Immune responses in liver and spleen against Plasmodium yoelii pre-erythrocytic stages in Swiss mice model

Abstract

Though the immunity to malaria has been associated with cellular immune responses, the exact function of the phenotypic cell population is still unclear. This study investigated the host immune responses elicited during the pre-erythrocytic stage, post-Plasmodium yoelii sporozoite infection in Swiss mice model. For this purpose, we analyzed the dynamics of different subsets of immune cells population and cytokine levels in the hepatic mononuclear and splenic cells population during pre-erythrocytic liver-stage infection. We observed a significant reduction in the effectors immune cells population including CD8⁺ T cell, F4/80⁺ macrophage and in plasmacytoid dendritic cells (CD11c⁺ B220⁺). Interestingly, substantial down-regulation was also noted in pro-inflammatory cytokines (i.e. IFN-γ, TNF-α, IL-12, IL-2, IL-17 and iNOS), while, up-regulation of anti-inflammatory cytokines (i.e. IL-10, IL-4 and TGF-β) during asymptomatic pre-erythrocytic liver-stage infection. Collectively, this study demonstrated that during pre-erythrocytic development, Plasmodium yoelii sporozoite impaired the host activators of innate and adaptive immune responses by regulating the immune effector cells, gene expression and cytokines levels for the establishment of infection and subsequent development in the liver and spleen. The results in this study provided a better understanding of the events leading to malarial infection and will be helpful in supportive treatment and vaccine development strategy.

Keywords: Immune responses; Plasmodium yoelii; Pre-erythrocytic stage; Splenic cells; Swiss mice; T cells.

Graphical abstract

Fig. 1

Quantification of liver-stage and blood-stage parasite load following P. yoelii sporozoite infection. (a) Relative expression levels of Py18S rRNA normalized to the mouse GAPDH. The bar graph represents Mean ± SD (n = 5) Py18S rRNA copy number. Statistical significance between control and sporozoite infected mice was determined using the student’s t-test (*** = p < 0.001). (b) Blood stage patency in sporozoite infected mice. The line graph shows the day-wise blood-stage parasitemia of individual mice from the sporozoite infected group.

Fig. 2

Phenotypic analysis of T cell, B cell and macrophage populations in HMNCs after P. yoelii sporozoite induced infection in mice. Percentage CD4⁺ and CD8⁺ population were determined in HMNCs isolated from different groups (naive and sporozoite infected mice) using flow cytometry. Dot plots show representative images from one experiment and show the percentage population of CD4⁺, CD8⁺, CD19^+, and F4/80⁺ cell populations, while the bar graph shows the Mean ± SD percentage population (n = 6). Statistical significance between different groups were determined using the student’s t-test (*p < 0.05, **=p < 0.01, ***=p < 0.001). (Sp.PI = Sporozoite post-infection).

Fig. 3

Phenotypic analysis of T cell, B cell and macrophage populations in spleen cells after P. yoelii sporozoite induced infection in mice. Percentage CD4⁺ and CD8⁺ population were determined in spleen cells isolated from different groups (naive and Sp. infected mice) using flow cytometry. Dot plots show representative images from one experiment and show percentage population of CD4⁺, CD8⁺, CD19^+, and F4/80⁺ cell populations, while the bar graph shows the Mean ± SD percentage population (n = 6). Statistical significances between different groups were determined using student’s t-test (*p < 0.05, **=p < 0.01, ***=p < 0.001). (Sp.PI = Sporozoite post-infection).

Fig. 4

Phenotypic analysis of dentritic cells (DC) and DCs subset populations in HMNC after P. yoelii sporozoite induced infection in mice. Percentage NK1.1⁻ CD11c⁺ B220⁺ CD8⁺, NK1.1⁻ CD11c⁺ B220⁺ CD8⁻, NK1.1⁻ CD11c⁺ B220⁺ CD4⁺, NK1.1⁻ CD11c⁺ B220⁺ CD4⁻, NK1.1⁻ CD11c⁺ B220⁺ CD11b⁺ and NK1.1⁻ CD11c⁺ B220⁺ CD11b⁻ DCs subset population was determined in HMNCs isolated from different groups (naive and Sp. infected mice) using flow cytometry. Dot plots show representative images from one experiment, while bar graph shows Mean ± SD percentage population (n = 6). Statistical significances between different groups were determined using student’s t-test (*p < 0.05, **=p < 0.01, ***=p < 0.001). (Sp.PI = Sporozoite post-infection).

Fig. 5

Phenotypic analysis of DC and DC subset populations in spleen during PE stage development after P. yoelii sporozoite induced infection in mice. Percent NK1.1⁻ CD11c⁺ B220⁺ CD8⁺, NK1.1⁻ CD11c⁺ B220⁺ CD8⁻, NK1.1⁻ CD11c⁺ B220⁺ CD4⁺, NK1.1⁻ CD11c⁺ B220⁺ CD4⁻, NK1.1⁻ CD11c⁺ B220⁺ CD11b⁺ and NK1.1⁻ CD11c⁺ B220⁺ CD11b⁻ DCs subset population was determined in splenic cells isolated from different groups (naive and Sp. infected mice) using flow cytometry. Dot plots show representative images from one experiment, while bar graph shows Mean ± SD percentage population (n = 6). Statistical significances between different groups were determined using student’s t-test (*p < 0.05, **=p < 0.01, ***=p < 0.001). (Sp.PI = Sporozoite post-infection).

Fig. 6

Relative mRNA expression of pro-inflammatory cytokines in HMNCs and spleen cells. HMNCs and spleen cells were isolated from control uninfected and P. yoelii sporozoites infected mice at different time points i.e. 20 h and 40 h. Each bar represents the Mean ± SD (n = 6) fold changes of respective cytokines (a) IFN-γ, (b) IL-12, (c) TNF-α, and (d) iNOS in HMNCs and splenocytes. Statistical significances between different groups were determined using student’s t-test (*p < 0.05, **=p < 0.01, ***=p < 0.001). (pi = post-sporozoite inoculation).

Fig. 7

Relative mRNA expression of anti-inflammatory cytokines in HMNCs and spleen cells. HMNCs and spleen cells were isolated from control uninfected and P. yoelii sporozoites infected mice at different time points i.e. 20 h and 40 h. Each bar represents the Mean ± SD fold changes of respective cytokines (a) IL-10, (b) IL-4, (c) IL-13 and (d)TGF-β in HMNCs and splenocytes. Statistical significances between different groups were determined using student’s t-test (*p < 0.05, **=p < 0.01, ***=p < 0.001). (pi = post-sporozoite inoculation).

Fig. 8

Measurement of pro-inflammatory cytokines in culture supernatants of HMNCs and spleen cells. HMNCs and spleen cells were isolated from control uninfected and P. yoelii sporozoites infected mice at different time points (i.e. 20 h and 40 h). The cells were seeded into 48-well tissue culture plates, culture and stimulated with either lipopolysaccharide (LPS) (1 µg/well). Subsequently, supernatants were collected from LPS stimulated cells and used for cytokines estimation. Each bar graph shows concentration (pg/ml) of the respective cytokines (a) IFN-γ, (b)TNF-α, (c) IL-2, (d) IL-6, and (e) IL-17. Statistical significances between different groups were determined using student’s t-test (*p < 0.05, **=p < 0.01, ***=p < 0.001). (pi = post-sporozoite inoculation).

Fig. 9

Measurement of anti-inflammatory cytokine level in culture supernatants of HMNCs and spleen cells. HMNCs and spleen cells were isolated from control uninfected and P. yoelii sporozoites infected mice at different time points (i.e. 20 h and 40 h). The cells were seeded into 48-well tissue culture plates, culture and stimulated with either lipopolysaccharide (LPS) (1 µg/well). Subsequently, supernatants were collected from LPS stimulated cells and used for cytokines estimation. Each bar graph shows the Mean ± SD concentration (pg/ml) of the respective cytokines (a) IL-10 and (b) IL-4. Statistical significance between different groups were determined using student’s t-test (*p < 0.05, **=p < 0.01, ***=p < 0.001). (pi = post-sporozoite inoculation).

Fig. 10

Nitric oxide (NO) and cell proliferation response in HMNCs and splenocytes. (a) Nitric oxide released in culture supernatants of hepatic and splenic macrophages from control uninfected and P. yoelii sporozoites infected mice at different time points (i.e. 20 h and 40 h) was determined by Griess reagent. (b) Cell proliferation response in HMNcs and spleen cells from control uninfected and P. yoelii sporozoites infected mice at different time points (i.e. 20 h and 40 h). Absorbance was determined at 450 nm using ELISA reader. Each bar graph shows the Mean ± SD values from different groups. (pi = post-sporozoite inoculation).