Limits...
Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis, but Not by Leishmania (Viannia) guyanensis.

DaMata JP, Mendes BP, Maciel-Lima K, Menezes CA, Dutra WO, Sousa LP, Horta MF - PLoS ONE (2015)

Bottom Line: We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy.None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells.We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

ABSTRACT
Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.

No MeSH data available.


Related in: MedlinePlus

Lesion progression in mice infected with L. amazonensis or L. guyanensis and in vitro infection of macrophages.Groups of 5 BALB/c (open circles) or C57BL/6 (filled circles) mice were infected in one of the hind footpads with 1x106 stationary phase promastigotes of L. amazonensis (A) or L. guyanensis (B). Lesion progression was assessed weekly for up to 20 weeks by measuring the hind footpads. Lesion size was expressed as the difference between infected and non-infected footpad. Each point represents mean ± SE obtained from 5 mice. Macrophages were infected with L. amazonensis (C, E, G) or L. guyanensis (D, F, H) at a 10:1 parasite:macrophage ratio and incubated for up to 4 days. Infected cells were counted and results are expressed as percentage of infected macrophages (C, D), number of parasite/cell (E, F) and infection index (G, H). Infection index was estimated as the mean percentage of infected cells x the mean number of parasites per infected cells. Bars represent mean ± SE of two to six (depending on the time point) independent experiments. Statistically significant differences between the two groups at a P < 0.05 are represented by (*). Statistically significant differences between values of the same group as compared to values at 24h at a P < 0.05 are represented by (#).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4626090&req=5

pone.0141196.g001: Lesion progression in mice infected with L. amazonensis or L. guyanensis and in vitro infection of macrophages.Groups of 5 BALB/c (open circles) or C57BL/6 (filled circles) mice were infected in one of the hind footpads with 1x106 stationary phase promastigotes of L. amazonensis (A) or L. guyanensis (B). Lesion progression was assessed weekly for up to 20 weeks by measuring the hind footpads. Lesion size was expressed as the difference between infected and non-infected footpad. Each point represents mean ± SE obtained from 5 mice. Macrophages were infected with L. amazonensis (C, E, G) or L. guyanensis (D, F, H) at a 10:1 parasite:macrophage ratio and incubated for up to 4 days. Infected cells were counted and results are expressed as percentage of infected macrophages (C, D), number of parasite/cell (E, F) and infection index (G, H). Infection index was estimated as the mean percentage of infected cells x the mean number of parasites per infected cells. Bars represent mean ± SE of two to six (depending on the time point) independent experiments. Statistically significant differences between the two groups at a P < 0.05 are represented by (*). Statistically significant differences between values of the same group as compared to values at 24h at a P < 0.05 are represented by (#).

Mentions: Epidemiological and experimental studies have shown that different species of Leishmania cause recognizably different disease outcomes. These differences are easily shown in murine models of infection, particularly in C57BL/6 and BALB/c strains of mice. Here we compare the course of infection with L. amazonensis and L. guyanensis in these two strains of mice, in which lesion growth was followed weekly (Fig 1). As observed, BALB/c mice are totally susceptible to L. amazonensis with an increasing lesion over time. Unable to control infection, mice would die if not euthanized at the 10th week of infection (Fig 1A). C57BL/6 mice are much less susceptible to infection, starting with lesions comparable to those of BALB/c up to 5 weeks of infection, which, although do not progress, are persistent (Fig 1A). It is already well documented that C57BL/6 mice are not able to resolve L. amazonensis, giving rise to a chronic infection [9]. In contrast, both strains of mice completely control the lesion caused by L. guyanensis. In Fig 1B, we can see that in BALB/c, infection results in a very small footpad swelling from the 5th week, which recedes to no swelling at the 20th week, whereas C57BL/6 shows no lesion whatsoever during the same period.


Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis, but Not by Leishmania (Viannia) guyanensis.

DaMata JP, Mendes BP, Maciel-Lima K, Menezes CA, Dutra WO, Sousa LP, Horta MF - PLoS ONE (2015)

Lesion progression in mice infected with L. amazonensis or L. guyanensis and in vitro infection of macrophages.Groups of 5 BALB/c (open circles) or C57BL/6 (filled circles) mice were infected in one of the hind footpads with 1x106 stationary phase promastigotes of L. amazonensis (A) or L. guyanensis (B). Lesion progression was assessed weekly for up to 20 weeks by measuring the hind footpads. Lesion size was expressed as the difference between infected and non-infected footpad. Each point represents mean ± SE obtained from 5 mice. Macrophages were infected with L. amazonensis (C, E, G) or L. guyanensis (D, F, H) at a 10:1 parasite:macrophage ratio and incubated for up to 4 days. Infected cells were counted and results are expressed as percentage of infected macrophages (C, D), number of parasite/cell (E, F) and infection index (G, H). Infection index was estimated as the mean percentage of infected cells x the mean number of parasites per infected cells. Bars represent mean ± SE of two to six (depending on the time point) independent experiments. Statistically significant differences between the two groups at a P < 0.05 are represented by (*). Statistically significant differences between values of the same group as compared to values at 24h at a P < 0.05 are represented by (#).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4626090&req=5

pone.0141196.g001: Lesion progression in mice infected with L. amazonensis or L. guyanensis and in vitro infection of macrophages.Groups of 5 BALB/c (open circles) or C57BL/6 (filled circles) mice were infected in one of the hind footpads with 1x106 stationary phase promastigotes of L. amazonensis (A) or L. guyanensis (B). Lesion progression was assessed weekly for up to 20 weeks by measuring the hind footpads. Lesion size was expressed as the difference between infected and non-infected footpad. Each point represents mean ± SE obtained from 5 mice. Macrophages were infected with L. amazonensis (C, E, G) or L. guyanensis (D, F, H) at a 10:1 parasite:macrophage ratio and incubated for up to 4 days. Infected cells were counted and results are expressed as percentage of infected macrophages (C, D), number of parasite/cell (E, F) and infection index (G, H). Infection index was estimated as the mean percentage of infected cells x the mean number of parasites per infected cells. Bars represent mean ± SE of two to six (depending on the time point) independent experiments. Statistically significant differences between the two groups at a P < 0.05 are represented by (*). Statistically significant differences between values of the same group as compared to values at 24h at a P < 0.05 are represented by (#).
Mentions: Epidemiological and experimental studies have shown that different species of Leishmania cause recognizably different disease outcomes. These differences are easily shown in murine models of infection, particularly in C57BL/6 and BALB/c strains of mice. Here we compare the course of infection with L. amazonensis and L. guyanensis in these two strains of mice, in which lesion growth was followed weekly (Fig 1). As observed, BALB/c mice are totally susceptible to L. amazonensis with an increasing lesion over time. Unable to control infection, mice would die if not euthanized at the 10th week of infection (Fig 1A). C57BL/6 mice are much less susceptible to infection, starting with lesions comparable to those of BALB/c up to 5 weeks of infection, which, although do not progress, are persistent (Fig 1A). It is already well documented that C57BL/6 mice are not able to resolve L. amazonensis, giving rise to a chronic infection [9]. In contrast, both strains of mice completely control the lesion caused by L. guyanensis. In Fig 1B, we can see that in BALB/c, infection results in a very small footpad swelling from the 5th week, which recedes to no swelling at the 20th week, whereas C57BL/6 shows no lesion whatsoever during the same period.

Bottom Line: We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy.None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells.We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

ABSTRACT
Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.

No MeSH data available.


Related in: MedlinePlus