Effect of cyclosporine on parasitemia and survival of Plasmodium berghei infected mice

Biochemical and Biophysical Research Communications 376 (2008) 494–498

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Effect of cyclosporine on parasitemia and survival of Plasmodium berghei infected mice Diwakar Bobbala 1, Saisudha Koka 1, Camelia Lang, Krishna M. Boini, Stephan M. Huber, Florian Lang * Physiologisches Institut, der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany

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Article history: Received 1 September 2008 Available online 24 September 2008

Keywords: Malaria Cell volume Phosphatidylserine Red blood cells Apoptosis Cell death

a b s t r a c t Cyclosporine triggers suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. The present study explored whether cyclosporine influences eryptosis of Plasmodium infected erythrocytes, development of parasitemia and thus the course of the disease. Annexin V binding was utilized to depict phosphatidylserine exposure and forward scatter in FACS analysis to estimate erythrocyte volume. In vitro infection of human erythrocytes with Plasmodium falciparum increased annexin binding and decreased forward scatter, effects potentiated by cyclosporine (P0.01 lM). Cyclosporine (P0.001 lM) significantly decreased intraerythrocytic DNA/ RNA content and in vitro parasitemia (P0.01 lM). Administration of cyclosporine (5 mg/kg b.w.) subcutaneously significantly decreased the parasitemia (from 47% to 27% of circulating erythrocytes 20 days after infection) and increased the survival of P. berghei infected mice (from 0% to 94% 30 days after infection). In conclusion, cyclosporine augments eryptosis, decreases parasitemia and enhances host survival during malaria. Ó 2008 Elsevier Inc. All rights reserved.

Cyclosporine elicits eryptosis [1], the suicidal death of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling resulting in exposure of phosphatidylserine at the cell surface [2–6]. The cell membrane scrambling is stimulated by increased cytosolic Ca2+ activity [2,4,5,7] and ceramide [8]. Ca2+ enters into the cells via Ca2+ permeable cation channels, activated by osmotic shock, oxidative stress and energy depletion [7,9–11]. In addition, Ca2+ activates Ca2+ sensitive K+ channels [12,13] resulting in exit of KCl and osmotically obliged water and thus in cell shrinkage [14]. Thus, sustained increase of cytosolic Ca2+ does not only stimulate apoptosis of nucleated cells [15] but similarly triggers suicidal erythrocyte death. Phosphatidylserine exposing cells are bound to specific receptors of macrophages [16,17], and subsequently phagocytosed [18,19]. Eryptosis could be similarly stimulated by infection of erythrocytes with Plasmodium falciparum [20,21]. Plasmodia-induce oxidative stress to host erythrocytes, which activates the Ca2+ permeable cation channels [22] thus leading to cell membrane scrambling and phosphatidylserine exposure at the erythrocyte surface [23]. During malaria, the clearance of infected erythrocytes by eryptosis may lead to removal of erythrocytes prior to the development of trophozoites enabled to intoxicate macrophages [24]. Accelerated eryptosis has thus been considered a protective mechanism in malaria [25]. Sickle-cell trait, b-thalassemia trait, homozygous * Corresponding author. Fax: +49 7071 29 5618. E-mail address: fl[email protected] (F. Lang). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.09.005

Hb-C and G6PD-deficiency lead to premature senescence and/or eryptosis upon infection with Plasmodium and may thus be protective due to accelerated clearance of ring stage-infected erythrocytes [6,26–30]. The present study explored whether cyclosporine could accelerate phosphatidylserine exposure of P. falciparum infected erythrocytes and thus influence parasitemia and survival following infection.

Materials and methods Animals, cells and solutions. Animal experiments were performed according to German animal protection law and approved by the local authorities (registration number PY 2/06). Healthy SV129/J wild type mice (aged 4 months, both male and female) were used for the experiments. The animals had free access to standard chow (C1310, Altromin, Lage, Germany) and drinking water. Mouse erythrocytes were drawn from animals by retroorbital venopuncture or by incision of the tail vein. Human erythrocytes were drawn from healthy volunteers. Experiments were performed at 37 °C in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES)/NaOH (pH 7.4), 5 glucose, 1 CaCl2 Cyclosporine was added to the NaCl Ringer at final concentrations varying from 0.001 to 1 lM (Sigma, Taufkirchen, Germany). For in vitro cyclosporine treatment, the final haematocrit was adjusted to 0.3%.

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binding cells within 24 h to 5.21 ± 0.57% (n = 6). Infection of erythrocytes with P. falciparum significantly enhanced the percentage of annexin binding erythrocytes (Fig. 1). Phosphatidylserine exposure of infected erythrocytes was augmented by cyclosporine (Fig. 1), an effect reaching statistical significance at 0.001 lM cyclosporine. The parasite DNA amplification in infected erythrocytes was slightly, but significantly decreased by cyclosporine (P0.001 lM) (Fig. 2A). Similarly, cyclosporine exposure (P0.01 lM) decreased the in vitro growth of P. falciparum (Fig. 2B). Infection of erythrocytes decreased erythrocyte forward scatter, an effect slightly, but significantly augmented in the presence of cyclosporine (Fig. 3). In a next step, mice were infected with P. berghei to determine the influence of cyclosporine treatment on the course of malaria in vivo. Up to 8 days after infection parasitemia was less than 3% irrespective of treatment (Fig. 4). The following days, the percentage of infected erythrocytes increased gradually in both, treated and untreated mice. The percentage of parasitized erythrocytes increased up to 47% (within 20 days) in animals without cyclosporine treatment, and to 27% (within 20 days) in animals treated with 5 mg/kg b.w. of cyclosporine subcutaneously. Thus, cyclosporine treatment significantly decreased parasitemia (Fig. 4). Cyclosporine treatment further increased the survival of P. berghei infected mice. All non-treated animals died within 24 days after the infection. In contrast 94% of the cyclosporine treated animals survived the infection for more than 30 days (Fig. 4). Discussion The present study discloses a significant effect of cyclosporine on the course of malaria. Cyclosporine treatment significantly decreases the parasitemia and significantly enhances the percentage of surviving animals after infection with P. berghei. While all nontreated animals died from infection with P. berghei, most of the cyclosporine treated animals survived the same infection. P. berghei infection has previously been shown to follow an invariably lethal course in mice [35]. Cyclosporine stimulates the machinery leading to suicidal death of erythrocytes [36], an effect, which could at least in theory con-

60

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50

Annexin binding (%)

Determination of phosphatidylserine exposure. FACS analysis was performed as described [7]. After incubation in the presence or absence of cyclosporine, suspensions of non-infected erythrocytes were stained with annexin V-FLUOS (Roche, Mannheim, Germany), suspensions of P. falciparum infected erythrocytes were stained with annexin V-APC (BD Biosciences Pharmingen, Heidelberg, Germany) and/or with the DNA/RNA specific dye Syto16 (Molecular Probes, Göttingen, Germany) to identify phosphatidylserine exposing and infected erythrocytes, respectively. For annexin binding, erythrocytes were washed, resuspended in annexin-binding buffer (Ringer solution containing 5 mM CaCl2 pH 7.4), stained with annexin V-APC (dilution 1:20) or annexin V-FLUOS (dilution 1:100), incubated for 20 min at room temperature, and diluted 1:5 with annexin binding buffer. Syto16 (final concentration of 20 nM) was added directly to the diluted erythrocyte suspension or coincubated in the annexin binding buffer. Cells were analyzed by flow cytometry (FACS-Calibur, Becton Dickinson, Heidelberg, Germany) in FL-1 for Syto16 or annexin V-FLUOS fluorescence intensity (detected at 530 nm) and in FL-4 for annexin V-APC fluorescence intensity (detected at 660 nm). In vitro cultivation of Plasmodium falciparum. For infection of human erythrocytes the human pathogen ( P. falciparum) strain BinH [31] was grown in vitro [32] in human erythrocytes. Parasites were cultured as described earlier [33,34] at a haematocrit of 2% and a parasitemia of 2–10% in RPMI 1640 medium supplemented with Albumax II (0.5%; Gibco, Karlsruhe, Germany) in an atmosphere of 90% N2, 5% CO2, 5% O2. In vivo proliferation of Plasmodium berghei. For infection of mice Plasmodium berghei ANKA-parasitized mouse erythrocytes (1  106) were injected intraperitoneally [35] into wildtype mice. Where indicated cyclosporine (5 mg/kg b.w) was administered subcutaneously from the eighth day of infection. Blood was collected from the mice 8 days after infection by the incision of the tail. Parasitemia was determined by Syto-16 staining in FACS analysis. In vitro growth assays of P. falciparum infected human erythrocytes. The P. falciparum BinH strain was cultured and synchronized to the ring stage by sorbitol treatment as described previously [22]. For the in vitro growth assay, synchronized parasitized erythrocytes were aliquoted in 96-well plates (200 ll aliquots, 1% haematocrit, 0.5–2% parasitemia) and grown for 48 h in the presence or absence of cyclosporine (0.001–1 lM). The parasitemia was assessed at time 0 h and after 48 h of culture by flow cytometry. Parasitemia was defined by the percentage of erythrocytes stained with the DNA/RNA specific fluorescence dye Syto16. To estimate DNA/RNA amplification, the culture was ring stagesynchronized, and resynchronized after 6 h of culture (to narrow the developmental parasite stage), aliquoted (200 ll aliquots, 2% haematocrit and 10% parasitemia) and cultured for further 16 h in the presence or absence of cyclosporine (0.001–1 lM). Thereafter, the DNA/RNA amount of the parasitized erythrocytes was determined by Syto16 fluorescence as a measure of intraerythrocytic parasite copies. Statistics. Data are expressed as arithmetic means ± SEM and statistical analysis was made by paired or unpaired t-test or ANOVA using Tukey’s test as post hoc test, where appropriate. p < 0.05 was considered as statistically significant.

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0 0 Results To identify phosphatidylserine exposing erythrocytes, annexin V binding has been determined in FACS analysis. The percentage of annexin-binding erythrocytes was low in fresh erythrocytes (0.96 ± 0.06%, n = 6). Exposure of uninfected erythrocytes to cyclosporine-free Ringer solution increased the percentage of annexin

0.001

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Conc. of Cyclosporine (μM) Fig. 1. Effects of cyclosporine on phosphatidylserine exposure of infected and noninfected erythrocytes. Arithmetic means ± SEM (n = 10) of annexin binding of infected (closed symbols) and noninfected (open symbols) erythrocytes before and after treatment with cyclosporine as a function of the cyclosporine concentration. * Indicates significant difference (p < 0.05) from absence of cyclosporine, #Indicates significant difference to noninfected erythrocytes.

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noninfected infected early stages

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Conc. of Cyclosporine (μM) Fig. 2. Effects of cyclosporine on intraerythrocytic amplification and in vitro growth of the parasite. (A) Intraerythrocytic DNA amplification in erythrocytes as a function of the cyclosporine concentration (arithmetic means ± SEM, n = 8). (B) In vitro growth of Plasmodium falciparum in human erythrocytes as a function of cyclosporine concentration (arithmetic means ± SEM, n = 8). *Indicates significant difference (p < 0.05) from absence of cyclosporine.

tribute to or even account for the blunted parasitemia and the survival of the infected mice. Accelerated phosphatidylserine exposure enhances the engulfment of infected erythrocytes by macrophages [18,19]. Cyclosporine further decreases cell volume, a further hallmark of eryptosis [37]. The cell shrinkage is due to increase of cytosolic Ca2+ activity, activation of Ca2+ sensitive K+ (GARDOS) channels [14], K+ exit, hyperpolarization of the erythrocyte membrane, Cl exit [38] and subsequent loss of osmotically obliged water [39]. Eryptosis is enhanced by several other endogenous mediators and xenobiotics including hemolysin Kanagawa [40], listeriolysin [41], PGE2 [42], azathioprine [43], Bay-5884 [44], platelet activating factor [45], chlorpromazine [46], anandamide [47], methylglyoxal [48], paclitaxel [36], curcumin [49] amyloid peptides [50], valinomycin [51], aluminium [52], lead [53], mercury [54] and copper ions [55]. Moreover, eryptosis is enhanced in a variety of clinical conditions including sickle-cell anemia [56,57], b-thalassemia [6], glucose-6-phosphate dehydrogenase (G6PD)-deficiency [6], phosphate depletion [58], iron deficiency [20], Hemolytic Uremic Syndrome [59], sepsis [60], malaria [25] and Wilson disease [55]. At least in theory, the effect of cyclosporine could result from a direct toxic action on the pathogen, as cyclosporine significantly decreased the intraerythrocytic DNA/RNA content. In addition, the scrambling effect of cyclosporine accelerates the clearance of

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Conc. of Cyclosporine (μM) Fig. 3. Effects of cyclosporine on forward scatter of infected and noninfected erythrocytes. (A) Arithmetic means ± SEM (n = 6) of forward scatter of the early stage-infected erythrocytes (closed symbols) and noninfected (open symbols) erythrocytes as a function of the cyclosporine concentration. *Indicates significant difference (p < 0.05) from absence of cyclosporine, #Indicates significant difference to noninfected erythrocytes. (B) Arithmetic means ± SEM (n = 6) of forward scatter of late stage-infected erythrocytes (closed symbols) and noninfected (open symbols) erythrocytes as a function of the cyclosporine concentration. *Indicates significant difference (p < 0.05) from absence of cyclosporine, #Indicates significant difference to noninfected erythrocytes.

infected erythrocytes from circulating blood. Along those lines, iron deficiency [20] and treatment with lead [21] have been shown to accelerate eryptosis of infected erythrocytes and to protect against a severe course of malaria. In conclusion, cyclosporine accelerates eryptosis of infected erythrocytes. The effect contributes to or even accounts for the effect of cyclosporine on parasitemia and survival of the host. Acknowledgments This study has been supported by the Deutsche Forschungsgemeinschaft (Hu 781/4-3, La 315/6-1 and La 315/13-1). The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic and Tanja Loch.

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Days after infection Fig. 4. Parasitemia and survival of Plasmodium berghei infected mice. (A) Original histograms of parasitemia in untreated animals (upper panels) and animals treated from day 10 until day 20 with 5 mg/kg b.w. of cyclosporine s.c. (lower panels) 10 (left panels) and 20 (right panels) days after infection with Plasmodium berghei. (B) Arithmetic means ± SEM of parasitemia in mice without treatment (open circles, n = 10) or with 5 mg/kg b.w. of cyclosporine s.c. (closed circles, n = 16) as a function of days after infection with Plasmodium berghei. *Indicates significant difference from the untreated animals. (C) Survival of mice without treatment (open circles) or with daily 5 mg/ kg b.w. of cyclosporine s.c. (closed squares) as a function of days after infection with Plasmodium berghei.

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