Preliminary Study On The Innate Immunity Induced By Schistosoma Japonicum Heat Shock Protein 70 In Mice

Previous studies have shown that in mice model vaccinated with radiation attenuated?RA?schistosomula,a high level of protective immunity can be induced by RA larvae which provoke innate immune response and provide micro-environment for CD4⁺T-cells-mediated adaptive immunity.And stress molecules?e.g heat shock proteins?derived from RA schistosomula might play an important role in conferring aforementioned Th1-polarized immune response.In this present study,we aim at exploring effects of Schistosoma japonicum heat shock protein 70?SjHSP70?on innate immunity in mice,and provide the foundation for further clarifying the mechanism of protective immunity elicited by RA schistosomula vaccines.1.Exploring the polarization of macrophages induced by SjHSP70.After the RAW264.7 cells were stimulated with SjHSP70 for 24⁴8 h,and the M1,M2 positive control groups were set up.Detect the secretion of NO by Griess assay,the quantitative detection of arginase was used to analyse enzyme activity,flow cytometry?FCM?to detect macrophage marker molecules,the expression of macrophages marker molecules in mRNA level were detected by RT-PCR.The results show that SjHSP70 could significantly promote the NO release by macrophage?P<0.001?;the detection of arginase activity showed arginase activity reached 6.15U/L,significantly higher than the control group?P<0.001?;FCM results showed that the expression of iNOS and CD16/32 of macrophages was significantly increased in M1and SjHSP70 stimulated group,but CD206 of M2 marker did not change significantly;RT-PCR results showed that iNOS was significantly increased?P<0.05?,Arginase I was up-regulated?P<0.01?,while CD206 was down-regulated?P<0.05?.It suggests that SjHSP70 could induce the polarization of macrophages,and the polarization was biased to M1 type in early stage,while after 48 h the polarization was inclined to M2 type.2.Analysis of lymphocyte responses induced by SjHSP70 in sensitized mice.The mice were immunized with recombinant SjHSP70,and after verification of immunogenicity the remains were infected with 50 cercariae for 42d.The ratio of CD4⁺and CD8⁺T cells from splenic lymphocytes in both sensitized and sensitized-and-infected?and some sensitized mice were infected with 50 S.japonicum cercariae,on day 42 after infection lymphocytes were collected?mice were detected by FCM.The sensitized lymphocytes were re-stimulated with SjHSP70 for 72 h.MTT method was used to assay the proliferation of sensitized lymphocyte,FCM detection of cytokine expression,RT-PCR detection of cytokine expression.The results showed that SjHSP70 could stimulate sensitized lymphocyte's proliferation with the proliferation index 2.59?SI>2?;The value of ratio of CD4⁺/CD8⁺T from group immunized with SjHSP70 was 2.83,significantly higher than that of control group which was2.46?P<0.001?;intracellular IL-4 in the CD4⁺T cells from sensitized lymphocytes re-stimulated with rSjHSP70 was significantly higher than that from control group;The expression of IL-4,TNF-?and IL-10 was significantly increased?P<0.05?,while IFN-?significantly decreased?P<0.01?;After infection of cercaria there is no significant difference in the ratio of CD4+/CD8+T?value of the ratio is 2.66?,compared with control group?value of the ratio is 2.44?.The results suggest that SjHSP70 can induce Th2-type immune response in mice.3.Investigating molecular mechanism of activation of SjHSP70-stimulated mouse bone marrow-derived dendritic cells via TLR2/TLR4 signaling pathway using knockout mice(TLR2^-/-and TLR4^-/-)and wild-type mice?C57BL/6J?.Mouse bone marrow-derived dendritic cells were stimulated with recombinant SjHSP70.Cell surface morphology was observed by scanning electron microscopy?SEM?,the expressions of TLR2 and TLR4 on the surface of DCs was detected by FCM,The expression levels of cytokine and I?B?at mRNA in DCs were detected by RT-PCR.The results showed that the structure of dendrites on the cell surface of DCs from TLR2^-/-mice can not be observed,while the structure of dendrites on the cell surface of DCs from both TLR4^-/-and wild-type mice can be observed,indicating that the activation of DC by SjHSP70 might be attributed to TLR2 pathway;the results of expression of TLR2 and TLR4 on the surface of DCs from both knockout and wild-type mice by FCM also suggest that the TLR2 pathway is dominant in the activation of DC by SjHSP70;after activation of DC by SjHSP70 via TLR2 pathway,the Th1-type inflammatory cytokine secretion of IFN-?was significantly decreased,while that of IL-6 and IL-1?significantly increased?P<0.05?,suggesting that the inflammatory micro-environment of interactions between DCs activated by SjHSP70and CD4+T cells might be polarized predominantly to Th2 type;the result of the expression levels of I?B?at mRNA indicated that activation of DC by SjHSP70 might be dependent on NF-?B signaling pathway.These data suggest that activation of DC by SjHSP70 is likely to be involved in activation of NF-?B via TLR2.In summary,we found that SjHSP70 could induce mouse macrophage polarization,and the macrophage was polarized to M1 type at early stage of stimulation,and predominantly M2 type after48h of stimulation;and SjHSP70 could activate DCs and promote differentiation of CD4+T cells from mice into Th2 subset;the activation of SjHSP70-stimulated mouse bone marrow-derived dendritic cells might be involved in activation of NF-?B via TLR2 signaling pathway.These findings in this present study clarify preliminarily effects of SjHSP70 on innate immunity in mice,and will provide a basis for further elucidating the mechanism of protective immunity elicited by RA schistosomula vaccines.