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Plant Hormone Salicylic Acid Produced by a Malaria Parasite Controls Host Immunity and Cerebral Malaria Outcome

  • Ryuma Matsubara,

    Affiliations Department of Parasitology, National Institute of Infectious Diseases, Shinjyuku, Tokyo, Japan, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

  • Hiroka Aonuma,

    Current address: Department of Tropical Medicine, The Jikei University School of Medicine, Minato, Tokyo, Japan.

    Affiliation Department of Parasitology, National Institute of Infectious Diseases, Shinjyuku, Tokyo, Japan

  • Mikiko Kojima,

    Affiliation Center for Sustainable Resource Science, RIKEN, Yokohama, Kanagawa, Japan

  • Michiru Tahara,

    Affiliation Department of Parasitology, National Institute of Infectious Diseases, Shinjyuku, Tokyo, Japan

  • Syed Bilal Ahmad Andrabi,

    Affiliation Department of Parasitology, National Institute of Infectious Diseases, Shinjyuku, Tokyo, Japan

  • Hitoshi Sakakibara,

    Affiliation Center for Sustainable Resource Science, RIKEN, Yokohama, Kanagawa, Japan

  • Kisaburo Nagamune

    nagamune@niid.go.jp

    Affiliations Department of Parasitology, National Institute of Infectious Diseases, Shinjyuku, Tokyo, Japan, Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

Plant Hormone Salicylic Acid Produced by a Malaria Parasite Controls Host Immunity and Cerebral Malaria Outcome

  • Ryuma Matsubara, 
  • Hiroka Aonuma, 
  • Mikiko Kojima, 
  • Michiru Tahara, 
  • Syed Bilal Ahmad Andrabi, 
  • Hitoshi Sakakibara, 
  • Kisaburo Nagamune
PLOS
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Abstract

The apicomplexan parasite Toxoplasma gondii produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite Plasmodium spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in P. berghei ANKA and T. gondii cell lysates. However, addition of salicylic acid to P. falciparum and T. gondii culture had no effect. We transfected P. falciparum 3D7 with the nahG gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting Pseudomonas sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E2, which potentially modulates host immunity as an adaptive evolution of Plasmodium spp. To investigate the function of salicylic acid and prostaglandin E2 on host immunity, we established P. berghei ANKA mutants expressing nahG. C57BL/6 mice infected with nahG transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The nahG-transfectant also significantly increased the mortality rate of mice. Prostaglandin E2 reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the nahG transfectant. Thus, salicylic acid of Plasmodium spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E2.

Introduction

Malaria is a dominant human infectious disease that affects more than 200 million people and caused 627,000 deaths in 2012 [1]. Plasmodium is an apicomplexan parasitic protist that causes malaria. Apicomplexan parasites are thought to originate from the second endosymbiosis of ancestral red algae [2]. All apicomplexan parasites except Cryptosporidium and some Gregarine possess a plastid, apicoplast, which is thought to be derived from an ancient endosymbiont [2]. Therefore, apicomplexans have conserved plant-like signaling mechanisms. We previously reported that Toxoplasma gondii, an apicomplexan parasite, produces a plant hormone, abscisic acid (ABA) [3]. ABA promotes the calcium-dependent egress of parasites from the host cell, and inhibition of the biosynthesis of ABA leads the parasite to differentiate into a chronic infection cycle. This hormone is a key molecule that determines parasite pathogenicity, and the presence of a plastid suggests that other phytohormones might be found in apicomplexans. Plasmodium spp. is also likely to have conserved phytohormone signaling that may contribute to pathogenicity. In this study, we examined phytohormones of malaria parasites as potential pathogenic factors, and identified salicylic acid (SA) as a phytohormone in T. gondii and Plasmodium spp. cell lysates.

In higher plants, SA regulates the pathogen-resistance system through a mechanism referred to as systemic acquired resistance [4]. Pathogen-induced SA is delivered to the whole plant to upregulate expression of pathogenesis-related (PR) genes [4]. Recently, the receptors for SA were identified in Arabidopsis thaliana and named as nonexpresser of PR genes (NPR) 3 and 4 [5]. NPR3 and 4 are adaptors of Cullin 3 ubiquitin E3 ligase, which degrades the transcription cofactor, NPR1. SA is synthesized by two pathways: a major benzoic acid (BA) pathway, in which L-phenylalanine is converted to SA by phenylalanine ammonia lyase and BA 2-hydroxylase [6]; and a minor isochorismate synthase (ICS) pathway [7], which is thought to be dominant in A. thaliana and requires ICS as the key enzyme.

Metazoans do not synthesize SA, but SA has a strong activity in animals. SA and its acetylated derivative, acetylsalicylic acid, inhibit prostaglandin (PG) synthesis in animals and are used as non-steroidal anti-inflammatory drugs (NSAIDs) [8]. The cyclooxygenase (COX), target of NSAIDs is a key enzyme in synthesis of PGs, including PGE2, the main molecule acting on the thermoregulatory center to cause fever and pain [9]. PGE2 may also suppress host immunity by switching cytokines to a T helper-2 (Th2) phenotype [10]. Kubata et al. [11] demonstrated production of prostanoids in P. falciparum lysates and culture supernatants in vitro. P. falciparum also releases PGD2, E2, and F2α into the infecting milieu. The PG-synthesizing enzyme of Plasmodium has not been identified, but is resistant to NSAIDs including indomethacin and acetylsalicylic acid [11]. This finding suggests that the PG synthase of parasites evolved specifically to adapt the host immune response.

In this study, we identified production of SA by malaria parasites and investigated the role of SA in vitro and in vivo. We found that the PGE2 concentration is controlled by parasite SA in vitro and that induction of an SA-degrading enzyme in the parasite altered cytokine levels and increased mouse mortality in vivo. Based on these results, we propose a novel immunomodulatory pathway that includes parasitic SA and PGs.

Materials and Methods

Parasites and animals

P. falciparum strain 3D7 was provided by The Malaria Research and Reference Reagent Resource Center (MR4) and cultivated in vitro as described previously [12]. Human red blood cells (RBCs) and serum were provided by the Japan Red Cross. P. berghei strain ANKA was also provided by MR4. Passage, gene manipulation, and cloning of the parasite were performed as previously described [13] in ddY mice purchased from Japan SLC (Shizuoka, Japan). We passaged the parasite when parasitemia reached approximately 5%. One drop of blood (~5 μL) was suspended in 100 μL of phosphate-buffered saline (PBS) and injected intraperitoneally into another mouse. Parasite specimens for mass spectrometry analysis of SA were collected from whole blood of infected ddY mice using the same method of infection. All other experiments, including survival, experimental cerebral malaria (ECM) assessment, and cytokine and prostaglandin measurements were performed in the C57BL/6 mouse strain. Female mice aged 6–9 weeks old were purchased from Japan SLC. T. gondii strain RH and 2F were used with in vitro conditions [14]. T. gondii was cultivated with human foreskin fibroblast (HFF) cells (Millipore, Darmstadt, Germany). All animal experiments were conducted in accordance with the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science and were approved by the Institutional Animal Care and Use Committee of the National Institute of Infectious Diseases (permission numbers: 112142, 213010, and 214004). All surgery was performed under isoflurane (Merck, Whitehouse Station, NJ) anesthesia and all efforts were made to minimize suffering.

Salicylic acid determination

ddY mice were treated intraperitoneally with 50 μg/kg phenylhydrazine 1 day before infection to increase the ratio of reticulocytes and increase parasitemia. P. berghei ANKA was intraperitoneally injected into mice and parasitemia was checked daily. When parasitemia was >40%, whole blood was collected from the heart under deep anesthesia. The total parasitized RBCs were 1–1.7×109 cells per sample. Blood parasites were purified by treatment with 1.5 mg/mL saponin in PBS to remove host contamination [15]. After three washes with PBS, the pellet was weighed and rapidly frozen in liquid nitrogen. Samples were stored at −80°C until use. T. gondii was collected as previously described [14]. Briefly, T. gondii tachyzoites were cultivated with HFF cells. Approximately 3×109 parasites were purified by filtration with a 3-μm pore polycarbonate membrane. Few host cells contaminated the parasite sample based on observation under a microscope. The same procedure without T. gondii infection resulted in almost no weight gain of harvested cells (data not shown). After purification, parasites were washed three times with Hank’s balanced salt solution supplemented with HEPES buffer (10 mM final) and ethylene glycol tetra-acetic acid (EGTA, 1 mM final) and immediately frozen as above.

Extraction of SA was performed as described elsewhere [16,17]. Briefly, 1 ml of prechilled (-30°C) extraction solvent (methanol: formic acid: water, 15:1:4) and internal standards were added to each parasite pellet and the mixture was kept at -30°C for at least 16 h. After centrifugation at 10,000×g for 15 min, the supernatant was transferred to a 96-well collection plate (Waters, Milford, MA) that was placed on an automated solid phase extraction system (SPE215; Gilson, Middleton, WI). SA purification was performed by passing the extract through an Oasis HLB 96-Well Plate (30 mg; Waters) equilibrated with 1 M formic acid, followed by washing with 0.3 ml of extraction solvent. The SA-containing fraction was then passed through an Oasis MCX 96-Well Plate (30 mg; Waters) equilibrated with 1 M formic acid. After washing with 1 M formic acid, SA was eluted with 1 ml methanol. The SA fractions were then evaporated and reconstituted with 1 ml of water. Finally, 50 μl of the SA fraction was combined with 1 μl of 5% formic acid for liquid chromatography (LC)-mass spectrometry analysis.

The mass spectrometry-based identification was performed with a Nexera UHPLC/HPLC (Shimadzu)/Triple TOF 5600 (AB SCIEX) system with a ZORBAX Eclipse XDB-C18 column (Agilent). Elution condition of LC is described previously [16].

Quantification of purified SA was performed with an ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (Aquity UPLC System/XEVO-TQS; Waters, Milford, MA) with an ODS column (Aquity UPLC BEH C18, 1.7 μm, 2.1×50.0 mm; Waters) [16,17]. Quantification of SA from P. falciparum was performed using a commercial salicylate diagnosis kit (Roche, Basel, Switzerland) with direct use of culture supernatants.

Addition of salicylic acid to a parasite culture in vitro

P. falciparum strain 3D7 and T. gondii strain 2F were cultured with media containing SA in vitro. After synchronization twice with 5% (w/v) D-sorbitol in Milli-Q water (Merck KGaA, Darmstadt, Germany) [11], P. falciparum was treated with 100 μM SA. Parasitemia was observed every 24 h. T. gondii 2F, a bacterial β-galactosidase (β-gal) expressing clone with a RH background, was allowed to infect HFF cells and immediately treated with 10−1 to 105 nM SA. After cultivation for 48 h, parasite growth was quantified based on β-gal activity [14].

Establishment of salicylic acid-deficient parasites

P. falciparum was transfected with the SA-degrading gene, nahG, using an episomal expression system [18]. Briefly, nahG was driven by P. falciparum chloroquine-resistant transporter gene 5′UTR (pCRT) and a P. berghei dihydrofolate reductase gene 3′UTR (PbDT) fused into an attR4-attR3 site of the destination vector pCHD43 (II) [19]. The constructed pCRT::nahG-cmyc vector was transfected by electroporation (S1 Fig). Parasites were selected with 5 nM WR99210 (a gift from Jacobus Pharmaceuticals). For P. berghei ANKA, the nahG expressing vector was constructed with the pL0006 vector (distributed from MR4) and nahG was also driven by pCRT and PbDT (S2 Fig). The vector was linearized and recombined into the 230p gene locus, which has no known function [20]. The P. berghei ANKA transfectants were selected with 70 μg/mL pyrimethamine in drinking water (MP Biomedicals, Solon, OH) and cloned by limiting dilution [13]. Control parasites transfected with gfp were made in the same way. Expression of nahG was confirmed by Western blotting with rabbit anti-c-Myc polyclonal antibody (1:100 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA), using the mouse anti-HSP-90 monoclonal antibody as control (Sigma Aldrich, St. Louis, MO). The goat anti-rabbit secondary antibody HRP conjugate (1:1,000) (Sigma Aldrich) was used for c-myc and the goat anti-mouse secondary antibody HRP conjugate (1:5,000) (Molecular Probes, Eugene, OR) was used for HSP-90. Signals were identified with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA).

Mice mortality assay

C57BL/6 mice received 104 parasitized red blood cells intravenously. Survival, weight, parasitemia, and clinical signs were checked twice a day, and survival was recorded daily to prepare survival curves. Moribund mice in a coma, with no movement, no body extension and no response to any stimulus were euthanized by deep isoflurane anesthesia. The decision to apply euthanasia followed previous criteria [21]. Brain hemorrhage was examined as a potential cause of death. At 21 days after infection, surviving mice were sacrificed to minimize pain using the same method of euthanization.

Evaluation of ECM

To evaluate disruption of the blood brain barrier (BBB), a dye leakage test was performed [22]. Briefly, mice at 6 days post-infection were intravenously injected with 100 μL of Evans blue dye (1% w/v in PBS; Sigma Aldrich). After 1 h, mice were anesthetized and euthanized to collect whole brains. The brains were photographed, weighed, and transferred into 2 mL formamide (Wako Chemicals, Osaka, Japan). Samples were incubated for 48 h at 37°C and dye extravasation was determined by measurement of optical density (OD) at 640 nm. Values were normalized using tissue weight. To evaluate sequestration of vessels in brains, samples were observed histologically. Mice 6 days post-infection were anesthetized, perfused through the heart with 5 mL of PBS, and then with 5 mL of ice-cold phosphate-buffered 4% paraformaldehyde solution (PFA; Wako Chemicals) for fixation. Brains were transferred into 4% PFA and fixed overnight at 4°C. Brain slices stained with hematoxylin and eosin were observed by microscopy.

Prostaglandin and cytokine quantification

PGE2 was measured using a Prostaglandin E2 EIA Kit Monoclonal (Oxford Biochemical Research, Oxford, MI). Culture supernatant and plasma from heparin-treated whole blood were used for measurements. Mouse plasma samples from animals at 6 days post-infection were collected as above and cytokines were analyzed using a Bio-Plex Pro Mouse Cytokine 23-plex Assay and Th1/Th2 Assay Kit (Bio-Rad, Hercules, CA).

Statistics

All statistical tests were performed using R software (ver. 3.0.0; R Foundation for Statistical Computing, Vienna, Austria [http://www.R-project.org/]). A Mann-Whitney U-test was used for non-parametric comparison of Evans blue leakage, plasma prostaglandins, cytokine quantification, and clinical signs (weight, parasitemia, and hematocrit). A log-rank test was used to compare survival curves of infected mice. A non-paired two-tailed Student t-test was used to compare in vitro P. falciparum results. Kaplan-Meier survival curves of parasite-challenged mice and box graphs of in vivo results were depicted using R software.

Results

Identification of salicylic acid in apicomplexan parasites

SA was extracted from P. berghei ANKA and analyzed by a LC-triple TOF mass spectrometry. Identical fragments to those for standard SA were detected in the parasite lysate, indicating that SA is present in this organism (Fig 1). For quantification, SA was extracted from P. berghei ANKA and T. gondii, respectively, and analyzed by an automated MS quantification system. Both parasites contained SA at remarkably high concentrations (P. berghei: 9.18 μM; T. gondii: 23.6 μM, respectively) (Fig 2A). Exogenous addition of SA did not alter parasite growth (Fig 2B and 2C), probably because the parasite already contained sufficient levels of SA.

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Fig 1. Identification of salicylic acid from Plasmodium berghei ANKA.

P. berghei ANKA was purified from infected mice blood, and salicylic acid (SA) was extracted, and analyzed by LC-triple TOF mass spectrometry. (A) Structural formula of SA. (B) LC chromatogram of SA standard (control) and P. berghei ANKA sample. (C) Fragmentation analysis of peaks in (B) (colored in aqua). Collision energy was 20 eV.

https://doi.org/10.1371/journal.pone.0140559.g001

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Fig 2. Quantification, in vitro function and genetic manipulation of salicylic acid (SA).

(A) SA concentrations identified by automated quantitative mass spectrometry. Black and white bars show the SA concentrations in fresh cell lysates of Plasmodium berghei ANKA (Pb) purified from infected mouse blood in vivo, and Toxoplasma gondii RH (Tg) cultured in vitro, respectively. The concentrations were calculated based on the weight of parasite lysate. (B) Effect of addition of SA to culture medium on growth of T. gondii 2F. Growth was quantified by β-galactosidase activity after cultivation for 48 h. Each value was normalized using control parasites treated with DMSO. The graph shows the mean of two independent experiments. (C) Effect of addition of SA to culture medium on growth of P. falciparum. The growth rate was monitored by counting parasitemia by Giemsa staining. (D) Western blotting of nahG fused with a cmyc2-tag. HSP-90 was used as an internal control (bottom panel). (E–G) Biological changes of P. falciparum due to SA deficiency in vitro. (E) Secretion of SA into the culture supernatant by P. falciparum transfected with nahG-cmyc2 or GFP (control). (F) Growth effect of intracellular SA depression. Growth kinetics of nahG-containing mutants were compared with the control (gfp-transfectant). (G) SA affected the synthesis of prostaglandin E2 (PGE2) in parasites (p<0.01, Student t-test). All experiments with P. falciparum were performed after synchronization twice with 5% (w/v) D-sorbitol solution (n = 3). Bars indicate the data range (B) or standard error (C, E-G).

https://doi.org/10.1371/journal.pone.0140559.g002

Establishment and analysis of SA-deficient parasites

Because addition of SA produced no significant phenotype, we transfected P. falciparum with the nahG gene, which encodes an SA-degrading enzyme found in the plant pathogenic bacteria Pseudomonas putida [23]. Expression of this gene product was confirmed by Western blotting using 2× myc-tag at the carboxyl-terminus of nahG (Fig 2D). The SA concentration of nahG-transfectants was lower than that in gfp-transfected controls, although the difference was not significant (Fig 2E). The growth kinetics and stage conversion of the parasites were similar (Fig 2F, S3 Fig). Taken together, these results indicate that the concentration of SA had no effect on parasite growth.

SA is an NSAID that modulates PGE2 levels in mammals. Plasmodium produces its own PGs, of which PGE2 is predicted to interfere with the immune system of a host [11]. We hypothesized that malaria parasites modulate the concentration of PGE2 in hosts through SA and their own production of PGE2. Therefore, we quantified the concentration of parasite prostaglandins. Surprisingly, the PGE2 concentration decreased significantly when the nahG gene was transfected into parasites (Fig 2G).

Influence of in vivo SA deficiency on mouse survival

To investigate the effect of SA in vivo, we established SA-deficient P. berghei ANKA by transfection with nahG. First, we examined the pathogenic effect of the SA deficiency. C57BL/6 mice were intravenously challenged with 104 parasitized RBCs. As indicated in Fig 3A, mice challenged with nahG parasites had significantly increased mortality compared with the gfp parasite group (p<0.05, Log-rank test). We used three independent nahG-transfected clones and obtained similar results (data not shown). Both nahG and gfp parasites induced ECM, resulting in a neurological syndrome characterized by clinical signs including ataxia, reduced reflex, convulsions, and coma. Dissection of the heads of dead mice showed obvious brain hemorrhage. Thus, the final cause of death was concluded to be ECM for all mice in the nahG and gfp groups. However, onset of ECM was shortened by 1 or 2 days by infection with the nahG-transfectant compared with control parasites. One mouse survived for 21 days after infection and was then sacrificed to minimize the pain.

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Fig 3. In vivo influence of SA depression.

(A) Survival following infection of mice with nahG- or gfp-expressing parasites. Data are from two independent experiments, n = 13. *: p<0.05. (B, C) Parasite growth (B) and animal weight gain/loss (C). Each graph indicates an individual mouse infected by nahG (red) or gfp (blue)-expressing parasites. (D) Parasitemia of nahG- or gfp-expressing parasites at day 6 post-infection, when clinical signs were most significant. Plasma PGE2 and cytokines were quantified at this time point. (E) Hematocrit levels in mice at 6 days post-infection. Hematocrit did not differ between the groups, whereas the survival curves and clinical behavior shifted at the same time point. Statistics were calculated by Log-rank and general Wilcoxon test (A) and Mann-Whitney U-test (E).

https://doi.org/10.1371/journal.pone.0140559.g003

Influence of in vivo SA deficiency on ECM

Since there seemed to be a difference in severity of ECM between mice with nahG- or gfp-expressing parasites, we analyzed the brains of infected mice histologically to evaluate ECM, including sequestration of microvessels and hemorrhage (Fig 4). The brain was stained with hematoxylin and eosin and the pathology was observed. The cerebellum of mice infected with nahG transfectants showed leucocyte sequestration in small vessels (Fig 4A). This pathological observation is a typical symptom of ECM [24]. Mice infected with gfp transfectants showed slight hemorrhage, but no sequestration of leukocytes, suggesting less severe ECM (Fig 4B). There were no pathological changes in vessels in uninfected control mice (Fig 4C). The midbrain, frontal cortex, occipital cortex and olfactory bulb had similar features (S4S6 Figs). The sequestration of nahG-transfectants was significant compared to gfp-transfectants or uninfected control (S7 Fig): two of 3 nahG-transfectants showed significant differences, whereas one mouse (nahG-3) showed similar level of sequestration comparing to the gfp-transfectants (S7 Fig).

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Fig 4. Parasite SA influences the cerebral malaria outcome.

(A-C) Histological observation of infected mouse cerebellums. (A) Cerebellum infected by nahG-expressing parasites. Note the sequestrated leukocytes in microvessels. The inset image shows a higher magnification of the boxed portion. Phagocytized hemozoin is observed (arrowhead). (B) Brain of a mouse infected with gfp-expressing parasites. Slight microbleeding was observed, but no sequestrated vessels were found. (C) Brain of an uninfected control. Sections were stained by hematoxylin and eosin. (D) Evans blue leakage analysis of the severity of cerebral malaria. Photographs of brains from mice infected with nahG- (left upper) and gfp- (left middle) expressing parasites and uninfected controls (left bottom), and quantification of dye leakage (right). Mice (n = 5) were sacrificed at 6 days post-infection. Solid line, p<0.01; dashed line, p<0.05. C57BL/6 mice at 6 days post-infection were used for all experiments. Bar: 50 μm.

https://doi.org/10.1371/journal.pone.0140559.g004

Next, we quantified the severity of ECM by Evans blue leakage assay (Fig 4D). Evans blue leakage was significantly higher in infected mice (p<0.01, nahG vs. control and gfp vs. control; p<0.05, nahG vs. gfp), indicating that nahG transfectants severely disrupted the BBB in ECM. Mouse parasitemia and body weight kinetics were also measured (Fig 3B and 3C). Parasitemia at day 6 after infection when mice infected with nahG transfectant but not gfp transfectant showed ECM signs, was not significantly different, suggesting that differences in the growth capacity of parasites did not explain differences in clinical changes (Fig 3D). Hematocrit scores on the same day were also equivalent (Fig 3E). Mouse weight did not differ and was even somewhat higher with nahG transfectant infection (Fig 3C). Thus, the differences in mortality may be explained by occurrence of ECM due to infection with nahG transfectants.

Prostaglandins and cytokines in infected mouse plasma

The ECM evaluation showed enhanced pathogenicity of nahG-transfected parasites and the in vitro results indicated that a deficiency in SA affected production of PGE2 by the parasite. Therefore, we investigated the plasma concentrations of PGE2 and several cytokines influenced by PGE2. Whole blood (6 days post-infection) was collected for quantification of PGE2 and cytokines. We chose to detect the difference of PGE2 and cytokines on the sixth days post-inoculation because at this time point, only nahG-transfected parasites induced ECM. On the contrary, no change in parasitemia or hematocrit was detected as above (Fig 3B–3E), suggesting that the ECM could be induced by the differentially controlled PGE2 and cytokines. However, the plasma PGE2 level of mice infected with nahG-transfected parasites showed no decrease compared with gfp-transfectant mice or uninfected controls (Fig 5A), in contrast to the in vitro experiment.

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Fig 5. Plasma levels of cytokines, chemokines, and prostaglandin E2 (PGE2) in infected mice.

(A) PGE2. (B-F) Proinflammatory cytokines: IFN-γ (B), TNF-α (C), IL-2 (D), IL-1β (E), and IL-12 (F). (G-I) Anti-inflammatory cytokines: IL-10 (G), IL-4 (H), and IL5 (I). (J) Inflammatory chemokine MCP-1. Plasma from heparin-treated whole blood of mice infected by nahG- or gfp-expressing parasites was used in all experiments. Control sample of IFN-γ was analyzed at the different day and indicated by dashed line. Statistics were calculated by Mann-Whitney U-test. Bonferroni correction was used for multiple tests.

https://doi.org/10.1371/journal.pone.0140559.g005

The level of proinflammatory (T helper-1, Th1) cytokines in nahG transfectant-infected mice changed markedly. Interferon (IFN)-γ and interleukin (IL)-2 increased significantly (p < 0.05 and 0.01, Fig 5B and 5D) and IL-1β was also increased by infection with nahG parasites (Fig 5E, p < 0.05). Two other major proinflammatory cytokines, tumor necrosis factor (TNF)-α and IL-12, showed a similar shift, but the change was not significant (Fig 5C and 5F). The concentration of a T helper-2 (Th2) cytokine, IL-10, did not change significantly (Fig 5G), but IL-4 and IL-5 were significantly elevated in mice infected with nahG parasites (Fig 5H and 5I). MCP-1, an inflammatory chemokine, was significantly decreased by nahG-transfectant infection (p < 0.05, Fig 5J).

Discussion

In this study, we found that Plasmodium spp. produces SA and may modulate the host immune response via PG production. The presence of SA was proven by mass spectrometry. Because we did not detect synthetic enzymes or receptors for SA in Plasmodium spp. genomes, we transfected P. falciparum and P. berghei ANKA with a SA-degrading gene, nahG, which was originally identified as a pathogenic factor in plant-infecting Pseudomonas putida. This gene encodes SA hydroxylase, which catalyzes SA to biologically inactive catechol [23]. nahG transfected P. falciparum had an approximately 50% decrease in SA concentration, which was a relatively small effect compared with results for plant transformants [23]. This might reflect differences in the intracellular environment (e.g., ions, pH, etc.) between land plants and Plasmodium spp. The SA concentration did not differ significantly at the 95% confidence level, but mutants of P. falciparum had significantly decreased endogenous production of PGE2, suggesting that the deficiency could be sufficient to explain the role of SA in Plasmodium spp. as a signaling molecule. Thus, production of PGE2 is probably dependent on SA in P. falciparum.

In animals, PGE2 usually functions as an active mediator of inflammation. It is released from whole cells, mainly vascular endothelial cells, fibroblasts and mast cells. Primary onset of fever is triggered through PGE2 receptor 3 expressed by neurons in the median preoptic nucleus [9]. PGE2 has complex activities in inflammation. It induces fever and pain, and has suppressive effects on both innate and acquired immune reactions. During innate immunity, PGE2 suppresses the functions and differentiation of natural killer cells, macrophages, granulocytes, and mast cells [10]. For example, the cytolytic activity of natural killer cells is suppressed by PGE2 through a mechanism involving suppression of IL-2, IL-12, and IL-15 [25,26]. Furthermore, macrophage functions are directly suppressed by PGE2 receptor 2-dependent signaling [27]. In acquired immunity, PGE2 inhibits production of IL-2 by T cells and IL-2 responsiveness [10]. It also suppresses T-cell activation and proliferation, and shifts the pattern of CD4+ T-cell responses from Th1 to Th2 and Th17 cells [10]. PGE2 directly prevents CD4+ T-cell production of IFN-γ, but not Th2 cytokines such as IL-4 and IL-5, in mice and humans [28,29]. Additionally, PGE2 prevents differentiation of CD4+ T-cell to Th1 by suppressing monocyte and dendritic cell production of IL-12 [10, 30]. Because PGE2 has such a complex role in immune activity, we hypothesized that the influence of a decrease in PGE2 in nahG-transfected parasites would lead to a distinct phenotype in vivo.

To investigate the function of parasite SA and induced PGE2, we established nahG-expressing P. berghei ANKA, and found that parasite virulence was significantly enhanced. The cause of death in infected mice was diagnosed as ECM based on the presence of neurological signs, clinical behavior, histological observation, and a dye leakage test. The severe pathogenicity caused by a malaria parasite is characterized by neurological signs in CM caused by sequestration of blood cells or disruption of the BBB in small vessels of the brain. Our result showed that microvessels of the brain, especially in the cerebellum, were sequestrated by many leucocytes and erythrocytes, and that the severity of sequestration was dependent on the transfected gene (nahG or gfp) (Fig 4, S4S7 Figs). Disruption of the BBB was quantified by a dye leakage test, in which the significant difference in leakage indicated a difference in damage to the BBB. These results show that introduction of nahG, a SA-degrading enzyme, significantly enhanced the pathogenicity of P. berghei ANKA in vivo. However, we could not detect a significant difference in SA levels in nahG+ or gfp+ parasite infection, in contrast to the tendency for a decrease in SA level found in vitro. The in vivo environment may have influenced this finding due to egestion from the host or degradation by host metabolism, or the SA effect may have been locally restricted around the parasite.

Together with physical damage to brain vessels, an aggravating role of proinflammatory cytokines such as IFN-γ, TNF-α, and IL-12 in CM development is also widely accepted [31,32]. The mechanisms of CM development have been investigated by clinical observation of human patients and laboratory animal models of ECM. Elevation of serum IFN-γ is associated with the severity of acute malaria in Asian and African patients [33,34], and both in vivo IFN-γ neutralization and knock-out of IFN-γ receptor enhanced resistance against ECM caused by a rodent malarial parasite, P. berghei ANKA [35,36]. TNF-α also has a crucial role in the pathogenesis of CM since administration of an anti-TNF-α antibody completely protected against P. berghei ANKA infection [37]. Our study demonstrated that infection with the nahG-transfectant induced upregulation of proinflammatory cytokines including IFN-γ, IL-1β, IL-2, and IL-12, while parasitemia and hematocrit were almost uniform at the same time point (Fig 3D and 3E). This result suggests that these proinflammatory cytokines might play a role in aggravation of ECM with nahG+ parasite infection.

The different elevation of proinflammatory cytokines suggests that the parasite actively modulates the host immunity. Taking into account the in vitro result, this modulation may occur via PGE2 produced by the parasite, which suppresses host immunity by switching cytokines to a T helper-2 phenotype [10]. Furthermore, PGE2 has a protective role in CM, with an inverse relationship between the concentration of plasma PGE2 and the severity of disease by P. falciparum infection found in a study in Gabonese children [38]. In a mouse model, the protective role of PGE2 was proven by administration of NSAIDs to infected mice [39,40]. These findings indicate the importance of PGE2 in protection against CM, and this effect can be explained by the suppressive activity of PGE2 on production of proinflammatory cytokines [10]. Therefore, elevation of proinflammatory cytokines due to the decrease of PGE2 in nahG+ parasites may have resulted in enhancement of pathogenicity.

Another proinflammatory chemokine, MCP-1, underwent a decrease that differed from the changes in other proinflammatory cytokines. Generally, at the site of infection, MCP-1 is cooperatively elevated with other inflammatory substances and involved in the establishment of inflammation. In addition, release of MCP-1 from mast cells is promoted by PGE2 stimulation [41] and mast cells are thought to be the dominant source of MCP-1 [42]. Thus, the decrease of MCP-1 in nahG+ parasite infection suggests that reduction of PGE2 production by the parasite may allow other cytokine levels to increase, but might reduce MCP-1 induction. Enhanced IL-4 and IL-5 in plasma from nahG-transfectant-infected mice is controversial because Th2 cytokines are generally protective against CM development, especially IL-4. However, a previous study showed that IL-4 was not associated with ECM pathogenesis [43]. Furthermore, few studies have focused on the role of IL-5 in severe malaria, including CM, and IL-5 production did not differ between ECM-susceptible and -resistant mouse strains [44]. The significant induction of IL-4 and IL-5 in the current study might reflect unknown cytokine functions in ECM development, which might be highlighted by nahG+ parasite infection. Further analysis is needed to understand the roles of these cytokines in the pathogenesis of CM.

Plasmodium produces and releases PGs into the infecting milieu. In animals, the key enzyme in PG synthesis is COX, which catalyzes conversion of arachidonic acid to PGH2 [45]. Orthologs of COX have not been identified in the Plasmodium spp. genome in silico. In contrast to mammalian COX, the activity of which is inhibited by SA and other NSAIDs, an unidentified enzyme from Plasmodium spp. catalyzing the same reaction as COX was not inhibited by NSAIDs [11]. The current study showed that SA actually activates PGE2 synthesis, which suggests the presence of a novel signaling pathway. Additionally, we found that the change in plasma PGE2 levels was not significant in mutants of P. berghei ANKA in vivo, despite the in vitro result showing a significant decrease (p < 0.01). This suggests that the mechanism of PGE2 induction is different between P. falciparum and P. berghei ANKA, and the changes in mice mortality and ECM were caused by another mechanism. Otherwise, the difference of PGE2 level could be present only in the infection microenvironment. According to the previous report, the major source of IFN-γ in P. berghei ANKA infection is CD4+ T-cells at the site of infection [46]. Since PGE2 inhibits production of IFN-γ by CD4+ T-cells via direct and indirect ways [10,2830], it is possible that the locally-concentrated PGE2 of parasite at the site of infection could influence the function of immigrating CD4+ T-cells. Investigation of this hypothesis will require analysis using a PGE2-receptor knock-out mouse.

SA was detected by quantitative UPLC-MS/MS for multi-phytohormone detection. This system allows identification of several plant hormones with high specificity [16,17]. The concentrations of SA in P. berghei ANKA and T. gondii were remarkably high compared with that in another phytohormone ABA (~200 nM) [3]. A high level of SA of almost 37 μg/g fresh weight is also found in the rice Oryza sativa, in which the concentration of SA is more than 1000-fold higher than that in the tobacco Nicotiana tabacum [47]. The high SA concentration in parasites is comparable and explains why there was no growth inhibition with exogenous addition of SA. In higher plants, such inhibition occurs when a compound is added at concentrations many times higher than the endogenous level. For example, the 90% inhibitory concentration (IC90) of ABA on O. sativa growth is 10 μM, which is almost 300-fold higher than the native level of 9.2 ng/g fresh weight (approximately 0.03 μM) [48].

SA has been studied for 100 years, but most studies have focused on anti-inflammatory effects in animals. In contrast, there have been few studies of signaling pathways and biosynthesis compared with other plant hormones [49]. The BA and ICS pathways have been identified as SA synthetic routes in higher plant, but the enzymes in these pathways have not been identified in any apicomplexan genome. Additionally, although NPR3 and NPR4 were recently identified as SA receptors [5], we could not identify the orthologs of NPR1, 3, or 4 in any apicomplexan genome database. This may be because of the diversity of higher plants and Apicomplexa or endosymbionts derived from early-branched red algae, as previously suggested for apicomplexan ABA synthesis [3].

In conclusion, a newly detected plant hormone SA may control endogenous PGE2 levels in Plasmodium in vitro. Our studies showed that introduction of nahG in P. berghei ANKA led to early onset of ECM in vivo. From our results, the most reasonable explanation is that this phenomenon is caused by overproduction of proinflammatory cytokines due to suppression of PGE2 production in parasites. This may be a new mechanism of Plasmodium spp.-induced modification of host immunity and this finding increases our understanding of the pathology of this threatening parasite.

Supporting Information

S1 Fig. Construction of a nahG-cmyc2 expression vector for Plasmodium falciparum 3D7.

(A) Promoter of P. falciparum chloroquine-resistant transporter gene (pCRT), terminator of P. berghei DHFR gene (PbDT), and nahG-cmyc2 were amplified by PCR. (B) These fragments were joined by PCR with overlapping primers. The outermost primers contained 3′- and 5′- extensions that correspond to 20 bp sequences of the pCHD43 (II) vector, respectively. (C) The cassette of pCRT-nahG-cmyc2-PbDT was fused into pCHD43 (II) by homologous integration using a Geneart seamless cloning kit (Invitrogen) to give pCRT::nahG-cmyc2 (D).

https://doi.org/10.1371/journal.pone.0140559.s001

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S2 Fig. Insertion of nahG-HA into the Pb230p locus in P. berghei genome.

500 bp sequences corresponding to the 5′ and 3′ regions of the Pb230p gene (which has no known function) were amplified by PCR and inserted into the pL0006 vector. Promoter of P. falciparum chloroquine-resistant transporter gene (pCRT), terminator of P. berghei DHFR gene (PbDT), and nahG-HA were amplified and joined by PCR with overlapping primers. The amplicon was also ligated into pL0006. For the control experiment, gfp-HA was amplified and introduced as for nahG. The constructed vector was cut by SacII and NotI, and electroporated into P. berghei ANKA. Transfectants were selected by pyrimethamine. Expression was confirmed by western blotting with anti-HA antibody (lower panal).

https://doi.org/10.1371/journal.pone.0140559.s002

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S3 Fig. Influence of nahG or gfp expression on the parasite development stage.

Plasmodium falciparum 3D7 expressing nahG or gfp was established as shown in S1 Fig The parasites were cultured in vitro, synchronized twice with 5% sorbitol, and the stages were observed under microscopic observation. Parasites were examined at 72, 96 h after synchronization and the percentage was calculated based on 100 parasites per sample. There was no significant difference between the two transfectants. (p>0.05, Student T test, Bar: SD., n = 4).

https://doi.org/10.1371/journal.pone.0140559.s003

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S4 Fig. Histological observation of mouse brain infected by nahG-HA expressing parasites.

Brains of infected mice were perfused with PBS and fixed with 4% PFA. Sliced sections were stained by hematoxylin and eosin. The file includes pictures of the cerebellum, midbrain, frontal cortex, occipital cortex and olfactory bulb.

https://doi.org/10.1371/journal.pone.0140559.s004

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S5 Fig. Histological observation of mouse brain infected by gfp-HA expressing parasites.

Methods are same as S4 Fig.

https://doi.org/10.1371/journal.pone.0140559.s005

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S6 Fig. Histological observation of uninfected mouse brain.

Methods are same as S4 Fig.

https://doi.org/10.1371/journal.pone.0140559.s006

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S7 Fig. Quantification of sequestrated brain vessels.

Brains of infected or uninfected (control) mice were perfused with PBS and fixed with 4% PFA. Sliced sections were stained by hematoxylin and eosin. Cerebellums of the brains were photographed at the same magnification of S4S6 Figs Five to 10 pictures per mice were counted (n>100 for each picture). A Mann-Whitney U-test with the Bonferroni's correction was used, and significant (p<0.05) differences were denoted by asterisks.

https://doi.org/10.1371/journal.pone.0140559.s007

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Acknowledgments

We thank Dr. Geoffrey I. McFadden (University of Melbourne) for vector pCHDR-3/4; Jacobus Pharmaceuticals for WR99210; Dr. Fumie Kobayashi (Kyorin University), Dr. Mamoru Niikura (Kyorin University) and Dr. Makoto Hirai (Gumma University) for their suggestions and instructions on animal handling; and Dr. Kazuo Ebine (University of Tokyo) for his support for the molecular experiments. We also thank the members of the Nagamune laboratory for valuable discussions. Human serum and RBC were provided by the Japan Red Cross. P. berghei ANKA line (deposited by Dr. T.F. McCutchan, MRA-311) and pL0006 plasmid (deposited by Dr. A.P. Waters, MRA-775) were obtained through the MR4.

Author Contributions

Conceived and designed the experiments: KN. Performed the experiments: RM HA MK MT SBAA HS. Contributed reagents/materials/analysis tools: MK HS. Wrote the paper: RM KN.

References

  1. 1. World Health Organization. The World Malaria Report 2013. Available: http://www.who.int/malaria/publications/world_malaria_report_2014/en/. Accessed 14 January 2015.
  2. 2. Janouskovec J, Horák A, Oborník M, Lukes J, Keeling PJ. A common red algal origin of the apicomplexan, dinoflagellate, and heterokont plastids. Proc Natl Acad Sci U S A. 2010;107: 10949–10954. pmid:20534454
  3. 3. Nagamune K, Hicks LM, Fux B, Brossier F, Chini EN, Sibley LD. Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii. Nature. 2008;451: 207–210. pmid:18185591
  4. 4. Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY, Hunt MD. Systemic Acquired Resistance. Plant Cell. 1996;8: 1809–1819. pmid:12239363
  5. 5. Fu ZQ, Yan S, Saleh A, Wang W, Ruble J, Oka N, et al. NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature. 2012;486: 228–232. pmid:22699612
  6. 6. Lee HI, Leon J, Raskin I. Biosynthesis and metabolism of salicylic acid. Proc Natl Acad Sci U S A. 1995;92: 4076–4079. pmid:11607533
  7. 7. Métraux JP. Recent breakthroughs in the study of salicylic acid biosynthesis. Trends Plant Sci. 2002;7: 332–334. pmid:12167322
  8. 8. Boynton CS, Dick CF, Mayor GH. NSAIDs: an overview. J Clin Pharmacol. 1988;28: 512–517. pmid:3047176
  9. 9. Lazarus M, Yoshida K, Coppari R, Bass CE, Mochizuki T, Lowell BB, et al. EP3 prostaglandin receptors in the median preoptic nucleus are critical for fever responses. Nat Neurosci. 2007;10: 1131–1133. pmid:17676060
  10. 10. Kalinski P. Regulation of immune responses by prostaglandin E2. J Immunol. 2012;188: 21–28. pmid:22187483
  11. 11. Kilunga Kubata B, Eguchi N, Urade Y, Yamashita K, Mitamura T, Tai K, et al. Plasmodium falciparum produces prostaglandins that are pyrogenic, somnogenic, and immunosuppressive substances in humans. J Exp Med. 1998;188: 1197–1202. pmid:9743538
  12. 12. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science. 1976;193: 673–675. pmid:781840
  13. 13. Janse CJ, Ramesar J, Waters AP. High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nat Protoc. 2006;1: 346–356. pmid:17406255
  14. 14. Nagamune K, Moreno SNJ, Sibley LD. Artemisinin-resistant mutants of Toxoplasma gondii have altered calcium homeostasis. Antimicrob Agents Chemother. 2007;51: 3816–3823. pmid:17698618
  15. 15. Rosenthal PJ, McKerrow JH, Aikawa M, Nagasawa H, Leech JH. A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum. J Clin Invest. 1988;82: 1560–1566. pmid:3053784
  16. 16. Kojima M, Sakakibara H. Highly sensitive high-throughput profiling of six phytohormones using MS-probe modification and liquid chromatography-tandem mass spectrometry. Methods Mol Biol. 2012;918: 151–164. pmid:22893291
  17. 17. Kojima M, Kamada-Nobusada T, Komatsu H, Takei K, Kuroha T, Mizutani M, et al. Highly sensitive and high-throughput analysis of plant hormones using MS-probe modification and liquid chromatography-tandem mass spectrometry: an application for hormone profiling in Oryza sativa. Plant Cell Physiol. 2009;50: 1201–1214. pmid:19369275
  18. 18. Zhu X, Yahata K, Alexandre JSF, Tsuboi T, Kaneko O. The N-terminal segment of Plasmodium falciparum SURFIN4.1 is required for its trafficking to the red blood cell cytosol through the endoplasmic reticulum. Parasitol Int. 2013;62: 215–229. pmid:23287798
  19. 19. Sakura T, Yahata K, Kaneko O. The upstream sequence segment of the C-terminal cysteine-rich domain is required for microneme trafficking of Plasmodium falciparum erythrocyte binding antigen 175. Parasitol Int. 2013;62: 157–164. pmid:23268338
  20. 20. Lin JW, Annoura T, Sajid M, Chevalley-Maurel S, Ramesar J, Klop O, et al. A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites. PLoS One. 2011;6: e29289. pmid:22216235
  21. 21. Carroll RW, Wainwright MS, Kim K- Y, Kidambi T, Gómez ND, Taylor T, et al. A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria. PLoS One. 2010;5: e13124. pmid:20957049
  22. 22. Penet MF, Abou-Hamdan M, Coltel N, Cornille E, Grau GE, de Reggi M, et al. Protection against cerebral malaria by the low-molecular-weight thiol pantethine. Proc Natl Acad Sci U S A. 2008;105: 1321–1326. pmid:18195363
  23. 23. Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, et al. Requirement of salicylic Acid for the induction of systemic acquired resistance. Science. 1993;261: 754–756. pmid:17757215
  24. 24. White NJ, Turner GD, Medana IM, Dondorp AM, Day NP. The murine cerebral malaria phenomenon. Trends Parasitol. 2010;26: 11–15. pmid:19932638
  25. 25. Walker W, Rotondo D. Prostaglandin E2 is a potent regulator of interleukin-12- and interleukin-18-induced natural killer cell interferon-gamma synthesis. Immunology. 2004;111: 298–305. pmid:15009430
  26. 26. Joshi PC, Zhou X, Cuchens M, Jones Q. Prostaglandin E2 suppressed IL-15-mediated human NK cell function through down-regulation of common gamma-chain. J Immunol. 2001;166: 885–891. pmid:11145664
  27. 27. Aronoff DM, Canetti C, Peters-Golden M. Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP. J Immunol. 2004;173: 559–565. pmid:15210817
  28. 28. Betz M, Fox BS. Prostaglandin E2 inhibits production of Th1 lymphokines but not of Th2 lymphokines. J Immunol. 1991;146: 108–113. pmid:1845802
  29. 29. Snijdewint FG, Kaliński P, Wierenga EA, Bos JD, Kapsenberg ML. Prostaglandin E2 differentially modulates cytokine secretion profiles of human T helper lymphocytes. J Immunol. 1993;150: 5321–5329. pmid:8390534
  30. 30. van der Pouw Kraan TC, Boeije LC, Smeenk RJ, Wijdenes J, Aarden LA. Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production. J Exp Med. 1995;181: 775–779. pmid:7836930
  31. 31. Hunt NH, Grau GE. Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol. 2003;24: 491–499. pmid:12967673
  32. 32. Idro R, Marsh K, John CC, Newton CR. Cerebral malaria: mechanisms of brain injury and strategies for improved neurocognitive outcome. Pediatr Res. 2010;68: 267–274. pmid:20606600
  33. 33. Ho M, Sexton MM, Tongtawe P, Looareesuwan S, Suntharasamai P, Webster HK. Interleukin-10 inhibits tumor necrosis factor production but not antigen-specific lymphoproliferation in acute Plasmodium falciparum malaria. J Infect Dis. 1995;172: 838–844. pmid:7658079
  34. 34. Ringwald P, Peyron F, Vuillez JP, Touze JE, Le Bras J, Deloron P. Levels of cytokines in plasma during Plasmodium falciparum malaria attacks. J Clin Microbiol. 1991;29: 2076–2078. pmid:1774338
  35. 35. Amani V, Vigário AM, Belnoue E, Marussig M, Fonseca L, Mazier D, et al. Involvement of IFN-gamma receptor-medicated signaling in pathology and anti-malarial immunity induced by Plasmodium berghei infection. Eur. J Immunol. 2000;30: 1646–1655. pmid:10898501
  36. 36. Yañez DM, Manning DD, Cooley AJ, Weidanz WP, van der Heyde HC. Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria. J Immunol. 1996;157: 1620–1624. pmid:8759747
  37. 37. Grau GE, Fajardo LF, Piguet PF, Allet B, Lambert PH, Vassalli P. Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science. 1987;237: 1210–1212. pmid:3306918
  38. 38. Perkins DJ, Kremsner PG, Weinberg JB. Inverse relationship of plasma prostaglandin E2 and blood mononuclear cell cyclooxygenase-2 with disease severity in children with Plasmodium falciparum malaria. J Infect Dis. 2001;183: 113–118. pmid:11076710
  39. 39. Ball HJ, MacDougall HG, McGregor IS, Hunt NH. Cyclooxygenase-2 in the pathogenesis of murine cerebral malaria. J Infect Dis. 2004;189: 751–758. pmid:14767831
  40. 40. Xiao L, Patterson PS, Yang C, Lal AA. Role of eicosanoids in the pathogenesis of murine cerebral malaria. Am J Trop Med Hyg. 1999;60: 668–673. pmid:10348246
  41. 41. Nakayama T, Mutsuga N, Yao L, Tosato G. Prostaglandin E2 promotes degranulation-independent release of MCP-1 from mast cells. J Leukoc Biol. 2006;79: 95–104. pmid:16275896
  42. 42. Carollo M, Hogaboam CM, Kunkel SL, Delaney S, Christie MI, Perretti M. Analysis of the temporal expression of chemokines and chemokine receptors during experimental granulomatous inflammation: role and expression of MIP-1alpha and MCP-1. Br J Pharmacol. 2001;134: 1166–1179. pmid:11704636
  43. 43. Yañez DM, Manning DD, Cooley AJ, Weidanz WP, van der Heyde HC. Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria. J Immunol. 1996;157: 1620–1624. pmid:8759747
  44. 44. de Kossodo S, Grau GE. Profiles of cytokine production in relation with susceptibility to cerebral malaria. J Immunol. 1993;151: 4811–4820. pmid:8409439
  45. 45. Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol. 2011;31: 986–1000. pmid:21508345
  46. 46. Villegas-Mendez A, Greig R, Shaw TN, de Souza JB, Gwyer Findlay E, Stumhofer JS, et al. IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain. J Immunol. 2012;189: 968–979. pmid:22723523
  47. 47. Raskin I, Skubatz H, Tang W, Meeuse BJD. Salicylic acid levels in thermogenic and non-thermogenic plants. Annals Botany. 1990;66: 369–373.
  48. 48. Hoffmann-Benning S, Kende H. On the role of abscisic acid and gibberellin in the regulation of growth in rice. Plant Physiol. 1992;99: 1156–1161. pmid:16668983
  49. 49. Kelley DR, Estelle M. Ubiquitin-mediated control of plant hormone signaling. Plant Physiol. 2012;160: 47–55. pmid:22723083