Changes On The Immunosuppressive Status Of Mice Chronically Infected With Schistosoma Japonicum After Praziquantel Chemotherapy

Schistosomiasis,a parasitic disease,is one of the most important challenges to the population living in the epidemic areas.At present,the main problem is the long-term persistence of pathogen in the host and no strong protective immunity available.Although adult worms of schistosome can be effectively killed by praziquantel,reinfection is difficult to be interrupted.Accordingly,vaccine strategies on control of this disease have been expected.For several decades of efforts,however, there are still few effective vaccines against schistosomiasis.Studies on human populations and experimental models showed that the specific host immunity to schistosome is gradually down-regulated along with the progress of schistosomiasis,which is similar to other chronic infections, including tuberculosis and leishmaniasis.Recently,growing evidence has been presented to support the association between the chronic infections and CD4~+CD25~+ regulatory T cells,but to date,most data focused on the role of CD4~+CD25~+ regulatory T cells in some diseases while the functions of these subset T cells in schistosomiasis japonica are unclear. Our previous study demonstrated that there were some relationships between the down-regulated immunity of the chronic infections and CD4~+CD25~+ regulatory T cells in murine schistosomiasis.This study focused on the changes of CD4~+CD25~+ regulatory T cells and immunosuppression of mice chronically infected with Schistosoma japonicum after treatment with praziquantel.In this study,murine models infected with Schistosoma japonicum were established.Thirteen weeks after the infection,the mice were treated with praziquantel.At different time points,foxp3~+ cells distribution in the hepatic granulomas were examined by immunohistochemistry method.Then the numbers of CD4~+CD25~+ regulatory T cells were detected by flow cytometry and foxp3 mRNA expression on CD4~+ T cells in splenocytes was analyzed by real-time PCR.The inhibitory function of CD4~+CD25~+ regulatory T cells was assessed by the[~3H]-thymidine incorporation method.Finally,the serous specific antibody responses after OVA inoculation were assessed by ELISA.The main results are as follows:1.Foxp3~+ cells in the hepatic egg granulomas decreased when mice chronically infected with Schistosoma japonicum were treated with praziquantel In the hepatic sections of mice with the chronic infection,foxp3~+ cells were aggregated in the egg granulomas.On the day 3 after treatment,there were few changes on the number of foxp3~+ cells.Howerer,on the day 7 after treatment,the foxp3~+ cells were significantly decreased.On day 14 after treatment,almost no foxp3~+ cells could be found in the granulomas.The results demonstrated that praziquantel chemotherapy played roles on reduction of local foxp3~+ cells in the egg granulomas of mice infected with Schistosoma japonicum.2.The relative ratio of CD4~+CD25~+Foxp3~+ T cells from splenocytes was significantly decreased in the chronically infected mice after treatment with praziquantel Flow cytometry analysis showed that the ratio of CD4~+CD25~+Foxp3~+T cells in CD4~+T cells was higher in the chronic infection group than that in normal control.After praziquantel treatment,the ratio was decreased slightly on day 3 and significantly on day 7.Till day 14,the ratio of CD4~+CD25~+ Foxp3~+T cells fell to the level of normal control.The results indicated that praziquantel intervention also caused markedly reduction of splenic CD4~+CD25~+ Foxp3~+T cells.3.The expressions of foxp3 mRAN in splenic CD4~+T cells were down-regulated after mice with chronic infection were treated with praziquantel Real-time PCR analysis showed that in the infection group,the expression of foxp3 mRNA was significantly down-regulated on day 3 after treatment,compared with that in the normal control and sustained the same levels on day 7 and day 14. This indicated that the changes of foxp3 mRNA expressions were prior to the reduction of the CD4~+CD25~+ Foxp3~+T cell numbers.4.SWAP injection may cause reduction of splenic CD4~+CD25~+ Foxp3~+T cell numbers Flow cytometry analysis showed that praziquantel per se didn't cause any significant changes of splenic CD4~+CD25~+Foxp3~+ T cell numbers.However,the splenic CD4~+ CD25~+Foxp3~+T cell numbers decreased obviously when mice were injected i.p with SWAP.We presumed that the antigen injection may mimic the circumstance which the adult worms were killed by praziquantel,and consequently released lots of antigens in a short period of time.The mechanism in detail of how SWAP causes decrease of CD4~+CD25~+Foxp3~+T cell remains unclear.5.Suppressive potential of CD4~+CD25~+ T cells on the proliferation of CD4~+CD25~- T cells was attenuated in the chronically infected mice after treatment with praziquantel ~3H-TdR incorporation method was used to evaluate the Suppressive ability of CD4~+CD25~+ T cells.The experiments showed that the CD4~+CD25~+ T cells isolated from mice with chronic infection presented higher suppressive potential on the proliferation of CD4~+CD25~- T cells from the same mice under ConA stimulation,compared with the CD4~+CD25~+T cells isolated from normal mice.However,these suppressive capabilities began to subside gradually after CD4~+CD25~+ T cell donated mice were treated with praziquantel.It became obvious on day 7 after the treatment.On the day 14,the suppressive capabilities of CD4~+CD25~+ T cells decreased to the level approximately resembling that from normal mice.The results suggests that praziquantel chemotherapy could not only reduce both the numbers and foxp3 mRNA expression of CD4~+CD25~+ T cells,but also degrade the suppressive functions of this cell subset.6.Specific anti-OVA antibody was increased in chronically infected mice after treatment with praziquantel ELISA detection showed that the level of specific anti-OVA antibody IgG and its isotype IgG1 as well as IgG2a were higher in treated group than those in chronic infection group.Meanwhile,the ratio of IgG2a to IgG1 was rose in treated group compared to that in chronic infection group.The results demonstrated that schistosome infection could induce immunosuppression of the hosts and the immune responses of the infected mice were rebounded and skewed to Th1 type shortly after praziquantel chemotherapy.In conclusion,our study demonstrated that the numbers and the inhibitory activities of CD4~+CD25~+ regulatory T cells of mice chronically infected with Schistosoma japonicum were down-regulated after praziquantel chemotherapy.The same changes in the numbers of CD4~+CD25~+ regulatory T cells could also be induced by SWAP injection.Furthermore,the immunosuppressive status of mice caused by the infection could be reversed by the chemotherapy.The results suggested that CD4~+CD25~+ regulatory T cells may play important roles in sustaining the immunosuppressive status of mice chronically infected with Schistosoma japonicum.This also revealed that praziquantel,as a drug of the first choice for anti-schistosomiasis,could terminate the persistent infection by effectively killing the adult worms and simultaneously restore the immunologic balance that once was broken by the infection.