Date Published: March 5, 2015
Publisher: Public Library of Science
Author(s): Benoit Stijlemans, Jennifer Cnops, Peter Naniima, Axel Vaast, Viki Bockstal, Patrick De Baetselier, Stefan Magez, Jayne Raper. http://doi.org/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.
Partial Text: 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. Despite the incredibly wide range of trypanosomosis-associated diseases and pathological complications, one common feature of trypanosomosis is the occurrence of anemia, which is seen in human infections  as well as non-human primate trypanosomosis [2,3], and most other wildlife and livestock trypanosome infections . Anemia is a condition in which an imbalance occurs between erythropoiesis and red blood cell clearance. RBC are either destined for clearance (e.g. by senescence, antibody coating or damage) or are cleared as “innocent bystanders” (e.g. during hemorrhage) . A loss of membrane phospholipid asymmetry has been recognized as a key trigger that can lead to recognition and extravascular removal of senescent RBCs, disordered RBCs, and transfused RBC that have been stored for a long time, by cells of the myeloid phagocyte system [6–8]. Hence, infection-associated complications that affect either of these processes will lead to anemia. Trypanosomosis is suggested to 1) hamper erythropoiesis, 2) enhance erythrophagocytosis (also termed extravascular hemolysis) and 3) in some cases, eg T. vivax infections, cause intravascular hemolysis. 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, reminiscent of ‘anemia of chronic infection’ [9–13]. Interestingly, 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 . 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 hyper-activation and enhanced erythrophagocytosis [12,13,17–20]. However, till now two main obstacles have hampered the in depth assessment of this hypothesis as (i) previous methods for RBC phagocytosis have difficulties differentiating between actual RBC uptake and RBC adherence to phagocytozing cells, and (ii) quantification of phagocytozed RBC numbers with simultaneous characterization of in vivo RBC phagocytozing cells has been virtually impossible. In order to address these issues, we now used a newly developed pHrodo based in vitro erythrophagocytosis assay, as well as an ex vivo FACS based analysis using the same substrate. Unique in this approach is that the visualization of RBC labeling is pH dependent and only becomes traceable in the acidic environment of the lysosome of phagocytozing cells. Hence, we were able to show ex vivo that activated liver monocytes, monocyte-derived macrophages as well as neutrophils are the main cells contributing to trypanosomosis-associated acute stage erythrophagocytosis. In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.
Infection-associated anemia is considered one of the most important pathological features of trypanosomosis and constitutes a major cause of death in bovine African trypanosomosis . Anemia is also a prominent pathological feature of murine trypanosome infection, offering a good model to identify the mechanisms that mediate this phenomenon. T. b. brucei infection of C57Bl/6 mice elicits a severe reduction of RBCs between day 5 and day 8 post infection. Given the acuteness of this phenomenon, a consumptive process seems to be implicated, in particular, as no hemolysis appears to occur. Previously, we have reported a strong increase of cell surface receptors involved in uptake of RBCs and iron-containing compounds in liver tissue. Therefore, liver-associated erythrophagocytosis mediated by cytokine-activated macrophages (M1 cells) is a likely contributor to the aggressive anemia during the acute phase of infection .