| Staphylococcus aureus（S.aureus）is a mammalian commensal and opportunistic pathogen that colonizes in skin.S.aureus can cause a wide spectrum of diseases in humans,such as endocarditis,pneumonia,osteomyelitis and bacteremia.In addition,mastitis caused by S.aureus is a common disease among lactating women and dairy animals.S.aureus-induced mastitis results in major economic losses to producers and the global dairy industry,and so far,commercially available vaccines have shown limited success to prevent S.aureus-induced mastitis.So far,the immune mechanism against S.aureus has not been fully ascertained.Studies over the past several years have shown that“unconventional”T cells not only mediated immune responses to pathogenic microbial but also play a key role in regulating the type and intensity of immune responses.Gamma-delta（γδ）T cells are an important subset of“unconventional”T lymphocytes,which present in much higher numbers（10–100%）in epithelial tissues such as the epidermis of the skin,the gastrointestinal tract and the reproductive track.As a bridge between innate and adaptive immune responses,γδT cells play a key role in mucosal immunity.The mammary gland is an immune-tolerant mucosal organ,and during S.aureus infection,the dynamic changes,functions and mechanisms of mammary glandγδT cells are still unclear.In this study,we investigated the dynamics of theγδT cells in mammary gland challenged with S.aureus during the early infectious stages and also the profiles of cytokines were analyzed.TCRδ-/-mice were used to explore the main functions of mammary glandγδT cells after S.aureus infection.Finally,the Vγ4 T-cell depletion and IL-17A-/-mice were used to clarify the role and mechanism ofγδT cells in S.aureus infection.The main research contents and methods:C57BL/6 mice were challenged with S.aureus and lymphocytes of mammary gland and spleen were collected at the indicated times（within 24 h）post-infection.The dynamics of NK cells,NK T cells,CD4+T cells,CD8+T cells andγδT cells were examined.The phenotype and cytokine profiles ofγδT cells in mammary gland tissue were detected during S.aureus infection.To explore the role of mammary glandγδT cells in the early stage of S.aureus infection,TCRδ-/-mice were used and the bacteria load,pathology,chemokines and neutrophils in mammary gland were measured.Furthermore,mammary gland and splenic lymphocytes were collected for analysis of specific Th1,Th2,Th17,Tc and Treg response by intracellular staining in WT and TCRδ-/-mice.The dynamics and cytokine profiles of Vγ1 and Vγ4γδT cells were detected by flow cytometry.To deplete Vγ1 and Vγ4γδT cells,mice were implemented by intramammary injection of InVivoMAb anti-mouse TCR Vγ1（clone 2.11）and InVivoMAb anti-mouse TCR Vγ4（clone UC3-10A6）.Then the bacteria load,cytokines and neutrophils were measured for analysis the role of Vγ1 and Vγ4γδT cells in the mammary gland.Finally,to explore the mechanism of mammary glandγδT cells in S.aureus infection,IL-17A-/-mice were used and the bacteria load,pathology,chemokines and neutrophils in mammary gland were measured.The results as following:1)Following S.aureus infection,NK cells,NK T cells,CD4+T cells and CD8+T cells were no significant changes in mammary gland.γδT cells was expanded from 1h p.i.and reach a peak at 3 h p.i.and then decreased.At 24 h p.i.,the proportion ofγδT cells in the mammary gland decreased to 1.95%,which was no significant difference with the control group.Following S.aureus infection,γδT cells exhibit increased CD69 and CD44 expression.Effect of S.aureus infection on cytokine profiles ofγδT cells was investigated.IFN-γ-and IL-17-producingγδT cells were the dominant population.2)TCRδ-/-mice were infected with S.aureus.The results showed that the bacterial colonization in mammary gland of TCRδ-/-mice was significantly increased,and the pathological degree of mammary glands of TCRδ-/-mice was aggravated.It is suggested thatγδT cells play an immune protective role in the early stage of mammary gland S.aureus infection.Following S.aureus infection in TCRδ-/-mice,the expression of KC,MIP-2 and IL-8 in mammary gland decreased significantly.At the same time,the percentage of neutrophils in mammary gland of WT mice and TCRδ-/-mice was detected by flow cytometry.The results showed that the absolute numbers of neutrophils in mammary glands of TCRδ-/-mice was significantly decreased than that of WT group.In addition,compared to the WT mice,the expression of MPO,IL-17A and IFN-γin TCRδ-/-mice was significantly decreased.Mammary gland and splenic lymphocytes from TCR-δ-/-mice showed significantly lower Th1,Th17,Tc and Treg cell responses than those from WT mice,while the proportion of Th2 cells had no significant changes.3)Following S.aureus infection,the proportion of Vγ1 and Vγ4γδT cells increased significantly at 3 h and 6 h post infection.Flow cytometry analysis showed that Vγ1γδT cells mainly produced IFN-γin the early stage of S.aureus infection,while Vγ4γδT cells mainly produced IL-17A in the early stage of S.aureus infection.The Vγ1 and Vγ4γδT cells were depleted by anti-mouse TCR Vγ1（clone 2.11）MAb and anti-mouse TCR Vγ4（clone UC3-10A6）Mab.Compared with the control group,the bacterial colonization in mammary gland of Vγ1 T-cell depletion and Vγ4 T-cell depletion mice was significantly increased,and the pathological degree of mammary glands of Vγ1 T-cell depletion and Vγ4 T-cell depletion mice was aggravated.Unsurprisingly,compared to the control group,the absolute numbers of neutrophils in mammary glands of Vγ1 T-cell depletion and Vγ4 T-cell depletion mice was significantly decreased.These results suggest that Vγ1 and Vγ4γδT cells are the mainγδT cell subtypes in mammary gland against S.aureus infection.4)To further analyze the mechanism ofγδT cells,the present study investigates the bacteria load in mammary gland of IL-17A-/-mice.The results showed that,compared to the WT group,the bacterial colonization in the mammary gland of IL-17A-/-mice was significantly increased.In addition,the pathological degree of IL-17A-/-mice was aggravated,and the expression of KC,MIP-2 and IL-8 in mammary gland of IL-17A-/-mice was significantly decreased.At the same time,the percentage of neutrophils in mammary gland of WT mice and IL-17A-/-mice was detected by flow cytometry.The results showed that the absolute numbers of neutrophils in mammary glands of IL-17A-/-mice was significantly decreased than that of WT group.These results suggest thatγδT cells play an immune-protective role in mammary gland against S.aureus infection.The main conclusions of this study are as follows:1)In the early stage of S.aureus infection,mammary glandγδT cells,which highly express CD44 and CD69 molecules and secrete IL-17A and IFN-γ,were the dominantγδT cell population.2)Deletion ofγδT cells led to attenuated production of chemokines and neutrophils in mammary gland.On the contrary,mammary gland inflammation and S.aureus colonization were increased in TCRδ-/-mice.It is suggested thatγδT cells play a key protective role in the early stage of S.aureus infection.Deletion ofγδT cells led to weakened the immune responses of Th1,Th17,Tc and Treg in mammary gland,which revealed thatγδT cells had a certain regulatory effect on adaptive immune response.3)Following S.aureus infection,the proportion of Vγ1 and Vγ4γδT cells were increased,while Vγ1γδT cells mainly secrete IFN-γand Vγ4γδT cells mainly secrete IL-17A.Vγ1γδT cells and Vγ4γδT cells play a positive role in bacterial clearance and tissue damage in the early stage of mammary gland S.aureus infection,and they are the mainγδT cell subtypes in mammary glands to resist S.aureus infection.4)Vγ4γδT cells play an immune protective role by rapidly secrete IL‐17A that contributes to neutrophil recruitment and the elimination of S.aureus.In conclusion,this study provides new insights of local immune responses against S.aureus infection in mammary gland,and provides theoretical basis and experimental data for the prevention and treatment of S.aureus mastitis. |
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