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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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Anemia development during T. brucei infection and pHrodo erythrophagocytosis assay on liver and spleen cells during the acute stage of infection.Upper panel (A): Anemia profile during the course of T. brucei infection, whereby the boxed area indicates the time point of interest. Lower panels: Histograms showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells in presence of unlabeled RBCs from cells in presence of pHrodo-labeled RBCs following in vitro incubation (B and C). Different cell populations (neutrophils, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) were identified for liver and spleen as described in S2 Fig and S3 Fig, respectively, from naïve (white bars) or 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.g004: Anemia development during T. brucei infection and pHrodo erythrophagocytosis assay on liver and spleen cells during the acute stage of infection.Upper panel (A): Anemia profile during the course of T. brucei infection, whereby the boxed area indicates the time point of interest. Lower panels: Histograms showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells in presence of unlabeled RBCs from cells in presence of pHrodo-labeled RBCs following in vitro incubation (B and C). Different cell populations (neutrophils, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) were identified for liver and spleen as described in S2 Fig and S3 Fig, respectively, from naïve (white bars) or 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: During the course of trypanosome infection anemia develops, which can be divided into different stages. During the acute stage of T. b. brucei infection, C57Bl/6 mice develop acute anemia, which is typically observed between day 5–8 post infection (Fig. 4A). After a slight recovery phase (i.e. between day 8–10), the reduction in the RBC percentage persists throughout the chronic phase of infection. Here, using the pHrodo in vitro erythrophagocytosis assay we determined whether enhanced erythrophagocytosis could be responsible for this severe reduction in RBCs observed during the early stage of infection. Whole liver and spleen cells of day 6 infected mice were put in co-culture with labeled RBCs of the corresponding mice and the erythrophagocytozing potential was analyzed and compared to that of non-infected animals. Trypanosome infection causes an alteration of the liver myeloid cell composition, therefore a more elaborate gating strategy (S3 Fig) allows to distinguish monocyte-derived macrophages (CD11b+ Ly6C+ MHC-II+) and resident macrophages (CD11b+ F4/80+ Ly6C- MHC-II+) in addition to neutrophils and monocytes (CD11b+ Ly6C+ MHC-II-). In the liver, neutrophils and monocytes show a remarkable increase in their phagocytozing potential (Fig. 4B & S4 Fig) during infection while the phagocytozing potential of the monocyte-derived macrophages was unaltered. In contrast, the phagocytozing potential of resident macrophages diminished and the Rest fraction (consisting of NK cells and patrolling monocytes) only played a minor role in erythrophagocytosis during infection. In contrast to the situation in the liver, spleen neutrophil-mediated erythrophagocytosis was not enhanced upon infection (Fig. 4C). Monocytes on the other hand exhibited increased erythrophagocytosis and the CD11b+ F4/80+ myeloid cell-mediated erythrophagocytosis was reduced during infection (Fig. 4C).


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)

Anemia development during T. brucei infection and pHrodo erythrophagocytosis assay on liver and spleen cells during the acute stage of infection.Upper panel (A): Anemia profile during the course of T. brucei infection, whereby the boxed area indicates the time point of interest. Lower panels: Histograms showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells in presence of unlabeled RBCs from cells in presence of pHrodo-labeled RBCs following in vitro incubation (B and C). Different cell populations (neutrophils, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) were identified for liver and spleen as described in S2 Fig and S3 Fig, respectively, from naïve (white bars) or 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.g004: Anemia development during T. brucei infection and pHrodo erythrophagocytosis assay on liver and spleen cells during the acute stage of infection.Upper panel (A): Anemia profile during the course of T. brucei infection, whereby the boxed area indicates the time point of interest. Lower panels: Histograms showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells in presence of unlabeled RBCs from cells in presence of pHrodo-labeled RBCs following in vitro incubation (B and C). Different cell populations (neutrophils, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) were identified for liver and spleen as described in S2 Fig and S3 Fig, respectively, from naïve (white bars) or 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: During the course of trypanosome infection anemia develops, which can be divided into different stages. During the acute stage of T. b. brucei infection, C57Bl/6 mice develop acute anemia, which is typically observed between day 5–8 post infection (Fig. 4A). After a slight recovery phase (i.e. between day 8–10), the reduction in the RBC percentage persists throughout the chronic phase of infection. Here, using the pHrodo in vitro erythrophagocytosis assay we determined whether enhanced erythrophagocytosis could be responsible for this severe reduction in RBCs observed during the early stage of infection. Whole liver and spleen cells of day 6 infected mice were put in co-culture with labeled RBCs of the corresponding mice and the erythrophagocytozing potential was analyzed and compared to that of non-infected animals. Trypanosome infection causes an alteration of the liver myeloid cell composition, therefore a more elaborate gating strategy (S3 Fig) allows to distinguish monocyte-derived macrophages (CD11b+ Ly6C+ MHC-II+) and resident macrophages (CD11b+ F4/80+ Ly6C- MHC-II+) in addition to neutrophils and monocytes (CD11b+ Ly6C+ MHC-II-). In the liver, neutrophils and monocytes show a remarkable increase in their phagocytozing potential (Fig. 4B & S4 Fig) during infection while the phagocytozing potential of the monocyte-derived macrophages was unaltered. In contrast, the phagocytozing potential of resident macrophages diminished and the Rest fraction (consisting of NK cells and patrolling monocytes) only played a minor role in erythrophagocytosis during infection. In contrast to the situation in the liver, spleen neutrophil-mediated erythrophagocytosis was not enhanced upon infection (Fig. 4C). Monocytes on the other hand exhibited increased erythrophagocytosis and the CD11b+ F4/80+ myeloid cell-mediated erythrophagocytosis was reduced during infection (Fig. 4C).

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