Macrophages are critical for cross-protective immunity conferred by Babesia microti against Babesia rodhaini infection in mice

Abstract

Although primary infection of mice with Babesia microti has been shown to protect mice against subsequent lethal infection by Babesia rodhaini, the mechanism behind the cross-protection is unknown. To unravel this mechanism, we investigated the influence of primary infection of mice with nonlethal B. microti using different time courses on the outcome of subsequent lethal B. rodhaini infection. Simultaneous infections of mice with these parasites resulted in rapid increases in parasitemia, with 100% mortality in BALB/c mice, as observed with control mice infected with B. rodhaini alone. In contrast, mice with acute, resolving, and chronic-phase B. microti infections were completely protected against B. rodhaini, resulting in low parasitemia and no mortalities. Mice immunized with dead B. microti were not protected from B. rodhaini infection, although high antibody responses were induced. Interestingly, the protected mice had significantly decreased levels of antibody response, cytokines (including gamma interferon [IFN-γ], interleukin-2 [IL-2], IL-8, IL-10, and IL-12), and nitric oxide levels after infection with B. rodhaini. SCID mice and IFN-γ-deficient mice with chronic B. microti infections demonstrated protective responses comparable to those of immunocompetent mice. Likewise, in vivo NK cell depletion did not significantly impair the protective responses. Conversely, macrophage depletion resulted in increased susceptibility to B. rodhaini infection associated with changes in their antibody and cytokines profiles, indicating that macrophages contribute to the protection against this challenge infection. We conclude that future development of vaccines against Babesia should include a strategy that enhances the appropriate activation of macrophages.

Fig 1

Parasitemia and survival rates after B. microti inoculation and B. rodhaini challenge infection of BALB/c mice. Parasitemia course (A, C, E, G, and I) and survival rates (B, D, F, H, and J) of mock and test mice are presented. Test mice were initially infected with B. microti then challenged with B. rodhaini on days 0, 7, 14, 28 and 56 after primary infection. Mock mice received B. rodhaini alone. Arrows indicate the time of challenge infection with B. rodhaini (A, C, E, G, and I). The parasitemia course (K) and survival rate (L) of mice immunized with either dead B. microti (pRBCs) or nonparasitized murine RBCs (npRBCs) and then challenged with B. rodhaini. The results are expressed as a mean percent parasitemias ± the standard deviations (SD) of five mice.

Fig 2

Kinetics of serum IgGs and cytokines of protected and susceptible mice after B. rodhaini challenge infection. The production of IgG (A), IgG1 (B), IgG₂a (C), IL-8 (D), IL-12 (E), IL-2 (F), IFN-γ (G), IL-10 (H), TNF-α (I), and NO (J) in mice after challenge infection with B. rodhaini was determined. Test mice with acute and chronic B. microti infection, control mice (which received npRBC), or mock mice (which received no injection) were infected with B. rodhaini. Detection of IgGs, cytokines, and NO was performed in the mice at days 2, 4 and 6 after challenge infection. Asterisks indicate statistically significant differences (*, P < 0.05; **, P < 0.005; ***, P < 0.0001 [compared to mock and control mice]). The results are expressed as mean values ± the SD for five mice.

Fig 3

Parasitemia and survival rates of IFN-γ^−/− mice and SCID mice after B. rodhaini challenge infection. Parasitemia profiles and survival rates of chronically B. microti-infected IFN-γ^−/− mice (A and B) and SCID mice (C and D), respectively, over a period of 20 days after challenge infection are presented. Test mice were initially infected with B. microti and then challenged with B. rodhaini on day 28 after the primary infection. Mock mice received B. rodhaini alone. Arrows indicate the time of challenge infection with B. rodhaini. The results are expressed as mean percent parasitemias ± the SD for five mice.

Fig 4

Parasitemia and survival rates of NK cell- and macrophage/monocyte-depleted mice after B. rodhaini challenge infection. (A and B) BALB/c mice with chronic B. microti infection were treated by i.p. injections with either anti-asialo-GM1 antibody to deplete NK cells or control rabbit antibody. (C and D) BALB/c mice were treated by i.p injections with either clodronate liposome to deplete macrophages or PBS-liposome as a control. The results are expressed as mean percent parasitemias ± the SD for five mice monitored over a period of 20 days after challenge infection with B. rodhaini.

Fig 5

Kinetics of serum IgGs and cytokines of macrophage/monocyte-depleted and control mice after B. rodhaini challenge infection. (A) Production of IgGs. (B to D) Production of IL-8, IL-12, TNF-α, IL-2, IFN-γ, and IL-10 (B and C) and NO (D) at day 8 postchallenge infection with B. rodhaini. Asterisks indicate statistically significant differences (*, P < 0.05; **, P < 0.005; ***, P < 0.0001 [compared to the control]). The results are expressed as mean values ± the SD of five mice.