Study On The Role Of RPSA-positive Neutrophils Against SS2 Infection

Objective:Streptococcus suis type 2(SS2)is an important pathogen of zoonosis in humans and pigs,causing non-reversible meningitis,shock and even death.Neutrophils,also known as polymorphonuclear leukocytes(PMN),as the first line of defense against bacterial infections,play an important role in bacterial meningitis.Ribosomal protein SA(RPSA),as a cell surface receptor,plays a vital role in the process of pathogenic organisms adhering to host cells.Our previous study found that the infiltration of neutrophils and the expression of RPSA on neutrophil surface in mice brain tissue increased when SS2 induced meningitis.Therefore,the purpose of this study was to explore the function of RPSA-positive neutrophils in meningitis induced by SS2 and its effects on blood-brain barrier(BBB).Methods:In this study,C57BL/6 mice were selected for SS2 infection test,The expression of neutrophils and RPSA-positive neutrophils in mice brain were analyzed by flow cytometry,and the bacterial load in brain tissue was counted by plate colony counting;Mouse bone marrow neutrophils were isolated in vitro,flow cytometry,laser confocal microscope and qPCR were used to verify RPSA-positive neutrophils;Flow cytometry,immunofluorescence microscope and qPCR were used to detect the functions of PMN RPSA~+and PMN RPSA~-,including phagocytosis,the neutrophil extracell?Lar trapping net(NET)formation and secretion of cytokines IL-6 and TNF-?;The effects of PMN RPSA~+on BBB were detected by human brain microvasc?Lar endothelia cells(hCMEC/D3)model and qPCR;Finally,promyelocytic leukemia cell lines(HL-60 cells)were induced into neutrophils to verify the expression of RPSA on neutrophils.Res?Lts:(1)Animal experiments in vivo showed that neutrophil infiltration and the expression of RPSA-positive neutrophil increased significantly after SS2 infection in C57BL/6 mice,and the bacterial load in brain tissue increased at first and then decreased;(2)SS2 infected the neutrophils isolated from mouse bone marrow in vitro and induced by HL-60 cell lines,which further proved that SS2 can induce expression of RPSA on PMN;(3)The functional differences between PMN RPSA~+and PMN RPSA~-were detected.The res?Lts showed that the phagocytic function of PMN RPSA~+was stronger than that of PMN RPSA~-,after blocking RPSA with antibody,the phagocytic ability of the two kinds of cells have no change.At the same time,the ability of PMN RPSA~+to form NET was stronger than that of PMN RPSA~-,but the ability of PMN RPSA~+to secrete proinflammatory cytokines such as TNF-?and IL-6 was weaken than that of PMN RPSA~-;(4)The res?Lts of BBB model showed that PMN co?Ld destroy the integrity of BBB,while PMN RPSA~+co?Ld aggravate the destruction of BBB integrity,decrease the transendothelial cell resistance(TEER)value,increase the permeability and destroy the expression of tight junction associated membrane protein.In addition,after blocking RPSA with antibody,the TEER value of BBB increased,the permeability of the BBB reduced,which further indicated that the RPSA-positive neutrophils co?Ld aggravate the destruction of BBB.Conclusion:(1)The expression of RPSA-positive neutrophils was increased in meningitis induced by SS2 infection;(2)The ability of RPSA-positive neutrophils to kill and remove bacteria was enhanced;(3)RPSA-positive neutrophils aggravate the destruction of the integrity BBB,and the inhibition of RPSA expression can be used as a therapeutic target for SS2 meningitis.