Immunological interactions between 2 common pathogens, Th1-inducing protozoan Toxoplasma gondii and the Th2-inducing helminth Fasciola hepatica

Abstract

Background: The nature of the immune response to infection is dependent on the type of infecting organism. Intracellular organisms such as Toxoplasma gondii stimulate a Th1-driven response associated with production of IL-12, IFN-gamma, nitric oxide and IgG2a antibodies and classical activation of macrophages. In contrast, extracellular helminths such as Fasciola hepatica induce Th2 responses characterised by the production of IL-4, IL-5, IL-10 and IgG1 antibodies and alternative activation of macrophages. As co-infections with these types of parasites commonly exist in the field it is relevant to examine how the various facets of the immune responses induced by each may influence or counter-regulate that of the other.

Principal findings: Regardless, of whether F. hepatica infection preceded or succeeded T. gondii infection, there was little impact on the production of the Th1 cytokines IL-12, IFN-gamma or on the development of classically-activated macrophages induced by T. gondii. By contrast, the production of helminth-specific Th2 cytokines, such as IL-4 and IL-5, was suppressed by infection with T. gondii. Additionally, the recruitment and alternative activation of macrophages by F. hepatica was blocked or reversed by subsequent infection with T. gondii. The clinical symptoms of toxoplasmosis and the survival rate of infected mice were not significantly altered by the helminth.

Conclusions: Despite previous studies showing that F. hepatica suppressed the classical activation of macrophages and the Th1-driven responses of mice to bystander microbial infection, as well as reduced their ability to reject these, here we found that the potent immune responses to T. gondii were capable of suppressing the responses to helminth infection. Clearly, the outcome of particular infections in polyparasitoses depends on the means and potency by which each pathogen controls the immune response.

Figure 1. Mice infected with both Toxoplasma gondii and Fasciola hepatica produce both anti-T. gondii IgG2a and anti-F. hepatica IgG1 indicating a Th1 response to the T. gondii infection and a Th2 response to the F. hepatica infection.

BALB/c mice were infected with F. hepatica (Fh) 5 days prior to infection with T. gondii (A,C) or T. gondii 3 days prior to infection with F. hepatica (B,D). Mice infected with either F. hepatica alone or T. gondii alone or uninfected mice were used as controls. Serum was collected by cardiac puncture on: (A,C) day 10 post T. gondii infection (day 15 post F. hepatica infection) or (B,D) day 14 post T. gondii infection (day 11 post F. hepatica infection). T. gondii-specific IgG1 and IgG2a (A,B) and F. hepatica-specific IgG1 and IgG2a (C,D) antibodies were measured by ELISA. The results represent the mean±SE of one experiment with five animals (A,C) or eight animals (B,D) per group. ^a indicates groups where the values obtained were significantly different to values obtained for the T. gondii-only infected mice and ^b represents groups where the values obtained were significantly different to those obtained for the F. hepatica-only infected group (P<0.05, Mann-Whitney non-parametric test).

Figure 2. Toxoplasma gondii infection suppresses the recruitment of cells to the site of infection normally associated with F. hepatica infection.

(A) BALB/c mice were infected with F. hepatica (Fh) then 5 days later were infected with T. gondii (Tg) or (B) mice were infected with T. gondii 3 days prior to infection with F. hepatica. Mice infected with either F. hepatica alone or T. gondii alone or uninfected mice were used as controls. On days 10 (A) and 14 (B) post infection T. gondii (days 15 and 11 post F. hepatica infection respectively) peritoneal exudate cells were recovered from the peritoneal cavity by flushing with PBS. Total cell numbers were determined by counting using a Neubauer slide. Microscopic analysis showed macrophages to be the dominant cell type in all the mice making up 89–91% of cells; other cells present included lymphocytes (4–5%) and neutrophils (5–6%). Results are presented as mean±SE for five animals per group (A) or eight animals per group (B). ^a represents groups where the values obtained were significantly different to values obtained for the T. gondii-only infected mice and ^b represents groups where the values obtained were significantly different to those obtained for the F. hepatica-only infected group (P<0.05, Mann-Whitney non-parametric test).

Figure 3. Macrophages recovered from the peritoneal cavity of mice co-infected with T. gondii and F. hepatica are classically activated.

Mice were infected with F. hepatica 5 days prior (A) or 3 days subsequent (B) to infection with T. gondii. Mice infected with either F. hepatica alone or T. gondii alone were used as controls. Fifteen (A) or eleven (B) days post F. hepatica infection (day 10 (A) or 14 (B) post T. gondii infection respectively) peritoneal exudate cells were recovered from the peritoneal cavity by flushing with PBS. Expression levels of inducible nitric oxide synthase (iNOS), Arginase 1 (Arg 1) and β-actin in adhered peritoneal exudate cells were analysed by RT-PCR. The data shown are from single mice and are representative of results obtained from all mice in each group. Serum levels of NO were determined in the same mice. Serum was collected by cardiac puncture on (A) day 10 post T. gondii infection (day 15 post F. hepatica infection) or (B) day 14 post T. gondii infection (day 11 post F. hepatica). A modified Griess reaction was used to measure NO (see Materials and Methods). The results represent the mean±SE of one experiment with five animals per group (A) or eight animals per group (B). ). ^a represents groups where the values obtained were significantly different to values obtained for the T. gondii-only infected mice and ^b represents groups where the values obtained were significantly different to those obtained for the F. hepatica-only infected group (P<0.05, Mann-Whitney non-parametric test).

Figure 4. Infection with Fasciola hepatica does not inhibit IL-12 production early in infection with Toxoplasma gondii.

BALB/c mice were infected with F. hepatica 5 days prior to infection with T. gondii. Mice infected with either F. hepatica alone or T. gondii alone were used as controls. On day 4 post T. gondii infection (day 9 post F. hepatica infection) the peritoneal cavity was rinsed with PBS and the resulting lavage was spun at 500 g to pellet the exudate cells. The supernatant was retained to measure the level of IL-12 being secreted into the peritoneal cavity by cytokine ELISA (B). Adherent peritoneal exudate cells (PEC) were cultured for 48 h and supernatants assayed for secretion of IL-12 by cytokine ELISA (A). Serum was collected by cardiac puncture and levels of IL-12 determined by cytokine ELISA (C). The results represent the mean±SE of one experiment with five animals per group. ^a represents groups where the values obtained were significantly different to values obtained for the T. gondii-only infected mice and ^b represents groups where the values obtained were significantly different to those obtained for the F. hepatica-only infected group (P<0.05, Mann-Whitney non-parametric test).

Figure 5. Production of T. gondii-specific Th1 cytokines is not suppressed by F. hepatica infection but T. gondii infection inhibits the ability of splenocytes from F. hepatica-infected mice to produce Th2 cytokines.

Mice were infected with F. hepatica 5 days prior or 3 days subsequent to infection with T. gondii. Mice infected with either F. hepatica alone or T. gondii alone were used as controls. Cytokine production of the Th1-associated cytokine IFN-γ (A, B) and the Th2-associated cytokines, IL-4 (C, D) and IL-5 (E, F) by spleen cells was assessed on days 10 and 14 post T. gondii infection (day 15 or 11 post F. hepatica infection respectively) by stimulation in vitro with ES antigen or T. gondii lysate. Cytokine concentrations represent mean±SE after subtraction of background control values with medium only for five mice (A, C, E) or eight mice (B, D, F) per group. ^a indicates groups where the values obtained were significantly different to values obtained for the T. gondii-only infected mice and ^b represents groups where the values obtained were significantly different to those obtained for the F. hepatica-only infected group (P<0.05, Mann-Whitney non-parametric test).

Figure 6. Toxoplasma gondii suppresses Fasciola hepatica-driven non-specific Th2 responses in co-infected mice.

Mice were infected with F. hepatica 5 days prior to infection with T. gondii to allow time for a Th2 response to be induced. Mice infected with either F. hepatica alone or T. gondii alone were used as controls. Fifteen days post F. hepatica infection (day 10 post T. gondii infection) spleen cells were stimulated in vitro with PMA plus anti-CD3 and medium alone was included as a negative control. Levels of the Th1-associated cytokine IFN-γ (A) and the Th2-associated cytokines, IL-4 and IL-5 (B, C) were assessed in supernatants 3 days later. Cytokine concentrations represent mean±SE after subtraction of background control values with medium only for five mice per group. ^a represents groups where the values obtained were significantly different to values obtained for the T. gondii-only infected mice and ^b represents groups where the values obtained were significantly different to those obtained for the F. hepatica-only infected group (P<0.05, Mann-Whitney non-parametric test).