Malaria infection is known to be age-related, with children being more susceptible than adults [19, 20, 21, 22]. This study aimed to investigate whether the susceptibility to malaria infection in childhood and adulthood is associated with cellular and humoral immune responses, using a mouse model of lethal P.y17XL and non-lethal P.y17XNL infections in different age groups. childhood mice were found to be more susceptible to P.y17XNL infection, with lower survival rate and higher parasitemia at various time points. The adult group was more resistant to P.y17XL infection with lower parasitemia during the early stage of malaria infection. Importantly, enhanced cellular and humoral immunity, especially MSP-1 specific antibody, might contribute to rapid clearance of malaria in the adult group.
Malaria infections have various clinical phenotypes, ranging from a mild febrile illness to life-threatening severe anemia and acidosis, as well as end-organ failure among individuals with little or no acquired anti-malarial immunity. In part, this is explained by heritable differences in susceptibility to malaria infections, including different parasite proliferation rates governed by erythrocyte and hemoglobin polymorphisms [23]. Both the strain and host were thought to be important determinants of the disease profile [24]. In this study, 4-week-old and 8-week-old mice were used to mimic infancy and adulthood, respectively. We successfully established the age-related malaria infection mouse model to study the age-related anti-malaria immunity. Compared with 8-week-old group, the survival rate and parasitemia at different time points indicated that the 4-week-old group was same to both lethal and non-lethal parasite infections. After non-lethal P.y17XNL infection, parasitemia was significantly higher in the 4-week-old mice than the 8-week-old mice during the acute and chronic stages of infection. After lethal P.y17XL infection, a significant difference in parasitemia was observed in the early stage of infection. In accordance with the parasitemia, enhanced Th1 immune responses were only observed in the early stage (day 3) in adult mice after lethal P.y17XL infection and enhanced adaptive immune responses were detected in adult mice during both the early and late stages of non-lethal P.y17XNL infection. These data suggested that the difference in response to non-lethal and lethal Plasmodium infections was associated with the pattern of immune cell responses in the host. Thus, clinical phenotypes of malaria infections can be determined by age and immune states from host.
Similar to other infectious diseases, accumulating evidences have indicated that CD4+ T cells are essential to control malaria infection [25, 26, 27, 28]. Numerous studies have highlighted the role of Th1/Th2 cells or related signaling mechanisms in controlling malaria infection [29, 30, 31, 32, 33]. In this study, enhanced Th1 and Th2 responses were displayed in 8-week-old mice after malaria infection. Significantly higher percentage of Th1/Th2 cells and level of IFN-γ/IL-4 were observed in the 8-week-old mice as compared to the 4-week-old mice. In vitro studies also showed an enhanced Th1 cell response, which indicated an important role of Th1/Th2 cell-mediated age-related anti-malarial response. However, many studies suggested a shift from Th2 to Th1 cell responses with age. Li et al. found that IFN-γ level increased with age but not Th-related transcription factors, while IL-4 expression in plasma and CD4+ splenocytes declined with age [34]. A shift from Th2 towards Th1 immune responses was also observed in children with tertian or tropical malaria infection [35]. These studies partly supported our conclusion that enhanced Th1 cells might contribute to malaria clearance during the early stage of malaria infection. However, we observed enhanced Th2 cells during the late stage/chronic stage of malaria infection. Further studies are needed to investigate if any shift exists during the early stage of malaria infection. In addition, follicular T helper (Tfh) cells are essential for Plasmodium infection clearance by activating germinal center B cell responses [36, 37, 38, 39]. Relative research found that the preferential localization of Tfh cells in the germinal center (GC) suggests a unique, intimate relationship between the Tfh cell and the B cell. The help signals provided by Tfh cells to GC B cells consist of both cytokines and cell-surface receptors. While the help signals are incompletely characterized, CD40L, IL-21, and IL-4 are major ‘‘help’’ molecules produced by GC Tfh cells to keep GC B cells alive and induce their proliferation [40,41]. In this study, the percentage and absolute number of CD4+CXCR5+ Tfh cells peaked on day 5 p.i., and then decreased to normal level on day 10 p.i. in the 4-week-old mice. However, in the 8-week-old mice, the percentage and absolute number of CD4+CXCR5+ Tfh cells were significantly increased on day 10 p.i. as compared to the 4-week-old mice (Fig. 3E, 3F). These results indicated that GC has collapsed in young mice during the early stage of plasmodium infection, because Tfh cells increased rapidly. In addition, the GC is the primary site of B cell affinity maturation [41]. Thus The addition of Tfh cells induces GC collapse, result for damage of B cells. Moreover, Crompton PD et al. found that the activation of PD-1+CXCR5+ Tfh cells in malaria-infected children did not correlate with antibody responses indicating that Tfh cells may exhibit impaired B cells in children, which is different from Tfh cells in adults [40]. These studies supported our findings that the impaired function of antibody-secreting B cells and Tfh cells in childhoods and children may account for their susceptibility to malaria infection.
We also observed a dampening of PD-1 signaling on activated CD4+ T cells after non-lethal P.y17XNL infection but not lethal P.y17XL infection in the 8-week-old mice. PD-1 co-inhibitory signaling was reported to regulate helper T cell differentiation and anti-Plasmodium humoral immunity [41], and PD-1 deficiency could enhance humoral immunity during malaria infection [42]. PD-1 was also a marker of T-cell exhaustion [43]. Several studies have also proven that chronic malaria infection drives T cell exhaustion through PD-1 signaling [44, 45]. Therefore, we speculated that during non-lethal infection, humoral immunity plays an essential role in the late stage of malaria clearance, perhaps correlated with enhanced PD-1 signaling on activated CD4+ T cells, which may help to drive CD4+ effector T cell exhaustion and promote persistent infection in childhoods. Therefore, differences in PD-1 signaling could be observed in different age groups after non-lethal but not lethal malaria infection.
Several studies have confirmed that immune effector mechanisms are required to eliminate malarial parasites, and B cells secrete specific antibodies supported by Th2 cells, which can effectively remove the parasites to prevent the recidivation and recrudescence [46, 47]. Similarly, infusion of malaria hyperimmune serum resulted in rapid clearance of parasitized erythrocytes [47]. Anti-Plasmodium antibodies can prevent merozoites of infected red blood cells (RBCs), block cytoadherence to endotheliar capillary of infected RBCs (iRBCs), and promote phagocytosis by mononuclear cells [48, 49, 50]. However, persistence of significant levels of antimalarial antibodies relies on the continuous challenge resulting from chronic exposure to infection [51], probably as a consequence of impaired establishment of B cell memory [52]. This might explain the short-lived antibody responses [53, 54], mainly in young children [55, 56]. In this study, we detected the levels of B cell-related total IgG, IgG1 and IgG2a in P.y17XNL-infected BALB/c mice. The results showed a difference in antibody production between adult and childhood mice, and the levels of total antibody might contribute to rapid clearance of malarial parasites in the adult group during the chronic stage of non-lethal P.y17XNL infection. Moreover, IgG1 and IgG3 antibodies against merozoite surface proteins (MSPs) are thought to be instrumental in protection, which is considered as a major vaccine candidate [57]. Therefore, we detected the levels of P.y MSP-1 specific antibody. Consistently, the dynamics of P.y MSP-1 specific antibody was the same as total antibody. These data implied that an enhanced antibody response during chronic stage of non-lethal P.y17XNL infection might contribute to rapid clearance of malaria in the adult group.