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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: Leishmania is an intracellular parasite in vertebrate hosts, including man.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.L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice.

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

Exposure of PS by macrophages after in vitro infection with L. amazonensis or L. guyanensis.Peritoneal macrophages of BALB/c (A) or C57BL/6 (B) mice infected or not with L. amazonensis or L. guyanensis were labeled with AnnV-FITC and counterstained with PI. Analysis was carried out by flow cytometry and only initial moments of apoptosis (AnnV+/PI-) were considered at the indicated time points. Bars represent mean ± SE of two or three (depending on the time point) independent experiments. Statistically significant differences between the two groups indicated, at a P < 0.05, are represented by (*). A typical experiment is shown in dot plots after macrophage gating and analysis by FlowJo (C). Gating strategy is shown in S1A Fig.
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pone.0141196.g004: Exposure of PS by macrophages after in vitro infection with L. amazonensis or L. guyanensis.Peritoneal macrophages of BALB/c (A) or C57BL/6 (B) mice infected or not with L. amazonensis or L. guyanensis were labeled with AnnV-FITC and counterstained with PI. Analysis was carried out by flow cytometry and only initial moments of apoptosis (AnnV+/PI-) were considered at the indicated time points. Bars represent mean ± SE of two or three (depending on the time point) independent experiments. Statistically significant differences between the two groups indicated, at a P < 0.05, are represented by (*). A typical experiment is shown in dot plots after macrophage gating and analysis by FlowJo (C). Gating strategy is shown in S1A Fig.

Mentions: To investigate whether these Leishmania species were causing death of host cells, infected macrophages were treated with PI and cell permeability was assessed by flow cytometry (Fig 3). Membrane integrity of both BALB/c (Fig 3A) and C57BL/6 (Fig 3B) macrophages infected with each of the two species shows distinct patterns, as infection progresses. L. guyanensis clearly causes loss of macrophage membrane integrity, inducing a massive macrophage death as early as 24h of infection, culminating with most cells dead at 72h. On the contrary, although L. amazonensis also may cause some loss of macrophage membrane integrity in 24h of infection, this is not observed at 48 or 72h. In fact, there are less L. amazonensis-infected cells from both strains of mice permeable to PI than uninfected cells that spontaneously die, losing membrane integrity. A typical cytometry plot can be seen in Fig 4C, in which case cells were also labeled with AnnV, as described in the following section. Simultaneously, we sought to investigate whether macrophage death caused by L. guyanensis was through apoptosis. We initially looked for the following features shown by apoptotic cells: exposure of PS, degradation and loss of DNA.


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)

Exposure of PS by macrophages after in vitro infection with L. amazonensis or L. guyanensis.Peritoneal macrophages of BALB/c (A) or C57BL/6 (B) mice infected or not with L. amazonensis or L. guyanensis were labeled with AnnV-FITC and counterstained with PI. Analysis was carried out by flow cytometry and only initial moments of apoptosis (AnnV+/PI-) were considered at the indicated time points. Bars represent mean ± SE of two or three (depending on the time point) independent experiments. Statistically significant differences between the two groups indicated, at a P < 0.05, are represented by (*). A typical experiment is shown in dot plots after macrophage gating and analysis by FlowJo (C). Gating strategy is shown in S1A Fig.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141196.g004: Exposure of PS by macrophages after in vitro infection with L. amazonensis or L. guyanensis.Peritoneal macrophages of BALB/c (A) or C57BL/6 (B) mice infected or not with L. amazonensis or L. guyanensis were labeled with AnnV-FITC and counterstained with PI. Analysis was carried out by flow cytometry and only initial moments of apoptosis (AnnV+/PI-) were considered at the indicated time points. Bars represent mean ± SE of two or three (depending on the time point) independent experiments. Statistically significant differences between the two groups indicated, at a P < 0.05, are represented by (*). A typical experiment is shown in dot plots after macrophage gating and analysis by FlowJo (C). Gating strategy is shown in S1A Fig.
Mentions: To investigate whether these Leishmania species were causing death of host cells, infected macrophages were treated with PI and cell permeability was assessed by flow cytometry (Fig 3). Membrane integrity of both BALB/c (Fig 3A) and C57BL/6 (Fig 3B) macrophages infected with each of the two species shows distinct patterns, as infection progresses. L. guyanensis clearly causes loss of macrophage membrane integrity, inducing a massive macrophage death as early as 24h of infection, culminating with most cells dead at 72h. On the contrary, although L. amazonensis also may cause some loss of macrophage membrane integrity in 24h of infection, this is not observed at 48 or 72h. In fact, there are less L. amazonensis-infected cells from both strains of mice permeable to PI than uninfected cells that spontaneously die, losing membrane integrity. A typical cytometry plot can be seen in Fig 4C, in which case cells were also labeled with AnnV, as described in the following section. Simultaneously, we sought to investigate whether macrophage death caused by L. guyanensis was through apoptosis. We initially looked for the following features shown by apoptotic cells: exposure of PS, degradation and loss of DNA.

Bottom Line: Leishmania is an intracellular parasite in vertebrate hosts, including man.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.L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice.

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