Malaria blood stage suppression of liver stage immunity by dendritic cells

Abstract

Malaria starts with Plasmodium sporozoites infection of the host's liver, where development into blood stage parasites occurs. It is not clear why natural infections do not induce protection against the initial liver stage and generate low CD8+ T cell responses. Using a rodent malaria model, we show that Plasmodium blood stage infection suppresses CD8+ T cell immune responses that were induced against the initial liver stage. Blood stage Plasmodium affects dendritic cell (DC) functions, inhibiting maturation and the capacity to initiate immune responses and inverting the interleukin (IL)-12/IL-10 secretion pattern. The interaction of blood stage parasites with DCs induces the secretion of soluble factors that inhibit the activation of CD8+ T cells in vitro and the suppression of protective CD8+ T cell responses against the liver stage in vivo. We propose that blood stage infection induces DCs to suppress CD8+ T cell responses in natural malaria infections. This evasion mechanism leaves the host unprotected against reinfection by inhibiting the immune response against the initial liver stage of the disease.

Figures

Figure 1.

Liver stage Plasmodium infection induces CD8+ T cell responses that are suppressed by blood stage parasites. ELISPOT results show IFN-γ–secreting CD8+ T cells specific for the CD8+ T cell P. yoelii CS epitope detected in the spleens of two mice. Results are expressed as average ± standard deviations for duplicates cultures. (a) Mice were immunized with 105P. yoelii–irradiated sporozoites, 105 nonirradiated sporozoites, or a mixture of both. (b) Mice were immunized with 105 irradiated sporozoites followed by 4 × 106P. yoelii–infected erythrocytes (iRBC) 7 d after immunization. (c) Mice were immunized with 105 irradiated or 105 nonirradiated sporozoites followed or not by treatment with artemisinin. *, significant difference (P < 0.01) in numbers of IFN-γ spots compared with aremisinin-treated group. (d) Mice were infected or not with 4 × 106P. yoelii–infected erythrocytes (iRBC), followed by immunization with 105 irradiated sporozoites at different times after infection. Results are expressed as percentage inhibition of IFN-γ–secreting CD8+ T cells as compared with noninfected immunized mice.

Figure 2.

P. yoelii–infected erythrocytes inhibit the expression of costimulatory and MHC molecules on DCs in vitro and in vivo. (a) Expression of costimulatory and MHC molecules on DCs alone (black bars), preincubated with uninfected erythrocytes (gray bars) or P. yoelii–infected erythrocytes (white bars) 24 h after the addition or not of LPS. Results are expressed as mean fluorescence intensity as determined by FACS® analysis of gated CD11c+ cells. Error bars indicate standard deviation of duplicated samples. Significant difference (*, P < 0.05; **, P < 0.01) in surface expression compared with expression on DCs preincubated with uninfected erythrocytes. Representative results from one of four independent experiments are shown. (b) Expression of MHC and costimulatory molecules on DCs isolated from the spleens of mice 7 d after infection with 4 × 106P. yoelii–infected erythrocytes (iRBC, white bars) and uninfected mice (black bars) 24 h after the addition or not of LPS. Representative results from one of three independent experiments are shown. Error bars indicate standard deviation of duplicated samples. Significant difference (*, P < 0.05; **, P < 0.01) in surface expression compared with expression on DCs from uninfected mice.

Figure 3.

P. yoelii-infected erythrocytes prevent DC death and reverse the IL-12/IL-10 secretion pattern. (a) Percentage of survival of DCs (FACS® analysis of CD11c+, propidium iodide−) alone (•), preincubated with uninfected erythrocytes (○), or with P. yoelii–infected erythrocytes (▴) after the addition of LPS. (b) Concentration of IL-12p70 (top) and IL-10 (bottom) secreted by DCs alone, preincubated with uninfected erythrocytes (RBC), or P. yoelii–infected erythrocytes (iRBC) after the addition (black bars) or not (white bars) of LPS. Representative results from one of three independent experiments are shown. Error bars indicate standard deviation of duplicated samples. Concentration of (c) IL-12p70 and (d) IL-10 in sera of malaria-infected mice. Sera were obtained from heparinized blood at different time points after infection with 4 × 106P. yoelii–infected erythrocytes. Representative results from one of three mice are shown. Error bars indicate standard deviation of duplicated samples.

Figure 4.

P. yoelii–infected erythrocytes do not affect antigen uptake by DCs. (a) Macropinocytosis of FITC-dextran and (b) phagocytosis of ethidium bromide–labeled P. yoelii–infected erythrocytes by DCs alone, preincubated with uninfected erythrocytes (RBC) or with P. yoelii–infected erythrocytes (iRBC). DCs were incubated for 2 h with FITC-dextran at 4°C (filled histogram) or 37°C (solid line). Histograms show CD11c+ gated cells. (c) DCs after incubation with FITC-dextran at 37°C. Fluorescence shows dextran internalization and no staining was observed after incubation at 4°C (not depicted). (d) DCs labeled with Cell Tracker (green) after phagocytosis of a P. yoelii–infected erythrocyte labeled with ethidium bromide (red).

Figure 5.

Blood stage infection induces DCs to secrete soluble factors that inhibit the activation of CD8+ T cells. Results of ELISPOT showing IFN-γ–secreting cells from the CD8+ T cell clone specific for P. yoelii CS epitope. Bone marrow–derived DCs preincubated with uninfected erythrocytes (RBC) or P. yoelii–infected erythrocytes (iRBC) were incubated with a peptide spanning the (a) CS CD8 epitope or (b) recombinant influenza virus expressing the same epitope before the addition of clone cells. As control, DCs without peptide or virus were incubated with clone cells. Representative results from one of three independent experiments are shown. (c) Proliferation of CFDA SE–stained CD8+ T cell clone cells after 5 d of incubation with DCs pulsed with the CD8 epitope peptide and preincubated with uninfected erythrocytes (RBC) or P. yoelii–infected erythrocytes (iRBC). Representative results from one of two independent experiments are shown. (d) DCs were isolated from the spleens of mice 7 d after infection with 4 × 106P. yoelii–infected erythrocytes (iRBC) or noninfected mice (control) before incubation with the CD8 epitope peptide and the addition of clone cells. Representative results from one of three mice are shown. (e and f) The incubation medium of cocultures of DCs and uninfected or P. yoelii–infected erythrocytes was added to (e) A20.2J cells or (f) DCs loaded with the peptide spanning the CS epitope before the addition of clone cells. Incubation medium (IM) from DCs and uninfected erythrocytes (IM1), DCs and uninfected erythrocytes with LPS (IM2), DCs and P. yoelii–infected erythrocytes (IM3), and DCs and P. yoelii–infected erythrocytes with LPS (IM3) are shown. As control, DCs or A20.2J cells without peptide were incubated with clone cells. Results are expressed as percentage inhibition of IFN-γ–secreting cells from CD8+ T cell clone incubated with each IM as compared with those incubated with normal medium. Representative results from one of two independent experiments are shown. (g) DCs pulsed with the CD8 epitope and preincubated with medium (black bars), uninfected erythrocytes (gray bars), or P. yoelii–infected erythrocytes (white bars) were mixed with CD8+ T cell clone cells in the presence of recombinant mouse IL-12 or anti–mouse IL-10 blocking monoclonal antibody. Significant difference (*, P < 0.1; **, P < 0.05) in IFN-γ spots compared with DCs preincubated with uninfected erythrocytes. Error bars indicate standard deviation of duplicated samples.

Figure 6.

Protective immune responses against malaria liver stage are suppressed by the transfer of DCs incubated with P. yoelii–infected erythrocytes. (a) Results of ELISPOT showing the numbers of IFN-γ–secreting CD8+ T cells from the CD8+ T cell clone specific for P. yoelii CS epitope. DCs preincubated with uninfected erythrocytes (DC + RBC) or P. yoelii–infected erythrocytes (DC + iRBC) were incubated with clone cells at a 2:1 ratio for 7 d before the addition of A20.2J loaded with the peptide spanning the CD8 epitope. As control, A20.2J cells without peptide were incubated with cocultures of DCs/clone cells. Representative results from one of two independent experiments are shown. (b) Results of ELISPOT showing the numbers of IFN-γ–secreting CD8+ T cells detected in the spleens of mice immunized with irradiated sporozoites and inoculated or not (control) 7 d later with DCs preincubated with uninfected erythrocytes (DC + RBC) or P. yoelii–infected erythrocytes (DC + iRBC) and stimulated with LPS. Another group of mice was inoculated with P. yoelii–infected erythrocytes alone (iRBC). All cells were irradiated before injection into mice to avoid the development of blood stage malaria infection. *, significant difference (P < 0.01) in IFN-γ spots compared with the other groups. ELISPOT was performed 7 d after the transfer of DCs. (c) P. yoelii development in the livers of mice after challenge with sporozoites. Mice were immunized with irradiated sporozoites, followed by inoculation 7 d later of DCs preincubated with uninfected erythrocytes (DC + RBC) or P. yoelii–infected erythrocytes (DC + iRBC), and challenged 7 d later. *, significant difference (P < 0.01) in the number of RNA molecules compared with the other groups. Representative results from one of three independent experiments are shown.