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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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Different myeloid sub-populations expressed as percentage within the CD45+ hematopoietic compartment or as absolute numbers within the organ.Left panels: Percentage of the different cell populations (neutrophils/PMN, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) within the CD45+ hematopoietic compartment obtained following gating of liver (A) and spleen (B) as described in S1 Fig and S2 Fig, respectively, from non-infected (white bars) or T. brucei infected (day 6 p.i.) animals. Right panels: Absolute numbers of the different cell populations (neutrophils/PMN, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) in total liver and spleen of non-infected (white bars) and T. brucei infected (day 6 p.i.) mice. Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05; **: p-values ≤ 0.01; ***: p-values ≤ 0.005 and if nothing is mentioned the differences were not significant.
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pntd.0003561.g005: Different myeloid sub-populations expressed as percentage within the CD45+ hematopoietic compartment or as absolute numbers within the organ.Left panels: Percentage of the different cell populations (neutrophils/PMN, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) within the CD45+ hematopoietic compartment obtained following gating of liver (A) and spleen (B) as described in S1 Fig and S2 Fig, respectively, from non-infected (white bars) or T. brucei infected (day 6 p.i.) animals. Right panels: Absolute numbers of the different cell populations (neutrophils/PMN, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) in total liver and spleen of non-infected (white bars) and T. brucei infected (day 6 p.i.) mice. Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05; **: p-values ≤ 0.01; ***: p-values ≤ 0.005 and if nothing is mentioned the differences were not significant.

Mentions: Besides anemia development, trypanosomosis infection has been shown to have a vast effect on spleen and liver immune cell populations [26–28]. Indeed, as mentioned earlier we observed that during the early stage of infection there was an increase (in percentage as well as absolute numbers) of monocytic cells and neutrophils in the liver and a decrease in resident macrophages (Fig. 5A). In the spleen, a similar increase in monocytes and neutrophils is observed as well as an increase in CD11b+ F4/80+ myeloid cells (Fig. 5B). Therefore, at the level of the spleen it seems that besides monocytes also the CD11b+ F4/80+ myeloid cells (i.e. red pulp macrophages) can significantly contribute to erythrophagocytosis during infection. Hence both organs seem to contribute to erythrophagocytosis during the acute stage of infection.


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)

Different myeloid sub-populations expressed as percentage within the CD45+ hematopoietic compartment or as absolute numbers within the organ.Left panels: Percentage of the different cell populations (neutrophils/PMN, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) within the CD45+ hematopoietic compartment obtained following gating of liver (A) and spleen (B) as described in S1 Fig and S2 Fig, respectively, from non-infected (white bars) or T. brucei infected (day 6 p.i.) animals. Right panels: Absolute numbers of the different cell populations (neutrophils/PMN, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) in total liver and spleen of non-infected (white bars) and T. brucei infected (day 6 p.i.) mice. Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05; **: p-values ≤ 0.01; ***: p-values ≤ 0.005 and if nothing is mentioned the differences were not significant.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003561.g005: Different myeloid sub-populations expressed as percentage within the CD45+ hematopoietic compartment or as absolute numbers within the organ.Left panels: Percentage of the different cell populations (neutrophils/PMN, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) within the CD45+ hematopoietic compartment obtained following gating of liver (A) and spleen (B) as described in S1 Fig and S2 Fig, respectively, from non-infected (white bars) or T. brucei infected (day 6 p.i.) animals. Right panels: Absolute numbers of the different cell populations (neutrophils/PMN, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) in total liver and spleen of non-infected (white bars) and T. brucei infected (day 6 p.i.) mice. Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05; **: p-values ≤ 0.01; ***: p-values ≤ 0.005 and if nothing is mentioned the differences were not significant.
Mentions: Besides anemia development, trypanosomosis infection has been shown to have a vast effect on spleen and liver immune cell populations [26–28]. Indeed, as mentioned earlier we observed that during the early stage of infection there was an increase (in percentage as well as absolute numbers) of monocytic cells and neutrophils in the liver and a decrease in resident macrophages (Fig. 5A). In the spleen, a similar increase in monocytes and neutrophils is observed as well as an increase in CD11b+ F4/80+ myeloid cells (Fig. 5B). Therefore, at the level of the spleen it seems that besides monocytes also the CD11b+ F4/80+ myeloid cells (i.e. red pulp macrophages) can significantly contribute to erythrophagocytosis during infection. Hence both organs seem to contribute to erythrophagocytosis during the acute stage of infection.

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