| Neutrophils,as the typical innate immune cells,exert the irreplaceable role as firstline effector cells.Actually,they also exert vital role in adaptive immunity,such as they take up antigens in immunization sites and then migrate to lymphoid organs for B cell activation.However,neutrophils,the major producers of B cell-activating factor(BAFF),it was less known whether and how the neutrophils that first recruit to the immunization site of vaccines will affect the antigen-specific antibody response.When evaluating the vaccine efficacy in the study,we unexpectedly found that the vaccine could confer the ability of the locally recruited neutrophils to initiate and induce an enhanced antibody response through early producing BAFF just at the immunization site.We hope that the data could provide a timely and novel idea for evaluating the vaccine efficacy.1 Construction of the subunit vaccine and detection their immunological activityAt the beginning,considering that Porcine circovirus 3(PCV3)was raging at that time,we were going to design a subunit vaccine of PCV3 which could induce a strong humoral immune response.Using the amino acid sequence of PCV3 capsid protein(CP)as the template and combing the bioinformatics methods,we predicted and picked out four possible PCV3-CP linear B cell epitopes(Epitope,Epi),and marked as Epi A-Epi D respectively.Combined with the reports on PCV2-CP and comparative analysis with PCV3-CP,we predicted that Epi A as the optimal B cell epitope and Epi C as the escape epitope.Considering that escape epitopes inducing ineffective antibodies,we replaced the Epi C with the Epi A.Finally,constructed,expressed and purified the recombinant PCV3-CP(PCV3-r CP)with doble Epi A,named as r CPd A and used for vaccine antigen formulated with an oil-in-water emulsion(E)to form the subunit vaccine r CPd A-E.During testing its immunological activity,we found mice immunized with r CPd A-E producing high level antigen-specific antibodies,indicating that r CPd A-E had promising immunological activity and successfully induced a strong humoral immune response.When explored the activation of B cells in the early stage of primary immunization by flow cytometry,we found the proportion of CD69+ B cells in the draining lymph nodes(DLNs)and spleen was significantly increased,revealing that r CPd A-E could stimulate B cell activation in lymphoid organs in the early stage.Considering that antigen presenting cells(APCs)are important in vaccine-induced humoral immune responses,we observed typical APC,dendritic cells(DCs),in DLNs and muscle sites after intramuscular injection(i.m.).As atypical APCs,the distribution of neutrophils was also tentatively tested.The results showed that DCs and neutrophils were massive in the immunization site;however,in the DLNs,DCs increased but neutrophils not,showing that r CPd A-E might promote the uptake of antigens by DCs from the immunization site and entered the DLNs for antigen presenting,rather than the massive neutrophils recruited in the immunization site as reported.It made us deeply curious.After all,the massive recruitment of neutrophils in the immunization site were positively correlated with r CPd A-E induced antibody level.2 Exploration of the relationship between neutrophils in immunization sites and enhanced B cell responsesTo clarify the relationship between recruited neutrophils and enhanced antibody responses,we did further exploration by using intraperitoneally(i.p.)immunized mice to conveniently investigate recruited neutrophils.Eager to know where the driving force coming from,and what these recruited neutrophils produced at the immunization site were related to antibody responses,we detected the m RNA levels of neutrophil chemokines CXCL2,CXCL10 and B cell activation-related cytokines BAFF and IL-21 in peritoneal lavage cells(PLCs)of the i.p.immunized mice by q RT-PCR.We found the m RNA levels of CXCL2 and BAFF were significantly upregulated,suggesting that CXCL2 might be the strongest neutrophil chemokine,BAFF might be the most relevant B cell-activating factor with the enhanced antibody responses.Further tracking the CXCL2 and BAFF-producing cells in PLCs by flow cytometry we found that,CXCL2 was mainly came from peritoneal resident macrophages,and the recruited neutrophils also expressed CXCL2 to amplify the driving signal.Importantly,we found the recruited neutrophils produced high level of BAFF at the immunization site,which undoubtedly tightly linked neutrophils to B cell responses.Considering that B cells also present in the peritoneal cavity and they are also producers of BAFF,we compared the expression of BAFF in B cells and neutrophils in PLCs after r CPd A-E immunization by flow cytometry and found that,the recruited neutrophils expressed BAFF earlier and more than B cells,and with the increase of neutrophil-BAFF,the peritoneal B cells expressed more CD69 and CD40,indicating that the recruited neutrophils were the major BAFF producer in the immunization site and played the important role in initiating local B cell activation.In order to explore whether the neutrophil-BAFF could activate B cells for antibody production in lymphoid organs,we detected the expressions of CD69,CD40 and BAFF on/in B cells in DLNs and spleen of mice after immunization and found all of them were increased,showing that BAFF produced by neutrophils in the immunization site also activated B cells in lymph organs.So,we further speculated that the neutrophils in the immunization site could both initiate the local and systemic B cell activation after r CPd A-E immunization.3 Exploration of the mechanism of how recruited neutrophils in immunization sites initiate B cell responsesTo confirm the hypothesis above,we designed experiments to mimic neutrophil depletion as the following: stimulated isolated neutrophil-free PLCs with r CPd A-E to mimic neutrophil depletion prior to immunization,a group which neutrophils was added as control,simulating neutrophil recruitment after r CPd A-E immunization in vivo;the supernatants of the above culture system were obtained and continued to culture the mouse splenocytes for detecting B cell activation.The results showed :1)r CPd A-E significantly upregulated the expression levels of CD69,CD40 and BAFF in/on B cells in neutrophil-containing PLCs,and the BAFF level in the medium was dramatically increased,demonstrating the presence of neutrophils played a decisive role in the local B cell activation;2)the expression levels of CD69,CD40,and BAFF were significantly upregulated in splenic B cells cultured with neutrophil-containing system supernatants,demonstrating that the presence of neutrophils was essential for r CPd A-E-induced systemic B cell responses.In addition,curious about whether immunization with r CPd A-E simultaneously induced the BAFF-related TLR9-IRF5 signaling pathway activation,thereby enhancing local and systemic B cell responses,we incidentally explored the expression of key molecules of the pathway in PLCs of i.p.immunized mice and found that,the m RNA and protein levels of TLR9 and IRF5 were increased,indicating that the TLR9-IRF5 signaling pathway in the immunization site was activated.The expressions of TLR9 and IRF5 in both of PLCs and splenocyte CD19+ cells were significantly increased,however,in Ly6G+ cells were not,which revealed that immunization with r CPd A-E activated the TLR9-IRF5 signaling pathway in B cells in the immunization site and lymphoid organs of mice.It might be the activation of the TLR9-IRF5 signaling pathway in B cells leading to B cells more sensitive to BAFF producing by recruited neutrophils,thereby enhancing B cell responses.To deeply explore whether the early activation of B cells was initiated by BAFF produced by neutrophils,we attempted to inhibit BAFF production in neutrophils with BAFF si RNA and observed the effect on B cell responses.The results of in vitro experiments showed: the m RNA and protein level of BAFF in neutrophils incubated with r CPd A-E plus BAFF si RNA were significantly down-regulated,the BAFF level in the supernatant was significantly reduced,the expressions of BAFF,CD69,CD40,TLR9 and IRF5 in the cultured splenic B cells of mice were also significantly reduced,demonstrating that BAFF inhibition in neutrophils led to the inhibition of B cell activation.Inhibition results of in vivo experiments showed that mice i.p.immunized with CPd A-E plus BAFF si RNA had significantly reduced production of neutrophil-BAFF in the immunization site,and the expressions of CD69,CD40 and BAFF on/in B cells both in the PLCs and spleens were inhibited.Importantly,the level of antigen-specific antibodies in serum was halved.All of the results demonstrated that inhibition of BAFF expression by neutrophils at the immunization site attenuated B cell responses both in immunization sites and lymphoid organs.Overall,in the process of evaluating the vaccine efficacy in mice elicited by our self-constructed subunit vaccine,we found that the recruited neutrophils in immunization sites were positively correlated with vaccine-induced antibody levels.The neutrophils produced BAFF earlier and more than local B cells to initiate B cell activation in immunization sites and lymph organs.The neutrophil-BAFF also activated the B cell TLR9-IRF5 signaling pathway,which prompted activated B cells to produce more BAFF,thereby amplifying vaccine-induced B cell responses. |
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