Characterization Of Natural Killer Cells In Mice Infected With Schistosoma Japonicum

Schistosomiasis in Asia is an important zoonotic parasitic disease caused by schistosoma japonicum(S.japonicum),seriously affecting the health of human being.The main pathological damage is caused by schistosome eggs deposited in the hosts’livers,which produce a granulomatous reaction and further develop into liver fibrosis and cirrhosis.The interaction between the host and S.japonicum parasite causes schistosomiasis,and lymphocytes are the cells where this interaction occurs.There are a large number of lymphocytes such as natural killer cells(NK cells),T cells,B cells and NKT cells in liver.NK cells produce an immediate response to the target without prior sensitization.Hepatic stellate cells(HSCs)are the main source of Myo-Fibroblaster(MFB)that forms hepatic fibrosis in schistosomiasis.Many factors are involved in activating resting HSCs to MFB,and a high expression of alpha-smooth muscle actin(α-SMA)and accumulation of extracellular matrix proteins including Collagen Ⅰ and CollagenⅢ,may faciliate the development of cirrhosis.In recent years,it has been found that NK cells could alleviate liver fibrosis by killing HSC.The development of liver fibrosis is closely related to the activation state of NK cells,while the functional status of NK cells depends on the expression of surface receptors and effector molecules.Activated NK cells in the liver after infection with S.japonicum could negatively regulate liver fibrosis by killing effector molecules such as interferon gamma(IFN-γ),perforin and granzyme,and kill early activated HSC.NK cell surface receptors mainly include activated and inhibitory receptors,which ultimately determine the functional activity of NK cells by precisely regulating NK cell surface receptors,thereby alleviating or even reversing the progression of schistosomiasis liver fibrosis.In this study,a mouse model with schistosomiasis infection was established,and the dynamic changes of NK cells in the liver and spleen of infected mice were examined by flow cytometry.CBA technique was used to determine the dynamic changes of cytokines in sera of infected mice.Immunohistochemistry(HE staining and Masson staining)was used to detect the pathological changes of the liver of infected mice.The dynamic changes of NK cell surface receptors and effector molecules were detected by q-PCR,and the selected receptors were verified by q-PCR in vitro.In this study,immune changes,liver pathological changes and dynamic changes of NK cell ratio and surface receptors in infected mice were studied,laying a foundation for finding potential targets of anti-schistosomiasis liver fibrosis on the surface of NK cells.I.Immunological characteristics and pathological changes of liver in mice infected with S.japonicumObjective:To determine immunological characteristics,pathological changes of liver and immunitic situation in mice infected with S.japonicum.Methods:A mouse model with S.japonicum infection was established,and each mouse in the infected group was infected with 20±2 cercariae via abdominal patch.Two,4,6,8,10 weeks and uninfected mice liver and serum were collected.The expression changes of α-SMA,Collagen Ⅰ,Collagen Ⅲ at different time points after infection were quantified with qPCR.Pathological dyeing was used to determine the pathological changes of the liver.Flow microsphere capture protein quantification(CBA)method was used to measure the expression of cytokines in serum at different time points after infection to investigate the immune response of S.japonicum infected mice.Results:The results of HE staining showed that a large number of egg granuloma appeared in the mouse liver at the 6th week post infection.The granuloma merged at the 8th week,and gradually entered the chronic phase at the 10th week.The area of hepatic ranuloma in the mouse gradually narrowed and the liver became fibrotic.The results of Masson staining showed that local blue collagen fibers were visible from the 4th week post infection,and the area of blue collagen fibers began to expand at the 6th week,and the area of collagen fibers was the largest from 8th to 10th week.The results of q-PCR showed that α-SMA and Collagen Ⅲ were significantly increased from 6th to 10th week post infection in mice.Collagen Ⅰ increased significantly at 6 and 10 weeks post infection,Collagen Ⅰ,Collagen Ⅲ expression increased significantly 6 weeks post infection,and at this time the liver began to appear fibers.The results of CBA showed that pro-inflammatory cytokines such as IL-12,IL-6 and IFN-γ showed a increasing trend at the 6th week post infection and anti-inflammatory cytokines such as IL-21,IL-9 and IL-10 an increasing trend at the 8th week post infection.Conclusion:At the 6th week post infection,a large number of egg granuloma appeared in the mouse liver,the area of blue collagen fibers began to expand and pro-inflammatory cytokines exhibited an increasing trend,suggesting that the body was in an acute inflammatory reaction period.Eight weeks post infection,the area of collagen fibers was the largest,and anti-inflammatory cytokines showed an increasing trend,suggesting that the body was in the stage of liver fibrosis and inflammatory repair period.Part 2.The dynamic pattern of hepatic and splenic NK cells in mice infected with S.japonicum.Objective:To investigate the dynamic change of the proportion of NK cells in the liver and spleen of mice infected with S.japonicum.Methods:Hepatic and splenic cell samples were collected from mice at 2,4,6,8,and 10 weeks post infection,and the proportion of NK cells in the liver and spleen of mice was examined by flow cytometry.The proportion and functional status of each lymphocyte in infected mice were compared to that of uninfected group.The differences between groups were examinerd using the t-test and by ANOVA test.Results:Flow cytometry results showed that the proportions of NK cells in liver of infected mice 24.53%,64.87%,67.30%,67.00%from 4th to 10th week post infection,significantly higher than the uninfected group 13.80%(P<0.05).NK cells in spleen from 6th to 10th week post infection were 32.50%,58.27%,62.75%,significantly higher than the uninfected group 17.13%(P<0.01).Conclusion:The results suggested that NK cells play an important role in the establishment of S.japonicum infection.The specific mechanism needs further investigation.Part 3.The surface receptor and functional molecule changes of NK cells in mice infected with S.japonicum.Objective:To explore the changes of surface receptor(activation and inhibitory receptors)and functional molecules(perforin,granular enzyme,IFN-y)of NK cells in liver and spleen after infection with S.japonicum in mice.Method:The BALB/c mice infected with S.japonicum were established.Flow cytometry and real-time PCR were used to examine the dynamic changes of cell surface receptor(activation and inhibitory receptors)and functional molecules(perforin,granular enzyme,IFN-y)of NK cells in mouse liver and spleen.Results:The results of NK cell surface receptors showed that the expression of inhibitory receptors PD-L1,Trail and Tim-3 showed an increasing trend from 2 to 4 weeks post infection,and the expression of surface activated receptors CD226,NKG2D and 2B4 also showed an increasing trend.In particular,2B4 was significantly higher than the control group at 4 week post infection(P<0.05).The expression of all inhibitory receptors was significantly decreased 6 to 10 weeks post infection,the expressions of activated receptors CD226,NKG2D,NKp46 and 2B4 were significantly decreased,but the expression of CD 16 in liver was significantly increased.Functional test results showed that IFN-γ,granulozyme and perforin in NK cells increased 2 to 4 weeks post infection and decreased significantly 6 to 10 weeks post infection.Conclusion:NK cells were activated 2 to 4 weeks after infection,and incapacitation occured 6 to 10 weeks post infection.Combined with the expression of previously studied receptors,it was hypothesized that the expression of surface activated receptors CD226,NKG2D,2B4 and the inhibitory receptors PD-L1,Trail and Tim-3 2 to 4 weeks post S.japonicum infection were closely related to the function of NK cells.Part 4 The surface receptor and functional molecule changes of NK cells stimulated by SEA.Objective:To determine the changes of the expression of receptors and effector molecules of NK cells during several important periods(SEA and AWA)in mice infected by S.japonicum.Methods:The NK cells isolated from the liver and spleen of healthy mice were stimulated by S.japonicum adult worm antigen(AWA)and soluble egg antigen(SEA)and cytokines.The real-time PCR method was used to examine the changes of NK cells function in S.japonicum infection during several important infection time points.Results:In vitro results showed 24-36 hours after SEA stimulation,the surface inhibitory receptors(Trail,PD-L1 and Tim-3),the activated receptors(CD 16 and 2B4)and the effector molecules(perforin,granular enzyme and IFN-y)of NK cells showed an increasing trend.Conclusion:The effector molecules secreted by NK cells played a killing role after SEA stimulation.The surface-activated receptors 2B4 and CD 16 of NK cell may be important receptors for NK to recognize SEA.