Macrophage-mediated but gamma interferon-independent innate immune responses control the primary wave of Plasmodium yoelii parasitemia

Abstract

In most models of blood-stage malaria infection, proinflammatory immune responses are required for control of infection and elimination of parasites. We hypothesized therefore that the fulminant infections caused in mice by the lethal strain of Plasmodium yoelii (17XL) might be due to failure to activate a sufficient inflammatory response. Here we have compared the adaptive CD4+ T-cell and innate immune response to P. yoelii 17XL with that induced by the self-resolving, nonlethal strain of P. yoelii, 17X(NL). During the first 7 to 9 days of infection, splenic effector CD4+ T-cell responses were similar in mice with lethal and nonlethal infections with similar levels of activation in vivo and equivalent proliferation in vitro following mitogenic stimulation. Nonspecific T-cell hyporesponsiveness was observed at similar levels during both infections and was due, in part, to suppression mediated by CD11b+ cells. Importantly, however, RAG-/- mice were able to control the initial growth phase of nonlethal P. yoelii infection as effectively as wild-type mice, indicating that T cells and/or B cells play little, if any, role in control of the primary peak of parasitemia. Somewhat unexpectedly, we could find no clear role for either NK cells or gamma interferon (IFN-gamma) in controlling primary P. yoelii infection. In contrast, depletion of monocytes/macrophages exacerbated parasite growth and anemia during both lethal and nonlethal acute P. yoelii infections, indicating that there is an IFN-gamma-, NK cell-, and T-cell-independent pathway for induction of effector macrophages during acute malaria infection.

FIG. 1.

Course of PyL and PyNL infections in C57BL/6 mice. C57BL/6 mice were infected i.v. with 10⁴ lethal P. yoelii 17XL (PyL) or nonlethal P. yoelii 17X (PyNL) parasites and monitored for parasitemia (A), anemia (B), and survival (C). Groups consisted of four or five mice, and the results are representative of four independent experiments. Values that are significantly different (P < 0.05) in PyL- and PyNL-infected mice are indicated (#).

FIG. 2.

Ex vivo splenic CD4 T-cell activation is comparable in PyL- and PyNL-infected mice. Mice were infected i.v. with PyL or PyNL parasites, and on selected days postinfection, the mice were sacrificed, and their spleens were removed to assess ex vivo T-cell activation. (A to F) The frequencies (A to C) and absolute numbers (D to F) of activated splenic CD4⁺ T cells were determined by expression of CD69 (A and D), CD44 (B and E), or CD25 (IL-2 receptor α chain) (C and F) in Foxp3⁻ cells. (G) On days 3, 5, and 7 p.i., CD4⁺ T cells were purified, and their phenotype was determined by real-time PCR (TaqMan). Expression levels were normalized to GAPDH and are presented as changes in the increase compared to CD4⁺ T cells from uninfected animals. Groups consisted of three to five mice, and the results are representative of three independent experiments. Symbols represent significant differences (P < 0.05) between groups: #, PyL-infected group versus PyNL-infected group; *, PyL-infected group versus uninfected group; ∼, PyNL-infected group versus uninfected group. IFN-g, IFN-γ; TNF-a, TNF-α.

FIG. 3.

Comparable responses of in vitro-restimulated splenic CD4 T cells from PyL- and PyNL-infected mice. On selected days postinfection, splenocytes were isolated and restimulated in vitro with ConA and analyzed after 72 h for proliferation (A) and after 18 h for CD69 upregulation on all CD4⁺ T cells (B) or CD4⁺ T cells expressing specific TCR Vβ genes (C). Groups consisted of three to five mice, and the results are representative of two independent experiments. Symbols represent significant differences (P < 0.05) between groups: *, infected (PyL and PyNL) versus uninfected groups in panels A and C; *, PyL-infected group versus PyNL-infected group in panel B.

FIG. 4.

P. yoelii-induced T-cell hyporesponsiveness is due to defects in both CD11b⁺ antigen-presenting cells and T cells. CD4⁺ T cells and CD11b⁺ cells were purified from PyL- and PyNL-infected mice (on day 7 p.i.) and uninfected control mice. CD4⁺ T cells were marked with CFSE, cocultured with CD11b⁺ cells from uninfected mice or from mice infected 7 days earlier with PyL or PyNL, and stimulated for 18 h with ConA. (A) Representative plot showing recovery of CFSE-labeled CD4⁺ T cells after coculture and ConA stimulation. (B) Representative plots showing the purity of unpurified (left) and purified (right) CD11b⁺ APCs. PE, phycoerythrin. (C) Activation of purified CD4⁺ T cells following ConA stimulation was determined by assessing CD25 expression on CFSE-labeled cells. MFI, mean fluorescence intensity, N. lethal, not lethal. (D to G) The levels of IL-2 (D), IFN-γ (E), IL-12 (F), and IL-10 (G) in the supernatants of T-cell-APC cocultures were assessed by ELISAs. The results are representative of two independent experiments with three to five mice per group. Significant differences (P < 0.05) between groups are indicated (*).

FIG. 5.

Neither T nor B lymphocytes are required for the control of the primary wave of P. yoelii parasitemia. The course of parasitemia (A) and survival (B) were compared in PyL- and PyNL-infected C56BL/6 (WT) and RAG-1^−/− mice. Groups consisted of three to five mice, and the results are representative of four independent experiments. Symbols indicate significant differences (P < 0.05) between groups: #, PyL-infected RAG-1^−/− group versus WT group; *, PyNL-infected RAG-1^−/− group versus WT group.

FIG. 6.

Minimal role for NK cells in the control of the primary wave of P. yoelii parasitemia in C57BL/6 mice. (A and B) NK cell activation (CD69 expression on CD3⁻ NK1.1⁺ cells) in peripheral blood (A) and spleens (B) of PyL- and PyNL-infected mice on various days postinfection with PyL or PyNL. (C to F) Parasitemia (C and D) and survival (E and F) in WT (C and E) and RAG^−/− (D and F) mice treated i.p. on days −1, 3, and 6 relative to PyL or PyNL infection with asialo GM-1 antibody or PBS (control). Groups consisted of four or five mice, and the results are representative of two independent experiments. Symbols represent significant differences (P < 0.05) between groups as follows: in panels A and B, #, PyL-infected group versus PyNL-infected group; *, PyL-infected group versus uninfected group; ∼, PyNL-infected group versus uninfected group; in panels C and D, #, PyL-infected, asialo GM-1 antibody-treated group versus control group; *, PyNL-infected, asialo GM-1 antibody-treated group versus control group.

FIG. 7.

Early T-cell-independent IFN-γ response differentiates PyL and PyNL infection but is not required for control of acute P. yoelii infection. (A) Plasma levels of IFN-γ in PyL- and PyNL-infected WT and RAG-1^−/− mice. (B to D) The course of parasitemia (B), anemia (C), and survival (D) in PyL- and PyNL-infected WT and IFN-γ^−/− mice. Groups consisted of three to five mice, and the results are representative of three independent experiments. Symbols represent significant differences (P < 0.05) between groups: in panel A, #, PyL-infected RAG-1^−/− group versus WT group; *, PyNL-infected RAG-1^−/− group versus WT group; ∼, PyL-infected RAG-1^−/− group versus PyNL-infected group; +, PyL-infected WT group versus PyNL-infected group; for panels B and C, *, PyNL-infected IFN-γ^−/− group versus WT group.

FIG. 8.

Macrophages contribute to control of the primary wave of P. yoelii parasitemia. C57BL/6 mice were treated i.p. on days −2 and 3 relative to P. yoelii infection with 300 μl of clodronate-liposome suspension. (A) Representative dot plots showing efficacy of splenic F4-80⁺ MHC class II⁺ macrophage depletion 3 days after clodronate liposome treatment (day 1 p.i.). (B) Efficacy of macrophage depletion (percentage of F4-80⁺ MHC class II⁺ splenocytes) over the initial 7 days of malaria infection. (C to E) Parasitemia (C), anemia (D), and mortality (E) in clodronate liposome-treated and untreated PyL- and PyNL-infected mice. Groups consisted of five mice, and the results are representative of two independent experiments. Symbols represent significant differences (P < 0.05) between groups: in panel B, #, untreated PyL-infected group versus untreated PyNL-infected group; *, untreated PyNL-infected group versus uninfected group; in panels C and D, #, PyL-infected, treated group versus control group; *, PyNL-infected, treated group versus control group.