Profiles Of Lymphocyte Subpopulations And Cytokines In Mastitis

Mastitis, the inflammation of the mammary gland when affected by pathogen, is theimmune response to the invasions. Understanding the innate immune response to the bacterialcauses of mastitis is a prerequisite to the development of novel and effective diagnostics andtherapeutics. The inflammation following mastitis are mainly considered to be mediated byvarious proinflammatory cytokines and immunocytes induced by the bacteria, especially Tlymphocytes and its effector cytokines. However, However, systematic research on theresponse of T lymphocytes and cytokines in early inflammation of mammary gland have noreports. In this study, the systematic dynamics of Th1, Th17, IFN-γ~+γδ T cells, IL-17~+γδ Tcells, and cytokines in sera, spleen and mammary tissue were analysed via ELISA,Quantitative Real-time PCR and cytometry.Part I Dynamics of IL-17and IL-17associated cytokines in sera and milk of goatmastitis induced with E. coli and S. aureus experimentallyThe concentrations of IL-17, IL-6, IL-1β, TGF-β were assayed in sera and milk ofGuanzhong dairy goat mastitis induced with S. aureus or E. coli by the enzyme-linkedimmunosorbent assay (ELSA). The results showed that the levels of all cytokines fluctuatedslightly in sera, while raised markedly in milk whether infected with S. aureus or E. coli at4h、8h、24h、48h and72h post intramammary injection. However, the different kinetics wereresults by S. aureus and E. coli. The results display that the proinflammation cytokines can beinduced in mammary gland by S. aureus and E. coli, and that the cytokines level in milk wasmore meaningful than those in sera in mastitis diagnosis and prognosis.Part II Systemic T lymphocytes and cytokines responses in subclinic mice mastitisinduced with S.aureusThe concentrations of IL-17in sera increased during24h post challenge with S.aureusand its value was higher than control at18h. The level of sera TGF-β and IL-6wereunchanged compared to control. The percent of IL-17~+cells in spleen lymphocytes rised at2h, and higher than control signicantly at8h and48h, respectively. Further analysis displayedthat Th17cells, but not IL-17~+γδ cells, contributed to the IL-17~+cells elevation. Similarly,IFN-γ~+cells percent in spleen lymphocytes rised at2h, and significance were seen at12h,18h,24h and36h post challenge. IFN-γ~+γδ T cells and IL-17~+γδ T cells in spleen decresedcompared with control. The results suggested that IFN-γ producing cells and IL-17producing cells in spleen, especially Th1and Th17, were activated rapidly to produce effector cytokineand mediate systemic response following challenge with S.aureus, and IFN-γ~+γδ T cells andIL-17~+γδ T cells in spleen had no response to S. aureus infected in mammary gland.Part III Profiles of T lymphocytes and cytokines of mammary gland in subclinic micemastitis induced with S.aureusConcentrations of TGF-β and IL-6in mammary gland tissue increased at8~60h postchallenge with S.aureus. The levels of IL-17were higher than control at12~60h postchallenge. Expressions of IL-1β, IL-6, IL-12, IL-17, IFN-γ,IL-23and TGF-β mRNA inmammary gland tissue increased, while IL-10mRNA decreased compared with control postchallenge. The results demonstrated that IL-1β, IL-6, IL-12, IL-17, IFN-γ,IL-23and TGF-βwere induced following intramammary injection of S.aureus, and these proinflammatorycytokines were important in mediating mammary inflammation. Decreasing of IL-10mRNAin mammary gland may beneficial to secretion of proinflammatory cytokines and the S.aureusclearing in the chronic mastitis.The immunohistochemistry displayed that no IL-17-positive cells were observed incontrol and challenged tissue at2h and4h post challenge. The IL-17-positive cells appearedat8h and were increased during the following stadges. Especially, a large numberIL-17-positive lymphocyte-like cells were observed at48h and60h.Percent of IL-17~+cells in mammary gland lymphocytes increased significantly at8h,18h,48h and60h post challenge compared with control. Further analysis showed that Th17elevated at8h,12h,18h and48h, and IL-17~+γδ T cells increased at8h,18h,48h and60hpost challenge. At the same time, expressions of CCR6and RORγt in mammary gland tissueincreased markedly.Percent of IFN-γ~+cells in mammary gland lymphocytes increased significantly at12hand18h post challenge compared with control, while decreased significantly at48h,60hand72h. Further analysis displayed that Th1and IFN-γ~+γδ T cells decreased compared tocontrol in this study. Transcription factor T-bet mRNA level in mammary gland tissuedecreased compared with control.The results demonstrated that IL-17~+cells resided in mammary gland were important indefense against pathogens by secreting IL-17. IL-17~+cells, including Th17and IL-17~+γδ Tcells, were activated and produced IL-17by TGF-β, IL-6and IL-23following S. aureusintramammary injection. Th17and IL-17~+γδ T cells are key mediators in mammary glandimmunity and S. aureus clearing. Conclusion1. IL-17concentrations in sera and mammary gland increased markedly, whichsuggested that IL-17may be an immportant cytokine in mediating the inflammationof mammary gland when S. aureus infected.2. The expressions of IFN-γ, IL-1β, IL-6, IL-12, IL-17, IL-23and TGF-β mRNA inmammary gland play important role in mammary gland inflammation caused by S.aureus. Decreased IL-10were beneficial for differentiation of lymphocytes andproducing of proinflammatory cytokines.3. The ratio of Th1and Th17in spleen elavated markedly during36h postintramammary injectin with S. aureus, while the ratio of γδ T cells in spleen had anychanged. The results suggested that Th1and Th17may be an immportant mediator ofinflammation in mastitis.4. IL-17~+cells, including Th17and IL-17~+γδ T cells, were activated and producedIL-17by TGF-β, IL-6and IL-23following S. aureus intramammary injection. Th17and IL-17~+γδ T cells are key mediators in mammary gland immunity and S. aureusclearing.