| Previous studies have shown that irradiation-attenuated schistosoma vaccine can induce a high protective immune effect,which may be achieved through irradiation-attenuated schistosomia-derived molecules stimulating host immune cells to form an inflammatory environment dominated by Th1 in the lungs.Therefore,the discovery and identification of these protective molecules and the elucidating of their immunobiological functions have important guiding significance for the development of effective and safe molecular vaccine against schistosomiasis.Previous studies in our laboratory have shown that the cells from irradiation-attenuated schistosomule present immunogenic cell death phenotypes,and the release of dangerous(stress)signaling molecules such as HSP70,CRT,HMGB1 and ATP may play an important role in inducing high protective immunity in the host.This study aims to investigate the mechanism of extracellular adenosine triphosphate(ATP)combined with Schistosoma japonicum(SjCRT)in innate and adaptive immune responses,in order to provide a theoretical basis for elucidating the mechanism of high protective effect induced by irradiation-attenuated Schistosoma japonicum vaccine.1.Extracellular ATP release pattern and intracellular ATP expression in normal and irradiation-attenuated schistosomule.The normal and irradiation-attenuated schistosomule were cultured in vitro,and the culture medium and cells were collected,respectively.The extracellular and intracellular ATP content of schistosomule was detected by enhanced ATP detection kit.The experimental results showed that with the passing of culture time,the extracellular and extracellular ATP content of the irradiated group gradually decreased,while the normal group showed a trend of increasing at first and then decreasing.The extracellular ATP content of the irradiated group was higher than that of the normal group at 1 d and 2 d in vitro culture,and the difference was extremely significant(P < 0.01).However,The normal cell group had more ATP inventory during the 4 day and 5 day training than the irradiated group,and the difference between the two groups was statistically consistent(P<0.01).At 3 d,6 d and 7 d,there was no significant difference in the extracellular ATP content between the irradiation group and the normal group.Detected the intracellular ATP content of the worm body,we found that the intracellular ATP content of the irradiated group showed a trend of gradual decrease with the increase of culture time,while the intracellular ATP content of the normal group with the trend of increasing first and then decreasing,showing a general trend of decrease.In vitro cultivation of 1d,children’s insect cells intracellular ATP irradiation group was obviously higher than normal group,and extremely significant difference(P< 0.01),2 d,3 d,4 d,5 d,6 d and 7 d,the cells from schistosomule intracellular ATP content in normal group was obviously higher than that of irradiation group,extremely significant difference(P < 0.01),and training to the 8 d,compared with normal group,the irradiation group no obvious difference was found between schistosomule extracellular ATP content.The results indicated that the contents of intracellular ATP and extracellular ATP were high at the early 1 d of irradiation,and the contents of intracellular and extracellular ATP decreased with the passing of culture time.2.Study on the function and mechanism of activation of mouse DC by using extracellular ATP combined with SjCRT.The expression of marker receptors CD80,CD86 and MHCII on Dendritic cells derived from bone marrow(BMDC)surface stimulated by 250 n M or 1 m M Bz ATP(P2X7R agonist,an ATP simulant)or r SjCRT by flow cytometry;The expression level of cytokines in the supernatant of BMDC cultured with Bz ATP or r SjCRT was detected by ELISA.CCK-8 assay was used to detect the proliferation of CD4+T cells induced by stimulant pulse.The expression of IFN-γ,TNF-α and IL-4 in CD4+T cells was detected by flow cytometry.Results of flow cytometry showed that both 250 n M ATP and 1 m M ATP could significantly up-regulate the expression of DC surface marker molecules CD80,CD86 and MHCII.However,when combined with r SjCRT,CD80,CD86 and MHCII could be extremely significantly increased.ELISA results showed that the stimulation of ATP at 250 n M and 1 m M significantly increased IFN-γ,and TNF-α in DC(P < 0.05),but the stimulation of ATP at 250 n M significantly increased the secretion of IL-4 compared with the PBS group,while the stimulation of ATP at 1m M significantly decreased the expression of IL-10,and there was no significant difference between the two groups.The expression trends of IFN-γ,TNF-α,IL-4 and IL-10 were the same when ATP at 250 n M and 1 m M combined with SjCRT as when ATP alone stimulated.CCK-8results showed that 250 n M ATP,1 m M ATP,250 n M ATP+SjCRT and 1 m M ATP+SjCRT all stimulated CD4+T cell proliferation,but the combined stimulation group promoted CD4+T cell proliferation more than the single stimulation group.Flow cytometry showed that 250 n M ATP,1m M ATP and 250 n M ATP+ SjCRT could significantly up-regulatev IFN-γ expression(P<0.05),there was no statistical difference between 1 m M ATP+ SjCRT group and comparison(control group)group IFN-γ.250 n M ATP+ SjCRT significantly decreased the expression of IL-4(P<0.05),but there was no significant difference between 250 n M ATP,1 m M ATP and 1 m M ATP+ SjCRT.The results suggest that 250 n M ATP+ SjCRT can promote phenotype and functional maturation of DCs,and mature DCs can induce proliferation and activation of CD4+T.3.The characterization of mixed lymphocyte immune response induced by extracellular ATP combined with SjCRT in the spleen of sensitized mice.MTT assay was used to detect the proliferation of splenic mixed lymphocytes of Bz ATP or r SjCRT restimulated sensitized mice.Flow cytometry was used to detect the lymphocyte subsets of sensitized mice and the expression of intracellular cytokines in splenic mixed lymphocytes of sensitized mice after restimulation.The expression of extracellular cytokines in restimulated lymphocytes of Bz ATP or r SjCRT sensitized mice was detected by ELISA.MTT assay showed that 250 n M ATP+ SjCRT significantly promoted the proliferation of spleen mixed lymphocytes(P<0.05).Lymphatic subsets showed that CD4+ /CD8+Tcell were 2.12 in the mixed lymphocytes of 250 n M ATP immunization group and 1.47 and 250 n M in the mixed lymphocytes of 1 m M ATP immunization group CD4+ /CD8+Tcell was 2.19 in the mixed lymphocytes of the ATP +SjCRT group and 2.93 in the CD4+T cell of the 1 m M ATP +SjCRT group.Compared with the control group,ATP+SjCRT at 250 n M and ATP+SjCRT at 1 m M could extremely significantly(P<0.01)promote the expression of IFN-γ in spleen mixed lymphocytes.ATP at 250 n M and 1 m M significantly down-regulated the expression of IL-4(P<0.01).ELISA results showed that the stimulation of 250 n M ATP,250 n M ATP+SjCRT or 1 m M ATP+SjCRT significantly increased the expression of extracellular proinflammatory cytokines IFN-γ,and TNF-α,in spleen mixed lymphocytes(P<0.05).The secretion of IL-10 in 1 m M ATP+ SjCRT stimulated group was significantly higher than that in the negative control group(P<0.05).There was no significant difference in the expression of extracellular cytokine IL-4 between the experimental group and the negative control group.The results suggest that 250 n M ATP+ SjCRT can induce CD4+Th1type immune response in mice.In conclusion,ATP can be highly expressed in the intracellular stage of Schistosoma japonicum at the early stage of irradiation,and is released into the extracellular stage in large quantities.Extracellular 250 n M ATP combined with SjCRT can promote the phenotypic and functional maturation of bone marrow derived dendritic cells,activate CD4+T cells and induce their differentiation to inflammatory Th1 type cell immune response.ATP combined with SjCRT induces activation of dendritic cells(DC),and polarize the direction of acquired immunity.This study laid a foundation for further clarifying the role and mechanism of damage-related molecular patterns(SjCRT,Sj HMGB1,Sj HSP70 and ATP)in the immunogenic death induced by radiation in the irradiation-attenuated vaccine model. |
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