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

Loss of nuclear DNA 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. After the indicated time points cells were lysed with hypotonic lysis buffer containing PI and nuclei were analyzed by flow cytometry. Bars represent mean ± SD of three independent experiments. A typical experiment is shown in histograms after macrophage nuclei gating and analysis by FlowJo. Gating strategy of cell nuclei is shown in S1B and S1C Fig.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141196.g005: Loss of nuclear DNA 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. After the indicated time points cells were lysed with hypotonic lysis buffer containing PI and nuclei were analyzed by flow cytometry. Bars represent mean ± SD of three independent experiments. A typical experiment is shown in histograms after macrophage nuclei gating and analysis by FlowJo. Gating strategy of cell nuclei is shown in S1B and S1C Fig.

Mentions: Loss of nuclear DNA from infected macrophages was evaluated with PI staining of cell nuclei only and quantified by flow cytometry. This protocol is based on the principle that apoptotic cells have their DNA degraded by the action of endonucleases, and PI binding to DNA emits fluorescence proportional to nuclear DNA content [36]. Fig 5 shows the percentage of hypodiploidic macrophage nuclei of BALB/c (Fig 5A) and C57BL/6 (Fig 5B) mice after infection with each species of Leishmania. Corroborating the previous results, we could observe that L. guyanensis induces a very small loss of DNA content, whereas L. amazonensis induces a substantial DNA loss in both strains of mice, indicating that L. amazonensis-infected cells are indeed undergoing apoptosis. Typical histograms are shown in Fig 5C. To confirm that classic apoptosis was taking place, we carried out agarose gel electrophoresis to investigate how loss of DNA was occurring.


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)

Loss of nuclear DNA 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. After the indicated time points cells were lysed with hypotonic lysis buffer containing PI and nuclei were analyzed by flow cytometry. Bars represent mean ± SD of three independent experiments. A typical experiment is shown in histograms after macrophage nuclei gating and analysis by FlowJo. Gating strategy of cell nuclei is shown in S1B and S1C Fig.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141196.g005: Loss of nuclear DNA 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. After the indicated time points cells were lysed with hypotonic lysis buffer containing PI and nuclei were analyzed by flow cytometry. Bars represent mean ± SD of three independent experiments. A typical experiment is shown in histograms after macrophage nuclei gating and analysis by FlowJo. Gating strategy of cell nuclei is shown in S1B and S1C Fig.
Mentions: Loss of nuclear DNA from infected macrophages was evaluated with PI staining of cell nuclei only and quantified by flow cytometry. This protocol is based on the principle that apoptotic cells have their DNA degraded by the action of endonucleases, and PI binding to DNA emits fluorescence proportional to nuclear DNA content [36]. Fig 5 shows the percentage of hypodiploidic macrophage nuclei of BALB/c (Fig 5A) and C57BL/6 (Fig 5B) mice after infection with each species of Leishmania. Corroborating the previous results, we could observe that L. guyanensis induces a very small loss of DNA content, whereas L. amazonensis induces a substantial DNA loss in both strains of mice, indicating that L. amazonensis-infected cells are indeed undergoing apoptosis. Typical histograms are shown in Fig 5C. To confirm that classic apoptosis was taking place, we carried out agarose gel electrophoresis to investigate how loss of DNA was occurring.

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