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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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pHrodo erythrophagocytosis assay using naïve PECs.A (left panel): CD11b versus F4/80 profile following gating on CD45+ cells allows identifying two distinct populations; (i) CD11b+F4/80+ cells which represent macrophages within the PEC population and (ii) cells which are negative or low for CD11b and F4/80 expression referred by as the Rest fraction. A (right panel): histogram showing the PE signal obtained following gating on CD11b+ F4/80+ cells when PECs are incubated alone (black line), with unlabeled RBCs (blue line), with pHrodo-labeled RBCs (orange line) or stimulated with LPS and incubated with pHrodo-labeled RBCs (green line). B: Histogram showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells incubated with unlabeled RBCs from cells incubated with pHrodo-labeled RBCs. PECS were unstimulated (white bars) or stimulated with 1μg/ml LPS (black bars). Results are presented +/- SEM and are representative of 4 independent experiments (for each point triplicates were used). (*: p-values ≤ 0.05) C: Microscopic pictures from PECs incubated with pHrodo-labeled RBCs.
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pntd.0003561.g002: pHrodo erythrophagocytosis assay using naïve PECs.A (left panel): CD11b versus F4/80 profile following gating on CD45+ cells allows identifying two distinct populations; (i) CD11b+F4/80+ cells which represent macrophages within the PEC population and (ii) cells which are negative or low for CD11b and F4/80 expression referred by as the Rest fraction. A (right panel): histogram showing the PE signal obtained following gating on CD11b+ F4/80+ cells when PECs are incubated alone (black line), with unlabeled RBCs (blue line), with pHrodo-labeled RBCs (orange line) or stimulated with LPS and incubated with pHrodo-labeled RBCs (green line). B: Histogram showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells incubated with unlabeled RBCs from cells incubated with pHrodo-labeled RBCs. PECS were unstimulated (white bars) or stimulated with 1μg/ml LPS (black bars). Results are presented +/- SEM and are representative of 4 independent experiments (for each point triplicates were used). (*: p-values ≤ 0.05) C: Microscopic pictures from PECs incubated with pHrodo-labeled RBCs.

Mentions: Multiple assays for erythrophagocytosis have been developed in the past, each set-up having its own drawbacks such as the need for radioactive reagents. Here we use the pHrodo dye for the labeling of RBCs. The dye reacts with the primary amines on the RBC to yield a covalently linked pH probe, which increases in fluorescence as the pH of the surroundings becomes more acidic. Due to the low pH of the phagolysosome, phagocytozed RBCs can be visualized without being mistaken for RBCs merely sticking to the outside of the phagocytozing cell. Hence this technique enables the straightforward quantification of erythrophagocytosis using FACS and, if required, confirmation of the obtained result by fluorescence microscopy. As shown in Fig. 1, we used this technique to monitor erythrophagocytosis in different cellular contexts, always using the same experimental layout. To validate this erythrophagocytosis assay, pHrodo labeled RBCs and PECs from non-infected mice were put in co-culture overnight with or without LPS stimulation and subsequently stained and analyzed via flow cytometry to distinguish phagocytozing cells. Following gating on peritoneal macrophages, i.e. CD11bhi F4/80hi cells (Fig. 2A, left panel), a shift in the fluorescence signal in co-cultures of PECs with pHrodo labeled RBCs occurs (Fig. 2A, right panel) indicating erythrophagocytosis by these cells. The shift in fluorescent signal expressed as delta median fluorescence intensity (ΔMFI) clearly shows that the peritoneal macrophages are the only cells involved in RBC uptake (Fig. 2B). Treatment of PECs with LPS enhances the erythrophagocytozing ability of these cells. Uptake of labeled RBCs by PECs was confirmed by fluorescence microscopy (Fig. 2C).


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)

pHrodo erythrophagocytosis assay using naïve PECs.A (left panel): CD11b versus F4/80 profile following gating on CD45+ cells allows identifying two distinct populations; (i) CD11b+F4/80+ cells which represent macrophages within the PEC population and (ii) cells which are negative or low for CD11b and F4/80 expression referred by as the Rest fraction. A (right panel): histogram showing the PE signal obtained following gating on CD11b+ F4/80+ cells when PECs are incubated alone (black line), with unlabeled RBCs (blue line), with pHrodo-labeled RBCs (orange line) or stimulated with LPS and incubated with pHrodo-labeled RBCs (green line). B: Histogram showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells incubated with unlabeled RBCs from cells incubated with pHrodo-labeled RBCs. PECS were unstimulated (white bars) or stimulated with 1μg/ml LPS (black bars). Results are presented +/- SEM and are representative of 4 independent experiments (for each point triplicates were used). (*: p-values ≤ 0.05) C: Microscopic pictures from PECs incubated with pHrodo-labeled RBCs.
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Related In: Results  -  Collection

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pntd.0003561.g002: pHrodo erythrophagocytosis assay using naïve PECs.A (left panel): CD11b versus F4/80 profile following gating on CD45+ cells allows identifying two distinct populations; (i) CD11b+F4/80+ cells which represent macrophages within the PEC population and (ii) cells which are negative or low for CD11b and F4/80 expression referred by as the Rest fraction. A (right panel): histogram showing the PE signal obtained following gating on CD11b+ F4/80+ cells when PECs are incubated alone (black line), with unlabeled RBCs (blue line), with pHrodo-labeled RBCs (orange line) or stimulated with LPS and incubated with pHrodo-labeled RBCs (green line). B: Histogram showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells incubated with unlabeled RBCs from cells incubated with pHrodo-labeled RBCs. PECS were unstimulated (white bars) or stimulated with 1μg/ml LPS (black bars). Results are presented +/- SEM and are representative of 4 independent experiments (for each point triplicates were used). (*: p-values ≤ 0.05) C: Microscopic pictures from PECs incubated with pHrodo-labeled RBCs.
Mentions: Multiple assays for erythrophagocytosis have been developed in the past, each set-up having its own drawbacks such as the need for radioactive reagents. Here we use the pHrodo dye for the labeling of RBCs. The dye reacts with the primary amines on the RBC to yield a covalently linked pH probe, which increases in fluorescence as the pH of the surroundings becomes more acidic. Due to the low pH of the phagolysosome, phagocytozed RBCs can be visualized without being mistaken for RBCs merely sticking to the outside of the phagocytozing cell. Hence this technique enables the straightforward quantification of erythrophagocytosis using FACS and, if required, confirmation of the obtained result by fluorescence microscopy. As shown in Fig. 1, we used this technique to monitor erythrophagocytosis in different cellular contexts, always using the same experimental layout. To validate this erythrophagocytosis assay, pHrodo labeled RBCs and PECs from non-infected mice were put in co-culture overnight with or without LPS stimulation and subsequently stained and analyzed via flow cytometry to distinguish phagocytozing cells. Following gating on peritoneal macrophages, i.e. CD11bhi F4/80hi cells (Fig. 2A, left panel), a shift in the fluorescence signal in co-cultures of PECs with pHrodo labeled RBCs occurs (Fig. 2A, right panel) indicating erythrophagocytosis by these cells. The shift in fluorescent signal expressed as delta median fluorescence intensity (ΔMFI) clearly shows that the peritoneal macrophages are the only cells involved in RBC uptake (Fig. 2B). Treatment of PECs with LPS enhances the erythrophagocytozing ability of these cells. Uptake of labeled RBCs by PECs was confirmed by fluorescence microscopy (Fig. 2C).

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