Cytokine responses of CD4+ T cells during a Plasmodium chabaudi chabaudi (ER) blood-stage infection in mice initiated by the natural route of infection

Abstract

Background: Investigation of host responses to blood stages of Plasmodium spp, and the immunopathology associated with this phase of the life cycle are often performed on mice infected directly with infected red blood cells. Thus, the effects of mosquito bites and the pre-erythrocytic stages of the parasite, which would be present in natural infection, are ignored In this paper, Plasmodium chabaudi chabaudi infections of mice injected directly with infected red blood cells were compared with those of mice infected by the bites of infected mosquitoes, in order to determine whether the courses of primary infection and splenic CD4 T cell responses are similar.

Methods: C57Bl/6 mice were injected with red blood cells infected with P. chabaudi (ER) or infected via the bite of Anopheles stephensi mosquitoes. Parasitaemia were monitored by Giemsa-stained thin blood films. Total spleen cells, CD4+ T cells, and cytokine production (IFN-gamma, IL-2, IL-4, IL-10) were analysed by flow cytometry. In some experiments, mice were subjected to bites of uninfected mosquitoes prior to infectious bites in order to determine whether mosquito bites per se could affect a subsequent P. chabaudi infection.

Results: P. chabaudi (ER) infections initiated by mosquito bite were characterized by lower parasitaemia of shorter duration than those observed after direct blood challenge. However, splenomegaly was comparable suggesting that parasitaemia alone does not account for the increase in spleen size. Total numbers of CD4 T cells and those producing IFN-gamma, IL-10 and IL-2 were reduced in comparison to direct blood challenge. By contrast, the reduction in IL-4 producing cells was less marked suggesting that there is a proportionally lower Th1-like response in mice infected via infectious mosquitoes. Strikingly, pre-exposure to bites of uninfected mosquitoes reduced the magnitude and duration of the subsequent mosquito-transmitted infection still further, but enhanced the response of CD4 T cells producing IFN-gamma and IL-4.

Conclusion: The data in this paper suggest that studying early host responses in blood stage malaria infections measured after direct blood challenge of mice may not completely reflect the natural situation, and more detailed investigations of blood-stage immunity after mosquito transmission in experimental models should be considered.

Figure 1

Blood stage infections of P. chabaudi (ER) in C57BlC57BL/6 mice initiated by bite of infected mosquitoes and injection of parasitized erythrocytes (pRBCs). a) Course of infection after direct challenge with pRBC, and after infectious mosquito bites as described in the materials and methods (representative experiment of 3 performed). The values shown are the geometric means and standard errors of the parasitaemia from a minimum of five mice. b) Duration of the pre-patent period, peak parasitaemia and duration of infection. The values shown are the means and standard errors of the mean of a minimum of 15 mice.

Figure 2

Total numbers of splenic cells (a), CD4⁺ T cells (b) and CD4⁺ T cells producing cytokines, IFN-γ (c), IL-4 (d), IL-2 (e) and IL-10 (f) during a primary infection with P. chabaudi (ER). Spleen cells taken from mice at days 13 and 28 of infection were counted as described in the materials and methods. CD4⁺ T cells and cytokines, IFN-γ, IL-2, IL-4 and IL-10, were enumerated by surface and intracellular staining and analysed by FACS. The values shown represent the mean and standard errors of the means from 3 to 5 mice submitted to bites by infected mosquitoes (inf. bites), injected with 10⁵ parasitized erythrocytes (pRBC), submitted only to uninfected mosquito bites (uninfected bites) and mice not bitten or injected (naive).

Figure 3

Effects of prior exposure to the bites of uninfected mosquitoes on a blood stage infection of P. chabaudi (ER) transmitted by infected mosquitoes. a) Experimental procedure for the infection of mice with P. chabaudi (ER) through the bites of infected mosquitoes, after being exposed to four weekly sessions of bites by uninfected mosquitoes. One group of mice was only submitted to infectious mosquito bites. b) Course of infection in mice submitted first to uninfected followed by infected mosquito bites (▲), and infected mosquito bites only (■). The infection was followed for 13 days at which time mice were sacrificed and FACS analysis performed (see Figure 4). c) Length of pre-patent period, and peak parasitaemia of mice submitted to uninfected and infected mosquito bites (□), and infected mosquito bites only (■). The values represent the means and standard errors of the means of at least 3 mice per group.

Figure 4

Total numbers of splenic cells (a), CD4⁺ T cells (b) and CD4⁺ T cells producing cytokines, IFN-γ, IL-2, IL-4 and IL-10 (c-f respectively) in mice infected with P. chabaudi (ER) on day 13 after infection. Mice were submitted to bites by infected mosquitoes (infect), previous bites by uninfected mosquitoes before infected mosquitoes (uninfect. + infect.), only bites by uninfected mosquitoes (uninfect.) and not bitten (naive). The values represent the means and standard errors of the means of 3 mice.