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Development of a pHrodo-based assay for the assessment of in vitro and in vivo erythrophagocytosis during experimental trypanosomosis.

Stijlemans B, Cnops J, Naniima P, Vaast A, Bockstal V, De Baetselier P, Magez S - PLoS Negl Trop Dis (2015)

Bottom Line: Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts.Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia.In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium; Department of Myeloid Cell Immunology, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.

ABSTRACT
Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts. One common feature of trypanosomosis is the occurrence of anemia, caused by an imbalance between erythropoiesis and red blood cell clearance of aging erythrocytes. In murine models for T. brucei trypanosomosis, anemia is marked by a very sudden non-hemolytic loss of RBCs during the first-peak parasitemia control, followed by a short recovery phase and the subsequent gradual occurrence of an ever-increasing level of anemia. Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia. In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.

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Flow chart of the in vitro erythrophagocytosis assay protocol.
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pntd.0003561.g001: Flow chart of the in vitro erythrophagocytosis assay protocol.

Mentions: Peritoneal exudate cells (PECs), spleen and liver were harvested from CO2 euthanized non-infected and day 6 infected mice. Livers were minced in 10 ml digestive media (0.05% collagenase type A in Hanks’ Balanced Salt Solution (HBSS) without calcium or magnesium; Invitrogen) and incubation at 37°C for 30 minutes, the digested tissue was homogenized and filtered (40 μm pore filter). Spleen cells were obtained by homogenizing the organs in 10 ml RPMI medium containing 5% foetal calf serum (FCS) with or without collagenase type A, Next, the liver and spleen cell suspension was centrifuged (7 minutes, 300×g, 4°C) and the pellet treated with RBC lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2-EDTA). Subsequently, the cells were resuspended in ME—medium (RPMI medium, 5% FCS, 1% L-glutamine and non essential amino acids, 1% Penicillin-Streptomycin and β-mercaptoethanol) and 4 105 cells were put in co-culture with or without 2 107 labeled or unlabeled RBCs in polypropylene tubes (BD Biosciences). Co-cultures were incubated overnight at 37°C and 5% CO2 with or without lipopolysaccharide (LPS) stimulation (1μg/ml). An overview of the isolation protocol and erythrophagocytosis assay is given in Fig. 1.


Development of a pHrodo-based assay for the assessment of in vitro and in vivo erythrophagocytosis during experimental trypanosomosis.

Stijlemans B, Cnops J, Naniima P, Vaast A, Bockstal V, De Baetselier P, Magez S - PLoS Negl Trop Dis (2015)

Flow chart of the in vitro erythrophagocytosis assay protocol.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352936&req=5

pntd.0003561.g001: Flow chart of the in vitro erythrophagocytosis assay protocol.
Mentions: Peritoneal exudate cells (PECs), spleen and liver were harvested from CO2 euthanized non-infected and day 6 infected mice. Livers were minced in 10 ml digestive media (0.05% collagenase type A in Hanks’ Balanced Salt Solution (HBSS) without calcium or magnesium; Invitrogen) and incubation at 37°C for 30 minutes, the digested tissue was homogenized and filtered (40 μm pore filter). Spleen cells were obtained by homogenizing the organs in 10 ml RPMI medium containing 5% foetal calf serum (FCS) with or without collagenase type A, Next, the liver and spleen cell suspension was centrifuged (7 minutes, 300×g, 4°C) and the pellet treated with RBC lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2-EDTA). Subsequently, the cells were resuspended in ME—medium (RPMI medium, 5% FCS, 1% L-glutamine and non essential amino acids, 1% Penicillin-Streptomycin and β-mercaptoethanol) and 4 105 cells were put in co-culture with or without 2 107 labeled or unlabeled RBCs in polypropylene tubes (BD Biosciences). Co-cultures were incubated overnight at 37°C and 5% CO2 with or without lipopolysaccharide (LPS) stimulation (1μg/ml). An overview of the isolation protocol and erythrophagocytosis assay is given in Fig. 1.

Bottom Line: Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts.Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia.In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium; Department of Myeloid Cell Immunology, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.

ABSTRACT
Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts. One common feature of trypanosomosis is the occurrence of anemia, caused by an imbalance between erythropoiesis and red blood cell clearance of aging erythrocytes. In murine models for T. brucei trypanosomosis, anemia is marked by a very sudden non-hemolytic loss of RBCs during the first-peak parasitemia control, followed by a short recovery phase and the subsequent gradual occurrence of an ever-increasing level of anemia. Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia. In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.

Show MeSH
Related in: MedlinePlus