Development of a pHrodo-Based Assay for the Assessment of In Vitro and In Vivo Erythrophagocytosis during Experimental Trypanosomosis

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.

Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia

“…Human erythrocytes incubated with EVs showed an increase in membrane rigidity as indicated by a narrowing and shift of the emission spectra of Laurdan towards shorter wavelengths (Figure 6D). Recently it has been shown that erythrocyte lipid composition was altered during trypanosomiasis, and these cells were preferentially phagocytosed by myeloid cells (Stijlemans et al, 2015). Incorporating parasite lipid via EV fusion may explain altered erythrocyte lipid composition.…”

“…These host immune responses have been implicated in the pathology of trypanosomiasis-associated anemia (Stijlemans et al, 2015). Anemia is markedly severe during acute infection and is the major cause of cattle death due to Nagana (Naessens, 2006).…”

“…Recently, erythrophagocytosis by activated liver and spleen myeloid cells has been identified as a major contributor to erythrocyte clearance. In addition, the lipid composition of erythrocytes is altered during trypanosome infection and these erythrocytes are preferentially phagocytosed by the host’s myeloid cells (Stijlemans et al, 2015). While host responses that contribute to anemia have been characterized, the parasite factors that elicit erythrocyte clearance are unknown.…”

Vesicles as Vehicles for Virulence

“…The transfer of VSG to host erythrocytes was previously observed by [5] but the mechanism behind it and its functional relevance remained a mystery. Szempruch et al demonstrate that fusion of EVs with host erythrocytes affects erythrocyte rigidity and size and postulate that this results in preferential clearance by host myeloid cells [6], though this is not tested directly. Consistent with this model, the intravenous injection of parasite EVs into naïve mice results in a 5–10% decrease in the number of host erythrocytes within 1hr, demonstrating a potential role for parasite EVs in infection-associated anemia.…”

“…Consistent with this model, the intravenous injection of parasite EVs into naïve mice results in a 5–10% decrease in the number of host erythrocytes within 1hr, demonstrating a potential role for parasite EVs in infection-associated anemia. The transfer of VSG from the parasite surface to the recipient erythrocyte raises the possibility that erythrocytes could become targets of antibody responses directed against parasite VSGs, which might also be expected to cause increased erythrocyte clearance, though [6] show preferential clearance of trypanosome-altered erythrocytes even in uninfected mice. It remains to be seen whether T. brucei EVs fuse with other host cell types, and what, if any, modulatory effect they have.…”