelial cells (BMEC). In this study, we detected the expression of autophagy-related proteins during infection and assessed the effect of autophagosome formation and degradation on the proliferation of intracellular Staph. aureus. Infection with Staph. aureus increased the protein expression of microtubule-associated protein 1 light chain 3-II (MAP1LC3, also called LC3-II) and sequestosome-1 (SQSTM1, also called p62) in BMEC. After infection, the formation of the autophagosomes increased but the autophagosomes and lysosomes could not fuse normally to form autolysosomes. When the formation of the autophagosomes was enhanced or the degradation of the autolysosomes was inhibited, the number of Staph. aureus in the BMEC increased. However, the intracellular proliferation of Staph. aureus was slowed when formation of autophagosomes was inhibited. Therefore, autophagy was induced in BMEC challenged by Staph. aureus but the autophagic flux was obstructed. Inhibiting the formation of autophagosomes in BMEC facilitated the clearance of intracellular Staph. aureus, which may offer a new strategy for the treatment of mastitis in cows.

Background The aim of the present study was to clarify the changes in complete blood count, blood biochemistry, and the gene expressions of pro-inflammatory cytokines of peripheral white blood cells in postpartum dairy cows with metritis. Results The cows were assigned to the control group (n = 28) or the metritis group (n = 28), retrospectively. Blood samples were taken 7 days before the estimated parturition (− 7 d), on the day of parturition (0 d), and 7 and 30 d after parturition. There was no difference in blood indexes between the control group and the metritis group at − 7 d. The WBC, granulocytes and monocytes were generally higher at 7 and 30 d in the metritis group than the control. In comparison with the controls, all liver function parameters and triglyceride levels at 0, 7 and 30 d, and the creatinine level at 7 and 30 d were higher in cows with metritis. The concentrations of Ca and P at 0, 7 and 30 d, and of glucose at 0 d were lower for cows in the metritis group compared with cows in the control group. Among these parameters, the WBC at 30 d, the aspartate aminotransferase activity (AST) at 7 d exceeded normal ranges (WBC: 5.0 ~ 16.0 × 109/L; AST: 42.5 ~ 98 U/L), whereas the concentrations of glucose and Ca from 0 to 30 d were below normal ranges (glucose: 2.5 ~ 4.5 mmol/L; Ca: 2.2 ~ 2.5 mmol/L) in the metritis group. The gene expressions of pro-inflammatory cytokines in the metritis group were higher than those in the control group, including the IL-1α at 7d, the IL-1β at − 7, 0 and 7 d, the IL-6 at − 7, 0, 7 and 30 d, the IL-8 at 0, 7 and 30 d, and the TNF-α at 7 and 30 d. Conclusion The cows with metritis experienced systemic inflammation for 4 weeks after calving, the impaired hepatic function, and the altered metabolic status with increased triglyceride level and decreased concentrations of glucose, Ca and P.

Bovine uterine infection is commonly caused by Escherichia coli (E. coli). Elevated concentrations of plasma cortisol have been reported in postpartum cows. However, the direct role of cortisol in the inflammatory response of bovine endometrial stromal cells (BESCs) remains unclear. Therefore, the aim of the study was to explore the regulatory effect of cortisol on lipopolysaccharide (LPS)-induced inflammatory response in BESCs. Both the primary and immortalized BESCs were used in this study. BESCs were treated with cortisol (5, 15, and 30 ng/mL) in the presence of LPS. The mRNA expression of inflammatory cytokines and chemokines was detected using RT-qPCR. Western blot and immunofluorescence were used to analyze the activation of the NF-κB and MAPK signaling pathways. The results revealed that cortisol downregulated the LPS-induced overexpression of interleukin(IL)-1β, IL-6, IL-8, TNF-α, COX-2, iNOS in BESCs. Moreover, cortisol inhibited LPS-induced phosphorylation levels of IκB, p65, ERK1/2, JNK and p38, and p65 nuclear translocation in BESCs. These results indicated that cortisol inhibited LPS-induced inflammatory response in BESCs, which may be mediated by suppressing the NF-κB and MAPK signaling pathways.

Postpartum uterine infection in dairy cows is commonly caused by pathogenic bacteria such as Escherichia coli (E. coli). Progesterone elicits immunosuppressive function within bovine endometrium, and has been suggested to be related to postpartum uterine infection. Endometrial stroma is exposed to bacteria due to the disruption of epithelium during parturition, but the effect and mechanism of progesterone on innate immune response of stromal cells has not been reported. This study evaluated the impact of progesterone on inflammatory response of primary endometrial stromal cells stimulated by lipopolysaccharide or heat-killed E. coli. Quantitative PCR analysis revealed that progesterone repressed mRNA induction of IL1B, IL6, TNF, CXCL8, NOS2, and PTGS2 in stromal cells in response to lipopolysaccharide or E. coli challenge. Consistently, Western blot and immunofluorescence staining results showed that progesterone suppressed lipopolysaccharide- or E. coli-induced MAPK and NF-κB activations characterized with decreased phosphorylations of ERK1/2, JNK, P38, IκBα, and P65, and inhibition of P65 nuclear translocation. In unstimulated stromal cells, progesterone alone did not affect the mRNA transcription for IL6, TNF, CXCL8, NOS2, and PTGS2, and the signaling cascade of MAPK and NF-κB, but decreased IL1B mRNA expression. These results revealed that the anti-inflammatory effect of progesterone in lipopolysaccharide- or E. coli-challenged endometrial stromal cells was probably mediated through MAPK and NF-κB pathways.

The bovine uterus is subject to infection after calving, which may lead to endometritis. Elevated cortisol levels have been observed in postpartum cattle. However, the role of cortisol in the inflammatory response of the uterus has not been reported. The aim of this study was to investigate the anti-inflammatory effects of cortisol on lipopolysaccharide (LPS)-induced primary bovine endometrial epithelial cells (BEECs). BEECs were treated with various concentrations of cortisol (5, 15 and 30 ng/mL) in the presence of LPS. The mRNA expression of TLR4 and proinflammatory cytokines was measured with qPCR. The activation of NF-κB and MAPK signalling pathways was detected with Western blotting and immunofluorescence. Cortisol induced the down-regulation of the mRNA expression of toll-like receptor 4 (TLR4) and proinflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor–α (TNF-α), cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS). Cortisol inhibited the activity of nuclear factor-κB (NF-κB) via blocking the phosphorylation and degradation of IκB. Cortisol suppressed the phosphorylation of mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase (ERK1/2), p38MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK). These results demonstrated that cortisol may exert its anti-inflammatory actions by regulating NF-κB activation and MAPK phosphorylation.

Postpartum uterine infections are common reproductive diseases in postpartum cows. Evidence has shown that plasma β-endorphins increase during bovine uterine inflammation. However, the effect of β-endorphins on the inflammatory response in bovine endometrium has not been clarified. The aim of this study was to investigate the effect of β-endorphins on the inflammatory response of bovine endometrial epithelial and stromal cells, and to explore the possible mechanism. The cells were treated with E. coli lipopolysaccharide (LPS) to simulate inflammation, which was characterized by the significant activation of NF-κB signaling pathway and the increased gene expression of the downstream proinflammatory cytokines (approximately 1.2- to 15-fold increase, P 0.05). By using Western blot and qPCR techniques, we found that β-endorphins inhibited the key protein expression of NF-κB pathway, and the gene expressions of TNF, IL1B, IL6, CXCL8, nitric oxide synthase 2, and prostaglandin-endoperoxide synthase 2 (P 0.05). The co-treatment of β-endorphins and opioid antagonists showed that the anti-inflammatory effect of β-endorphins could be blocked (P 0.05) by non-selective opioid antagonist naloxone or δ opioid receptor antagonist ICI 154129, but not the μ opioid receptor antagonist CTAP (P 0.05). In conclusion, β-endorphins may inhibit the inflammatory response of bovine endometrial epithelial and stromal cells through δ opioid receptor.

Glucocorticoids have been widely used as anti-inflammatory therapies. The mechanisms of cortisol action in goat does with endometritis, however, have not been reported. The aim of this study was to investigate the mechanism of cortisol in modulation of effects of E. coli-induced endometritis in the does. Does (n = 24) were assigned to four groups (n = 6): control, E. coli, cortisol, and E. coli + cortisol groups. Does in the cortisol and E. coli + cortisol group were treated with cortisol from 3 days before E. coli inoculations occurred to 36 days post-partum. Does in the E. coli and inoculation groups were administered via intrauterine infusion E. coli O55 (109 CFU/mL) at 0 h. Physical indicators, macroscopic and microscopic changes in the endometrium, uterine secretion cytology and bacteriology were evaluated before (0 h) and at 6, 12, 24, 48, and 72 h after E. coli inoculation. The TLR4 and pro-inflammatory cytokine mRNA transcripts were detected using qPCR. The activations of NF-κB and MAPK signaling pathways were detected using Western blot procedures. As a result, cortisol inhibited the inflammatory response of does by reducing the clinical symptoms, morphological endometrial damage, % PMN in uterine secretions, relative abundance of inflammatory gene mRNA transcripts in the endometrium of does. Cortisol inhibited NF-κB activity by reducing MyD88 and IκB phosphorylation. Treatment with cortisol suppressed the phosphorylation of ERK1/2, p38MAPK, and JNK. These results indicate the anti-inflammatory effect of cortisol in the endometrium of does may be regulated by NF-κB and MAPK pathways.

Progesterone suppresses the innate immune function of bovine endometrium, making the uterus susceptible to bacterial infection. The bovine endometrial epithelial cells (BEEC) are the first line of defense against bacteria, such as Escherichia coli (E.coli) that causes inflammation of endometrium through the recognition of lipopolysaccharide (LPS). The aim of this study was to investigate the effect of progesterone on the inflammatory response and its potential mechanism using E.coli- or LPS-induced BEEC. Concentrations of 1, 3 and 5 ng/mL progesterone were selected. The mRNA expressions of proinflammatory cytokines were determined using qPCR. The activations of NF-κB and MAPK pathways were detected by Western blot and immunofluorescence. An increase in the mRNA expression of IL-1β, IL-6, IL-8 and TNF-α was observed in BEEC treated with progesterone, LPS or E.coli alone. Progesterone inhibited the E.coli- or LPS-induced gene expression of these cytokines. Progesterone treatment alone showed little influence on NF-κB or MAPK pathway. In BEEC stimulated with E.coli or LPS, progesterone inhibited the phosphorylations of IκB, p65, ERK1/2, p38MAPK and JNK, and the translocation of p65 into the nucleus. These results suggested that progesterone has anti-inflammatory effect, which may be mediated by inhibiting NF-κB activation and MAPK phosphorylation in BEEC.