Fonseca et al. Malar J (2016) 15:410 DOI 10.1186/s12936-016-1465-5 Malaria Journal RESEARCH Open Access Quantifying the removal of red blood cells in Macaca mulatta during a Plasmodium coatneyi infection Luis L. Fonseca1,4*, Harnel S. Alezi1, Alberto Moreno2,4, John W. Barnwell3,4, Mary R. Galinski2,4 and Eberhard O. Voit1,4 Abstract Background: Malaria is the most deadly parasitic disease in humans globally, and the long-time coexistence with malaria has left indelible marks in the human genome that are the causes of a variety of genetic disorders. Although anaemia is a common clinical complication of malaria, the root causes and mechanisms involved in the pathogenesis of malarial anaemia are unclear and difficult to study in humans. Non-human primate (NHP) model systems enable the mechanistic study and quantification of underlying causative factors of malarial anaemia, and particularly the onset of severe anaemia. Methods: Data were obtained in the course of Plasmodium coatneyi infections of malaria-naïve and semi-immune rhesus macaques (Macaca mulatta), whose red blood cells (RBCs) were labelled in situ with biotin at the time the infections were initiated. The data were used for a survival analysis that permitted, for the first time, an accurate esti- mation of the lifespan of erythrocytes in macaques. The data furthermore formed the basis for the development and parameterization of a recursive dynamic model of erythrocyte turnover, which was used for the quantification of RBC production and removal in each macaque. Results: The computational analysis demonstrated that the lifespan of erythrocytes in macaques is 98 21 days. The model also unambiguously showed that death due to senescence and parasitaemia is not sufficient± to account for the extent of infection-induced anaemia. Specifically, the model permits, for the first time, the quantification of the different causes of RBC death, namely, normal senescence, age-independent random loss, parasitization, and bystander effects in uninfected cells. Such a dissection of the overall RBC removal process is hardly possible with experimental means alone. In the infected malaria-naïve macaques, death of erythrocytes by normal physiological senescence processes accounts for 20 % and parasitization for only 4 %, whereas bystander effects are associated with an astonishing 76 % of total RBC losses. Model-based comparisons of alternative mechanisms involved in the bystander effect revealed that most of the losses are likely due to a process of removing uninfected RBCs of all age classes and only minimally due to an increased rate of senescence of the uninfected RBCs. Conclusions: A new malaria blood-stage model was developed for the analysis of data characterizing P. coatneyi infec- tions of M. mulatta. The model used a discrete and recursive framework with age-structure that allowed the quantifica- tion of the most significant pathophysiological processes of RBC removal. The computational results revealed that the malarial anaemia caused by this parasite is mostly due to a loss of uninfected RBCs by an age-independent process. The biological identity and complete mechanism of this process is not fully understood and requires further investigation. Keywords: Macaca mulatta, Malarial anaemia, Mathematical model, Plasmodium coatneyi, Red blood cell removal *Correspondence: [email protected] 1 The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fonseca et al. Malar J (2016) 15:410 Page 2 of 15 Background anaemia are unknown, but have been suggested to be Malaria, caused by infection with parasites of the genus due to multiple possible factors leading to a reduction in Plasmodium, is responsible for over half a million deaths circulating RBCs including physicochemical membrane per year worldwide, with children being the main victims changes, reduced erythrocyte deformability, accelerated [1]. The immense severity of the disease and the long erythrocyte senescence, and immunological reactions coexistence of humans and Plasmodium parasites have that cause their removal [14, 15]. even led to the emergence and perpetuation of possible This paper presents a mathematical model that was protective genetic disorders like thalassaemias and sickle- developed to study RBC dynamics in circulating blood cell disease, as well as haemoglobin C, haemoglobin E, during malaria infections. The model was parameter- and G6P dehydrogenase and pyruvate kinase deficiencies ized using experimental results from Moreno et al. [14] [2–7]. In primates, Plasmodium sporozoites infect, trans- and implemented as a discrete recursive structure that form and multiply within parenchymal hepatocytes to was previously identified as best suited for this class of form merozoites; these developmental life cycle processes problems [16]. The choice of this framework was based are asymptomatic for the host. Once merozoite forms of on the need to account for the aging of the RBCs rather the parasite are released from the infected hepatocytes, accurately, which is problematic in delayed differen- the parasite begins its cyclical blood-stage development. tial and integro-differential equations that do not track Merozoites invade and multiply within red blood cells age directly, but instead approximate the time passed (RBCs) every 24, 48 or 72 h, depending on the species, since the cell was generated. Ordinary differential equa- and new merozoite progeny are released to invade other tions with age classes do not model the aging accurately RBCs [8]. During this cyclical process of invasion and since inspection of age-classes reveals a distribution of destruction of RBCs, the symptoms and clinical compli- ages within each class. Partial differential equation mod- cations associated with malaria emerge. The blood stage els like the Lotka–McKendrick age-structured popula- of an infection is characterized by repeated rounds of tion model [17, 18] do accurately model the aging of a RBC invasion which, if not kept under control by host population, but are difficult to implement, especially if immune responses or anti-malarial treatment, can lead the model includes variables with and without an age- to exponential growth of the parasite, with a concomi- structure and if the aging process is perturbed by events tant destruction of the parasitized RBCs. This destruc- like a malaria infection. The most effective alternative is a tion, however, is not the predominant mechanism of RBC discrete recursive framework with age-classes. In a sense, removal that leads to anaemia, and indeed seems to be this structure corresponds to the discretization of a PDE vastly surpassed by the destruction of uninfected RBCs model and hence shares all of its properties, but is more (uRBCs) [9–11]. easily implemented and faster to solve, with an accuracy Infections with Plasmodium coatneyi, a simian malaria that is readily tuned by stipulating a desired time-step. species that is closely related to Plasmodium knowlesi The model permits the quantification of the produc- [12, 13], mirror the biology and pathogenesis of falcipa- tion of newly generated RBCs and of the different pro- rum malaria, with severe forms of pathology including cesses leading to the removal of RBCs in the absence or anaemia. To study mechanisms of the onset and recov- presence of a P. coatneyi infection in malaria naïve or ery of anaemia, Moreno et al. [14] established procedures semi-immune rhesus macaques. Additionally, two alter- to measure the turnover of in vivo biotinylated RBCs in native mechanisms of uninfected RBC removal, namely rhesus macaques (Macaca mulatta). These macaques accelerated erythrocyte senescence and immunologic had been experimentally infected for the first time (i.e., removal, are modelled and compared on the basis of their when malaria naïve) with P. coatneyi infected RBCs and, respective predictions. The results demonstrate that the then again, while partially immune, 9 months after cura- destruction of uninfected RBCs was the dominant pro- tive anti-malarial drug treatment. Five infected and then cess underlying malarial anaemia in the P. coatneyi infec- re-challenged (semi-immune) animals were compared tions reported by Moreno et al. [14], and that the direct to five control rhesus with biotinylated RBCs, but no destruction of infected RBCs by the parasite accounted malaria infection. Microscopy-based counts of infected for only about 4 % of the total RBC loss. Beyond this RBCs and haemoglobin levels were monitored daily, and specific result, the model can be employed as a tool for the numbers of biotinylated RBCs were assessed using predicting and exploring disease severity and evaluating flow cytometry. This work demonstrated that malarial host-directed interventions. This capability includes the infections result in