The Role And Mechanism Of Acinetobacter Baumannii OMVs In Regulating Dendritic Cells To Participate In Anti-Infection

ObjectiveIt is well acknowledged as potential vaccines candidates that bacterial outer membrane vesicles(OMVs)are composed of numerous antigen derived from their parent bacteria and have adjuvant effects,which shows effective in activating host immune systems;at present,more attention have been paied on nosocomial infections associated with Acinetobacter baumannii(A.baumannii),multidrug resistance Acinetobacter baumannii(MDR-Ab),acted as“nightmares of Intensive Care Unit”,have extreme threats to resident patients and bring difficulties to the control of nosocomial infections.Our research aims at elucidating that how OMVs isolated from A.baumannii modulate dendritic cells,well known as playing vital roles in innate and adaptive immune responses,and investigating the mechanisms involving in functions of A.baumannii OMVs acted as vaccines and the possibilities of preventing A.baumannii infection via OMVs immunization.Methods(1)Isolation of OMVs from A.baumannii:A.baumannii were obtained by common culture methods and OMVs were acquired by differential centrifugation.These bacterial OMVs were identified by transmission electron microscope(TEM),sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),coomassie blue staining and other related methods.(2)Determination of stimulation concentration of bacterial OMVs:Isolated OMVs were series diluted to treat bone marrow-derived dendritic cells(BMDCs)at different concentrations and the survival rate of cells were detected by MTT.The intended stimulating concentration(10μg/mL)was established following the principle that the maximum dosages of OMVs but hardly affect the survival of dendritic cells.(3)Analysis on the function of BMDCs stimulated by OMVs derived from A.baumannii:After treating BMDCs with determined concentration(10μg/mL)of OMVs,the changes in biological activities of BMDCs,which including the markers on BMDCs and cytokines profiles,were analyzed by utilizing flow cytometry,qRT-PCR and ELISA.(4)Impacts on naive T cells stimulated by A.baumannii OMVs-primed BMDCs:Navie T cells were obtained by magnetic-activated cell sorting(MACS)and then co-cultured with A.baumannii OMVs-primed BMDCs,T cells differentiation were detected by flow cytometry and cytokines production in co-culture supernatant were analyzed by ELISA;in addition,flow cytometry was conducted respectively to analyze the percentages of activated dendritic cells and subsets of T cells,meanwhile,serum specific antibodies and changes of serum levels of related cytokines in mice were detected by ELISA.(5)Protection researches in vivo:A.baumannii induced sepsis models were established in mice in advance,the protection effects against A.baumannii infections were evaluated according to the survival rates of mice after A.baumannii OMVs immunization.Results(1)More OMVs production can be obtained by clinical isolation strain of A.baumannii:We analyzed the ability to produce OMVs of clinical isolation strain from A.baumannii(JU0126)using A.baumannii standard strain(ATCC 19606~T)as control.The results showed that both of them can produce OMVs in artificial culture medium but they had discrepancies in numbers of OMVs production.(2)A.baumannii OMVs have cytotoxicity:Low concentration(no more than10μg/mL)of A.baumannii OMVs have slight impacts on the survival of BMDCs,but have obvious cytotoxicity to BMDCs under the pressure of high concentration(more than 50μg/mL).(3)A.baumannii OMVs can promote BMDCs maturation:The expressions of markers on BMDCs including MHC-Ⅱ,CD80 and CD86 can be enhanced by10μg/mL OMVs treatments for 20 hours,which indicates the ability of antigen presenting is strengthened in BMDCs.Besides that,more various cytokines can be secreted by dendritic cells after OMVs stimulation.(4)A.baumannii OMVs can drive Th2 cells differentiation and enhance the abilities of antibodies production:A.baumannii clinical isolation(JU0126)derived OMVs can effectively induce dendritic cells activities and drive navie T cells differentiation into Th2 subsets.Meanwhile,immunized mice with OMVs can produce more antibodies.(5)A.baumannii OMVs have immune protection effects:It is obvious that immunization with OMVs derived from JU0126 strain can protect mice against sepsis induced by A.baumannii infection and lengthen survival of infected mice obviously,which indicates that OMVs have obvious immune protection bonus.ConclusionsClinical isolation strain of A.baumannii can produce more OMVs,enhance dendritic cells the abilities of antigen presentation to mediate the production of specific antibodies,and then play roles in immune protection against A.baumannii.On one hand,it can be elucidated by activating Th2 differentiation to upregulate the functions of specific humoral immune responses;on the other hand,it can be explained by innate immune responses mediated by related cytokines,which are secreted by dendritic cells after OMVs stimulation.