The Role Of TLR2-mediated Signaling Pathway In Streptococcus Uberis Infection Process

The wide type(TLR2+/+)and TLR2 knock out(TLR2-/-)B6 mice,the primary mice mammary epithelial cells(MMEC)as well as the Streptococcus uberis 0140J(S.uberis)were utilized in this study to investigate the function and anti-inflammation mechanism of TLR2 during S.uberis infection.The results were follows:1 The role of TLR2 mediated signaling pathways in S.uberis-induced mice mastitisIn order to investigate the inflammation in mammary gland induced by S.uberis and TLR2 mediated innate immune response,the wild type B6(TLR2+/+)mice and the TLR2-deficient(TLR2-/-)mice were utilized to perform our experiment,at the 72nd h after parturition.Mammary glands of female mice in experimental groups were infused with S.uberis,while control groups were infused with saline.Mammary gland were aseptically collected at the 24th h post-infusion by euthanatizing the mice.Firstly,the mammary gland were observed with microscopy in HE staining after the infusion of S.uberis.We examined the activities of NAGase and iNOS as well as the concentration of S.uberis next.Then,the proteins of TLR2 signaling pathway were analyzed by immunohistochemistry.Lastly,the secretion of cytokines and the indexes of anti-oxidant capability were detected.The results presented that the infusion of S.uberis induced obviously inflammatory changes in mammary gland of both TLR2+/+ and TLR2-/-mice,but TLR2-/-mice showed more severe bleeding and degeneration,adipose tissue hyperplasia in statistics(P<0.05).The concentration of S.uberis in TLR2-/-mice was higher than TLR2+/+ mice(P<0.05).The infusion of S.uberis into mammary gland elevated the activities of NAGase and iNOS in both TLR2+/+and TLR2-/-mice,however,the activities of NAGase and iNOS were higher in TLR2-/-mice(P<0.05).In TLR2+/+ mice,S.uberis activated proteins of TLR2 signaling pathway including MyD88,TAK1,TRAF6,ECSIT,while MyD88,TAK1,TRAF6,TRIF in TLR2-/-mice,and the expression of MyD88 and TAK1 in TLR2-/-mice were remarkable lower than TLR2+/+mice(P<0.05).The secretion of TNF-a,IL-1?,CXCL15 increased in TLR2+/+ mice,and TNF-?,IL-1?,IL-6,CXCL15 augmented in TLR2-/-mice,what's more,IL-1? in TLR2-/-mice was higher(P<0.05).Both the TLR2+/+ and TLR2-/-mice showed the decrease of total antioxidant capacity(T-AOC)and superoxide dismutases(SOD)and an increase of malondialdehyde,but there was no significant difference in the activity of SOD of TLR2+/+ mice.These results indicated that the deficiency of TLR2 induced more severe mastitis and the declined ability of bacteria clearance during S.uberis infection.Although S.uberis activated several proteins of TLR2-mediated signaling pathway and the secretion of cytokines,the expression proteins and the secretion of cytokines in TLR2-/-mice were relatively lower than that of TLR2+/+ mice.Of note,the oxidative stress responding to the treatment of S.uberis in both mice indicated that there were other factors which can recognize and response to S.uberis.2 The comparation of anti-oxidant capacity and production of cytokines in serum of TLR2+/+ and TLR2-/-mice after infusion of Streptococcus uberis into mammary glandIn order to investigate the role of TLR2 in anti-oxidant capacity and the production of cytokines in serum from mice infected by Streptococcus uberis(S.uberis),the wild type B6(TLR2+/+)mice and the TLR2-deficient(TLR2-/-)mice were utilized to perform our experiment,at the 72nd h after parturition.Mammary glands of female mice in experimental groups were infused with S.uberis,while control groups were infused with saline.Blood samples were aseptically collected at the 24th h post-infusion by euthanatizing the mice to produce serum.The results presented that the level of T-AOC in serum from TLR2-/-mice was lower than TLR2+/+ mice.After the infection of S.uberis,the level of T-AOC significantly decreased in both TLR2+/+and TLR2-/-mice compared with control groups(P<0.05).The activity of SOD in TLR2-/-mice showed no changes,however,it significantly reduced in TLR2+/+ mice.The production of MDA obviously increased in these two kinds of mice,and the increasement in TLR2+/+ mice was lower than TLR2-/-mice(P<0.05).The secretion of TNF-a in TLR2-/-mice and IL-6 in TLR2+/+ mice elevated after infection(P<0.05).These results indicated that TLR2 participated in mediating the level of T-AOC and the secretion of cytokines in S.uberis-induced mastitis,but it was not the unique factor.3 Study on the mechanism of TLR2 resistance to Streptococcus uberis in primary mice mammary epithelial cellsThe role of TLR2 in focal inflammatory and systemic inflammatory induced by S.uberis had already been investigated in vivo.In order to further explore the mechanism of TLR2 resistance to S.uberis,the mammary gland from TLR2+/+ and TLR2-/-pregnant mice were aseptically collected and digested in the mixture of collagenase I and trypsin to isolate the primary mice mammary epithelial cells.Then,the cells were incubated with S.uberis and the supernatant and cells were harvested at different time points.The results revealed that after the stimulation of S.uberis both TLR2+/+ and TLR2-/-MMEC showed cell shrinkage,and died at 5 h.The concentration of S.uberis in TLR2+/+ MMEC declined at 3 h and increased at 5 h,while it increased all the time in TLR2-/-MMEC,which was higher than that of TLR2+1+ MMEC at every time point.The incubation of S.uberis aggrandized the activities of NAGase and iNOS in both TLR2+/+ and TLR2-/-MMEC(P<0.05),the activities of NAGase(at 1 h,3 h)and iNOS(at 3 h)in TLR2-1-MMEC were higher than wide type MMEC(P<0.05).Total reactive oxygen species(ROS)in TLR2-1-MMEC was lower than TLR2+/+ MMEC in response to S.uberis infection,although it reached a maximum at the 2nd h.The determination of mitochondrial reactive oxygen species(mROS)showed that the production in TLR2-1-MMEC was lower than TLR2+/+ MMEC.What's more,the detection of uncoupling protein(UCP)2 showed the opposite trend of mROS.S.uberis increased the production of H2O2 in TLR2+/+ and TLR2-/-MMEC,and the yield in TLR2-/-MMEC was lower.S.uberis decreased the level of T-AOC and the activity of SOD,lifted the concentration of MDA in both TLR2+/+ and TLR2-/-MMEC,and the activity of SOD(at the 1st h)was higher and the concentration of MDA(at 1 h,3 h)was lower in TLR2-/-MMEC.The secretion of cytokines was promoted in response of S.uberis,the secretion of TNF-a(at 1 h,3 h),IL-1?(at 3 h),IL-6(at 1 h,3 h)in TLR2-/-MMEC were lower than TLR2+/+ cells,however,the CXCL15(at 1 h,3 h)in TLR2-/-MMEC was higher.These results showed that the knockout of TLR2 led to more severe damage and higher burden of bacteria during the S.uberis infection.TLR2 acted an indispensable role in the production of ROS and mROS in response to S.uberis.Furthermore,the anti-oxidant capability declined little compared with TLR2+/+ MMEC,as well as the secretion of cytokine except CXCL15.