| Mastitis is the most commonly diagnosed infectious disease reducing milk yield in cows accompanied by mammary tissue damage,and causes severe economic losses to the dairy industry and effect on human health.Staphylococcus aureus(S.aureus),a prevalent Gram-positive(G~+)bacteria,causes mastitis in humans and animals.S.aureus-induced mastitis is the most frequent,pathogenic and prevalent infection of the mammary gland in dairy cows.The ligand growth arrest-specific 6(Gas6)and Protein S(Pro S)are a secretory protein that are responsible to binds and activate TAM receptor kinase(Tyro3/Axl/MerTK,TAM).However,the role of TAM following S.aureus infection in mammary gland still remains elusive.This project was designed to understand the underlying signaling pathways that induced by S.aureus in mammary gland and to elucidate the cellular intrinsic feedback mechanism which negatively regulate the TLRs mediated inflammation for tissue homeostasis.The finding of this study will help to better understand novel targets for immune equilibrium and in developing future treatment strategies for mastitis.1. The Histological Diagnosis and Detection of Relative Inflammatory Cytokines and TAM Receptor Kinases Genes Up-regulation during Cow MastitisMastitis is a characteristic inflammatory condition affecting dairy cow mammary glands worldwide.Mammary gland inflammation is mostly associated by pathogenic bacteria.These mammary gland pathogenic bacteria elicit inflammatory mediator’s resulting in the production of pro-inflammatory cytokines(TNF-α,IL-1β,IL-6).However,this mammary host defense response needs equilibrium to prevent unnecessary inflammatory impairment.For histopathological investigation and analysis of various inflammatory mediators cow udder were collected from slaughter-house of Wuhan,Hubei,China.Hematoxylin and eosin stained mammary gland tissues of infected cow exhibited severe histopathological changes,structural deterioration of mammary alveoli,and mammary epithelial cells along with infiltration of inflammatory cells,edema and hyperemia.Lactiferous ductal lobules showed distended nucleoli with accumulation of cellular debris.Furthermore,infected cow mammary gland tissues q RT-PCR results suggested significant increase in m RNAs level of pro-inflammatory cytokines(TNF-α,IL-1βand IL-6).Moreover,up-regulation of gene expression of TAM receptor kinases(Tyro3,Axl and MerTK)along with SOCS1 and SOCS3 were also observed in infected tissues other than non-infected tissues.However,the role of TAM receptor kinase following infection in mammary gland still not fully elucidated.Here,we investigate upregulation of pro-inflammatory cytokines following MerTK,Axl,Tyro3 genes levels in cow mammary gland tissues(in vivo).Collectively,these results indicate towards the new insight intrinsic feedback regulator for investigation during cow mastitisAdditionally,S.aureus is reported most widespread bacterium in cows mastitis.Hence,to further validate our current finding results we procced our experiment with mastitis in vivo designed model in MerTK and Gas6 knocked down mice and for in-vitro model we used RAW 264.7 and also harvested mammary epithelial cell from MerTK and Gas6 knocked down mice to elucidate inflammation equilibrium in mastitis.2. The Molecular Mechanism of Mer-tyrosine-kinase Attenuating Macrophage Inflammation Induced by Staphylococcus aureusThe central feature of S.aureus-induced acute inflammation are the impeded host defense,which cause failure in inflammatory cytokines homeostasis and leads to serious damage.Furthermore,whether MerTK alleviates S.aureus induced uncontrolled inflammation through negatively regulating Toll-like receptor 2 and 6(TLR2/TLR6)via suppressor of cytokine signaling 1,3(SOCS1/SOCS3)in RAW 264.7(in vitro).To clarify the TAM receptor kinase and associated protein we decided to continue our consideration in RAW 264.7.We select macrophages because till date it was considered that TAM receptor only expressed on macrophages to perform phagocytosis and efferocytosis.RAW 264.7 macrophages upon S.aureus infection activates TLR2 and TLR6 driven mitogen-activated protein kinases(MAPKs)and nuclear factor kappa B(NF-κB)signaling pathways,resulting in production of inflammatory cytokines including tumor-αnecrosis factor(TNF-α),interleukin 1β(IL-1β),interleukin 6(IL-6).Furthermore,S.aureus-infection groups showed a significant up-regulation of MerTK which serves as mediator of SOCS1 and SOCS3.Subsequently,through feedback mechanism SOCS1 SOCS3 degrade Mal,resulting in inhibition of downstream TLR mediated inflammatory pathways.Moreover,silencing MerTK in RAW 264.7macrophages inhibited the S.aureus-induced activation of MerTK,which significantly upregulated the phosphorylation of crucial protein in MAPKs(ERK,JNK,p38)and NF-κB(I?Bα,p65)signaling pathways,as well as the production of pro-inflammatory cytokines.Collectively,these findings indicate the important in vitro role of MerTK in self-regulatory resolution of S.aureus-induced inflammatory pathways and cytokines through intrinsic SOCS1 and SOCS3 repressed feedback on TLR2,TLR6.3. MerTK Negatively Regulates Staphylococcus aureus-induced Inflammatory Response via Toll-like-receptor Signaling in the Mammary GlandIn combatting the S.aureus induced inflammatory response,we designed MerTK knocked down mice mastitis model to demonstrate here,that Mer-tyrosine kinase(MerTK)regulates the intrinsic negative feedback to balance the over-reaction of the host defense system.S.aureus elicited toll-like receptors 2 and 6(TLR2/TLR6)signaling,subsequently recruiting TRAF6,whose ubiquitination is intricate to the downstream signaling including mitogen-activated protein kinases(MAPKs)and nuclear factor kappa-B(NF-κB).We observed that TLR2/TLR6 activation,in response to S.aureus,was contemporaneous with induced MerTK activation,leading to raised expression of suppressor of cytokine signaling 1 and 3(SOCS1,SOCS3)in wild type mice mammary tissues and epithelial cells.Meanwhile,S.aureus infection in MerTK-/-mice showed significant increased phosphorylation of p65,IκBα,p38,JNK and ERK along with production of pro-inflammatory cytokines.Moreover,MerTK-/-evidently inhibited S.aureus induced phosphorylation of STAT1 and subsequent SOCS1/SOCS3 expression which are pivotal in the negative feedback mechanism for targeting TRAF6 to inhibit the TLR2/TLR6 mediated immune response.Taken together,our findings verified the importance of MerTK in the regulation of the intrinsic feedback during the inflammatory response induced by S.aureus through STAT1/SOCS1/SOCS3 in mice mammary tissues and mice mammary epithelial cells(MMECs).4. Gas6 Negatively Regulates the Staphylococcus aureus-induced Inflammatory Response via TLR Signaling in the Mouse Mammary GlandThis study explored the role of ligand Gas6 in S.aureus-induced mastitis.Our results revealed that TLR receptors initiate the innate immune response in mammary gland tissues and epithelial cells and that introducing S.aureus activates TLR2 and TLR6 to drive multiple intracellular mitogen-activated protein kinase(MAPK)and nuclear factor kappa-B(NF-κB)pathways.Moreover,S.aureus also induces Gas6,which then activates the TAM receptor kinase pathway,which is related to the inhibition of TLR2-and TLR6-mediated inflammatory pathways through SOCS1 and SOCS3 induction.Gas6 absence alone was found to be involved in the downregulation of TAM receptor-mediated anti-inflammatory effects by inducing significantly prominent expression of TRAF6 and low protein and m RNA expression of SOCS1 and SOCS3.S.aureus-induced MAPK and NF-?B p65 phosphorylation was also dependent on Gas6,which negatively regulated the production of pro-inflammatory cytokines(IL-1β,IL-6,and TNF-α)in S.aureus-treated mammary tissues and mammary epithelial cells.Our in vivo and in vitro study uncovered the Gas6-mediated negative feedback mechanism,which inhibits TLR2-and TLR6-mediated MAPK and NF-?B signaling by activating TAM receptor kinase(MerTK,Axl,and Tyro3)through the induction of SOCS1/SOCS3 proteins.Summarizing our in vivo and in vitro study,we come to following conclusion;TAM receptor tyrosine kinases(Tyro3,Axl,and MerTK)triggered during response to vital stimulator of inflammation.However MerTK,Axl,Tyro3 activation is dependent on it ligand Gas6 and Pro S.Upregulation of TAM receptor tyrosine kinases inhibits both-TLR2/6 induced MAPKs/NF-κB and pro-inflammatory cytokine cascades.Remarkably,this inhibition of inflammation by MerTK and Gas6 via SOCS1/SOCS3 as an intrinsic negative feedback for TLR2/TLR6 signaling network by degrading TRAF6/Mal in Staphylococcus aureus infected mammary tissues and mammary epithelial cells(MECs)... |
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