A parent-of-origin effect determines the susceptibility of a non-informative F1 population to Trypanosoma cruzi infection in vivo

Abstract

The development of Chagas disease is determined by a complex interaction between the genetic traits of both the protozoan parasite, T. cruzi, and the infected host. This process is regulated by multiple genes that control different aspects of the host-parasite interaction. While determination of the relevant genes in humans is extremely difficult, it is feasible to use inbred mouse strains to determine the genes and loci responsible for host resistance to infection. In this study, we investigated the susceptibility of several inbred mouse strains to infection with the highly virulent Y strain of T. cruzi and found a considerable difference in susceptibility between A/J and C57BL/6 mice. We explored the differences between these two mouse strains and found that the A/J strain presented higher mortality, exacerbated and uncontrolled parasitemia and distinct histopathology in the target organs, which were associated with a higher parasite burden and more extensive tissue lesions. We then employed a genetic approach to assess the pattern of inheritance of the resistance phenotype in an F1 population and detected a strong parent-of-origin effect determining the susceptibility of the F1 male mice. This effect is unlikely to result from imprinted genes because the inheritance of this susceptibility was affected by the direction of the parental crossing. Collectively, our genetic approach of using the F1 population suggests that genes contained in the murine chromosome X contribute to the natural resistance against T. cruzi infection. Future linkage studies may reveal the locus and genes participating on the host resistance process reported herein.

Figure 1. Inbred mouse strains vary in their susceptibility to infection by the Y strain of T. cruzi.

Male mice were infected i.p. with 1000 trypomastigotes when eight weeks old and included A/J (n = 8), BALB/c (n = 10), C3H/HePas (n = 8), C57BL/6 (n = 7) and DBA (n = 8) strains. (A) Mortality was evaluated by daily inspection of the cages and differed (P<0.05) between BALB/c and C57BL/6, BALB/c and DBA, BALB/c and A/J, BALB/c and C3H/HePas, C57BL/6 and A/J, C57BL/6 and C3H/HePas, DBA and A/J, and DBA and C3H/HePas. Data are representative of those found in three independent experiments. (B) Parasitemia was quantified microscopically by counting the parasites in 5 μl of citrated blood obtained from the tail lateral vein at days 7, 9, 11 and 13 days postinfection. At day 11, (*) indicates P<0.05 between A/J and all other groups. At day 13, (*) indicates P<0.05 between A/J and BALB/c, A/J and C57BL/6, and A/J and DBA; (#) indicates P<0.05 between C3H/HePas and BALB/c, C3H/HePas and C57BL/6, and C3H/HePas and DBA; and (&) indicates P<0.05 between DBA and C57BL/6. Data are representative of those found in three independent experiments.

Figure 2. Macrophages from A/J mice are more susceptible to T. cruzi infection than those from C57BL/6 mice.

Bone marrow-derived macrophages (BMDMs) from A/J and C57BL/6 mice were infected at a multiplicity of infection of 5 with trypomastigotes of the Y strain of T. cruzi. At 48 h postinfection, cells were fixed and stained with Giemsa. (A) Representative micrographs of infected cells. Arrows indicate the intracellular amastigotes; arrowheads indicate the macrophage nuclei. The scale bar represents 20 µm. (B) Percentage of parasite-infected BMDMs containing 1 to 3, 4 to 6, 7 to 9, 10 to 15, or >15 amastigotes per cell. A total of 100 infected cells were scored in each of the 6 distinguished replicates. Data are expressed as the mean ± standard deviation of the 6 replicates. The experiment shown is representative of those found in three independent experiments. (*) indicates P<0.05.

Figure 3. C57BL/6 and A/J mice infected with T. cruzi differ in cardiac and hepatic histopathology, tissue injury and parasite load.

A/J and C57BL/6 mice were infected i.p. with 1000 trypomastigotes of the Y strain of T. cruzi. Infected animals were euthanized 15 days postinfection, and cardiac muscle and liver were collected and processed as described in Material and Methods. (A) Tissue samples were stained with hematoxylin-eosin to determine the presence of inflammatory infiltrates. Representative micrographs of C57BL/6 and A/J tissues are shown. Arrows indicate the presence of infiltrates of mononuclear cells in the myocardium and among the hepatocytes; the scale bar represents 100 µm. (B–C) Inflammatory infiltrates in the heart (B) and liver (C) were determined by counting nuclei of infiltrating cells after staining the organ sections with hematoxylin-eosin. (D–E) Lesions in the heart (D) and liver (E) of infected mice were estimated by quantifying the levels of creatine kinase enzyme (CK-MB) and alanine transaminase (ALT), respectively. (F–G) Parasite loads in the heart (F) and liver (G) of infected mice were determined by quantitative real time PCR using specific primers for T. cruzi (as described in Material and Methods). Data are representative of those found in three independent experiments. (*) indicates P<0.05.

Figure 4. Gender influences susceptibility to infection in F1 offspring of A/J females crossed with C57BL/6 males.

Age-matched males and females of parental A/J and C57BL/6 mouse strains together with heterozygous F1 offspring (F1(AXB)) of A/J females crossed with C57BL/6 males were infected i.p. with 1000 trypomastigotes of the Y strain of T. cruzi. Mouse numbers were as follows: A/J male (n = 11), A/J female (n = 8), C57BL/6 male (n = 11) C57BL/6 female (n = 8), F1(AXB) male (n = 9), F1(AXB) female (n = 11). Mortality was evaluated by daily inspection of the cages. Data are representative of those found in seven independent experiments. (*) indicates P<0.05 in comparisons between F1(AXB) males and the other experimental groups.

Figure 5. Direction of the cross between A/J and C57BL/6 mice determines gender differences in the susceptibility to infection of the F1 offspring.

Age-matched A/J, C57BL/6, F1(AXB) (offspring of A/J females and C57BL/6 males) and F1(BXA) (offspring of C57BL/6 females and A/J males) mice were infected i.p. with 1000 trypomastigotes of the Y strain of T. cruzi. Mortality of female (A) and male (B) mice was evaluated by daily inspection of the cages. Mouse numbers were as follows: A/J male (n = 10), A/J female (n = 10), C57BL/6 male (n = 11), C57BL/6 female (n = 08), F1(AXB) male (n = 08), F1(AXB) female (n = 11), F1(BXA) male (n = 7) and F1(BXA) female (n = 8). (C) Parasitemia of male mice was quantified by microscopically counting the parasites in 5 μl of citrated blood obtained from the tail lateral vein on days 7, 9, 11 and 13 post-infection. Data shown is one representative experiment of those found in three independent experiments. (*), (#), and (&) indicate P<0.05 in relation to C57BL/6, A/J and F1(BXA) respectively. (D) Parasitemia of F1(AXB) (open triangle) and F1 (BXA) (closed triangle) male mice on days 7, 9, 11, 13 post-infection. The data shown is a pool of 8 independent experiments; each plotted symbol represents the parasitemia of a single mouse. (*) indicates P<0.05.

Figure 6. Analysis of macrophage infection, histopatology and parasite load of F1(AXB) and F1(BXA) male mice infected with T. cruzi.

(A) Bone marrow-derived macrophages (BMDMs) from A/J, C57BL/6, F1(AXB) and F1(BXA) mice were infected at a multiplicity of infection of 5 with trypomastigotes of the Y strain of T. cruzi. At 48 h postinfection, cells were fixed and stained with Giemsa for determination of the percentage of parasite-infected BMDMs containing 1 to 3, 4 to 6, 7 to 9, 10 to 15, or >15 amastigotes per cell. A total of 100 infected cells were scored in each of the 6 distinguished replicates. Data are expressed as the mean ± standard deviation of the 6 replicates. (*) and (#) indicate P<0.05 in relation to C57BL/6 and A/J BMDMs, respectively. (B–G) A/J, C57BL/6, F1(AXB) and F1(BXA) male mice were infected i.p with 1000 trypomastigotes of the Y strain of T. cruzi. Infected mice were euthanized 15 days postinfection, and cardiac muscle and liver were collected and processed as described in Material and Methods. (B–C) Tissue samples were stained with hematoxylin-eosin (H&E) for determination of inflammatory infiltrates in the heart (B) and liver (C). (D–E) Lesions in the heart (D) and liver (E) of infected mice were estimated by quantifying the levels of creatine kinase enzyme (CK-MB) and alanine transaminase (ALT), respectively. (F–G) Parasite loads in the heart (F) and liver (G) of infected mice were determined by quantitative real time PCR using specific primers for T.cruzi (as described in Material and Methods). (B–G) (*), (#), and (&) indicate P<0.05 in relation to C57BL/6, A/J and F1(BXA) respectively.