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

Benoit Stijlemans,Peter Naniima,Jennifer Cnops,Axel Vaast,Patrick De Baetselier,Stefan Magez,Viki Bockstal,Jayne Raper

doi:10.1371/journal.pntd.0003561

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.

Highlights

Extracellular trypanosomes including Trypanosoma brucei, T. evansi, T. congolense and T. vivax, are parasites that affect a very broad host range, and combined, threaten human and animal health throughout various continents
During the early phase of experimental murine trypanosomosis, acute anemia occurs as witnessed by a 50% reduction in red blood cells within a 48 hour time span
We developed a new erythrophagocytosis assay based on the labeling of red blood cells with the acid-sensitive dye pHrodo

Summary

Introduction

Extracellular trypanosomes including Trypanosoma brucei, T. evansi, T. congolense and T. vivax, are parasites that affect a very broad host range, and combined, threaten human and animal health throughout various continents. As anemia occurs in B-cell deficient μMT mice with similar kinetics as WT mice, the process involved appears antibody independent [14,15] This contrasts a previous in-vitro based hypothesis that cross-reactive anti-VSG antibodies might contribute to a complement-mediated hemolysis event [16]. Based on combined recent data, the most plausible explanation for the initiation of trypanosomosis-associated anemia is the occurrence of enhanced RBC phagocytosis, resulting from a pro-inflammatory cytokine storm occurring during the early stage of infection, leading to macrophage hyperactivation and enhanced erythrophagocytosis [12,13,17,18,19,20]. We show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis

Results

Discussion

Conclusion