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

Silva GK, Cunha LD, Horta CV, Silva AL, Gutierrez FR, Silva JS, Zamboni DS - PLoS ONE (2013)

Bottom Line: 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 effect is unlikely to result from imprinted genes because the inheritance of this susceptibility was affected by the direction of the parental crossing.Future linkage studies may reveal the locus and genes participating on the host resistance process reported herein.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of São Paulo, Medical School Ribeirão Preto, FMRP/USP, Ribeirão Preto, São Paulo, Brazil.

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.

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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.
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pone-0056347-g006: 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.

Mentions: To investigate if the increased susceptibility of F1(AXB) mice was a feature associated with macrophages resistance to infection, we determined the intracellular replication of T. cruzi in BMDMs obtained from these strains. BMDMs were generated from C57BL/6, A/J, F1(AXB) and F1(BXA) male mice and infected with T. cruzi for 48 hours to evaluate intracellular parasite replication. As previously demonstrated in Fig. 2, BMDM obtained from A/J were significantly more permissive to T. cruzi replication as compared to C57BL/6 (Fig. 6A). Moreover, both F1 crossings showed an intermediate phenotype between C57BL/6 and A/J (Fig. 6A). Of note, we found no significant differences between BMDMs from F1(AXB) and F1(BXA) for intracellular parasite replication, indicating that the increased susceptibility of the F1(AXB) mice is not macrophage associated (Fig. 6A). Thus, additional loci independent of those contained in the sex chromosomes may be involved in the macrophage resistance to T. cruzi infection. To further characterize the increased susceptibility of the F1(AXB) mice, we evaluated additional parameters related to T. cruzi infection in vivo: the replication of the parasite in the target organs, the inflammatory response and the extent of the tissue damage in the heart and liver. Examination of the inflammatory infiltrate in the heart and liver of infected mice showed that the F1(AXB) and F1(BXA) did not differ between each other and from the pattern observed in the organs of C57BL/6 mice (Fig. 6B and 6C). Analysis of the activity of the enzymes CK-MB and ALT indicated that tissue lesion in the heart and the liver of F1(AXB) and F1(BXA) were not statistically different (Fig. 6D and 6E). However, by quantifying parasite DNA in the heart and liver, we found an increased parasite load in the organs of the F1(AXB) as compared to F1(BXA) (Fig. 6F and 6G). Of note, the parasite load detected in the heart of F1(AXB) mice was not statistically different from that found in A/J parental strain (Fig. 6F). These observations support our findings related to the increased susceptibility of F1(AXB) mice compared with the F1(BXA) male mice.


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

Silva GK, Cunha LD, Horta CV, Silva AL, Gutierrez FR, Silva JS, Zamboni DS - PLoS ONE (2013)

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.
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Related In: Results  -  Collection

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pone-0056347-g006: 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.
Mentions: To investigate if the increased susceptibility of F1(AXB) mice was a feature associated with macrophages resistance to infection, we determined the intracellular replication of T. cruzi in BMDMs obtained from these strains. BMDMs were generated from C57BL/6, A/J, F1(AXB) and F1(BXA) male mice and infected with T. cruzi for 48 hours to evaluate intracellular parasite replication. As previously demonstrated in Fig. 2, BMDM obtained from A/J were significantly more permissive to T. cruzi replication as compared to C57BL/6 (Fig. 6A). Moreover, both F1 crossings showed an intermediate phenotype between C57BL/6 and A/J (Fig. 6A). Of note, we found no significant differences between BMDMs from F1(AXB) and F1(BXA) for intracellular parasite replication, indicating that the increased susceptibility of the F1(AXB) mice is not macrophage associated (Fig. 6A). Thus, additional loci independent of those contained in the sex chromosomes may be involved in the macrophage resistance to T. cruzi infection. To further characterize the increased susceptibility of the F1(AXB) mice, we evaluated additional parameters related to T. cruzi infection in vivo: the replication of the parasite in the target organs, the inflammatory response and the extent of the tissue damage in the heart and liver. Examination of the inflammatory infiltrate in the heart and liver of infected mice showed that the F1(AXB) and F1(BXA) did not differ between each other and from the pattern observed in the organs of C57BL/6 mice (Fig. 6B and 6C). Analysis of the activity of the enzymes CK-MB and ALT indicated that tissue lesion in the heart and the liver of F1(AXB) and F1(BXA) were not statistically different (Fig. 6D and 6E). However, by quantifying parasite DNA in the heart and liver, we found an increased parasite load in the organs of the F1(AXB) as compared to F1(BXA) (Fig. 6F and 6G). Of note, the parasite load detected in the heart of F1(AXB) mice was not statistically different from that found in A/J parental strain (Fig. 6F). These observations support our findings related to the increased susceptibility of F1(AXB) mice compared with the F1(BXA) male mice.

Bottom Line: 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 effect is unlikely to result from imprinted genes because the inheritance of this susceptibility was affected by the direction of the parental crossing.Future linkage studies may reveal the locus and genes participating on the host resistance process reported herein.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of São Paulo, Medical School Ribeirão Preto, FMRP/USP, Ribeirão Preto, São Paulo, Brazil.

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.

Show MeSH
Related in: MedlinePlus