Staphylococcus Aureus ?-hemolysin Promotes The Secretion Of IFN-? By Cd56^brightNK Cells

Staphylococcus aureus?S.aureus?is a gram-positive pathogen,which causes purulent infection,pneumonia,pseudomembranous colitis,pericarditis and other local infections,as well as septicemia,sepsis and other systemic infections^[1,2].Widespread use of antibiotics has led to an increase in drug resistance of S.aureus.Methicillin-resistant S.aureus has become a global public health problem to be solved^[3,4].IFN-?is a type II interferon,secreted mainly by natural immune NK cells,NKT cells,and T helper cell and cytotoxic T cells.Its main function is to clear pathogens by exerting immune activation and immune regulation.The role of IFN-?on S.aureus infection is infection-stage dependent In the early stage of infection,IFN-?can promote the clearance of S.aureus mediated byneutrophils,macrophages and mast cells^[5,6,7]and subsequently reduce the infection.However,in the later stage of infection,the production of IFN-?can aggravate the infection and increase the lethality of S.aureus^[8,9,10].To uncover the mechanism of IFN-?production during S.aureus infection will improve the study on the pathogenesis of S.aureus.The specific mechanism of IFN-?induced by S.aureus infection is still far from clear.It is usually considered that pathogen activates the host immune system and indirectly stimulates the production of IFN-?by inducing expression of cytokines such as IL-12,IL-15,IL-18 and type I interferon^{[11,12,13,14]}.However,Yoshihara R et al.observed in 1993 that S.aureus directly stimulated the secretion of IFN-?by human NK cells,but the molecular mechanism is not yet disclosed^[15].It is known that S.aureus can secrete many kinds of proteins that are involved in the process of infection by interacting with host molecules^[16].Herein,we have explored whether molecules expressed by S.aureus can directly induce the production of IFN-?.Firstly,we incubated human peripheral blood mononuclear cells?PBMCs?with a variety of S.aureus functional extracellular proteins alpha-hemolysin?Hla?,?-hemolysin?Hlb?,?-hemolysin?HlgA,HlgB?,eLtaS?extracellular domain of LtaS?already expressed in the laboratory.Of note,the hemolysin concentration in this experiment can not lead cell lysis.It showed that only?-hemolysin significantly increased the level of IFN-?in the PBMC supernatant.Further flow cytometry analysis showed that?-hemolysin specifically stimulated CD56^bright NK cells to secrete IFN-?,but had no effect on the production of IFN-?in peripheral T cells,which is consistent with the previous study^[15].?-hemolysin is a kind of Mg²⁺-dependent sphingomyelinase C,which can dissolve sphingomyelin on the cell membrane surface and break cell membranes^[16,17].The current studys show that?-hemolysin has other functions in addition to traditional hemolysis.It has been reported that?-hemolysin promote the formation of S.aureus biofilms by acting as a DNA binding protein^[18].?-hemolysin also can inhibit human endothelial cells secrete IL-8,which results in attenuating the initial immune response^[19].Meanwhile,?-hemolysin facilitates the colonization of microorganisms on the skin surface by inducing the injury of human keratinocytes^[20].Moreover,it has been identified that?-hemolysin can releases the plasma membrane protein Syndecan-1,and subsequently aggravates S.aureus-induced lung infection^[21].According to the level of CD56,human peripheral blood NK cells can be divided into two distinct functional subpopulations:CD56^bright and CD56^dim[22].CD56^brightNK cells play an immunomodulatory function through the secretion of cytokine IFN-?,while CD56^dimNK cells directly kill cells through secreting granzymes.Moreover,CD56^dimNK cells are the main effector cells for antibody-mediated cell killing^[22,23].In the further investigation,we used the CD56^brightNK cell line NK-92 cells to explore the molecular mechanism of?-hemolysin-promoting IFN-?levels.Our results indicate that?-hemolysin can increase IFN-?levels in the supernatant of NK-92 cells in a dose-dependent manner,which is inhibited by?-hemolysin neutralizing polyclonal antibodies.Further experimental results indicate that addition of?-hemolysin increases Ca⁺⁺flux and stimulates phosphorylation of ERK.Moreover,we show that?-hemolysin upregulates the transcription of?-hemolysin.the Inhibitor of ERK significantly blocks the effect of?-hemolysin.Previous studies have shown that IL12,IL15 and IL18 can stimulate STAT4 phosphorylation,thereby upregulate IFN-?expression^[14,24,25].we do not observe that?-hemolysin promotes STAT4 phosphorylation,which suggests that?-hemolysin promotes IFN-?expression independent of STAT4 phosphorylation.Bioinformatics analysis shows that c-Myc binds the IFN-?promoter which is detected by chromatin immunoprecipitation.The addition of the c-Myc inhibitor significantly blocked the promoting effect of?-hemolysin on IFN-?expression.Overall,our findings presentthat S.aureus?-hemolysin can directly and specifically induce the production of IFN-?in CD56^brightNK cells.