Effects Of Praziquantel On Macrophages In The Spleens Of Mice Infected With Schistosoma Japonicum

Schistosomiasis is a parasitic disease that caused by Schistosoma japonicum.It has led to a serious public health problem in China and worldwide.To the end of2012,there are still about 240,000 schistosomiasis patients in China.The pathological damage caused by schistosomiasis is mainly due to the deposition of eggs in the liver which results in granuloma formation,portal vein obstruction,liver fibrosis,and spleen abnormal enlargement and hypersplenism.Until now,reports about the treatment of spleen pathological damage caused by S.japonicum are rare.For those patients,splenectomy or partial splenectomy have to be used to alleviate hypersplenism.During chronic stage of schistosomiasis,spleen abnormal enlargement is obvious.However,splenomegaly is significantly relieved after praziquantel treatment.The change of immune cell function in the spleen and the portal hypertension are considered to be two key factors in causing splenomegaly.Praziquantel,a traditional anti-S.japonicum drug,is well known its insecticidal effect.Previous studies found that praziquantel could relieve portal hypertension,and inhibit the expression of both inflammatory factor and cytokine gene in spleen macrophage of mice infected with S.japonicum.But whether praziquantel has effect on macrophages,the key cells of splenomegaly,has not yet been reported.Our study focus on splenic macrophages,especially the functional and number changes of macrophages after infection and praziquantel treatment,in order to further revealed the mechanism of praziquantel treatment on the splenomegaly and hypersplenism caused by schistosomiasis.It is acknowledged that macrophages may differentiate into M1 and M2 types of cells when induced by different factors through the classical pathway and the alternative pathway.It is reported that M1 and M2 type of cells are associated with Th1-type and Th2-type immune responses,respectively.In various clinical conditions,M1 macrophages mediated activation of inflammatory immune response,often associated with diabetes,arthritis,atherosclerosis and other diseases,while M2type macrophages usually represent immune modulation,often involved in fibrosis,wound healing,cancer metastasis and other physiological phenomena.In schistosome infection,liver and peritoneal macrophages expressed M1 phenotype in early stage,and transfor to M2 phenogype in chronic stage.However,it is not clear that how it occur for splenic macrophages of mice with chronic schistosomiasis.For this reason,we observed changes on the population and the polarization of splenic macrophages of mice with chronic schistosomiasis with or without praziquantel treatment.At first,we established mice models of chronic schistosomiasis.The infected mice received continuous four weeks praziquantel treatment.Then,we observed the changes of splenic macrophages.Finally,we analysed the polarization of splenic macrophages after praziquantel treatment.The main findings are as follows:1.Praziquantel treatment significantly ameliorated spleen pathological condition of mice infected with S.japonicum.The infected mice were given praziquantel treatment in 300mg/kg dosage for continuous.The results showed that after treatment 3 days,the spleens of mice had a reduction.The spleen weight and body mass index had a reduction.After treatment4 weeks,the spleens of mice returned to the normal size.The spleen weight and body mass index were also restored to the normal level,in comparison with that of infected group of mice.In addition,H.E staining showed that the histological structure of spleen in mice infected with S.japonicum was disorganized,with chaotic red pulp and white pulp distribution and the visible part of splenic sinus congestion,irregular cells arranged and tissue necrosis.After praziquantel treatment 4 weeks,the splenic parenchymal structure repaired,with the re-formation of clear white pulp and red pulp and the regular cell distribution.The above results indicated that praziquantel treatment 4 weeks has a obvious effect on splenomegaly and have significantly improved in histological structure of spleen in mice infected with S.japonicum.2.After praziquantel treatment,the proportion of splenic macrophages and its secretion of inflammatory cytokine of mice infected with S.japonicum decreased significantly.In order to observe the effect of praziquantel on splenic macrophages,we used immunohistochemistry method to label surface molecule CD68 on splenic macrophages The proportion of the number of macrophages was increased significantly after the infection,after praziquantel treatment 3 days,had a reduction but declined significantly after praziquantel treatment 4 weeks,in comparison with the control group.Flow cytometric analysis further confirmed that the proportion of splenic macrophages obviously increased in infected mice,and decreased significantly after praziquantel treatment.Detection on the cultured supernatant of isolated macrophages revealed that the levels of inflammatory factor IL-6 also decreased after praziquantel treatment.These results suggested that praziquantel treatment 4 weeks may significantly decreased the number and the secretion of inflammatory factor of infected mice with splenomegaly.3.After praziquantel treatment,the polarization of splenic macrophages changed in mice infected with S.japonicum and the ratio of M1 macrophages decreased.In order to further evaluate the effect of praziquantel on macrophage polarization,we used flow cytometry to analyse the changes of M1/M2phenotype population.M1 positive macrophages were labeled CD16/32~+and M2macrophages labeled CD206~+.The results showed that M1 type of cells were predominant in splenic macrophages of mice after infection,and decreased after praziquantel treatment,and praziquantel treatment 4 weeks was more obvious than praziquantel treatment 3 days.However,M2 type cells did not change significantly either with praziquantel treatment of not.These results suggested that praziquantel 4weeks down-regulated M1 macrophages of mice with splenomegaly.4.After mefloquine treatment,spleen pathological condition,the number of macrophages and the ratio of M1 macrophage has not been changed in mice infected with S.japonicum.In order to discriminate the effects of praziquantel on macrophages and splenomegaly were not just the consequent event of helminthicide of this drug,we used mefloquine,the another strong helminthicide drug,to treat the chronically infected mice.The results showed that splenomegaly and pathological injury had not been changed after treated with mefloquine.Furthmore,the ratio of splenic macrophage were similar with the infected group,and still expressed M1 dominant cell population.These results even confirmed that praziquantel not only had the helminthicide effect,but also had a regulatory effect on splenic macrophages.