Staphylococcus aureus (S. aureus) internalization into bovine mammary epithelial cells (bMECs) is considered an important pathogenic mechanism for the establishment of mastitis. Given the interesting link between selenium (Se) status and mastitis, our objective was to prove that Se was essential to suppress pro-inflammatory mediators, in part, by modulation of Toll-like receptor2 (TLR2), nuclear factor kappaB (NF-κB) and mitogen activated protein kinase (MAPK) signal transduction pathway in bMECs. Results showed that Se (0~ 16 μM) did not affect the growth of bMECs. The mRNA expression of TLR2, Myeloid differentiation factor 88 (Myd88), Interleukin-1 receptor-associated kinase4 (Irak4), Interleukin-1 receptor-associated kinase1 (Irak1) and TNF receptor-associated factor6 (Traf6) in TLR2 signal pathway were increased or significantly increased by S. aureus. Se played an important role in regulating the genes expression of TLR2, Myd88, Traf6 but not in controlling the expression of Irak4 and Irak1. In addition, Se exerted strong inhibitory effects on the genes expression of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) induced by S. aureus. To further investigate the possible signaling mechanisms involved in the processes, we analyzed the role of MAPK and NF-κB signaling pathway in inflammation response in S. aureus-stimulated bMECs in vitro. Results showed that the phosphorylation of inhibitory kappaB alpha (IκBα), p65, p38 and extracellular regulated protein kinase (Erk) were significantly increased in S. aureus-stimulated bMECs. It indicated that S. aureus activated NF-κB and MAPK signaling pathway. We also examined the effects of Se on the phosphorylation of IκBα, p65, p38 and Erk in NF-κB and MAPK signaling pathway, which have well been proved to control the synthesis and release of pro-inflammatory mediators during inflammation. The findings are exciting, that pretreatment with Se (4, 8 μM) significantly suppressed the phosphorylation of IκBα, p65, p38 and Erk. These results suggest that Se down-regulates inflammatory mediators TNF-α, IL-1β and IL-6 gene expressions via TLR2, NF-κB and MAPK signaling pathway in S. aureus-stimulated bMECs, which may be responsible for the anti-inflammatory effect of Se.

Staphylococcus aureus causes subclinical mastitis as well as persistent and chronic infections in cattle. Bovine mastitis induced by S. aureus is often refractory to antibiotic treatment. Local innate immune defenses play an important role in eliminating the invading bacteria. TLR2 and NOD2 are important pathogen recognition receptors, but their functions have not been investigated in the context of early stages of mastitis. The present study examined TLR2, NOD2, and related cytokines in mammary glands infection induced by S. aureus at early stages in a rat mastitis model. All inoculated mammary glands developed mastitis. Acute changes were induced in mammary tissues infected with S. aureus at early stages and then chronic infections persisted until the end of the experiment. TLR2 and NOD2 mRNA expression increased significantly after inoculation with S. aureus. The expression levels of cytokine mRNAs, including TNF-α, IL-1β, IL-6, IL-10, and CXCL1, also increased. TGF-β1 expression was suppressed at early phase and IFN-γ mRNA expression increased significantly at a later stage. Mammary innate immune responses were activated after S. aureus inoculation. TLR2, NOD2, and inflammatory cytokines (TNF-α, IL-1β, IL-6, CXCL1, IL-10, TGF-β1, and IFN-γ) are involved in the response to mastitis induced by S. aureus.

Colibactin is synthesized by a 54-kb genomic island, leads to toxicity in eukaryotic cells, and plays a vital role in many diseases, including neonatal sepsis and meningitis. Avian pathogenic Escherichia coli (APEC) is speculated to be an armory of extraintestinal pathogenic Escherichia coli and can be a potential zoonotic bacterium that threatens human and animal health. In this study, the APEC XM meningitis mouse model was successfully established to investigate the effect of colibactin in in vivo infection. The clbH-deletion mutant strain induced lower γ-H2AX expression, no megalocytosis, and no cell cycle arrest in bEnd.3 cells, which showed that the deletion of clbH decreased the production of colibactin in the APEC XM strain. The deletion of clbH did not affect the APEC XM strain’s ability of adhering to and invading bEnd.3 cells. In vitro, the non-colibactin-producing strain displayed significantly lower serum resistance and it also induced a lower level of cytokine mRNA and few disruptions of tight junction proteins in infected bEnd.3 cells. Meningitis did not occur in APEC ΔclbH-infected mice in vivo, who showed fewer clinical symptoms and fewer lesions on radiological and histopathological analyses. Compared with the APEX XM strain, APEC ΔclbH induced lower bacterial colonization in tissues, lower mRNA expression of cytokines in brain tissues, and slight destruction of the brain blood barrier. These results indicate that clbH is a necessary component for the synthesis of genotoxic colibactin, and colibactin is related to the development of meningitis induced by APEC XM.