Activation And Function Of Antigen Presenting Cells In Response To Schistosoma Japonicum Calreticulin

Schistosomiasis japonica is still one of the widely spread zoonosesin China. Prophylactic vaccines with high effectiveness and safety areneeded urgently for sustainable control of schistosomiasis. Therefore, it isalways an urgent being solved problem for the researchers to developthe high effective, safe and useful anti-schistosomiasis vaccines. Manystudies have elucidated that high levels of protective immunity can beinduced in animals immunized with radiation-attenuated (RA)schistosoma cercariae or schistosomula, so it should be a promisingstrategy to identify candidated antigens that are capable of simulatingprotective mechanisms of RA schistosoma for the development ofmolecular vaccines against schistosomiasis. Therefore, it has becomeone of the hot study topics for researchers to explore high protectivemechanisms of the RA vaccine, and much more attention has be paidon the molecular and cellular mechanisms of RAV’s immunogenicity.The previous studies have shown that, Calreticulin (short for CRT),which is composed of the conserved globular N-terminal domain,proline enriched domain, as well as acidic C-terminal domains, is a typeof conservative molecule in the evolution. CRT is an essentialCa2+-binding／storage chaperone resident protein which is founded inendoplasmic reticulum, and participates in the process of folding, genetranscription and post-translational modification of protein. Accordingto the latest report, CRTs in apoptosis cells or in cancer cells, which aretreated by some drugs or ultraviolet rays, would translocate to the cellsurface. Then, it would send a signal of‘eat me’ to antigen presentingcells (APCs), such as macrophages and dendritic cells. Eventually, thecells are phagocytized and processed by APCs. Meanwhile,immunogenicity of tumor antigen is also improved by this process underthe combined action of CRT with other stress molecules of these cells. The cell surface of CRT exposure can cause DC phagocytosis, andinduce a much more stronger immune response. Moreover, Naglaa ElGengehi et. al. have identified that Schistosoma mansoni CRT is one ofimmunodominant T cell antigens of radiation-attenuated vaccine.Chen Lei, one member of this study group, also found that S. japonicumCRT (short for SjCRT) contains a promiscuous Th1cell epitope, whichsuggest that schistosome CRT molecules may be an important T-cellantigen in RA schistosome. Therefore, we hypothesize that, in the RAschistosome vaccine model, some conservative stress molecules fromschistosome, such as CRT and HSP70molecule may play a key role. It ispossible for them by the interaction with DC to provide the environment,which could trigger and drive protective innate and adaptive immuneresponses. However, whether Schistosoma CRT molecules can activatethe host specific innate immune response induced by DC or notremains to be further validated. Therefore, based on gene cloning andexpression of S. japonicum CRT, in this present research, we observedthe localization of this molecules in S. japonicum of differentdevelopmental stages and preliminarily analyzed the functionalcharateristics of mouse antigen presenting cells stimulated with theSjCRT, and the object of this research is to provide preliminaryinformation for further clarifying immunobiological functions andmechanisms of SjCRT in RAV.Firstly, SjCRT was cloned by the molecular coloning techniques andwas expressed in vitro E. coli expression system. The immunogenicity ofthe protein was analyzed by Western bloting. Immunoflurescence wasapplied to the location of SjCRT in parasites at different developmentalstages. The results showed that the recombinant protein (rSjCRT) wassuccessfully cloned and expressed. Western bolting analysis showedthat serum from mice immunized with rSjCRT protein could recognizethe counterpart molecule from schistosome worms at7d,14d,23d,32d and42d post-infection. Immunofluorescence analysis showed thatgreen fluorescence signals could be observed on the teguments ofschistosomule at7d,10d and14d post-infection. These results suggestthat SjCRT may be a good immunogen in normal schistosomula.In order to avoid the situation that recombinant protein expressedin vitro E. coli expression system may be contaminated bylipopolysaccharide (LPS), which will influence our subsequentexperiments, an eukaryotic cell expression system—Bac-To-Bacbaculovirus expression system was used to the expression of SjCRTprotein. The SjCRT protein was purified by using His?Tag fusion proteinpurification method and Western blot was used to analyze theimmunogenicity of the purified SjCRT protein. The results showed thatSjCRT was successfully expressed in the Bac-To-Bac Baculovirus systemand purified, and purified protein could be reacted with both anti Hismonoclonal antibody and the serum from the mice immunized withrecombinant SjCRT (rSjCRT) protein from the prokaryotic E. coliexpression system. It provides material for the further study of activationof DC cells..RAW264.7cell line is of a strong function of antigen presentation,and is a commonly used cell model in investigating the innate immunefunction. LPS contaminated in rSjCRT protein from prokaryotic E. coliexpression system was eliminated by endotoxin removing gel, andactivation of RAW264.7cells stimulated with purified rSjCRT protein wasassayed. The modified MTT method (CCK-8) was used to assay theproliferation of the RAW264.7cells with or without the stimulation ofSjCRT protein, and flow cytometry was used to assay expression levels ofthe cell surface markers—MHCII、TLR2and TLR4molecules. The resultsshowed that SjCRT can significantly stimulate the proliferation ofRAW264.7cells (SI>2); the protein also significantly enhance the expression of MHCII、TLR2and TLR4(P<0.05). These results suggest thatthe SjCRT protein could activate RAW264.7cells, and the TLR2/TLR4-mediated signal pathways are likely to be involved in this process.In order to know whether SjCRT has the function of activation ofhost dendritic cells, we analyzed phenotypic and functional maturationof mouse Bone marrow-derived DC in response to SjCRT protein fromeukaryotic cell expression system mentioned above. Immature DCswere obtained by conventional method, the cell surface markers MHCⅡ、CD40and CD86expression levels of DCs stimulated with SjCRT wereassayed by Flow cytometry, and supernatants of DCs stimulated withSjCRT were analyzed for IFN-γ, IL-10, TNF-α and IL-6secretion by ELISAmethod. The results showed that the SjCRT significantly promotedup-regulation of the MHCⅡ and CD86expression (P<0.05) in DCs; TheIFN-γ secretion increased significantly (P<0.05), while IL-10, TNF-α andIL-6secretion significantly reduced (P<0.05) in DCs stimulated with SjCRT.These results suggest that SjCRT protein can induce phenotypicmaturation of dendritic cells, and stimulate Th1-biased andproinflammatory cytokine IFN-γ production.Taking together, in this present study, we successfully cloned andexpressed SjCRT, and found that SjCRT protein could locate on theteguments of schistosomule at7d,10d and14d post-infection, andpreliminarily elucidated that SjCRT protein could activate murinedendritic cells and macrophages. These results provide experimentalbasis for further clarifying the immunobiological functions andmechanisms of SjCRT in RA schistosoma vaccine model.