Impairment of protective immunity to blood-stage malaria by concurrent nematode infection

Abstract

Helminthiases, which are highly prevalent in areas where malaria is endemic, have been shown to modulate or suppress the immune response to unrelated antigens or pathogens. In this study, we established a murine model of coinfection with a gastrointestinal nematode parasite, Heligmosomoides polygyrus, and the blood-stage malaria parasite Plasmodium chabaudi AS in order to investigate the modulation of antimalarial immunity by concurrent nematode infection. Chronic infection with the nematode for 2, 3, or 5 weeks before P. chabaudi AS infection severely impaired the ability of C57BL/6 mice to control malaria, as demonstrated by severe mortality and significantly increased malaria peak parasitemia levels. Coinfected mice produced significantly lower levels of gamma interferon (IFN-gamma) during P. chabaudi AS infection than mice infected with malaria alone. Concurrent nematode infection also suppressed production of type 1-associated, malaria-specific immunoglobulin G2a. Mice either infected with the nematode alone or coinfected with the nematode and malaria had high transforming growth factor beta1 (TGF-beta1) levels, and concurrent nematode and malaria infections resulted in high levels of interleukin-10 in vivo. Splenic CD11c(+) dendritic cells (DC) from mice infected with malaria alone and coinfected mice showed similarly increased expression of CD40, CD80, and CD86, but DC from coinfected mice were unable to induce CD4(+) T-cell proliferation and optimal IFN-gamma production in response to the malaria antigen in vitro. Importantly, treatment of nematode-infected mice with an anthelmintic drug prior to malaria infection fully restored protective antimalarial immunity and reduced TGF-beta1 levels. These results demonstrate that concurrent nematode infection strongly modulates multiple aspects of immunity to blood-stage malaria and consequently impairs the development of protective antimalarial immunity.

FIG. 1.

Mortality and levels of parasitemia in B6 mice infected with blood-stage P. chabaudi AS (Pc) or coinfected with the nematode H. polygyrus (Hp) and malaria. Groups of B6 mice (n = 5) were infected by oral inoculation with 200 H. polygyrus L3 for 2, 3, or 5 weeks. Nematode-infected mice and a group of normal mice were infected i.p. with 10⁶ P. chabaudi AS-parasitized RBC. Mortality (A) and malaria parasitemia (B) were monitored for 3 weeks after malaria infection. Mortality is calculated from pooled data (n = 13 to 15) from three experiments. Parasitemia results shown are from one of three experiments. Asterisk indicates significant difference (P < 0.05) in peak parasitemia between coinfected and singly malaria infected groups.

FIG. 2.

Levels of IFN-γ and P. chabaudi AS-specific antibody in sera. (A) Sera were collected from normal uninfected B6 mice, from mice singly infected with either 200 H. polygyrus (Hp) L3 (20 days postinfection) or 10⁶ P. chabaudi AS (Pc)-parasitized RBC (6 days postinfection), and from mice coinfected with H. polygyrus (20 days) and P. chabaudi AS (6 days). IFN-γ levels were determined by ELISA. (B) B6 mice were either singly infected with P. chabaudi AS or coinfected with H. polygyrus and P. chabaudi AS, and serum samples were collected 5 weeks post-P. chabaudi AS infection. Levels of P. chabaudi AS-specific total Ig, IgG1, and IgG2a were measured by ELISA. Data from individual mice and the median for each group (n = 4) are presented. Results shown are representative of four (A) and two (B) independent experiments. Asterisk indicates a significant difference between groups (P < 0.05).

FIG. 3.

IFN-γ and IL-4 production by spleen cells in vitro. (A) Spleens were collected from normal uninfected B6 mice, from mice singly infected with either 200 H. polygyrus (Hp) L3 (20 days postinfection) or 10⁶ P. chabaudi AS (Pc)-parasitized RBC (6 days postinfection), and from mice coinfected with H. polygyrus (20 days) and P. chabaudi AS (6 days). Spleen cells were cultured as described in Materials and Methods. IFN-γ levels in supernatants were determined by ELISA. Data from individual mice and the median for each group (n = 4) are presented. (B) Groups of B6 mice were infected with 200 H. polygyrus L3 for the indicated times. Spleen cells were cultured in medium or in the presence of Hp-Ag, and IL-4 levels in supernatants were measured by ELISA. Data are presented as medians, with minimum and maximum values (n = 4) indicated by error bars for each group. (C) Spleens were collected from the four groups of mice, and cells were cultured as described for panel A. IL-4 levels in supernatants were measured by ELISA. Data from individual mice and the median for each group (n = 4) are presented. Results shown are representative of two experiments. Asterisk indicates a significant difference (P < 0.05) between groups.

FIG. 4.

Levels of bioactive TGF-β1 in plasma and of IL-10 in serum. (A and B) Plasma samples were collected from B6 mice at the indicated times after blood-stage P. chabaudi AS (A) or H. polygyrus (B) single infection, and levels of bioactive TGF-β1 were determined by ELISA. Data are presented as medians, with minimum and maximum values (n = 4) indicated by error bars for each group. Single (P < 0.05) and double (P < 0.01) asterisks indicate significant differences from values for normal uninfected mice. (C and D) Plasma or serum samples were collected from normal uninfected B6 mice, from mice singly infected with either H. polygyrus (Hp; 20 days postinfection) or P. chabaudi AS (Pc; 6 days postinfection), and from mice coinfected with H. polygyrus (20 days) and P. chabaudi AS (6 days), and levels of bioactive TGF-β1 in plasma (C) and IL-10 in serum (D) were determined by ELISA. Data from individual mice and the median for each group (n = 4) are presented. Asterisks (P < 0.01) indicate significant differences between groups. Results are representative of three (A and B) or two (C and D) experiments.

FIG. 5.

Proliferation of enriched CD4⁺ T cells induced by splenic CD11c⁺ DC. CD11c⁺ DC were isolated by magnetic sorting from spleens of normal, P. chabaudi AS (Pc)-infected (3 days), H. polygyrus (Hp)-infected (17 days), and coinfected B6 mice and were then cocultured with CD4⁺ T cells isolated from spleens of normal mice in the presence of 10⁶ pRBC/ml for 48 h. During the last 6 h of culture, 1 μCi/well of [³H]thymidine was added, and incorporation of [³H]thymidine was determined. Single (P < 0.05) and double (P < 0.01) asterisks indicate significant differences between DC from coinfected mice and singly P. chabaudi AS infected mice.

FIG. 6.

Effects of deworming on antimalarial immunity and TGF-β1 levels in coinfected mice. (A) Two groups of B6 mice (n = 5) were infected with 200 H. polygyrus (Hp) L3. Two weeks after H. polygyrus infection, one group of H. polygyrus-infected mice and one group of normal mice were treated with pyrantel pamoate. One week after drug treatment, the three groups, as well as a group of normal mice, were infected with blood-stage P. chabaudi AS (Pc), and malaria parasitemia was determined. Asterisks (P < 0.05) indicate a significant difference in peak parasitemia from mice infected with H. polygyrus and treated with drug. (B) Four groups of B6 mice were infected with H. polygyrus and drug treated as described for panel A. All animals were sacrificed 1 week after drug treatment, and levels of bioactive TGF-β1 in plasma were determined. Data from individual mice and the median for each group (n = 4) are presented. Asterisks (P < 0.05) indicate significant differences between groups.