Plasmodium coatneyi in Rhesus Macaques Replicates the Multisystemic Dysfunction of Severe Malaria in Humans Alberto Moreno, Emory University Monica Cabrera-Mora, Emory University Anapatricia Garcia, Emory University Jack Orkin, Emory University Elizabeth Strobert, Emory University John W. Barnwell, Centers for Disease Control and Prevention Mary R Galinski, Emory University

Journal Title: Infection and Immunity Volume: Volume 81, Number 6 Publisher: American Society for Microbiology | 2013-06-01, Pages 1889-1904 Type of Work: Article | Final Publisher PDF Publisher DOI: 10.1128/IAI.00027-13 Permanent URL: https://pid.emory.edu/ark:/25593/s78xt

Final published version: http://dx.doi.org/10.1128/IAI.00027-13 Copyright information: © 2013, American Society for Microbiology. All Rights Reserved. Accessed October 6, 2021 3:29 PM EDT Plasmodium coatneyi in Rhesus Macaques Replicates the Multisystemic Dysfunction of Severe Malaria in Humans

Alberto Moreno,a,b Monica Cabrera-Mora,a AnaPatricia Garcia,c,d Jack Orkin,e Elizabeth Strobert,e John W. Barnwell,f Mary R. Galinskia,b Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, Georgia, USAa; Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USAb; Division of Pathology, Yerkes National Primate Research Center, Emory University, Atlanta, Georgia, USAc; Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Emory University Hospital, Atlanta, Georgia, USAd; Division of Animal Resources, Yerkes National Primate Research Center, Emory University, Atlanta, Georgia, USAe; Malaria Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia, USAf

Severe malaria, a leading cause of mortality among children and nonimmune adults, is a multisystemic disorder characterized by complex clinical syndromes that are mechanistically poorly understood. The interplay of various parasite and host factors is crit- ical in the pathophysiology of severe malaria. However, knowledge regarding the pathophysiological mechanisms and pathways leading to the multisystemic disorders of severe malaria in humans is limited. Here, we systematically investigate infections with Plasmodium coatneyi, a simian malaria parasite that closely mimics the biological characteristics of P. falciparum, and develop baseline data and protocols for studying erythrocyte turnover and severe malaria in greater depth. We show that rhesus ma- caques (Macaca mulatta) experimentally infected with P. coatneyi develop anemia, coagulopathy, and renal and metabolic dys- function. The clinical course of acute infections required suppressive antimalaria chemotherapy, fluid support, and whole-blood transfusion, mimicking the standard of care for the management of severe malaria cases in humans. Subsequent infections in the same animals progressed with a mild illness in comparison, suggesting that immunity played a role in reducing the severity of the disease. Our results demonstrate that P. coatneyi infection in rhesus macaques can serve as a highly relevant model to inves- tigate the physiological pathways and molecular mechanisms of malaria pathogenesis in naïve and immune individuals. To- gether with high-throughput postgenomic technologies, such investigations hold promise for the identification of new clinical interventions and adjunctive therapies.

alaria is the most prevalent parasitic disease globally, with tions of malaria parasites with other pathogens, are also con- Mthe World Health Organization (WHO) recent estimates founding factors, constituting another level of parasite-host dy- showing 216 million or more clinical episodes annually, resulting namics that is not well understood. in 655,000 deaths (1). Children under 5 years of age, pregnant The World Health Organization has established clinical crite- women, and nonimmune adults are particularly susceptible to ria to define severe malaria cases and facilitate the comparison of developing severe disease (2). Although the large dimension of the data obtained from ecologically diverse areas of the world with problem is well known, knowledge concerning the molecular different patterns of transmission (5). The WHO has defined life- mechanisms involved in the physiopathology of severe malaria is threatening clinical syndromes that are frequently associated with limited. patients infected with P. falciparum as follows: severe anemia, Severe malaria has been traditionally associated with Plasmo- neurological syndrome, thrombocytopenia accompanied by co- dium falciparum infections; however, there is mounting evidence agulation disorders, respiratory distress or pulmonary edema, indicating that P. vivax can also be responsible for severe and acute renal failure, circulatory collapse, and severe metabolic sometimes lethal cases of malaria (3, 4). While both species can complications. Recently, Lanca and colleagues have summarized cause severe disease, with various commonalities, the clinical clinical and epidemiological data indicating that some similar syn- spectrum is known to differ in a number of respects. Cerebral dromes can also define severe malaria caused by P. vivax (6). malaria with coma is a common complication of P. falciparum Experimental model systems are needed to investigate the un- severe malaria. This contrasts with the frequent presentation of derlying mechanism and pathophysiology of severe malaria in hu- respiratory distress in severe malaria caused by P. vivax infections. mans in order to facilitate the development of biological or phar- Severe anemia, multiorgan dysfunction, and thrombocytopenia macological tools for clinical intervention. Rodent malaria models are complications noted with either species. Sequestration of P. falciparum-infected erythrocytes (RBCs) bound to the endothe- lium of small vessels in various tissues and organs, particularly in Received 11 January 2013 Returned for modification 14 February 2013 the brain, has been associated with cerebral malaria and other Accepted 6 March 2013 disorders of severe malaria, although it has not been determined if Published ahead of print 18 March 2013 disease results from the sequestration of infected erythrocytes or if Editor: J. H. Adams infected erythrocyte adhesion is enhanced by the pathophysiolog- Address correspondence to Alberto Moreno, [email protected]. ical state. P. vivax-infected erythrocytes circulate and largely are Copyright © 2013, American Society for Microbiology. All Rights Reserved. not adhesive and do not sequester in the vasculature of particular doi:10.1128/IAI.00027-13 organs. P. falciparum and P. vivax coinfections, as well as coinfec-

June 2013 Volume 81 Number 6 Infection and Immunity p. 1889–1904 iai.asm.org 1889 Moreno et al. of cerebral malaria and severe anemia have been studied and the TABLE 1 Rhesus physiological score system (RPSS) immune-pathological pathways of causation dissected exten- Score sively. However, new effective clinical interventions have not come from this body of work, and it has been questioned if these Variable 01 2 rodent malaria models are relevant to human malaria (7, 8). Non- Dehydration None Mild Moderate to human primate (NHP) models of malaria infection are available, marked although certainly underutilized, to study malaria disease states Respiratory rate Normal Tachypnea Dyspnea (8–10). The potential of infections of various simian malaria par- Activity Good Fair (malaise) Poor (lethargy) Appetite Good Fair (malaise) Poor (lethargy) asites in macaques to identify mechanisms of pathogenesis com- Hemoglobin (g/dl) Ͼ14 10–14 Ͻ10 mon to human malaria has been studied to a limited extent (8– Platelets (no. of cells/␮l) Ͼ300,000 100,000–300,000 Ͻ100,000 10). P. coatneyi and P. falciparum, although not particularly close Parasitemiaa (no. of 0 0.1–1,000 1,000–10,000 phylogenetically, do closely share a number of nearly identical parasites/␮l) phenotypic characteristics. Both species are characterized by a 48- a For parasitemia levels, additional scores were assigned: 3, 10,000 to 20,000/␮l; 4, hour intraerythrocytic developmental cycle, electron-dense knob 20,000 to 40,000/␮l; 5, 40,000 to 60,000/␮l; 6, 60,000 to 80,000/␮l; 7, 80,000 to 100,000/ protrusions on the surface of their infected host cells, and pro- ␮l; 8, Ͼ100,000/␮l. found deep vasculature sequestration of trophozoite- and schi- zont-infected erythrocytes. The primary sites of infected erythro- cyte adhesion to endothelium for P. coatneyi were in the vessels of National Primate Research Center were assigned to this study. Procedures the heart, fatty tissues, omentum, intestines, and musculature, used were approved by Emory University’s Institutional Animal Care and very similar to tissue sites for P. falciparum in human malaria Use Committee and followed accordingly. The Hackeri strain of P. coat- (11–13). In this regard, infection with P. coatneyi has been pro- neyi used in these studies came from archived stocks maintained by cryo- posed as an optimal primate model for human cerebral malaria preservation at the Centers for Disease Control and Prevention (21). (13–16). Although these particular studies have shown histologi- Twenty animals were randomized into four groups of 5 animals; and two cal findings with sequestration of infected erythrocytes in cerebral of these groups were exposed to P. coatneyi at two different time points. vasculature, the multisystemic clinical data collected during the Ten animals were selected as controls to characterize normal hematolog- ical parameters. Three macaques were infected with P. coatneyi-infected course of infection have been sparse or nonexistent. Similarly, erythrocytes from a cryopreserved stabilate to serve as donors to obtain compartmentalization of P. coatneyi-infected erythrocytes in the freshly drawn infected blood for inoculation of the test animals with a placenta in experimentally infected macaques has suggested that comparable number of viable parasites. Each recipient was inoculated P. falciparum and P. coatneyi may share molecular mechanisms of with 2 ϫ 104 P. coatneyi-infected erythrocytes/kg of body weight and pathogenesis during pregnancy (17–19). However, as noted followed up daily for 30 days. Based on our preliminary data, we expected above, only limited clinical information is available from these to have detectable parasitemia in malaria-naïve individuals on day 3 and studies. Nevertheless, studies conducted after the discovery of P. peaks of parasitemia within 2 weeks after experimental infection. Follow- coatneyi have reported signs of severe disease in the majority of ing subcurative antimalarial treatment with a fast-acting drug (arte- animals and a mortality of 30% to 35% in rhesus monkeys if not mether), episodes of recrudescence were also expected within 2 weeks treated (20). after the intervention. To test the effect of antimalarial acquired immunity on disease progression, the animals were exposed to a second experimen- We report here a comprehensive characterization of the clini- tal infection using the same number of parasites/kg 9 months after the first cal outcomes of rhesus macaques experimentally infected with infection had been cleared. In the course of each infection, the animals virulent stocks of P. coatneyi-infected RBCs. We exposed the ani- were monitored daily for clinical symptoms including anorexia, respira- mals to sequential experimental infections to observe the develop- tory distress, and impaired consciousness. ment of antiparasite immunity and the associated consequences To be able to quantitatively assess the clinical severity daily and facil- with regard to the altered course of pathogenesis. Subcurative an- itate comparison between groups, we developed a rhesus physiological timalaria treatment was required with the first infection to avoid score system (RPSS) (Table 1). Tachypnea was defined as increase in re- deaths during the development of life-threatening conditions spiratory rate over 20 breaths per minute. Dyspnea was defined as short- during the acute course of the infections. All malaria-naïve rhesus ness of breath accompanied by intercostal and supraclavicular retractions, macaques infected in this study developed severe malaria with nasal flaring, and paradoxical abdominal respiration as previously defined (22). Capillary blood samples were obtained every day by standardized ear signs of multisystemic involvement. Hematologic disorders and prick procedures and collected into EDTA-coated capillary tubes. Blood metabolic dysfunctions including severe anemia and coagulopa- samples were used to determine hemoglobin concentration using a thy were the most common complications. Both accelerated turn- HemoCue photometer (HemoCue Inc., Lake Forest, CA) and to quantify over of uninfected erythrocytes and ineffective erythropoiesis ac- the parasite load using Giemsa-stained thin and thick smears. Reticulo- counted for severe anemia in the course of the initial infection. In cyte enumeration was achieved by manual counting of new methylene stark contrast, only mild anemia and minor metabolic changes blue-stained reticulocytes in blood smears. Serum levels of fibrinogen were observed in these animals during their subsequent infection, degradation products were determined using a direct latex agglutination 9 months later. Our results support the use of P. coatneyi infec- assay (Pacific Hemostasis, Middletown, VA). On indicated dates, venous tions in rhesus macaques as a robust host-parasite model to iden- blood samples were obtained for blood coagulation tests and blood chem- tify physiological and immunological mechanisms associated with istry analyses. Blood platelet counts were obtained by manual quantifica- tion using Giemsa-stained thin smears. To avoid life-threatening clinical severe malaria pathogenesis relevant to human disease. conditions, animals with low hemoglobin levels or respiratory distress received subcurative treatment with artemether at 2 mg/kg in a single MATERIALS AND METHODS intramuscular injection. The animals were followed up for 30 days and Study design and experimental infections. Twenty-three male rhesus received a curative antimalarial regimen of 4 mg/kg of artemether once, macaques (Macaca mulatta) that had been born and raised at the Yerkes followed by 2 mg/kg/day for 6 days.

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FIG 1 P. coatneyi induces severe anemia in the absence of compensatory reticulocytosis. (Left) Course of parasitemia in rhesus macaques experimentally infected with P. coatneyi. The values are expressed as mean numbers of parasites/␮l Ϯ standard deviation (SD) determined using Giemsa-stained thick smears (left y axis) () and plotted with mean hemoglobin levels expressed in g/dl Ϯ SD (right y axis) (Œ). (Right) Changes in reticulocyte counts expressed as mean absolute ␮ Ϯ ᭜ Œ numbers/ l SD in rhesus macaques experimentally infected with P. coatneyi ( ); the hemoglobin levels are also plotted for comparison ( ). Rx, subcurative treatment with artemether (n ϭ 5).

In vivo biotinylation. In vivo biotinylation was conducted by intrave- ferences between groups were evaluated by comparing the mean area nous infusion of EZ-Link Sulfo-NHS-biotin (Pierce, Rockford, IL) at 37.5 under the curve (AUC) values using the Mann-Whitney test. Pearson or 20 mg/kg in phosphate-buffered saline (PBS). The procedure was con- correlation coefficient analysis was performed to determine the associa- ducted under anesthesia using a Buretrol infusion set (Baxter Healthcare tion between hemozoin-containing leukocytes, hemoglobin, and para- Corp., Deerfield, IL). To avoid potential anaphylactic reaction, the ani- sitemia. Differences in clinical laboratory parameters, coagulation tests, mals were premedicated with diphenylhydramine at 1 mg/kg. The biotin and proinflammatory mediators were evaluated using one-way between- suspension was infused for 20 min at 40 to 50 microdrops/minute. The groups analysis of variance (ANOVA) with post hoc Bonferroni’s multi- system was then flushed using sterile saline solution. The same intrave- ple-comparison posttest. P values of Ͻ0.05 were considered significant. nous line was used for parasite inoculation of the macaques included in the experimental groups. After infusion, the intravenous cannula was re- RESULTS moved and blood samples were collected to determine the biotinylation efficacy using flow cytometry. Plasmodium coatneyi infection of naïve rhesus macaques in- Characterization of the erythrocyte life span. EDTA capillary blood duces severe anemia and a delay in erythroid compensatory samples were obtained daily using BD Microtainer capillary blood tubes mechanisms. P. coatneyi infections were comprehensively moni- (Becton, Dickinson, Franklin Lakes, NJ). The cells were washed with PBS tored in a group of five naïve rhesus macaques. We designed ex- and incubated at 3% hematocrit with streptavidin-Alexa Fluor 488 (In- perimental protocols to record clinical and clinical laboratory pa- vitrogen, Carlsbad, CA) at room temperature in the dark for 30 min. After rameters in the course of the infections. The animals were incubation, the cells were washed twice, resuspended in PBS, and analyzed experimentally challenged intravenously with 1 ϫ 104 P. coatneyi- by flow cytometry using a BD FACSDiVa LSR II (BD Biosciences, San infected erythrocytes/kg of body weight obtained from an active Jose, CA). The percentage of biotinylated cells was calculated as the ratio infection in a donor monkey. To ensure that these infections of positive cells to all erythrocytes. Results are presented as absolute eryth- rocyte numbers by normalization with erythrocyte counts determined mimicked the natural course of P. coatneyi infections in rhesus every day in the course of the follow-up period. macaques as originally described (20, 21), parasites were inocu- Bone marrow core biopsy specimens. Bone marrow core biopsy spec- lated from the original line of P. coatneyi (24), archived as cryo- imens were obtained using a 13-gauge Jamshidi biopsy needle and stan- preserved stocks at the CDC and confirmed to have no history of dard technique. The biopsy specimens were taken from the femur via the experimental passage in splenectomized animals. Parasitemias, trochanteric fossa or ileac crest. Biopsy imprints were made by gently hemoglobin levels, and reticulocyte counts were determined daily touching the core on slides. The cores were then fixed in 10% formalin from peripheral blood capillary samples as described previously (23) and decalcified in RDO Gold (Apex Engineering Products Corp., (25). Similar capillary samples were also taken and monitored ␮ Aurora, IL). The specimens were embedded in paraffin and cut at 6 m. from uninfected control animals. Infected erythrocytes were first Analysis of plasma cytokine concentration. Interleukin-1beta (IL- identified in peripheral blood smears 72 h after experimental chal- 1␤), IL-6, IL-8, macrophage inflammatory protein-1 alpha (MIP1␣)/ chemokine ligand 3 (CCL3), gamma interferon (IFN-␥), monocyte che- lenge (Fig. 1). Parasitemia in the five monkeys followed the ex- moattractant protein-1 (MCP-1)/CCL2, macrophage inflammatory pected pattern of peaks on alternating days followed by a sharp protein-1 beta (MIP-1␤)/CCL4, tumor necrosis factor alpha (TNF-␣), drop in parasite levels, coinciding with the maturation and se- metalloproteinase-3 (MMP3), MMP9, RANTES (regulated on activation questration of the parasites, comparable to that of P. falciparum normal T cell expressed), tissue inhibitory of matrix metalloproteinase-1 (21); presumably via predicted variant antigens analogous to the (TIMP1), TIMP2, MMP2, granulocyte-macrophage colony-stimulating var gene-encoded erythrocyte membrane protein-1 (PfEMP1) factor (GM-CSF), IL6R (IL-6 receptor), macrophage-derived chemokine and SICA antigens of P. knowlesi (references 26 and 27 and un- (MDC), and neutrophil-activating protein-2 (NAP2) levels were deter- published data). Parasite increases were recorded on days 3, 5, and mined using multiplex luminescent beads by Aushon Biosystems (Bil- 7 or 9 after experimental infection. The parasitemias peaked on lerica, MA) using primate-optimized reagents. IL-10 and erythropoietin day 9 or 11 postinfection with mean levels of Ͼ200,000 parasites/ (EPO) were assayed by sandwich enzyme-linked immunosorbent assay ␮ (ELISA) using commercial immunoassays according to the manufactur- l. To avoid the onset of life-threatening conditions, the animals er’s instructions (R&D Systems, Inc., Minneapolis, MN). received a subcurative dose of artemether (2 mg/kg) between days Statistical analysis. Statistical analyses were made using GraphPad 9 and 11. The subcurative antimalaria treatments resulted in a Prism 5.0 software (GraphPad Software Inc., San Diego, CA). For assess- sustained drop in parasitemia, which started to increase again by ment of clinical severity and kinetics of circulating biotinylated cells, dif- day 16 (Fig. 1).

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FIG 2 Characterization of the erythrocyte life span in malaria-naïve rhesus macaques. The numbers of biotinylated cells were determined by flow cytometry and expressed as numbers of cells/␮l, and the data are plotted for individual animals. Representative histogram plots are shown at different time points (5, 35, 72, 92, and 105 days) after in vivo biotinylation.

The kinetics of hemoglobin concentration showed a steady de- tify the erythrocyte life span in nonhuman primates have usually crease from the baseline levels and a minimum mean value of 6.4 involved the autotransfusion of in vitro-labeled cells (29). g/dl on day 11. Two animals developed severe anemia by day 11 To monitor the complete turnover of erythrocytes in the pe- with hemoglobin levels lower than 5 g/dl. In one of these subjects, ripheral blood in naïve primates and during the course of active P. the severe anemia was associated with thrombocytopenia and co- coatneyi malaria infections, we standardized conditions for the agulopathy, complications that ultimately led to disseminated in- safe and effective in vivo biotinylation in rhesus macaques by using travascular coagulation and peripheral gangrene (25). The mean a water-soluble derivative, EZ-Link Sulfo-NHS-biotin. Two dif- hemoglobin levels increased after subcurative antimalaria treat- ferent concentrations, 20 and 37.5 mg/kg in PBS, were initially ment but reached only 40 to 60% of the baseline hemoglobin evaluated in two healthy animals. The biotin suspension was de- levels. The animals received complete antimalaria treatment 25 livered intravenously using a Buretrol infusion set adjusted to de- days after the challenge infection (Fig. 1). The sharp drop in liver 40 to 50 microdrops/min. To prevent potential anaphylactic hemoglobin concentrations in association with relatively low par- reactions, the animals were premedicated with diphenhydramine asitemia in all animals suggested that the major portion of the (10 mg intravenously [i.v.]). Clear distinctions between positive anemia was not due to erythrocyte destruction by parasitism but and negative cells were observed by flow cytometry using strepta- rather by hemolysis or an accelerated removal of uninfected eryth- vidin-Alexa Fluor 488, with no significant differences observed rocytes. between doses (data not shown). Based on these results, we de- Reticulocyte counts were also determined daily after experi- cided to use the lower dose of 20 mg/kg in subsequent experi- mental infection, as an indirect readout of the efficiency of eryth- ments. To determine if the procedure was adequately sensitive to ropoiesis. Consistent with an adverse effect of malaria infection on successfully study the erythrocyte life span, the biotin infusion was erythroid progenitors, reticulocyte counts did not change from administered to a group of five malaria-naïve rhesus macaques, baseline levels. The proportion of reticulocytes in the peripheral and the proportions of biotinylated cells in the capillary blood blood increased only after rapid-acting antimalarial treatment was were determined daily for 120 days (Fig. 2). The total number of given and not in response to the severe anemia observed on day 11 biotinylated cells in the peripheral blood at different time points is (Fig. 1). These results indicated a delay in the bone marrow com- presented for the individual animals with representative histo- pensatory response to anemia. grams. The kinetics of erythrocyte clearance determined from Development of an in vivo biotinylation procedure to char- these experiments is in agreement with the reported erythrocyte acterize erythrocyte turnover. The experimental infection de- life span in rhesus macaques, estimated to be ϳ100 days (30), with scribed above suggested that two independent mechanisms may the percentage of Alexa Fluor-positive cells ranging between 1 and play a role in the pathogenesis of severe malarial anemia during a 3% of the total number of circulating cells in the peripheral blood primary infection in rhesus macaques: an ineffective erythropoi- at the end of the follow-up period. esis and the accelerated removal of uninfected erythrocytes. To Accelerated removal of uninfected erythrocytes in the course quantitatively assess the impact of the infection on the erythrocyte of experimental infection of rhesus macaques with P. coatneyi. life span, we developed methodologies to determine the fate of Intravenous infusions of biotin were administered immediately normal erythrocytes in vivo. Comparable procedures have been before the experimental challenge of a new group of malaria-naïve optimized in mice using in vivo biotinylation to quantify erythro- rhesus monkeys by using the optimized dose of 20 mg/kg of EZ- cyte removal during the course of experimental infection with Link Sulfo-NHS-biotin (noted above). The kinetics of parasitemia rodent malaria parasites (28) but not, to our knowledge, in naïve and the total number of biotinylated cells in the peripheral blood or malaria-infected nonhuman primates. Methodologies to quan- during the primary infection is summarized in Fig. 3. The para-

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FIG 3 Accelerated turnover of noninfected erythrocytes during acute primary infections with P. coatneyi-nonimmune rhesus monkeys. (Left) Course of parasitemia () and hemoglobin levels (Œ) in malaria-naïve rhesus macaques as described for Fig. 1. (Right) Kinetics of circulating biotinylated cells in P. coatneyi-infected rhesus macaques (Œ) in comparison with the kinetics of circulating biotinylated cells in noninfected monkeys (o). The results are presented as the mean numbers of biotinylated cells/␮l determined by flow cytometry. Mean hemoglobin levels are also included for comparative purposes. *, P ϭ 0.011.

Differences were evaluated by comparing mean area under the curve (AUC) values using the Mann-Whitney test. Rx, subcurative treatment with artemether (n ϭ 5). sitemias during these new infections were represented by slightly and 7 postinfection with average levels of only 30 and 10 parasites/ different kinetics from those described above. Maximum para- ␮l. The maximum peak of parasitemia was observed on day 14 sitemia occurred on day 10 with a mean of 38,000 parasites/␮l. with an average number of 40,000 parasites/␮l(Fig. 4). Clinically, Biotinylated cells gradually decreased during the first few days the outcome was also striking by comparison. The animals were following challenge infection, with a subsequent accelerated drop assessed to be in fair condition and did not require antimalaria in the numbers of labeled cells between days 6 and 10. Biotinylated treatment or fluid support. Hemoglobin levels were maintained cells were not detected in the peripheral blood after 26 days over 11 g/dl, with the lowest level recorded on day 17, when the postinfection. These results are consistent with an accelerated and animals reached 64% of the baseline hemoglobin levels with a complete turnover of all of the biotinylated cells (Fig. 3, right mean value of 11.05 g/dl. The quantifying of reticulocytes in the panel). This contrasts with the physiological clearance of 35% Ϯ peripheral blood showed an average peak of 17% reticulocytes on 5% of the erythrocytes in the uninfected control animals on day 26 day 20, which corresponds to an average number of 735,678 re- (Fig. 3; P ϭ 0.011). The premature removal of the vast majority of ticulocytes/␮l(Fig. 4, right panel). Interestingly, a progressive in- biotinylated erythrocytes in the infected animals clearly occurred crease in the number of circulating reticulocytes coincided with a at the expense of uninfected erythrocytes and not through de- progressive decrease in hemoglobin levels. These results therefore struction from parasitism, given the relatively low level of parasit- suggested efficient compensatory mechanisms leading to effective ized cells during this portion of the acute infection. erythropoiesis in the course of the second experimental infection. Partially immune rhesus macaques are resistant to severe The turnover of biotinylated erythrocytes was also quantified anemia in P. coatneyi infections. Clinical immunity to P. falcip- during the course of each subsequent second infection (conducted arum malaria is acquired after one or more homologous infec- 9 months after the first infection) and compared with the data tions (31). To characterize the effect of clinical and antiparasite recorded from uninfected macaques (Fig. 5). In contrast to what immunity on malaria severity, acquired by experimental exposure was observed in the course of the first infections, a slow decrease in to P. coatneyi, the same group of rhesus macaques described above biotinylated cell counts was recorded, with removal of 60% Ϯ (Fig. 1) was reexposed to the same strain of parasite 9 months after 15% of the biotinylated cells by day 30 after infection. No clinical the first infection in the previously naïve animals. The kinetics of signs of severe infection were observed during the follow-up pe- parasitemia in the partially immune rhesus macaques followed a riod (see below). Although the maximum peak of parasitemia was strikingly different pattern in comparison to the accelerated in- recorded on day 12 with 22,428 parasites/␮l, a significant reduc- crease in parasite loads observed during the first infection. After tion in the number of biotinylated erythrocytes was observed in being reinfected, the animals first exhibited parasitemia on days 5 comparison to the unchallenged control group that received only

FIG 4 P. coatneyi-semi-immune rhesus macaques are protected against severe anemia. (Left) Course of parasitemia in rhesus macaques exposed to a second infection with P. coatneyi. The parasite load values are compared with hemoglobin levels determined as described in the legend for Fig. 1. Parasitemia levels were determined using Giemsa-stained thick smears and are expressed as the mean numbers of parasites/␮l Ϯ SD (left y axis) (). Hemoglobin levels are expressed as mean g/dl Ϯ SD (right y axis) (Œ). (Right) Changes in reticulocyte counts in rhesus macaques exposed to a second infection with P. coatneyi (᭜); the hemoglobin levels are also plotted for comparison (Œ). n ϭ 5.

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FIG 5 Turnover of noninfected erythrocytes during secondary infections with P. coatneyi in semi-immune rhesus monkeys. (Left) Course of parasitemia () and hemoglobin levels (Œ) in rhesus macaques during the second infection as described for Fig. 4. (Right) Kinetics of circulating biotinylated cells in P. coatneyi-infected rhesus macaques (᭜) in comparison with the kinetics of circulating biotinylated cells in noninfected monkeys ({). Mean hemoglobin levels are also included for comparative purposes. *, P ϭ 0.01. For differences in the number of biotinylated cells during the first infection (summarized in the legend for Fig. 3) versus the second infection, P ϭ 0.031. Differences were evaluated by comparing mean area under the curve (AUC) values using the Mann-Whitney test. n ϭ 5. a biotin infusion (Fig. 5; P ϭ 0.01). Differences were also signifi- of 7.2 on day 10 (Fig. 6). The scores recorded during the second cant compared with the number of biotinylated cells recorded infection showed a significantly lower mean peak value of 4.6 on during the first infection (P ϭ 0.0317). The lower level of hemo- day 12. Differences in severity were noted between the first and globin was recorded 24 days after challenge with an average level second infections in these animals (Fig. 6; P ϭ 0.016). As expected, of 9.7 g/dl. The animals recovered spontaneously without antima- the scores recorded in the control group showed similar patterns larial chemotherapy or fluid support. with no significant differences among them. Clinical assessment of malaria severity in rhesus macaques Characterization of hemozoin deposition. Epidemiological experimentally infected with P. coatneyi. Additional clinical and and experimental data in humans have shown that hemozoin pig- laboratory data were collected during the course of P. coatneyi ment-containing leukocytes circulating in the peripheral blood infections in a new group of five malaria-naïve rhesus macaques, are sensitive markers for parasite burden and disease severity, par- which also included daily monitoring of the erythrocyte life span ticularly when hemozoin-containing parasite stages are seques- by use of in vivo biotinylation procedures as described above. A tered (32, 33). The proportion of hemozoin-containing mono- control group of rhesus macaques were included to evaluate the cytes and neutrophils was therefore determined daily in the course normal variability in hematological and clinical laboratory pa- of the infections. Although the proportion of hemozoin-contain- rameters after biotinylation. To generate a daily quantitative as- ing leukocytes increased with rising parasitemia, phagocytosis of sessment of the clinical severity and to facilitate comparison be- hemozoin pigment by monocytes or neutrophils was positively tween groups, we also developed a rhesus physiological score correlated with parasitemia only in the course of the first infection system (RPSS; Table 1). Figure 6 summarizes the time course of (P ϭ 0.007, r ϭ 0.4818, and P Ͻ 0.0001, r ϭ 0.717, respectively). the RPSS recorded during the first and second infections in com- Interestingly, drops in hemoglobin levels were negatively corre- parison to clinical parameters recorded in the control group after lated with hemozoin-containing monocytes (P ϭ 0.0187, r ϭ in vivo biotinylation. In the course of the first experimental infec- Ϫ0.426) but not with hemozoin-containing neutrophils. When tion, the RPSS progressively increased to reach a mean peak value the proportions of hemozoin-containing phagocytes were com- pared with the RPSS, statistically significant positive correlations were noted in the course of the first infection between phagocytes and the clinical scores (for monocytes, P Ͻ 0.0001, r ϭ 0.7385; for neutrophils, P ϭ 0.002, r ϭ 0.5373). In the course of the second infection, a positive correlation was noted only between the fre- quency of hemozoin-containing monocytes and the clinical scores (P Ͻ 0.0001, r ϭ 0.6720). Bone marrow abnormalities indicate dyserythropoiesis in the naïve rhesus macaques infected with P. coatneyi. Core bone marrow biopsy specimens were obtained at three different times during the course of the experimental infections to assess the pos- sible impact of the P. coatneyi infections on bone marrow progen- itors. The time points were selected based on the clinical response to the experimental infection: (i) baseline (BL); (ii) acute phase of FIG 6 Assessment of the clinical severity of rhesus macaques experimentally infected with P. coatneyi. Time course of the rhesus physiological scores system the infection (clinical malaria [CM]), which coincides with the (RPSS) during the first (᭜) or the second () infections. The results are com- highest clinical score (RPSS); and (iii) 7 days after the highest pared with those recorded with the corresponding control groups ({, Œ, re- RPSS was reached (after severe malaria [AM]). Figure 7 includes spectively). The results are presented as mean values Ϯ standard errors of the images of representative core biopsy specimens. means (SEM). **, P Ͻ 0.01 for infected versus control group in the course of the first or second infection; *, P ϭ 0.016 for first infection versus second Baseline bone marrow samples were normocellular with a 1:3 infection. Differences were evaluated by comparing mean area under the curve ratio of erythroid to myeloid series. During the acute phase of the (AUC) values using the Mann-Whitney test. infection, both the erythroid and myeloid series were expanded,

1894 iai.asm.org Infection and Immunity Nonhuman Primate Model of Severe Malaria

FIG 7 Bone marrow core biopsy specimens of rhesus macaques experimentally infected with P. coatneyi. (A) Normocellular baseline bone marrow biopsy specimens obtained on day 0 showing a normal ratio of bone marrow cells to adipocytes (thick arrow) and a normal proportion of megakaryocytes (thin arrow). Bar, 100 ␮m. (B) Moderate to severe erythroid and myeloid hyperplasia with mild megakaryocyte hyperplasia of a representative core biopsy specimen obtained when severe clinical malaria was recorded. Bar, 100 ␮m. (C) Higher magnification of panel B showing pyknosis/karyorrhexis (arrows). Bar, 50 ␮m. (D) After antimalaria chemotherapy, nuclear changes were not observed. (E) Bone marrow hyperplasia of erythroid, myeloid, and megakaryocytic (arrows) linesinthe course of the second infection. Bar, 200 ␮m. (F) Higher magnification showing efficient erythroid differentiation. Bar, 50 ␮m.

with a marked shift to immaturity consistent with erythroid and urea nitrogen (BUN) and CPK levels were also significantly differ- myeloid hyperplasia (Fig. 7B). Nevertheless, erythroid hyperplasia ent in comparison to the levels in the same monkeys during the was associated with morphological changes consistent with dys- course of the second infection. Interestingly, hypoalbuminemia erythropoiesis, including nuclear lobulation and nuclear frag- was also observed during the second infection, with significant mentation (karyorrhexis) (Fig. 7C). Seven days after antimalaria differences compared to baseline levels (Fig. 8). Although differ- treatment, these nuclear changes were no longer observed, sug- ences in the triglyceride levels during the second infection were gesting the return of effective erythropoiesis (Fig. 7D). Bone mar- not significant in comparison to baseline levels, there was a trend row biopsy specimens were also obtained in the course of the toward hypertriglyceridemia (Fig. 8). second experimental infection, using similar time points based on We have reported that thrombocytopenia and severe coagu- the RPSS scores. Histological analyses revealed erythroid hyper- lopathy can be induced in rhesus macaques experimentally in- plasia with efficient erythroid differentiation (Fig. 7E and F). fected with P. coatneyi (25). To determine whether the activation Severe malaria and multisystemic effect of P. coatneyi infec- of the coagulation cascade is a common finding in this experimen- tions. Peripheral blood samples for clinical chemistry, coagula- tal model, coagulation profiles were assessed in the course of the tion tests, and the characterization of proinflammatory mediators experimental infections using fresh plasma samples to record pro- were collected at the same time points as described above for bone thrombin time (PT), partial-thromboplastin time (PTT), fibrin- marrow core biopsy specimens and at the end of the follow-up ogen, fibrin degradation products, and inhibitors of coagulation. period (EF). Clinical laboratory tests confirmed progression to No significant changes were observed for PT and PTT. As ex- severe disease in the course of the first infection with azotemia, pected for an acute-phase reactant, plasma fibrinogen levels were elevated levels of creatinine phosphokinase (CPK), hypertriglyc- elevated in infected macaques, and these changes coincided with eridemia, and hypoalbuminemia, which were significantly differ- increases in the number of parasites in the peripheral blood (data ent in comparison to baseline levels (Fig. 8). Differences in blood not shown). Significant increases in the levels of D-dimers in com-

June 2013 Volume 81 Number 6 iai.asm.org 1895 Moreno et al.

FIG 8 P. coatneyi induces multisystemic disorder in rhesus macaques. Time course of blood urea nitrogen (BUN), creatinine phosphokinase (CPK), triglycer- ides, and albumin levels in rhesus macaques exposed to the experimental infection. Data are from individual animals and group means (bars) at the following time points: BL, baseline (parasite inoculation); CM, clinical malaria (when RPSS clinical malaria was recorded); EF, end of the follow-up period. Closed symbols, first infection; open symbols, second infection. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001, by one-way ANOVA with Bonferroni’s multiple-comparison posttest. parison to baseline levels were recorded for the first infection, isodes were recorded using the RPSS during the course of the first suggesting fibrin formation and fibrinolysis (Fig. 9). These infection (Fig. 10A). changes were also observed 8 days after subcurative antimalarial The mean concentration of IFN-␥ in serum was 105 pg/ml in drug treatment was given and at the end of the follow-up period. the first infection and 67 pg/ml in the second infection, which is Differences were also significant in comparison to the levels ob- significantly different for the first infection compared to baseline tained during the second infection (Fig. 9). Consistent with fibrin values (P Ͻ 0.0001). The mean level of IL-6 was 19.4 pg/ml in the formation, significant decreases in the levels of protein C and pro- first infection and 1.5 pg/ml in the second infection, which was tein S were observed in the course of the infection, indicating significantly different for the first infection compared to baseline consumption of coagulation inhibitors (Fig. 9). Significant reduc- values (P Ͻ 0.01). The mean level of TNF-␣ in serum was 175 tions of protein S from baseline levels were also detected during pg/ml in the first infection and 39 pg/ml in the second infection, the second infection. with a significant difference for the first infection compared to The clinical and laboratory tests described above suggested baseline values (P Ͻ 0.001). The mean concentration of MIP-1␤/ progression to severe disease in the course of the infections in the CCL4 was 547 pg/ml during the first infection and 90 pg/ml in the malaria-naïve individuals, and each of these animals required sub- second infection, which was a significant difference in the first curative antimalarial treatment to avoid life-threatening compli- infection compared to baseline values (P Ͻ 0.0001). The mean cations. Given the clinical prognosis, four of the five animals re- level of MCP-1/CCL2 in serum was 1,607 pg/ml in the first infec- quired intravenous fluid support and also received whole-blood tion and 65 pg/ml in the second infection, with significant differ- transfusion with the recommendations and oversight from the ences for the first infection compared to baseline values (P Ͻ veterinarian support staff. This clinical outcome is in sharp con- 0.01). The mean level of TIMP-1 was 2.27 ␮g/ml in the first infec- trast with the clinical evolution of the subsequent experimental tion and 0.4 ␮g/ml in the second infection, with a significant dif- infections of these animals. Like in the first group of rhesus ma- ference for the first infection compared to the baseline value (P Ͻ caques after a subsequent second infection was given, these ani- 0.0001). The mean fold increase of NAP-2 during the first infec- mals also did not require antimalarial chemotherapy or fluid sup- tions compared to baseline levels was 8 (P Ͻ 0.05) (Fig. 10A). port and they recovered naturally. Levels of IL-6, TNF-␣, MIP-1␤, and MCP-1 in serum decreased 7 Proinflammatory response in P. coatneyi-infected rhesus days after provision of antimalarial treatment with artemether in macaques. Severe malaria in humans is associated with a strong the first infection (Fig. 10B). Mean fold increases of IFN-␥, proinflammatory immune response. To evaluate the magnitude TIMP-1, and NAP-2 were significant in the first infection in com- of the proinflammatory response in the rhesus macaques experi- parison to baseline levels at this posttreatment time point (P Ͻ mentally infected with P. coatneyi, using multiplex assays, we de- 0.05, P Ͻ 0.01, and P Ͻ 0.0001, respectively). Interestingly, levels termined the levels of 18 factors, including cytokines, chemokines, of MMP-9 in serum increased by the end of the follow-up of the and metalloproteinases in serum samples from four time points. first infection to reach a mean fold increase of 16 with respect to Eight factors were significantly different compared to baseline lev- baseline levels (Fig. 10B; P Ͻ 0.01). els (Fig. 10). We identified increased levels of IFN-␥, IL-6, TNF-␣, Erythropoietin (EPO) levels in P. coatneyi-infected rhesus MIP-1␤/CCL4, MCP-1/CCL2, TIMP1, and NAP2. These proin- macaques. To determine if the indications of dyserythropoiesis flammatory mediators were upregulated when severe malaria ep- seen in the bone marrows from P. coatneyi-infected rhesus mon-

1896 iai.asm.org Infection and Immunity Nonhuman Primate Model of Severe Malaria

FIG 10 P. coatneyi induces strong inflammatory immune response in malaria- naïve rhesus macaques. Individual patterns of plasma inflammatory mediators are compared in the course of the first (closed symbols) or second (open symbols) infections. (A) Samples obtained when high score values were de- tected using RPSS. (B) Samples obtained 7 days after the RPSS peaked. The results are expressed as fold increases of the corresponding mediator over baseline levels. Horizontal bars represent arithmetic mean values for each group. ANOVA with Bonferroni’s multiple-comparison posttest was used to compare changes in baseline levels after log transformation. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001; ****, P Ͻ 0.0001.

hemoglobin levels in the course of the first infection but not in the second infection (r ϭϪ0.902, P Ͻ 0.0001). These results suggest that the homeostatic mechanism of production of EPO in re- sponse to the acute anemia stress is operational and that the ab- FIG 9 Increased coagulation activity in P. coatneyi-infected rhesus macaques. Time course levels of D-dimers, protein C, and protein S in rhesus macaques sence of EPO was not a factor in the dyserythropoiesis described in exposed to the experimental infections. Data are from individual animals and this model. group means (dotted bars) at the following time points: at baseline (BL), be- fore parasite inoculation; at the time when RPSS clinical malaria (CM) was DISCUSSION recorded; 7 days after the episode of severe malaria (AM); and at the end of the Severe malaria is the result of extensive and excessive metabolic follow-up period (EF). Closed symbols, first infection; open symbols, second dysfunction that leads to a variety of clinically heterogeneous syn- infection. ANOVA with Bonferroni’s multiple-comparison posttest was used to compare changes after log transformation. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001; ****, P Ͻ 0.0001. keys are due to a deficiency in EPO production, we quantified the levels of EPO in serum samples using a commercially available cross-reactive human immunoassay. The serum samples tested were obtained at the same time points as described above for bone marrow core biopsy specimens. Comparable baseline EPO levels were recorded before biotin infusion with values ranging between 13.5 and 40.2 mUI/ml. However, EPO levels were 6-fold higher in the P. coatneyi-infected rhesus macaques than in the naïve group at the time of the maximum drop in hemoglobin levels. EPO levels FIG 11 Ineffective erythropoiesis in P. coatneyi rhesus macaques is not depen- in serum in the first infections at this time ranged between 207 and dent on abnormal EPO production. Time course levels of erythropoietin in plasma samples obtained from rhesus macaques in the course of the first in- 307 mUI/ml, in contrast to the levels that ranged between 9 and 80 fection (closed symbols) or second infection (opened symbols) determined by mUI/ml during the second infection. EPO levels decreased sharply immunoassay. Data present levels for individual animals at baseline (BL), be- 7 days after the single artemether treatment was given, with values fore parasite inoculation; at the time when RPSS clinical malaria (CM) was ranging between 39 and 120 mUI/ml for the first infection. This is recorded; 7 days after the episode of severe malaria (AM); and at the end of the follow-up period (EF). Horizontal bars represent arithmetic mean values for in contrast with the 10- to 17-fold increases above baseline EPO each group. ANOVA with post hoc Bonferroni’s multiple-comparison posttest levels in two of five macaques during the course of the second was used to compare changes after log transformation. **, P Ͻ 0.01; ***, P Ͻ infection (Fig. 11). EPO levels showed an inverse correlation with 0.001.

June 2013 Volume 81 Number 6 iai.asm.org 1897 Moreno et al. dromes. Although adhesion-mediated compartmentalization of celerated turnover of erythrocytes occurred at relatively low par- infected erythrocytes in microvascular beds of the brain and pla- asite burdens and could not be accounted for by the cumulative cental milieu supports a mechanistic role for sequestration in the destruction from parasitism, which suggested that the majority of physiopathology of P. falciparum cerebral and placental malaria erythrocyte clearance happened at the expense of uninfected (34–37), the complexity of the clinical presentation of severe ma- erythrocytes. Our data showed that in the course of the first infec- laria clearly indicates that additional factors beyond vascular ad- tion the life span of uninfected erythrocytes was shortened by hesion are involved (38). In fact, for both P. falciparum in humans about 80%. To our knowledge, this is the first time that removal of or P. coatneyi in rhesus monkeys, profound sequestration of in- uninfected erythrocytes in the course of malaria infection has been fected erythrocytes occurs in many tissues in uncomplicated ma- experimentally determined in nonhuman primates. laria infections. It is not known for certain that enhanced seques- The massive destruction of uninfected erythrocytes greatly in- tration of infected erythrocytes in particular compartments such creased EPO production with the subsequent hyperplasia of ery- as brain or lung directly leads to cerebral malaria or acute respira- throid progenitors. However, distinct dyserythropoietic changes tory distress, respectively, or if other pathogenic factors that lead that coincided with an observed reticulocytopenia with relatively to the observed sequestration in these tissue compartments in low parasitemias were noted. These results demonstrate that severe malaria are activated (39). Severe malaria cases reported for erythropoiesis is severely diminished in primary infections of P. the nonsequestering human malaria species, in particular P. vivax coatneyi in rhesus macaques. Our results contrast with those re- and P. knowlesi (40–42), also suggest the role of unknown mech- ported in the P. chabaudi AS mouse model, in which suppression anisms and mediators that are difficult to discern based solely on of erythropoiesis has been associated to hyperparasitemia (50, 51). data derived from clinical cases. Our data are also in contrast to those reported in the P. berghei Experimental animal models have the potential to identify fac- ANKA semi-immune mouse model, in which destruction of un- tors involved in initiating and accelerating the pathogenesis of infected erythrocytes appears to be solely responsible for the se- severe malaria, which will be important for developing novel in- vere anemia (28, 52), perhaps because the spleen and, to a lesser terventions and adjunctive therapies (8–10). The most character- extent, the liver are major hemopoietic tissues in mice but not ized experimental animal model of severe malaria is the rodent P. primates. Suppression of erythropoiesis with bone marrow hyper- berghei ANKA mouse model that was proposed for human cere- plasia accompanied by dyserythropoiesis and inefficient reticulo- bral malaria (43, 44). However, the ANKA model has unique his- cyte production index has been described in patients with severe tological findings, such as the accumulation of leukocytes, that malarial anemia (53–55). Acquired immunity in P. coatneyi-in- have not been reported in human cerebral malaria, which is char- fected rhesus monkeys is not sterilizing and can be used to mimic acterized by the adhesion of infected erythrocytes to the endothe- chronic infections that reproduce what is seen in children in areas lium of brain vessels (8). The model is therefore appropriate to test of high stable transmission (21). Taken together, the data support interventions aimed to reduce local inflammatory reaction (8), the use of P. coatneyi in rhesus macaques to study the molecular but this model may not adequately mimic the pathological path- mechanisms involved in severe malarial anemia. ways responsible for the syndromes that are invoked in human Our data on the reduced erythrocyte life span in P. coatneyi- severe or even cerebral malaria and has been called into question infected rhesus macaques conform with epidemiological data sug- (7). This is in contrast with the evidence of infected erythrocyte gesting that the destruction of uninfected erythrocytes accounts sequestration and rosetting reported in nonhuman primate mod- for more than 90% of the erythrocytes lost in malaria infections els infected with P. fragile and P. coatneyi that mimic the patterns (56). Although reduced erythrocyte life span was also recorded in of tissue sequestration seen in humans infected with P. falciparum our study in the course of the second infection, the reduction was (14, 17, 45–47). These findings provide a level of support for the less severe; an average of only 10% of biotinylated cells were de- use of nonhuman primate models to study the complexity of the tected on day 68 after infection, indicating that the removal of host-parasite interaction. uninfected erythrocytes was still occurring even after completion Here we add further support by demonstrating that malaria- of antimalaria chemotherapy. Data derived from naturally in- naïve rhesus macaques experimentally infected with P. coatneyi- fected individuals also indicate that after parasite clearance the infected erythrocytes develop severe anemia, coagulopathy, renal erythrocyte life span is reduced in patients recovering from P. impairment, and general metabolic dysfunction, outcomes fre- falciparum or P. vivax malaria (57, 58). Hypotheses put forth for quently encountered in severe malaria in humans (48). During the the destruction of uninfected erythrocytes have focused on differ- course of the acute P. coatneyi infections, clinical complications ent possible mechanisms including reduced erythrocyte deform- developed and required antimalaria chemotherapy, fluid support, ability (59, 60), accelerated erythrocyte senescence (61), and and whole-blood transfusion, mimicking the clinical care pro- immunologic removal (62–66), although none have been conclu- vided to treat severe malaria in humans (5). Signs of severe malaria sively proven to be the mechanism. were observed with relatively low parasitemia, resembling situa- In Gabonese children with P. falciparum malaria, proinflam- tions observed in nonimmune children in areas of unstable ma- matory responses have been shown to activate macrophages to laria transmission (49). Subsequent infections showed that ac- release oxygen and nitrogen radicals that can cause oxidative dam- quired immunity partially protects against severe malaria. age of infected and uninfected erythrocytes (67). Erythrocyte To investigate the mechanistic basis of severe anemia using the clearance in these situations involves the removal of both infected P. coatneyi-rhesus monkey model, we studied the erythrocyte life and uninfected cells by erythrophagocytosis mediated by macro- span and bone marrow core biopsy specimens. The experimental phages. Erythrophagocytosis is an effector mechanism that can be infection of malaria-naïve rhesus macaques resulted in the pro- modulated via the engagement of several receptors expressed on nounced accelerated reduction in hemoglobin levels that led to mononuclear phagocytes. Antierythrocytic antibodies can engage severe anemia 10 to 12 days after experimental infection. The ac- CD16/Fc␥RIIIA on monocytes, leading to anemia. Interestingly,

1898 iai.asm.org Infection and Immunity Nonhuman Primate Model of Severe Malaria studies in Kenya have confirmed that children at risk of develop- (81). Similarly, high levels of TNF-␣, associated with different ing severe malarial anemia overexpressed CD16/Fc␥RIIIA in re- inflammatory entities, inhibit erythropoiesis (82), having an im- sponse to the infection, in contrast to children who develop other pact on cell cycle progression (83), modulating transcription fac- forms of clinical malaria (68). Erythrophagocytosis can also be tors essential for erythroid differentiation (84), and promoting modulated by activation of the complement cascade. This homeo- erythrophagocytosis (85). TNF-␣ is also associated with dysfunc- static process is down-modulated by regulatory proteins that in- tion of multiple organs, leading to cerebral malaria, respiratory clude complement receptor-1 (CR1), decay-accelerating factor distress, hypoglycemia, and placental pathology (86, 87). High (DAF or CD55), and membrane inhibitor of reactive lysis (MIRL levels of IL-6 have been reported in patients with severe malaria or CD59) (reviewed in reference 69). It has been demonstrated compared to the matched uncomplicated malaria cases (88, 89). that the levels of such regulatory proteins are inversely correlated Along with TNF-␣ and IL-1, IL-6 is a pyrogen and inducer of the with malaria severity, suggesting that erythrocytes from nonim- acute-phase response (90). A recent prospective cohort study mune individuals could be more susceptible to complement-me- showed that IL-6 is a strong predictor of malaria clinical infections diated lysis than erythrocytes of immune individuals (62). How- (91). Interestingly, genetic population studies in East Africa have ever, intravascular erythrocyte lysis aside from that caused by indicated a high-frequency distribution of genotypes associated parasitism is not evident in severe anemia in humans or monkey with low expression of IL-6 in children over 12 years old and infections, and Coombs’ positive reactions in malaria infections young adults, indicating a positive selection early in life (92). do not correlate with the excessive loss of erythrocytes, which Among the chemokines analyzed, high levels of MCP-1/CCL2, suggests the participation of different mechanisms in erythrocyte MIP-1␤/CCL4, and NAP-2 were recorded in P. coatneyi-infected clearance (70). Interestingly, biochemical modifications that rhesus macaques. High levels of MCP-1/CCL2 and MIP-1␤/CCL4 mimic the physiological aging process are induced in the mem- have been reported to be associated with hyperparasitemia and brane of infected and uninfected erythrocytes in vitro (71), and placental pathology (93–95). Children with severe P. falciparum similar changes have been reported in vivo in P. knowlesi-infected infections associated with cerebral malaria and mortality had rhesus macaques (72). The definition of the mechanistic basis in- higher serum levels of MIP-1␤/CCL4 than did children who sur- volved in erythrocyte clearance in severe malaria anemia requires vived (96). Consistent with the role of MIP-1␤/CCL4 in cerebral further investigation. malaria pathogenesis, high levels were reported in postmortem Erythropoietin (EPO) is an essential growth factor that pro- cerebrospinal fluid samples obtained from children with cerebral motes erythropoiesis by supporting proliferation, differentiation, malaria compared with samples obtained from children that died and maturation of erythroid progenitors. Results in humans have of severe malarial anemia (97). been reported indicating that levels of EPO are elevated in chil- Interestingly, to our knowledge this is the first time that the dren with uncomplicated P. falciparum malaria (73). However, chemotactic cytokine NAP-2 has been reported to be associated to levels of EPO seem to be reduced relative to the severity of anemia severe malaria. NAP-2 is a proteolytic product derived from the in adults with malarial anemia (74, 75). We have shown here that chemokine CXCL7, the most abundant platelet chemokine, pres- EPO levels were associated with malaria severity in rhesus ma- ent in the ␣-granules (98). NAP-2 induces neutrophil adhesion to caques experimentally infected with P. coatneyi. The inverse cor- endothelial cells and transendothelial migration and has been in- relation between hemoglobin and EPO in the course of the first volved in regeneration of vascular integrity after injury. Studies infection but not during the second suggests suboptimal erythro- with malaria-naïve volunteers experimentally infected with P. fal- poietic responses in the naïve animals. Differences in the severity ciparum have shown that levels of CXCL7 are elevated in the early of malarial anemia between malaria-naïve and partially immune stages of the infection (99). Given the evidence of thrombocyto- rhesus macaques can therefore be explained by erythropoietic penia and coagulopathy described here for P. coatneyi infections, suppression. Our results are in agreement with recent data from and as we have reported for a severe case of disseminated intra- an area of endemicity with perennial transmission indicating that vascular coagulation (DIC) with P. coatneyi (25), high levels of EPO levels increased according to malaria severity (76). NAP-2 in plasma could indicate massive platelet activation. In The production of several immune mediators is essential for fact, high levels of platelet membrane products in plasma have parasite clearance. However, proinflammatory mediators are also been used as evidence of in vivo platelet activation (100). involved in malaria pathogenesis. We investigated the production Matrix metalloproteinases (MMPs) and their inhibitors (tissue of 18 proinflammatory mediators. Significant differences in the inhibitors of matrix metalloproteinases [TIMPs]) have been re- plasma levels of eight proinflammatory mediators tested were de- ported to play a role in the inflammatory response (reviewed in tected in rhesus macaques in the course of the first P. coatneyi reference 101). MMPs are essential for leukocyte traffic, modula- infection in comparison to baseline levels (IFN-␥, IL-6, TNF-␣, tion of proinflammatory mediators, and tissue repair. High levels MIP-1␤/CCL4, MCP-1/CCL2, TIMP1, NAP2, and MMP-9). of TIMP-1 and MMP-8 have been reported in serum samples These changes were evident only at the time when clinical severity from patients with severe or uncomplicated P. falciparum malaria scores reached a peak. With the exception of IFN-␥, TIMP-1, and (102). Here we demonstrate that malaria severity in the course of NAP-2, no significant differences from baseline values were found the first P. coatneyi infection was associated with high levels of in samples obtained 7 days after reaching the maximum clinical TIMP-1. High levels of TIMP-1 in plasma were also recorded at scores. IFN-␥ is essential for protection in clinical malaria (77, 78); two subsequent time points after subcurative treatment. Plasma in fact, early production of IFN-␥ is a critical factor in the differ- levels of MMP-9 were elevated in the first infection at the end of ences in susceptibility to malaria reported among sympatric eth- the follow-up period. These results could indicate that in our NHP nic groups living in Mali (79, 80). However, overproduction of model TIMP-1 is involved in malaria pathogenesis, whereas IFN-␥ has also been associated with anemia, exemplified by bone MMP-9 could be involved in tissue regeneration (103). Interest- marrow suppression, dyserythropoiesis, and erythrophagocytosis ingly, MMP-9 has a platelet antiaggregation effect and plays a role

June 2013 Volume 81 Number 6 iai.asm.org 1899 Moreno et al. in thrombopoiesis (104). It is therefore feasible that increasing been described in patients infected with P. falciparum (120, 121). levels of MMP-9 after several days of subcurative antimalarial che- Interestingly, high levels of triglycerides were also reported to be motherapy were in response to thrombocytopenia and massive associated with severe malaria (122, 123). In these studies, signif- endothelial cell activation. MMP-9 has been proposed as a bio- icant differences in the levels of triglycerides were defined in trav- marker of severe malaria and is a good candidate for targeted elers returned from areas of endemicity with severe malaria in therapy (105). Our data suggest that MMP-9 is involved in a coun- comparison to returned travelers with nonsevere malaria. In our terregulatory activity of proinflammatory mediators. Such results experimental model, differences in mean triglyceride levels were coincide with the reported failure to demonstrate a correlation of not significant when first and second infections were compared, MMP-9 sera or cerebrospinal fluid levels in patients with severe suggesting that malaria severity was not associated with hypertri- malaria (102, 106). The clinical outcome of malaria infections glyceridemia in rhesus macaques infected with P. coatneyi (Fig. 8). depends on the appropriate balance between proinflammatory The mechanism underlying changes in triglycerides involves in- and counterregulatory mediators. Understanding their role in creases in the production of very-low-density lipoprotein (VLDL) clinical disease could provide important insights for the develop- as a result of mobilization of free fatty acids from adipose tissue in ment of adjunctive chemotherapy. response to stress (120). Metabolic alterations mediated by ma- Hemozoin, an insoluble by-product of hemoglobin proteoly- laria parasites such as the reduction in lipoprotein lipase (LPL) sis, has been reported to exhibit several biological effects that in- activity may also account for the reduction in triglyceride clear- volve induction of proinflammatory mediators (reviewed in ref- ance (124). Interestingly, TNF-␣ has been associated with LPL erence 107), dysfunction of monocyte/macrophages (108), suppression (125). Here we have shown that the first experimental modulation of the maturation of dendritic cells (109, 110), immu- infection with P. coatneyi resulted in a 9- to 61-fold increase in nosuppression (111, 112), and suppression of erythropoiesis TNF-␣ levels compared to baseline levels, whereas the second in- (113–117). To investigate the role of hemozoin deposition in the fection resulted in a 2- to 21-fold increase in four of the five ani- pathogenesis of severe malaria, we characterized the relationship mals tested. In vitro evidence indicates that hyperlipidemia mod- between pigment-containing leukocytes, hemoglobin levels, and ifies the pharmacokinetics of lipophilic antimalarial drugs (126). clinical severity. The proportion of hemozoin-containing phago- Hypertriglyceridemia in the course of malaria infection might cytes correlated with clinical severity and parasitemia in the course therefore have an impact on chemotherapy efficacy. of the first infection. This is consistent with reports of high mor- Hypoalbuminemia was identified in the course of the first and tality in patients with severe malaria associated with a high pro- second P. coatneyi infections of the rhesus macaques. In contrast portion of hemozoin-containing neutrophils and monocytes in to triglyceride levels, albumin levels were also significantly lower comparison to patients with severe malaria that survive (32). Sim- than baseline levels at the end of the follow-up period, confirming ilar findings have also been reported in children (118, 119). How- that the inflammatory disorder was more severe in the course of ever, most recently, in a comprehensive study involving six sites in the first infection. Severe malaria is associated with changes in five different countries, broad heterogeneity was shown among capillary permeability that mimic what has been described in se- the sites, suggesting that circulating hemozoin-containing phago- verely ill patients with sepsis (127). Increased vascular permeabil- cytes have limited value as a predictor of fatal outcome (33). Ab- ity has been previously demonstrated in rhesus macaques experi- normal erythroid progenitors have been associated with hemo- mentally infected with P. coatneyi (128). In addition, the decrease zoin-containing myeloid cells in postmortem bone marrow of the hepatic synthesis of albumin due to increased synthesis of biopsy specimens derived from children with severe malarial ane- acute-phase proteins may also play a role in decreased albumin mia (113). The inhibitory role of hemozoin on erythropoiesis has concentration in severe malaria (129). Hemodynamic changes also been characterized using in vitro culture systems showing a that resulted from hypoalbuminemia and vascular dysfunction direct effect through inhibition of erythroid cell development are critical factors for the development of pulmonary edema (114, 117), growth inhibition by down-modulation of the expres- (130). sion of several receptors critical for differentiation (117), induc- In conclusion, we have presented comprehensive clinical and tion of apoptosis (116), and indirectly through the activity of in- laboratory parameters from rhesus macaques experimentally in- flammatory mediators (114). To determine if suppression of fected with P. coatneyi. This study represents the first in-depth erythropoiesis in malarial anemia can be associated with hemo- assessment and evidence showing that this simian malaria parasite zoin deposits in bone marrow, we evaluated the accumulation of can induce severe anemia, coagulopathy, renal impairment, and a hemozoin in the rhesus bone marrow samples using cell image generalized metabolic dysfunction comparable to what is seen in analysis. Similar patterns of hemozoin accumulation were deter- human cases. Our data support the use of this parasite-host com- mined in bone marrow biopsy specimens derived from both the bination to study the molecular mechanisms involved in the first and the second experimental infections (data not shown). pathogenesis of severe malaria. Finally, it is worth noting that Our data suggest that hemozoin deposits were not responsible for other simian Plasmodium species such P. cynomolgi, a parasite the observed effect on erythroid progenitors. The robust erythro- biologically and phylogenetically closer to P. vivax (131), offers a poietic response after antimalaria chemotherapy also suggests that unique opportunity for future comparative and coinfection stud- unidentified bioactive molecule(s) could play a role in dyseryth- ies to better understand the complex parasite-host pathophysio- ropoiesis during severe malaria. The NHP model will be useful in logical interactions that operate in human and nonhuman pri- future studies aiming to identify potential parasite bioactive prod- mate malaria infections. ucts using high-throughput technologies. The mean triglyceride levels in infected animals were signifi- ACKNOWLEDGMENTS cantly higher than baseline levels in the course of the first P. coat- This research was supported by NIH/NHLBI grant number 1P01 neyi infections. In retrospective studies, hypertriglyceridemia has HL078826 (original project 3 led by M.R.G. and A.M.) and NIH/NIAID

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grants R01-AI064766 and R01-AI024710. The Yerkes National Primate 17. Davison BB, Cogswell FB, Baskin GB, Falkenstein KP, Henson EW, Research Center received support from the National Center for Research Tarantal AF, Krogstad DJ. 1998. Plasmodium coatneyi in the rhesus Resources P51RR000165, and it is currently supported by the Office of monkey (Macaca mulatta) as a model of malaria in pregnancy. Am. J. Research Infrastructure Programs/OD P51OD011132. Trop. Med. Hyg. 59:189–201. 18. Davison BB, Cogswell FB, Baskin GB, Falkenstein KP, Henson EW, We are grateful for the contributions of Eileen Breding, Stephanie Krogstad DJ. 2000. Placental changes associated with fetal outcome in Ehnert, Christopher Souder, and all the veterinary staff at the Yerkes Na- the Plasmodium coatneyi/rhesus monkey model of malaria in pregnancy. tional Primate Research Center. Am. J. Trop. Med. Hyg. 63:158–173. We also express our appreciation to NIAID and members of the Ma- 19. Yang C, Xiao L, Tongren JE, Sullivan J, Lal AA, Collins WE. 1999. laria Host-Pathogen Interaction Center (MaHPIC; NIAID contract num- Cytokine production in rhesus monkeys infected with Plasmodium coat- ber HHSN272201200031C) for ongoing discussions and the continued neyi. Am. J. Trop. Med. Hyg. 61:226–229. advancement of this project, developing NHP models to study malaria 20. Coatney RG, Collins WE, Warren M, Contacos PG. 1971. The primate caused by various species. malarias. US Goverment Printing Office, Washington, DC. 21. Collins WE, Warren M, Sullivan JS, Galland GG. 2001. Plasmodium coatneyi: observations on periodicity, mosquito infection, and transmis- REFERENCES sion to Macaca mulatta monkeys. Am. J. Trop. Med. Hyg. 64:101–110. 1. WHO. 2011. World malaria report 2011. World Health Organization, 22. Grizzard MB, London WT, Sly DL, Murphy BR, James WD, Parnell Geneva, Switzerland. WP, Chanock RM. 1978. Experimental production of respiratory tract 2. 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