Effect Of Schistosoma Japonicum Infection And Its Immune Response On Hematological Malignancies

Schistosomiasis is a major parasitic disease that seriously affects human health and economic and social development.Epidemic data shows that there were about 230 to 250million people worldwide infected with schistosomiasis,and the number of people threatened with schistosomiasis infection is about 779 million.Only Schistosomiasis japonicum is prevalent in China and its life history,pathogenic mechanism,and immunopathological characteristics are all complicated,which have not been fully elucidated so far.Research shows that the activation of CD4~+T cells and macrophages plays a key role in the formation of egg granuloma.The initial CD4~+T cells differentiate and proliferate into Th1,Th2,Th17,Treg cells and secrete relevant cytokines to participate in the process of Schistosoma japonicum infection.There is a complex relationship between parasitic infections and cancer.Studies have suggested that liver fluke and Plasmodium falciparum are associated with hepatobiliary cancer and endemic Burkitt lymphoma respectively,while Toxoplasma and Trypanosoma can inhibit the development of cancer.The main pathogenic factor of Schistosoma japonicum is its eggs,which cause egg granulomas and fibrosis by depositing in the liver,and then progress to cirrhosis.These are all carcinogenic factors of hepatocellular carcinoma.Although more than 70%of the population in some high-endemic areas in China were infected with Schistosoma japonicum in the 1960s and 1970s,while there is no authentic connection between Schistosoma japonicum infection and hepatocellular carcinoma by epidemiological investigations.The hypothesis proposed earlier in this laboratory indicated that schistosomiasis infection can induce the host to produce anti-tumor immunity,and it has been confirmed by conducting a large number of experiments in vivo and in vitro:Schistosoma japonicum eggs and their secretions can significantly inhibit or even remove various metastatic tumors in the lungs and liver,including B16 cell metastases,and indicated that this antitumor effect is dependent on tissue macrophages and their cytokines,including IL-1?,etc.Based on this,we explored the effect of Schistosoma japonicum infection and its immune response on hematological malignancies:we constructed Zs Green1~+-L1210-luc~+cells and e GFP~+-A20 cell lines,expressing green fluorescent protein and luciferase respectively,which were used to establish hematological malignancies model in mice;freshly isolated Schistosoma japonicum eggs injected into the tail vein and a natural mouse infection model were conducted to evaluate their effects on tumor growth.And then,we evaluated the dynamics of CD4~+T cell subsets and so on by flow cytometry.The results are as follows:1.Effect of eggs on survival time in lymphoma bearing miceFirst,we established two animal models of L1210 leukemia and A20 lymphoma,and initially tested the effect of freshly isolated Schistosoma japonicum eggs(F-egg)on the survival of tumor bearing mice.The experiment was divided into two groups,the F-egg group and the PBS group(control group).F-egg was prepared by using the method of isolation and treatment of eggs established in the laboratory,and were injected into the tail vein on days 0,7,and 14,2x10~7 tumor cells were injected on day 12.The results showed that in the L1210 leukemia tumor bearing model,the average survival time of the mice in the PBS group and the F-egg group was 21.3±2.9 days and 32.7±2.5 days respectively after tumor cell inoculation.The survival time in the F-egg group was significantly prolonged(P=0.011).In the A20 lymphoma bearing mouse model,the average survival time of the mice in the PBS group and the F-egg group was 27.7±1.7 days and 36.3±1.6days respectively after tumor cell inoculation.The survival time of the mice in the F-egg group was significantly prolonged(P=0.018).2.Construction of Zs Green1~+-L1210-luc~+and e GFP~+-A20 cell lines expressing luciferase and GFPTo better count the number of tumor cells and observe the distribution of tumor growth,we constructed two tumor cells expressing GFP and luciferase by using lentiviral vectors,named Zs Green1~+-L1210-luc~+and e GFP~+-A20 cell lines respectively.1)Zs Green1~+-L1210-luc~+cell line:L1210 cell line was infected with a lentiviral vector containing genes encoding Zs Green1 and Luciferase.After 96 hours,positive cells were sorted by a flow sorter and cultured in vitro.After 2 months,the cells still stably expressed Zs Green1~+green fluorescent protein by the detection of fluorescence microscopy and flow cytometry;the luciferase substrate was added to the lysed cells for the detection on fluorescein luminometer,we found that Zs Green1~+-L1210-luc~+cell numbers and the fluorescein OD value was positively correlated(R2=0.095,P<0.05),that is the fluorescence intensity increased as the OD value increased;the cell line was subcutaneously planted on the right back of DBA/2 mice and detected by a live imager,we found that biofluorescence was concentrated on the right back of the mouse,and the fluorescence intensity was related to tumor size.2)e GFP~+-A20 cell lines:A20 lymphoma cells were infected with a lentiviral vector containing an e GFP-encoding gene.72 hours later,positive cells were sorted using a flow sorter and cultured in vitro.After 2 months,the cells still stably expressed e GFP~+green fluorescent protein by the detection of fluorescence microscopy3.Effect of F-egg on tumor growth in miceWe injected Zs Green1~+-L1210-luc~+cells into the tail vein of DBA/2 mice,there wasn't any symptom of end stage tumor in mice in each group.This experiment was repeated and we got the same result which indicated the spontaneous remission of leukemia may occurr due to lentivirus infection.In this way,this experiment focused on observing the inhibitory effect of F-egg on e GFP~+-A20 cells in vivo.F-egg,D-egg,and PBS were injected into the tail vein of mice on days 0,7,and 14 respectively,and the tumor cells were injected on the12th day,the observations were as follows:1)Comparison of the formation of solid tumors in tumor bearing mice at the end stage:the tumor formation rates of PBS group,D-egg group and F-egg are 100%,83.3%and 0%.In this way,compared with the other two control groups,the tumor incidence of mice in the F-egg group was reduced by 100%and 83.3%(P<0.001).2)Proportion of tumor cells of peripheral blood:bone marrow and spleen in mice:The proportion of tumor cells which expressing e GFP~+green fluorescent protein was detected by flow cytometry:in peripheral blood,the proportion of tumor cells in the F-egg group was(0.4±0.2)%,which is 96.0%and 96.6%(P<0.001)lower than the ratio of PBS group(9.9±3.6)%and D-egg group(11.9±2.6)%respectively;in bone marrow,the proportion of F-egg group is(0.7±0.2)%,which is 78.1%and 80.6%(P<0.05)less than that in the PBS group(3.2±1.9)%and D-egg group(3.6±1.5)%respectively;in spleen,the proportion of tumor cells in the F-egg group was(1.1±0.2)%,which was 79.6%and78.8%lower than that in the PBS group(5.4±2.1)%and D-egg group(5.2±2.3)%respectively(P<0.01).3)The expression of molecular markers Ki-67 and CD20:q PCR was used to detect the expression of Ki-67 and CD20(markers for cell proliferation and diffuse large B lymphoma respectively).The results showed that Ki-67 expression in the F-egg group were33.0%and 37.3%lower than that in the PBS and the D-egg group(P<0.001)respectively;The expression of CD20 in the F-egg group were 77.1%and 76.2%(P<0.001)lower than that in the PBS and the D-egg group respectively.Immunohistochemical method was used to detect the spleen of mice,and the results showed that the CD20 area density of the F-egg group was reduced by 64.3%and 74.5%when it was compared with the PBS and the D-egg group(P<0.001);4)Survival time of tumor bearing mice:the experiment was divided into 3 groups,namely the F-egg,D-egg,and PBS groups,each group was 6 mice.The results showed that the average survival time of the mice in the F-egg,D-egg and PBS groups was(49.3±4.5)days,(32.8±3.7)days,and(31.0±3.2)days after tumor inoculation respectively.The survival time of the mice in the F-egg group was significantly longer(P<0.01).The above results show that F-egg can inhibit the growth,tumor formation of e GFP~+-A20 tumor cells in vivo and significantly prolong the survival of tumor bearing mice.4.Schistosoma japonicum infection inhibits e GFP~+-A20 lymphoma in vivoTo learn whether the natural infection of Schistosoma japonicum can inhibit hematological malignancies,we established a mouse model of natural infection with Schistosoma japonicum:we inoculated e GFP~+-A20 tumor cells on the 40th day after infection and collected samples on the 69th day after infection.The experiment was divided into 4 groups,namely the infection+tumor group,the non-infection+tumor group,the infection group and the blank group,and 6 mice were in each group.The results are as follows:1)Comparison of the formation of solid tumors at end stage of tumor-bearing mice:the results showed that the incidence of tumors in the abdominal cavity and the whole body of the infection+tumor group,the non-infection+tumor group,the infection group and the blank group were 0%,100%,0%,and 0%.In this way,compared with the non-infection+tumor group,Schistosoma japonicum infection can completely inhibit the formation of e GFP~+-A20 tumors in mice.2)Proportion of tumor cells in peripheral blood,bone marrow and spleen of 4groups of mice:The proportion of tumor cells expressing e GFP+green fluorescent protein was detected by flow cytometry.The ratio in the infection+tumor group was(0.4±0.2)%,which was 95.2%lower than that in the non-infection tumor group(8.4±6.6)%(P<0.05).In bone marrow,the proportion of tumor cells in the infection+tumor group was(0.5±0.1)%,which is 70.6%lower than that in the non-infection+tumor group(1.7±0.5)%(P<0.001);in the spleen,the proportion of tumor cells in the infection+tumor group was(0.5±0.2)%,which is 87.2%lower than that in noninfection+tumor group(3.9±1.3)%(P<0.001).3)Expression of Ki-67 and CD20 molecular markers:in bone marrow,the expression of Ki-67 in the infection+tumor group was reduced by 37.9%when it was compared with the non-infection+tumor group(P<0.001);The expression of CD20 in the infection+tumor group was 92.3%lower than that in the non-infection+tumor group(P<0.001).The above results indicate that infection with Schistosoma japonicum can inhibit the growth and tumorigenesis of e GFP~+-A20 lymphoma in vivo.5.To evaluates dynamics of CD4~+T cell subsets in peripheral blood of mice infected with Schistosoma japonicum by flow cytometryAs the schistosome infection progress,the number of the CD4~+T cell subsets in peripheral blood of the infected mice is significantly increased in various trends,among which the Th1 cells account for a higher proportion at 3 weeks post infection and is increased slowly,while both Th2 and Treg cells account for a lower proportion at 3 weeks post infection,after which Th2 and Treg cells are highly elevated and peaked at 8 weeks post infection;There is no change of Th17 cells during the first 3 weeks after infection but the percentage of Th17 cells is significantly increased at 5 weeks after infection.The relationship between the dynamics in the subgroups of CD4~+T cells caused by Schistosoma japonicum infection and their mediating antitumor effects remains to be clarified.In summary,on the basis of previous experiments in this laboratory that Schistosoma japonicum eggs and their infection can significantly inhibit lung and liver tumors,this project is to explore the inhibitory effect of Schistosoma japonicum infection and its immune response on hematological malignancies.First,we established tumor cell lines expressing GFP or luciferase to detect its quantity and distribution in vivo,and then established a mouse animal model to study Schistosoma japonicum eggs and the natural infection of mice with hematological malignancies.The results of this project showed that F-egg and natural infection with Schistosoma japonicum can significantly inhibit the growth and tumorigenesis of e GFP~+-A20 lymphoma in vivo,prolong the survival of tumor bearing mice,and reduce the expression of molecular markers Ki-67 and CD20.The results have enriched our understanding of the relationship between schistosomiasis infection and its immune response and anti-hematological tumors.It also provided an important basis for subsequent research to clarify the mechanism of anti-hematological tumors mediated by Schistosoma japonicum,to discover anti-tumor effector cells or molecules and to identify parasites-derived molecules that induce anti-tumor effects.