Effect Of Age-Related Immune Response During Lethal And Non-Lethal Parasite Infection

Background:Malaria,caused by infection with Plasmodium transmitted via mosquito bite,are popular with more than 90 countries in the world,resulting in 445 thousands deaths only in 2016,most of whom are young children under 5 years.Now it is validated that the development of disease was associated with the catalog of parasite.Most of the malaria infections are caused by Plasmodium falciparum and P.vivax.Plasmodium falciparum,a major cause of death,was considered the most deadly of the human parasites by invading mature red cells and account for 99%of malaria while P.vivax is usually thought to be causing benign malaria with lower incidence of complications and usually not required hospitalization.Growing evidences have indicated that the host immune system determines the risk of Plasmodium infection and the status of immune system was closely related to the age.The immune system is not fully developed in young children,both the humoral and celluar immune response are totally different with adults.In the present study,we observe that different immune cells take part in different stage of parasite infection.Studies showed that different immune cells play different roles at different stage of parasite infection.In the early stage of parasite infection,IL-12 produced by DC cells could induce IFN-?secretion and enhance Th1 cell immune response.Subsequently,Th2cells shifting from Th1 cells secreted IL-4 to help B cells to produce antigen-specific antibody against parasite and eliminate Plasmodium effectively.However,what kinds of immune cells in host with different ages determine anti-parasite immune responses still remains unknown.However,how the immune system in infected host with different age take part in regulating the anti-parasite immune responses and affecting the course and consequences of parasite infection.In the present study,P.y17XL and P.y17XNL were used to mimic Plasmodium falciparum and P.vivax and 4-week-old male mice and 8-week-old mice were used to mimic infancy and adult mice respectively.Survivate rate and parasitemia were calculated and flow cytometry,Real-time PCR,ELISA were used to measure the change of immune cells as well as cytokines different time point after infection.In-vitro studies were used to assess the different responses in mice of different age.Systematic evaluation was performed to discuss the relationship between characteristic of immune cells responses and host age,catalog of parasite.Materials and Methods:4-week-old male mice(BALB/c)and 8-week-old mice(BALB/c)were injected with 1×10~6P.y17XNL and P.y17XL infected red cells with intraperitoneal injection.Both the mortality and parasites load were monitored at various time points after P.y17XNL and P.y17XL infection.Splenic lymphocytes were isolated for the total counts,phenotype and the percentage of pDC,mDC,macrophage,T-cell subsets(Th1,Th2,Tfh and Treg),B cells at different time points by FACS.The marker of T-cell exhaustion,PD-1,was also analyzed by flow cytometry.Cultural supernatant was collected for inflammatory cytokines IL-4,IL-10 and IFN-?by ELISA.Finally,in-vitro study was performed to determine IL-2,IL-10 and IFN-?cytokine secretion of splenocytes 12 hours after LPS stimulation.Results:1.The onset of death occurred at day 5 after P.y17XL infection in both groups.8-week BALB/c mice and died within 11 days while 4-week BALB/c mice died with 7days after infected with P.y17XL.The onset of death occurred at day 8 with 77.8%of total survival rate after P.y17XNL infection in 4-week-old mice while the onset of death occurred at day 18 with 96.3%of total survival rate after P.y17XNL infection in 8-week-old mice.The parasitemia levels in 4-week-old mice were significantly higher than that in 8-week-old mice at day 5 after P.y17XL infection.Compared with 4-week-old mice,the parasitemia increased at day 3 after infection with faster rate at day 9,11,13,15 after P.y17XNL infection in 8-week-old mice.2.During the early stage of P.y17XL infection,both the proportions and absolute number of CD11c~+CD11b~+mDC and CD11c~+B220~+pDC was both up-regulated compared to baseline and significantly higher in 8-week-old mice than that in 4-week-old mice.In the late stage,there was no significant difference between two groups although still higher than baseline.The activation of pDC and mDC did not have any difference between two groups.After P.y17XNL infection,the activation marker of pDC and mDC(MHC II and CD86)was significantly up-regulated in 8-week-old mice after infection.TLR9expression on DC in both groups was significantly up-regulated 5 days after P.y17XL infection.There was no significant difference between two groups.The TLR4 on DC cells did not have any significant difference between two groups at different time points after infection.Both TLR4 and TLR9 on DC were significantly up-regulated compared to normal mice after P.y17XNL infection.3.The percentage of macrophage did not change compared with baseline 3 days after P.y17XL infection and significantly up-regulated at day 5 with no difference between two groups.Compared with normal health control,the percentage of macrophages was significantly up-regulated at day 5 after P.y17XNL infection and was higher in 8-week group.4.The percentage of CD4~+T-bet~+IFN-?~+Th1 cells were determined by flow cytometry and IFN-?of total splenocytes were measured by ELISA.Both the frequency and absolute number of CD4~+T-bet~+IFN-?~+Th1 cells were significantly higher in 8-week-old mice at day 5 after non-lethal P.y17XNL infection.IFN-?of total splenocytes measured concurrently was significantly higher in adult mice compared to infancy group.The percentage and absolute number of CD4~+T-bet~+IFN-?~+Th1 cells as well as IFN-?of total splenocytes were only significantly higher at day 3 after lethal P.y17XL infection.Although significantly increased compared to baseline,the percentage,absolute number of Th1 cells and IFN-?of total splenocytes have no difference between two groups.The T-bet mRNA expression level was significantly higher at day 3 and 5 after P.y17XL infection in 8-week-old mice and only up-regulated 5 day after infection in 4-week-old mice.The T-bet mRNA expression level was significantly higher at day 5 after P.y17XL infection and was significantly higher in 8-week-old group compared with 4-week-old group5.Neither the percentage of regulatory T cells and IL-10 of splenocytes in both groups have any difference compared to baseline 3 days after lethal P.y17XL infection.Although they up-regulated at 5 days,there was no difference between two groups.The percentage of CD4~+CD25~+Foxp3~+natural regulatory T cells as well as IL-10 of splenocytes was significantly higher in 8-week-old mice than that in 4-week-old mice 5 days after P.y17XNL infection.6.PD-1 expression on activated T cells was significantly up-regulated 3 days after P.y17XL infection.PD-1 signaling was up-regulated at day 5 on both total CD4+T cells and activated T cells compared to baseline in both groups after lethal P.y17XL infection and significantly higher in 8-week-old mice.PD-1 signaling was up-regulated at day 5 on both total CD4+T cells and activated T cells compared to baseline in both groups after non-lethal P.y17XNL infection with no difference between two groups.At day 10,the expression of PD-1 was significantly higher on both total and activated CD4~+splenic T cells after non-lethal P.y17XNL infection compared with baseline and was significant higher in 4-week-old mice.The level of PD-1 mRNA was significantly higher compared to control mice in both groups and significantly lower in 8-week-old mice compared with4-week-old group 5 days after infection after lethal P.y17XL infection.The PD-1 mRNA level was significantly higher in 4-week-old but lower in 8-week-old mice compared with baseline at day 5 and day 10 with significant difference between two groups after non-lethal P.y17XNL infection.7.The proportion and number of Th2 cells as well as IL-4 of total splenocytes were elevated in both groups at day 5 and day 10 after infection compared to baseline.As we expected,the percentage of Th2 cells and IL-4 of total splenocytes was significantly higher during acute and chronic stage of malaria infection in 8-week-old group.The percentage of CD4~+CXCR5~+Tfh cells,recognized as specialized providers of cognate B cell help,were also up-regulated at day 10 but not day 5 of infection in adult mice although significantly up-regulated compared with baseline.The percentage of CD138~+B220~-IgG~+plasma cells did not change but the absolute number was significantly up-regulated compared with baseline in 4-week old mice but still maintains a low level compared with control mice at various time points after infection.The percentage of plasma cells was significantly up-regulated at day 10 and higher than 4-week-old group.The absolute number of plasma cells did up-regulated at day 10 of infection compared with control group.In accord with the change of plasma cells,the percentage and absolute number of B220~+IgG~+memory B cells did up-regulated at day 10 after infection and was significantly higher in 4-week-old mice.Elisa analysis of B cell-related total IgG,IgG1 and IgG2a also showed a significant difference of antibody production in adult mice compared with infancy mice.8.A significant higher level of IFN-?,IL-2 and lower levels of IL-10 was detected in 8-week-old mice indicating an enhanced Th1 response in adult mice than infancy individuals 12 hours after LPS stimulation.Conclusion:1.Enhanced Th1 cells responses in adult mice promote the control of parasite infection during the early stage of both lethal and non-lethal parasite infection.2.After non-lethal parasite infection,Stronger activated innate and adaptive immune responses were displayed in adult mice in the early stage of infection.During late stage of parasite infection,increased effector T cells exhaustion and weaker humoral immune responses promote the persistence infection of parasite in infancy individuals.3.Although with similar number of splenocytes,the enhanced DC cells and T cells in adult mice determines the consequence of parasite infection of both groups.