Accepted Manuscript

Title: Differences in the modulation of reactive species, lipid

bodies, cyclooxygenase-2, 5-lipoxygenase and PPAR- in
cerebral malaria-susceptible and resistant mice

Author: Tatiana K.S. Borges Erica

A.R. Alves Henda A.R.
Vasconcelos Fabiana P. Carneiro Andre M. Nicola Kelly G.
Magalhaes Maria Imaculada Muniz-Junqueira
PII:
DOI:
Reference:

S0171-2985(16)30439-9
http://dx.doi.org/doi:10.1016/j.imbio.2016.11.010
IMBIO 51584

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15-11-2016

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Differences in the modulation of reactive species, lipid bodies, cyclooxygenase-2,

5-lipoxygenase and PPAR- in cerebral malaria-susceptible and resistant mice

Tatiana K.S. Borgesa, rica A.R. Alvesa,b, Henda A.R. Vasconcelosa, Fabiana P. Carneiroc, Andr
M. Nicolaa, Kelly G. Magalhesd, Maria Imaculada Muniz-Junqueiraa*

Laboratory of Cellular Immunology, Pathology, Faculty of Medicine, University of Brasilia,

Brasilia, Campus Darcy Ribeiro, Brasilia, Distrito Federal 70.910.900, Brazil.

b

Laboratory of Cellular and Molecular Immunology, Ren Rachou Research Center, Belo

Horizonte, Minas Gerais 31090002, Brazil.

c

Laboratory of Pathology, Pathology, Faculty of Medicine, University of Brasilia, Campus Darcy

Ribeiro, Brasilia, Distrito Federal 70.910.900, Brazil.

d

Laboratory of Immunology and Inflammation, Department of Cellular Biology, Biology Institute,

University of Brasilia, Campus Darcy Ribeiro, Brasilia, Distrito Federal 70.910.900, Brazil.

*Corresponding author: Maria Imaculada Muniz-Junqueira. Laboratory of Cellular Immunology,

Pathology, Faculty of Medicine, University of Brasilia, Brasilia, Campus Darcy Ribeiro, Brasilia,
Distrito Federal 70.910.900, Brazil. Tel.: +55 61 3107-1934.
E-mail: mimjunqueira@unb.br; mimjunqueira@gmail.com (M.I. Muniz-Junqueira).

Graphical Abstract

Highlights
1. Eicosanoid-producing-enzymes and PPAR- were assessed in cerebral malaria-susceptible and resistant mice
2. P. berghei ANKA infection increased lipid body biogenesis in all mouse strains
3. COX-2 and 5-LOX enhanced in brain tissue only in cerebral malaria-susceptible mice.
4. PPAR- translocate from cytoplasm-to-nucleus only in cerebral malaria-resistant mice.

ABSTRACT
Proinflammatory responses are associated with the severity of cerebral malaria. NO, H2O2,
eicosanoid and PPAR- are involved in proinflammatory responses, but regulation of these factors
is unclear in malaria. This work aimed to compare the expression of eicosanoid-forming-enzymes in
cerebral malaria-susceptible CBA and C57BL/6 and -resistant BALB/c mice. Mice were infected
with Plasmodium berghei ANKA, and the survival rates and parasitemia curves were assessed. On
the sixth day post-infection, cyclooxygenase-2 and 5-lipoxygenase in brain sections were assessed
by immunohistochemistry, and, NO, H2O2, lipid bodies, and PPAR- expression were assessed in
peritoneal macrophages. The C57BL/6 had more severe disease with a lower survival time, higher
parasitemia and lower production of plasmodicidal NO and H2O2 molecules than BALB/c.
Enhanced COX-2 and 5-LOX expression was observed in brain tissue cells and vessels from
C57BL/6 mice, and these mice expressed higher constitutive PPAR- levels. There was no
translocation of PPAR- from cytoplasm to nucleus in macrophages from these mice. CBA mice
had enhanced COX-2 expression in brain tissue cells and vessels and also lacked PPAR-
cytoplasm-to-nucleus translocation. The resistant BALB/c mice presented higher survival time,
lower parasitemia and higher NO and H2O2 production on the sixth day post-infection. These mice
did not express either COX-2 or 5-LOX in brain tissue cells and vessels. Our data showed that
besides the high parasite burden and lack of microbicidal molecules, an imbalance with high COX2 and 5-LOX eicosanoid expression and a lack of regulatory PPAR- cytoplasm-to-nucleus
translocation in macrophages were observed in mice that develop cerebral malaria.

Keywords: Eicosanoids, Hydrogen Peroxide, Inflammatory response, Lipid bodies, Malaria, Nitric
Oxide, PPAR-

1. Introduction
Malaria remains one of the most important public health problems in vast regions of the world
and a major cause of morbidity and mortality, mainly due to the severe forms of Plasmodium
falciparum infection (Maitland and Marsh, 2004; Lapouble et al. 2015; WHO, 2015). Unregulated
production of nitrogen and oxygen radicals has been considered among the main pathophysiological
mechanisms producing cerebral malaria (Schofield and Grau, 2005; Muniz-Junqueira et al., 2005;
Muniz-Junqueira and Tosta, 2007; Idro et al., 2010). NO and ROIs might crosstalk and signal in
eicosanoid-responsive pathways, which are also regulated by PPAR- (Kim, 2011; Morgan and Liu,
2011). Eicosanoids modulate the pro- and anti-inflammatory immune response, a key determinant
for the pathogenesis of cerebral malaria (Maitland and Marsh, 2004; Muniz-Junqueira and Tosta,
2007). PPARs are eicosanoid-activated receptors regulated by TNF-, a key molecule involved in
cerebral malaria development (Wymann and Schneiter, 2008; Ye, 2008). So that, a better
understanding of the production of nitrogen and oxygen radicals, eicosanoids and PPAR- in
cerebral malaria-susceptible CBA and C57BL/6 and -resistant BALB/c mice may help us
understand the phatophysiologycal pathways determining severe disease.
There have been controversial reports about the roles of NO and ROIs and related molecules in
malaria. Although reactive nitrogen and oxygen intermediates, as effector molecules of
macrophages in antimalarial defense, have been associated with protection against cerebral malaria,
these molecules might also contribute to disease pathogenesis through their cytotoxic effects and
have been suggested to be potentially related to the development of severe disease (MunizJunqueira et al., 2005). Furthermore, NO and ROS might crosstalk and signal in several metabolic
pathways, such as eicosanoids pathways, and they might also regulate and be regulated by several
molecules in these pathways (Morgan and Liu, 2011).
The eicosanoids, including prostaglandins and leukotrienes, are signaling molecules derived
from the enzymatic oxygenation of arachidonic acid (Bozza et al., 2011; Rogerio and Anibal, 2012;
Gil-de-Gmez et al., 2013). Leukocyte lipid bodies are intracellular organelles that control the
5

synthesis and secretion of several inflammatory mediators (Bozza et al., 2009). An increase in the
number of lipid bodies correlates with the increased generation of eicosanoids (Bozza et al., 2011).
However, there have been no reports about lipid bodies in macrophages during malaria infection.
Proinflammatory cytokine expression is enhanced in cerebral malaria, and proinflammatory
conditions affect lipid body formation. Moreover, these conditions can activate cyclooxygenases
(COX) and lipoxygenases (LOX), which can stimulate the production of eicosanoids such as
prostaglandins, leukotrienes and lipoxins by oxygenating arachidonic acid. These eicosanoidsignaling molecules can interfere with host defense and inflammation (Wymann and Schneiter,
2008).
At high concentrations, prostaglandin E2 (PGE2) is a potent inhibitor of Th1-type responses and
TNF and NO production and thus decreases the inflammatory response that is a key factor in
cerebral malaria. Therefore, it is possible that prostaglandins play a role in preventing the cerebral
disease that depends on the inflammatory response in malaria. In fact, it has been suggested that the
induction of COX-2 expression and prostaglandin synthesis might be protective in cerebral malaria
(Ball et al., 2004). On the other hand, leukotrienes are proinflammatory molecules that originate
from another arachidonic acid metabolism pathway; therefore, it is feasible that leukotrienes might
induce pathological cerebral involvement in malaria. The production of these molecules with
opposing functions from arachidonic acid depends on the cellular pathway that is stimulated; the
cyclooxygenase pathway produces prostaglandins, and the lipoxygenase pathway produces
leukotrienes. However, 5-lipoxygenase deficient C57BL/6 mice infected with P. berghei ANKA
had accelerated death and increased brain inflammation (Shryock et al., 2013), suggesting that this
pathway play also a role in evolution of the infection by P. berghei Anka. However, little is known
about whether and which of these pathways are activated in experimental severe malaria.
Eicosanoid products can interact biochemically at various levels with reactive species such as
nitric oxide and hydrogen peroxide (Weigel et al., 1997; Baker et al., 2009; Kim, 2011). The
nuclear receptor PPAR-, a member of the peroxisome proliferator-activated receptor family, is a
6

master transcriptional regulator of lipid metabolism (Ye, 2008). The peroxisome is an organelle that
contains the machinery with which to oxidize fatty acids and is a site of H2O2 production and
destruction (Wymann and Schneiter, 2008). PPARs are eicosanoid-activated receptors that induce
peroxisome formation (Wymann and Schneiter, 2008), and PPAR- activity is regulated by TNF-
(Ye, 2008), a key molecule involved in cerebral malaria development. PPAR- is a nuclear receptor
that heterodimerizes with the 9-cis-retinoic acid nuclear receptor retinoid X receptors (RXRs) upon
translocation to the cell nucleus to regulate gene expression. The natural endogenous ligands of
PPARs include fatty acids and eicosanoids, while pathogens such as Plasmodium can act as direct
or indirect exogenous ligands and trigger the translocation of PPAR from the cytoplasm to the
nucleus, where it suppresses the expression of genes involved in proinflammatory cytokine
production (Balanchandar and Katyal, 2011).
Pathophysiological mechanisms causing cerebral involvement may differ in patients with
malaria, as shown by the range of histopathology observed in brain tissue of individuals that died
from cerebral malaria (Maneerat et al., 2000; Clark et al., 2003; Dorovini-Zis et al., 2011). In
addition, nitric oxide has been shown to be increased (Maneerat et al., 2000; Clark et al., 2003) or
not significantly different (Clark et al., 2003) in brain tissue of these patients. The same is observed
in experimental cerebral malaria models, whereas the Plasmodium berghei infected-C57BL/6 mice
show low availability of NO (Gramaglia et al., 2006), the Plasmodium berghei infected-CBA mice
show increased production of this molecule (Muniz-Junqueira et al., 2005), and both models
develop cerebral disease. These facts suggest cerebral malaria is not a homogeneous syndrome but
rather a heterogeneous spectrum of severe disease with multiple points of variation. Therefore, the
downstream mechanisms causing cerebral involvement, mainly production of inflammatory
mediators, should differ in different individuals. Taking into consideration that different pathways
of eicosanoids differently regulate the balance between pro- and anti-inflammatory immune
responses, we hypothesized that these pathways might be differentially activated in each malaria
experimental model.
7

The present work aimed to compare the expression of eicosanoid-forming-enzymes in cerebral

malaria-susceptible CBA and C57BL/6 and -resistant BALB/c mice in order to broaden the
understanding of the immunopathogenic mechanisms that control the inflammatory responses in
mouse strains that develop different forms of malaria upon infection with the same parasite,
Plasmodium berghei ANKA.

To test whether different experimental cerebral malaria models

trigger differently the eicosanoid pathways or not, we compared CBA with C57BL/6 mice, which
are both susceptible to cerebral involvement. In addition, to test whether experimental cerebral
malaria-susceptible or -resistant mice trigger differently the eicosanoid pathways or not, these
pathways were compared among mice susceptible to cerebral malaria (CBA and C57BL/6) with the
BALB/c mice, which are resistant to cerebral disease.

2. Materials and methods

2.1. Ethics Statement
This study was conducted in strict accordance with the recommendations of the Brazilian
National Council for the Control of Animal Research (CONCEA) and all efforts were made to
minimize suffering. The Animal Research Ethical Committee of the University of Brasilia approved
the experimental protocol for this study (process number: 43050/2010).

2.2. Study groups

Groups of 812-week-old male infected or non-infected C57BL/6 and CBA mice, which are
susceptible to cerebral malaria, and BALB/c mice, which are resistant to cerebral involvement, were
evaluated to compare the pathways that regulate the P. berghei ANKA infection-induced
inflammatory response in different genetic backgrounds. All animals were bred under strict
cleaning conditions and housed in groups of five, in plastic cages, with autoclaved food and filtered
and autoclaved water ad libitum in a room with a 12 h light/dark cycle. The following study groups
were assessed: Group iC57 included C57BL/6 mice that were intraperitoneally inoculated on the
8

first day with 106 P. berghei ANKA infected-erythrocytes in a 100 l volume, Group iCBA
included CBA mice that were intraperitoneally inoculated on the first day with 106 P. berghei
ANKA infected-erythrocytes in a 100 l volume, and Group iBAL included BALB/c mice that
were intraperitoneally inoculated on the first day with 106 P. berghei ANKA infected-erythrocytes
in a 100 l volume. The following control groups were also assessed: C57, CBA and BAL mice that
were intraperitoneally inoculated on the first day with 100 l of saline.
These animals were then separated into subgroups: 43 mice were included in the survival rate
evaluation, and, in a second subgroup, mice were infected or not and sacrificed on the sixth day of
follow-up to assess peritoneal macrophage functions (n = 10) and perform cerebral tissue analyses
(n = 5). To evaluate phagocyte function, peritoneal macrophages were obtained by peritoneal
washing and quantified, after which the viability was assessed with nigrosin solution (> 95%
viability); the nitric oxide and hydrogen peroxide production, lipid bodies, cyclooxygenase-2, 5lipoxygenase and PPAR- expression were subsequently assessed. Immediately before obtaining
macrophages to these studies, the animals were euthanized in a CO2 chamber.
Cerebral tissue was obtained to evaluate cyclooxygenase-2 and 5-lipoxygenase expression by
immunohistochemistry, and the microglial cells were separated to evaluate cyclooxygenase-2 and 5lipoxygenase expression. To avoid pain during perfusion procedure, all animals from
immunohistochemistry experiments were anaesthetized with ketamine-xylazine and after they were
euthanized in a CO2 chamber. In all of the infected mice, parasitemia was evaluated immediately
after sacrifice to ensure infection.
To evaluate the fatality rates in the three different genetic backgrounds, a comparative survival
curve with 15 C57BL/6 mice, 10 CBA mice and 18 BALB/c mice was generated, and the number
of dead mice was registered twice daily until the death of the last animal. The animals died as a
direct result of the infection. Humane endpoints were not possible to use during the survival study
to avoid bias because differences in survival time among groups are small.

Parasitemia was determined on days 3, 5, 8, 15 and 21 post-infection by staining of thin-blood

smears prepared from tail vein blood with 10% Giemsa solution. Percentage of parasitized red
blood cells was assessed by counting at least 500 red blood cells per slide.
P. berghei ANKA-infected erythrocytes were kept frozen with liquid nitrogen and were used to
infect the mice in the study groups only after the parasites had been recovered twice during in vivo
infections.

2.3. Nitrite production

Nitric oxide production by peritoneal macrophages was indirectly assessed by means of nitrite
(NO2) determination (Muniz-Junqueira et al., 2005). The nitrite (NO2) quantification was
individually evaluated for each mouse (n = 10 per group) and performed in triplicate from the
supernatants of 1.5 x 105 peritoneal macrophages per well of a 96-well flat bottom microplate (TTP,
Trasadingen, Switzerland) after a 24-h incubation in a humidified chamber at 37 C and in the
presence of 5% CO2 in air. The supernatants (100 l) were incubated with equal volumes of Griess
reagent (1% sulphanilamide/0.1% N-1-naphthylethylene diamine dihydrochloride/2.5% H3PO4) at
room temperature (RT) for 10 min. The absorbance was read on a SpectraMax plus 384 microplate
reader (Molecular Devices, Sunnyvale, CA, USA), at 540 nm. The results were expressed as M
NO2.

2.4. Hydrogen peroxide production

Hydrogen peroxide (H2O2) production by peritoneal macrophages was assessed according to
the horseradish peroxidase-dependent phenol red oxidation method (Alves et al, 2015). Briefly,
macrophages were individually obtained from each mouse (n = 10 per group), and triplicate
samples of 1.5 x 105 cells in RPMI 1640 (Sigma) were placed into each well of 96-well plastic
microplates (TPP). After a 1 h incubation with a solution containing 5.5 mM dextrose, 0.5 mM
phenol red and 19 U/ml of horseradish peroxidase type 2 RZ 1.3 (Sigma, St. Louis, MO, USA), as
10

well as 20 nM phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO, USA), the reaction
was stopped by adding 10 l of 1 N NaOH solution per well. The absorbance was read at 620 nm
on a microplate reader (SpectraMax plus 384; Molecular Devices, Sunnyvale, CA, USA). The
results were expressed as MH2O2 / 1.5 x 105 macrophages/h.

2.5. Immunofluorescent staining of macrophages for lipid bodies and PPAR-

To analyze the lipid bodies and PPAR- expression, macrophages from five mice were pooled,
and 106 macrophages/ml in RPMI 1640 (Sigma) were incubated for 4 hours in duplicate on 13-mm
round glass coverslips in 24-well flat-bottom microplates (TPP) in a humidified chamber at 37C
and 5% CO2 in air. The experiment was repeated twice. After cell adhesion, the wells were washed
with PBS, pH 7.2, and the cells were fixed in 4% paraformaldehyde for 10 min at RT. The wells
were again washed, and the cells were permeabilized with 0.2% Triton X-100 in PBS for 20 min.
After washing, blocking was performed with 2% bovine serum albumin in PBS for 20 min.
For BODIPY staining, cells were incubated for 24 h with the fluorophore lipophilic 4,4difluoro-third-fourth-diazo-s-indacene (BODIPY Lipid Probes; Molecular Probes/Invitrogen,
Eugene, OR, USA), which labels neutral lipids. For PPAR- staining, the cells were incubated with
a rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and after a 24 h
incubation, the wells were washed three times with PBS, pH 7.2, and the cells were treated with an
Alexa Fluor 546-conjugated anti-rabbit secondary antibody for 1 h. After washing, the cells were
treated with a 1:5000 solution of DAPI in PBS for 5 min, washed and mounted with an anti-fade
medium (Prolong Gold; Invitrogen, Eugene, OR, USA). To exclude nonspecific binding, in all
immunostaining experiments, it was used a control in which the slides were incubated with
antibody diluent, without the primary antibody. This was followed by incubation with secondary
antibodies and detection reagents (Burry, 2011). Fluorescent images were captured on a Leica SP5
confocal microscope and processed with LAS AF software, version 2.6.0 (Leica Microsystems,
Mannheim, Baden-Wrttemberg, Germany). A 1.26 Airy pinhole (246.0 m), a scan speed of 100
11

Hz and averaging of three lines and one frame were used. The photomultiplier tube gain and offset
were adjusted to yield sub-saturating fluorescence intensity with an optimal signal-to-noise ratio.

2.6. Fluorescence image analysis

To quantify the total, cytoplasmic and nuclear PPAR- levels in the macrophages, a PPAR-
image analysis method was adapted from Alves et al. (2015) and performed with ImageJ 1.47q
software (http://rsb.info.nih.gov/ij). Briefly, from each study group, five homogeneous high-power
fields were selected for analysis, and the DAPI and PPAR- fluorescence images were acquired in
separate image files. In the DAPI images, a median filter with a 33 pixel radius was applied to
remove noise and to approximate the staining intensity distribution to a median value. Next, the
DAPI images were thresholded using the Isodata algorithm to generate a binary mask that included
all above-background fluorescence data. The resulting DAPI mask was applied by the image
calculator to the original PPAR- image to obtain the nuclear PPAR- data. Next, the total PPAR-
level was obtained by analyzing the original PPAR- image. The nuclear and total PPAR- values
generated from the histograms were exported to Microsoft Excel, and the cytoplasmic PPAR- was
obtained by subtracting the nuclear PPAR- from the total PPAR- values. To exclude nonspecific
binding, five high-power fields from control macrophages (stained only with the secondary Alexa
Fluor 546-conjugated antibody) were similarly analyzed, and the mean values for the total, nuclear
and cytoplasmic PPAR- levels were obtained and discounted from the previously analyzed images.
The results were expressed as the mean fluorescence intensity. To avoid bias due to differences in
the numbers of analyzed cells, the total, nuclear and cytoplasmic fluorescence intensities of PPAR-
for each image were expressed per 100 cells.

2.7. Oil red staining of peritoneal macrophages

Macrophages were stained with oil red to quantify lipid bodies. Briefly, peritoneal macrophages
were individually analyzed in duplicate for each animal (n = 9 per group). Macrophages (2 x
12

105/well) were allowed to adhere on 13-mm round glass coverslips in 24-well flat-bottom
microplates for 4 h, after which the non-adherent cells were removed by washing. The cells were
fixed with 4% paraformaldehyde in PBS for 30 min, washed twice for 5 min each with PBS, rinsed
once with 60% isopropanol and stained for 15 min with freshly prepared 0.5% Oil Red O in 80%
isopropanol. Next, the cells were washed once with 60% isopropanol and twice with distilled water,
and the nuclei were stained with Mayers hematoxylin for 5 min. The cells were again washed twice
with distilled water, and the coverslips were mounted with glycerin jelly. Light microscopy analysis
revealed that positively stained macrophages presented red-stained lipid bodies in the cytoplasm.
The lipid body index (LBI) was calculated as the average number of lipid bodies expressed by a
macrophage multiplied by the percentage of cells that expressed lipid bodies (adapted from MunizJunqueira et al. 2003).

2.8. Brain sections for immunohistochemistry

On the sixth day post-infection, mice were anaesthetized with 150 mg/kg of ketamine and 7.5
mg/kg of xylazine and sacrificed by intracardiac perfusion with 10 ml of 4% paraformaldehyde and
5 mM EDTA in PBS. Immediately, the brains were dissected from the skulls, washed with PBS, pH
7.2 and fixed for 24 h in 4% paraformaldehyde. Next, the brains were embedded in paraffin and
sliced into 5-m coronal sections that were located approximately in the bregmatic-2 and -3
regions; according to the guide atlas of mouse brain stereotaxic coordinates, described by Paxinos
and Franklin (2008), this area comprises the hippocampal regions and the motor cortex, visual and
auditory regions. For each animal, four samples spaced 20 m from each other were mounted on
two slides. To assess non-specific binding for COX-2 and 5-LOX, two other slides per animal were
also processed without the primary antibody.
Paraffin sections were placed on sylanized slides and subsequently dewaxed and rehydrated
with distilled water. Heat-induced antigen retrieval with 0.2% Tween 20 in citrate buffer, pH 6.0,
was used for antigen retrieval. Endogenous peroxidases were quenched with 10% hydrogen
13

peroxide in distilled water after the sections were cooled. The sections were washed with 0.2%
Tween 20 in Tris-buffered saline (TBS) and incubated for 24 h at 8C in a humidified chamber with
goat polyclonal anti-5-LOX IgG (1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA,
USA) or goat polyclonal anti-COX-2 IgG (1:100; Santa Cruz Biotechnology Inc.) in a 1% BSA in
TBS solution. No primary antibody was added in background control slides. Next, the sections were
washed with 0.2% Tween 20 in TBS and incubated for 1 h with a biotinylated anti-goat IgG (1:200;
Invitrogen, Carlsbad, CA, USA). The slides were washed again, incubated with a streptavidinperoxidase complex (Spring Bioscience Inc., Pleasanton, CA, USA) in TBS for 30 min at RT and
developed for visualization with a commercial buffer that contained diaminobenzidine and
hydrogen peroxide (Dako North America Ink, Carpinteria, CA, USA). The slides were
counterstained with hematoxylin prior to mounting with Entellan (Merck, Darmstadt, Germany).
Brown-stained cells were considered positive. To quantify COX-2 and 5-LOX expression in brain
tissues, 500 cells were analyzed in the total section area per animal, and the number of brownstained cells was expressed as a percentage.
The blood vessels were also analyzed according to a staining pattern based on the method of
Deininger et al. (2000). Briefly, pattern zero was considered to be no visible staining around and
inside the vessel, pattern 1 was defined as staining only on endothelial cells, pattern 2 was defined
as staining on endothelial cells and partially within the vessel and pattern 3 was defined as a fully
and strongly marked vessel. A total of 30 vessels per animal in the brain, comprising the cortex,
hippocampus and thalamus, were analyzed. Images were captured with an Aperio ScanScope
slide scanner and analyzed with ImageScope software, version 11.2.0.780 (Aperio Technologies
Inc., Vista, CA, USA).

2.9. Microglia harvest procedure

Microglia were obtained according to the method described by Moussaud and Draheimb
(2010). Briefly, aseptically removed brains were rinsed with PBS, pH 7.2, finely minced with a
14

scalpel and treated with a solution of 20 U/ml of papain (Sigma), 0.5 mM EDTA, 25 mM glucose in
5 ml RPMI 1640 for 90 min with continuous stirring at 37C and 5% CO2. The enzymatic reaction
was then quenched by the addition of 10 ml of 10% heat-inactivated fetal calf serum (FCS) in
RPMI 1640. The tubes were centrifuged at 200 x g for 7 min at RT, and the pellets were suspended
in 0.5 mg/ml of protease inhibitor (Complete Mini; Roche, Mannheim, Germany) in 2 ml of RPMI
1640 and incubated for 5 min at RT. The tissues were then gently disrupted with a syringe. The
homogenates were filtered through a 70-m nylon filter, and the resulting cell suspensions were
centrifuged again at 200 x g for 7 min at RT. The cells were subsequently suspended in 7.5 ml of
70% stock isotonic Percoll in HBSS (Pharmacia, Uppsala, Sweden). The cell suspension was
carefully overlaid with 7.5 ml of pure HBSS and centrifuged at 200 x g for 20 min. Next, the pellets
were washed once in RPMI 1640 with 10% FCS and 1 M EDTA. The resulting cells were counted
and the viability, which was assessed with nigrosin, always exceeded 95%.

2.10.

Cyclooxygenase-2 and 5-lipoxygenase expression in peritoneal macrophages and microglia

Cyclooxygenase-2 and 5-lipoxygenase expression in the peritoneal macrophages and microglia

were evaluated by flow cytometry. The cells were suspended to 2.5x105 and incubated in conical
tubes with 1% bovine serum albumin and 1 M EDTA in PBS (PBS/BSA) for 20 min. Next, the
cells were incubated for 2 h with 0.5 l of a rabbit polyclonal IgG anti-mouse F4/80 (Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA) in PBS/BSA. After washing with PBS, the cells were
again washed twice with PBS/BSA and then permeabilized with 0.1% triton X-100 (Sigma) in
PBS/BSA for 30 min, washed again and incubated for 2 h at RT with a goat polyclonal anti-5-LOX
IgG (Santa Cruz Biotechnology Inc.) or goat polyclonal anti-COX-2 IgG (Santa Cruz
Biotechnology Inc.). The cells were incubated again for 1 h at RT with the following secondary
antibodies diluted in PBS/BSA/Triton: anti-rabbit-APC or anti-rabbit-FITC (Southern Biotech,
Birmingham, AL, USA) for anti-F4/80 and anti-goat-PE (Southern Biotech) for COX-2 or 5-LOX.
Finally, the cells were washed twice and suspended in a final volume of 200 l. LPS (10 ng/ml)
15

(Sigma) stimulated cells were used as a positive control. Pilot experiments showed that there was
minimal non-specific binding and antibody cross-reactivity. The samples were analyzed on a BD
FACSCaliburTM flow cytometer (Becton Dickinson Corporation, Franklin Lakes, NJ, USA) that was
equipped with 488-nm and 633-nm lasers. The flow cytometric data were analyzed with FlowJo
software (Tree Star Inc., Ashland, OR, USA).

2.11.

Statistical analysis

The data were previously tested with Bartletts test for equal variances and the KolmogorovSmirnov test for normality of distribution. Statistical analyses were performed with the ANOVA
test, followed by the Student-Newman-Keuls method to compare multiple unrelated normal
samples. The Kruskal-Wallis test followed by Dunns method was used to compare multiple
unrelated non-normal samples. The Students t test was performed to compare two normal unrelated
samples.

For non-normal distributions or small groups, the Mann-Whitney test was used to

compare two unrelated groups. Students t test or Mann-Whitney test were used when it was
compared control and infected mice from the same strain. Kaplan-Meier curves were used to
represent survival, and the Mantel-Cox log-rank test was used to compare survival differences; the
p value in multiple comparison test for comparing survival curves was adjusted by the Bonferroni
method, considering three groups. The results were expressed as medians, quartiles and extreme
values for data with non-normal distributions and as the means SEM for data with normal
distributions. Differences with two-tailed values of P < 0.05 were considered statistically
significant. The Prism 5 software package (GraphPad Software, Inc., San Diego, CA, USA; 2005)
was employed for the statistical tests and graphical presentation of the data.

3. Results
3.1. C57BL/6 mice had a lower survival rate than that of CBA and BALB/c mice

16

The differences in survival rates following infection with the different strains were evaluated
with a cumulative percentage curve according to the Kaplan-Meier method, and the significant
differences between the groups were evaluated with the log-rank test (Mantel-Cox) (8.3, P =
0.016). The two models with cerebral disease (C57BL/6 and CBA) died earlier than did the mice
without cerebral involvement (BALB/c). However, while the C57BL/6 P. berghei infected-mice
began to die on day 6 post-infection, those in the CBA group began to die two days later (on day 8
post-infection). On the tenth day post-infection, only 57% of the C57BL/6 mice remained alive,
while 80% of the BALB/c and 70% of the CBA mice remained alive. Furthermore, all mice in the
C57BL/6 group had died by day 16 post-infection, while four days later, on day 20 post-infection,
all mice in the CBA group had died. Furthermore, while all cerebral malaria-susceptible mice had
died by the twentieth day post-infection, 45% of the BALB/c mice remained alive at this time. The
BALB/c mice began to die on day 8, but all mice were not dead until day 34 (Fig. 1A).

3.2. The C57BL/6 mice had a higher parasitemia level than did the CBA and BALB/c mice
The parasitemia curve showed that the blood parasitemia level was higher in the P. bergheiinfected iC57 (n = 25) mice than in the iBALB/c (n = 21) and iCBA (n = 14) groups, and this
difference became evident at the third day post-infection (day 3: P = 0.005, Kruskal-Wallis, iC57 >
iBAL; days 5 and 8: P < 0.0001, ANOVA followed by Student-Newman-Keuls, iC57 > iCBA and
iBAL). The BALB/c mice had median parasitemia rates of 42.6% on day 15 and 52.4% on day 21
(Fig. 1B).

3.3. Plasmodium berghei infection decreased NO production in macrophages from C57BL/6 mice
While BALB/c did not change NO production in response to infection, the C57BL/6 mice
exhibited decreased NO production after P. berghei infection. Macrophages from the C57BL/6
group had significantly reduced NO production at 6 days post-parasite infection. The C57BL/6 non-

17

infected group produced NO levels that were two-fold higher than that of the infected group (P =
0.011, Mann-Whitney test; Fig. 2A).
There were no significant differences among the basal NO production levels in the different
strains of mice before P. berghei ANKA infection (P = 0.64, Kruskal-Wallis test; Fig. 2A).
However, after infection, BALB/c infected-mice had the highest macrophage NO production level,
while the C57BL/6 mice had the lowest NO production level, and the P. berghei infected-CBA
group had an intermediate level of production (P = 0.007, ANOVA; iC57 < iBAL, Fig. 2A).

3.4. Plasmodium berghei infection increased H2O2 production in macrophages from C57BL/6 mice
Before infection, the baseline hydrogen peroxide production in macrophages already differed
among the three mouse strains. C57BL/6 mice produced significantly lower amounts of H2O2 than
did the CBA mice (P = 0.007, Kruskal-Wallis test; Fig. 2B). After P. berghei ANKA infection, the
same profile remained, with the infected C57BL/6 mice producing lower amounts of H2O2 than the
infected CBA mice (P = 0.02, Kruskal-Wallis; Fig. 2B). However, the post-infection H2O2
production in P. berghei ANKA C57BL/6-infected mice was significantly higher than that in noninfected C57BL/6 mice (P = 0.004, t test; Fig. 2B).

3.5. P. berghei ANKA infection caused an increase in lipid body biogenesis in all mouse strains
Macrophages from all strains of studied mice showed similar BODIPY staining after P. berghei
infection. In the control groups, only light staining was observed in cells from non-infected BALB/c
mice, while no staining was observed in cells from the CBA and C57BL/6 groups. After infection,
all groups presented with significant lipid body staining. Fig. 3 shows representative image of
BODIPY.
Lipid body quantification was performed with the Oil-red-O lysochrome dyed slides. Small
quantities of lipid bodies were observed in the control group macrophages, and no difference was
observed at baseline among the CBA, C57BL/6 and BALB/c groups (P = 0.996, ANOVA; Fig. 4).
18

P. berghei ANKA infection increased lipid body production in all groups of mice; however, no
differences were observed among the different mouse strains. This lipid body index increase was
due to an increased number of macrophages with lipid bodies and an increased number of
corpuscles within each macrophage for all strains.

3.6. COX-2 and 5-LOX immunohistochemical analysis

a. Cerebral malaria-resistant BALB/c mice did not express cyclooxygenase-2 or 5-lipoxygenase in
brain blood vessels
The non-infected animals of all strains only exhibited low levels of COX-2 and 5-LOX
expression on brain vessel endothelial cells. However, when mice were infected with P. berghei
ANKA, the numbers of vessels with COX-2-expressing endothelial cells increased in the
susceptible CBA and C57BL/6 groups. Patterns 2 and 3, which are associated with congested
vessels or endothelium-adherent cells, respectively, were more pronounced in cerebral malariasusceptible strains. However, P. berghei infection predominantly produced an increase in pattern 3
in CBA mice, with mainly fully and strongly marked vessels (Mann-Whitney test, p = 0.02), while
pattern 2 was predominant in infected C57BL/6 mice (Mann-Whitney test, p = 0.04), with only
partially marked vessels.
In contrast, 5-LOX expression on vessels significantly increased only in the infected C57BL/6
mice, which predominantly exhibited the enhanced pattern 3 (P = 0.005, Mann-Whitney test)
(Table 1).
Fig. 5 shows representative figures of these immunohistochemical staining patterns. A
predominance of congested, COX-2-labeled vessels was observed in CBA mice, whereas a
predominance of COX-labeled capillaries was observed in C57BL/6 mice. This pattern did not
apply to 5-LOX expression, for which both the venules and capillaries were marked in both CBA
and C57BL/6 mice. The non-susceptible BALB/c mice did express COX-2 or 5-LOX.

19

b. Plasmodium berghei infection increased cyclooxygenase-2 and 5-lipoxygenase expression in

brain cells from CBA and C57BL/6 mice
There were significant increases in the percentages of COX-2 and 5-LOX-positive neurons and
glial cells in tissues from the susceptible CBA and C57BL/6 mice. The numbers of COX-2-positive
brain cells increased from 3.77% to 14.76% in P. berghei ANKA-infected CBA mice (P = 0.028,
Mann-Whitney test) and from 3.2% to 14.34% in P. berghei ANKA-infected C57BL/6 mice (P =
0.018, Mann-Whitney test). The numbers of 5-LOX-positive brain cells increased from 2.8% to
7.4% in P. berghei ANKA-infected CBA mice (P = 0.02, Mann-Whitney test) and from 2.5% to
11.10% to P. berghei ANKA-infected C57BL/6 mice (P = 0.02, Mann-Whitney test; Fig. 6).
Cyclooxygenase-2 or 5-lipoxygenase did not change in brain cell in cerebral malaria-resistant
BALB/c mice.

3.7. COX-2 and 5-LOX quantification in macrophages and microglia using flow cytometry
Pre-infection, 5-LOX-stained macrophages from C57 mice had a higher fluorescence
intensity (2542) than did those from BAL (505) and CBA (1619) mice (P = 0.03; Kruskal-Wallis
test). Post-P. berghei infection, there was a significant decrease in the 5-LOX fluorescence intensity
of the macrophages from the iC57 group (1060) in comparison to that of macrophages from the
non-infected C57 (2542) mice (P = 0.02, Mann-Whitney test). Additionally, the 5-LOX
fluorescence intensity of the macrophages from the iC57 group (1060) was also lower than those of
the iCBA (1773) and iBAL (2371) groups (P = 0.03, Kruskal-Wallis test). Furthermore,
macrophages had higher COX-2 and 5-LOX fluorescent intensities when compared to those of
microglia (Table 2). Fig. 7 shows representative cytometry image.

3.8. PPAR- did not translocate to the nucleus in peritoneal macrophages from C57BL/6 and CBA
mice

20

The Fig. 8 shows representative confocal images of PPAR- expression in macrophages from
non-infected and infected BALB/c, CBA and C57BL/6 mice.
The total PPAR- expression decreased in BALB/c and CBA mice in response to Plasmodium
infection, but the expression level did not change in C57BL/6 mice (Fig. 9C). In BALB/c mice, an
increase in nuclear PPAR- was observed (Fig. 9A), which paralleled a decrease in cytoplasmic
PPAR-

(Fig. 9B) and suggested the post-infection translocation of this molecule from the

cytoplasm to the nucleus in BALB/c mice. This suggestion was confirmed by an increase in the
PPAR- nuclear/cytoplasmic ratio in BALB/c mice after infection (Fig. 9D). These findings suggest
that for BALB/c infected mice, in addition to destruction/impaired production, there was also
translocation of some PPAR- molecules from the cytoplasm to the nucleus. In contrast, in CBA
mice, there was a decrease in cytoplasmic PPAR- (Fig. 9B) without a parallel increase in nuclear
PPAR- after Plasmodium infection (Fig. 9A), along with a decrease in total PPAR-, suggesting
that the molecule was destroyed or its production was impaired in the cytoplasm in the absence of
translocation to the nucleus. This was confirmed by the nuclear/cytoplasmic ratio, for which no
difference was observed between the non-infected and infected CBA mice (Fig. 9D). In C57BL/6
mice, total PPAR- was not destroyed (Fig. 9D) and there was no translocation from the cytoplasm
to the nucleus (Figs 9A, B, C). However, the pre-infection level of nuclear PPAR- was higher in
non-infected C57BL/6 mice than in BALB/c mice (Fig. 9A), suggesting an higher basal nuclear
level of PPAR- in C57BL/6 mice, the cerebral malaria model with the most aggressive disease.
Figure 10 synthesize and correlate all these results.

4. Discussion
This work shows that P. berghei-infected C57BL/6 mice exhibited more severe disease and a
lower survival time, which corresponded with the increased blood parasitemia and reduced
production of the plasmodicidal NO and H2O2 molecules on the sixth day post-infection when
compared to those observed in infected CBA and BALB/c mice. Additionally, the cerebral malaria21

resistant BALB/c mice showed better disease evolution, with improved survival, reduced blood
parasitemia and increased production of the plasmodicidal NO and H2O2 molecules at the sixth day
post-infection.
The iC57 group produced lower amounts of hydrogen peroxide than the iCBA and iBAL
strains. Additionally, the C57BL/6 mice also produced lower levels of the plasmodicidal nitric
oxide molecule after Plasmodium infection. In contrast, the cerebral malaria-resistant BALB/c mice
produced significantly higher levels of NO after Plasmodium berghei infection than did the
C57BL/6 mice; these opposing effects of infection on NO production in P. berghei-infected
C57BL/6 and BALB/c mice were previously observed by Hanun et al. (2003) in spleen cells. The
lower production of molecules involved in anti-parasite defense might explain the high parasitemia
observed in the infected-C57BL/6 mice and might also contribute to the severity of the cerebral
disease observed in this model. However, the lack of differences with regard to parasitemia and NO
and H2O2 production between the cerebral malaria-susceptible CBA and resistant BALB/c strains
suggest that the lack of microbicidal action mediated by these molecules was not the sole cause of
the cerebral disease severity in these models.
The biogenesis of lipid bodies, which are the production sites of eicosanoid-forming enzymes,
was increased in all of the infected animals evaluated. This result suggests an inflammatory
condition in both cerebral malaria-susceptible and resistant mouse strains. Although Plasmodium
infection increased the production of lipid bodies in all assessed mice, there were differences
between the enzymes produced within the lipid bodies, evidenced by the differing levels of COX-2
and 5-LOX expression among the three strains. Whereas CBA mice exhibited higher COX-2
expression, C57BL/6 mice exhibited increased levels of both COX-2 and 5-LOX in cerebral vessels
(Table 1). Additionally, enhanced COX-2 and 5-LOX expression were observed in the neurons and
astrocytes from both the cerebral malaria-susceptible C57BL/6 and CBA mouse strains (Fig. 6).
Interestingly, BALB/c mice, which do not develop cerebral disease, did not exhibit enhanced COX-

22

2 or 5-LOX expression. These facts suggest that both pathways play a role in the development of
cerebral malaria in the susceptible mice.
The CBA group predominantly expressed COX-2, which triggers prostaglandin production; in
turn, prostaglandins stimulate Th1 lymphocytes and thus increase NO, TNF- and interferon-
production. In fact, the roles of TNF- (Muniz-Junqueira et al., 2001; Muniz-Junqueira et al., 2005)
and IFN- (Grau et al., 1989; Lou et al., 2001) in malaria immunity and pathogenesis have been
demonstrated. Increased IFN- mRNA levels were detected in cerebral malaria-susceptible mice
with the neurological syndrome, but not in resistant mice. IFN- promotes increased TNF mRNA
levels and upregulates TNF receptors on target cell surfaces (Grau et al., 1989; Lou et al., 2001).
The administration of an anti-IFN- antibody to P. berghei-infected CBA mice improved the
evolution of malaria by reducing the serum TNF levels and preventing the development of cerebral
pathology (Lou et al., 2001). Ball et al. observed enhanced COX-2 expression in the brain and
increased interferon- and TNF- production in CBA mice (Ball et al., 2004). Both the lack and the
excess of COX were related to malarial severity. Impaired systemic prostaglandin E2 production
were associated with adverse outcomes in children with cerebral malaria, and elevated PGE2 levels
were observed in cases of asymptomatic parasitemia (Perkins et al., 2005; Anyona et al., 2013).
Healthy malaria-exposed children had higher prostaglandin E2 levels than did children with malarial
anemia (Keller et al., 2006). Lymphocytes from Plasmodium-infected individuals show an
increased resistance to the antiproliferative effects of prostaglandin E2 (Alves et al., 1992).
Higher COX-2 expression was observed in endothelial cells and astrocytes from the brains of
individuals with cerebral malaria, compared to those from healthy controls (Deininger et al., 2000),
similar to our observations in CBA mice. Furthermore, COX inhibitors and the pharmacological
antipyretic-mediated inhibition of COX enzymatic activity, which are used during treatment of
human malaria, increased TNF- production (Keller et al., 2006). These data suggest that not only
the presence, but also the concentration of COX might influence the disease evolution. Our data

23

suggest that the exacerbated production of COX-2 pathway molecules may participate in the
development of cerebral malaria in CBA mice.
In addition to higher COX-2 expression, infected C57BL/6 mice exhibited enhanced 5-LOX
expression in cerebral tissues. 5-LOX increases the synthesis of leukotrienes and other hydroxyl
acids, which then enhance leukocyte adhesion and activation and increase vascular permeability and
proinflammatory cytokine production (Gerritsen, 1996); all of these effects are involved in cerebral
malaria pathogenesis. Xiao et al. (1999) observed that at the peak of cerebral malaria, Plasmodium
berghei ANKA-infected ICR mice had higher serum leukotriene B4 levels than did the controls.
These observations suggest that the 5-LOX pathway products are also involved in cerebral malaria
development. However, 5-lipoxygenase-deficient mice infected with Plasmodium berghei-ANKA
had lower survival, suggesting that molecules of this pathway also protect against cerebral malaria
(Shryock et al., 2013). Furthermore, the fact that there was minor expression of both COX-2 and 5LOX in the brains of BALB/c mice, a cerebral malaria-resistant strain, reinforces the importance of
the exacerbation of both pathways in cerebral disease development. Exacerbation of both the COX2 and 5-LOX pathways might enhance the production of inflammatory cytokines that are involved
in cerebral malaria.
Our data showed that after P. berghei infection, the PPAR- levels were enhanced in the
nucleus and decreased in the cytoplasm only in macrophages from infected BALB/c mice,
indicating that PPAR- had translocated from the cytoplasm to the nucleus only in macrophages
from the cerebral malaria-resistant P. berghei-infected BALB/c mice (Fig. 9). PPAR- translocation
to the nucleus downregulates proinflammatory cytokine production (Delerive et al., 2001), thus
delaying the disease severity in BALB/c mice; this possibility agrees with the better evolution of
these mice during the first six days of infection when compared to the other strains.
P. berghei-infected CBA mice showed a predominance of the cytoplasmic destruction and/or
inhibited production of PPAR-, without nuclear translocation. P. berghei-infected C57BL/6 mice
did not show any modifications in PPAR- levels in response to infection, and no translocation of
24

new PPAR- molecules into the nuclei occurred in the macrophages. It is possible that the absence
of new regulatory PPAR- molecules in the nuclei might have favored the unbalanced production of
proinflammatory cytokines, a hallmark of cerebral malaria, and it might also have contributed to the
poorer disease evolution in these cerebral malaria-susceptible mice. However, the C57BL/6 mice
showed constitutively higher levels of nuclear PPAR-. PPAR--mediated inhibition of iNOS
transcription (Torra et al., 2001) could explain the lower constitutive level of NO production in
macrophages from C57BL/6 that was observed in this work.
Two possibilities might explain the decrease in total and cytoplasmic PPAR- levels observed
in CBA mice: either the molecule was destroyed or its production was impaired. In fact, TNF-, the
expression of which is increased in severe malaria, has been shown to inhibit PPAR- activity. The
chronic influence of this cytokine reduces the IKK/NFB pathway-dependent expression of PPAR (Ye, 2008). Additionally, ubiquitin-proteasome-mediated PPAR degradation modulates the
intensity of the ligand response by controlling the PPAR protein levels in cells (Blanquart et al.,
2003). It is possible that these two mechanisms contributed to the reduced total and cytoplasmic
PPAR- levels in infected CBA mice.
Agonist drug-induced PPAR- modulation has been suggested as a novel target for adjunctive
cerebral malaria therapy (Balanchandar and Katyal, 2011; Serghides, 2012). The treatment of
C57BL/6 mice with rosiglitazone, a PPAR- agonist, modified the inflammatory response to
malarial infection and improved the survival rate of the animals (Serghides et al., 2009; Serghides,
et al., 2014). Possibly, this effect occurred because this drug also induced an increase in CD36mediated phagocytosis and a decrease in parasite-induced TNF- secretion (Serghides and Kain,
2001). The treatment of symptomatic P. falciparum malaria patients with rosiglitazone has already
been assessed, and an increase in parasite clearance and a decrease in the inflammatory biomarkers
associated with adverse malaria outcomes (e.g., TNF, IL-6 and MCP1) were observed (Boggild et

25

al., 2009). These findings agree with the higher parasitemia and unregulated PPAR- production
that we observed in the C57BL/6 mice.
Higher parasitemia and deficient plasmodicidal NO and H2O2 production played a role in the
disease severity in the C57BL/6 mice. Additionally, the later mortality that occurred in BALB/c
mice was also associated with increased parasitemia. These facts indicate that the parasite burden
plays an important role in disease severity and conforms to the idea of early, appropriate and
efficient antimalarial chemotherapy as the easiest and most important known approach to
preventing severe forms of malaria (Warrel, 1999). However, there were no differences with regard
to parasitemia or NO and H2O2 production between the cerebral malaria-susceptible CBA and
resistant BALB/c mice during the first six days of the infection. Suggesting that the high parasite
burden was not the sole cause of cerebral disease severity in these models and that, besides the
parasite burden, other factors are also involved in cerebral malaria induction. In fact, we observed
an unbalanced and unregulated inflammatory response in both cerebral malaria models. COX-2 and
5-LOX expression were increased in both cerebral malaria-susceptible strains (C57BL/6 and CBA),
whereas the cerebral malaria-resistant BALB/c mice showed no increases in either COX-2 or 5LOX, thus reinforcing the importance of imbalances in both the COX-2 and 5-LOX pathways
during cerebral disease development. Furthermore, nuclear translocation of the regulatory PPAR-
molecule occurred only in the cerebral malaria-resistant mice, while both cerebral malariasusceptible mice exhibited unregulated responses in the PPAR- pathway, suggesting that a lack of
PPAR- pathway-mediated regulation also promoted cerebral involvement.
Our paper contributes to a broader understanding of responses of the C57BL/6 and CBA
experimental cerebral malaria models to Plasmodium berghei ANKA infection. These models
triggered differently cellular inflammatory pathways involved in cerebral malaria development. We
found that, besides higher parasitemia, the inflammatory response was more unbalanced and
unregulated in cerebral malaria-susceptible models than in resistant models. It is possible these
phenomena also occur in cerebral human malaria. The fact that different mechanisms determine
26

cerebral malaria development in individuals with different genetic backgrounds might explain the
controversial results found during attempts to therapeutically modulate the immune system and thus
treat severe malaria (Muniz-Junqueira et al., 2001; Muniz-Junqueira, 2007) and increases the
difficulty associated with suggesting and evaluating the responses to immunomodulatory therapies
for severe malaria. These different responses should be considered during proper clinical
evaluations and to facilitate the better management of immunomodulatory drugs as adjunctive
therapies for severe malaria. Furthermore, our paper might facilitate the choice of a better
experimental model when conducting research on some aspects of malarial disease.
In conclusion, the C57BL/6 had more severe disease with a lower survival time, higher
parasitemia and lower production of plasmodicidal NO and H2O2 molecules. Enhanced COX-2 and
5-LOX expression was observed in their brain tissue, and these mice expressed higher constitutive
PPAR- levels. There was no translocation of PPAR- from cytoplasm to nucleus in macrophages.
CBA mice had enhanced COX-2 expression in brain tissue and lacked PPAR- cytoplasm-tonucleus translocation. The resistant BALB/c mice presented higher survival time, lower parasitemia
and higher NO and H2O2 production. They did not express either COX-2 or 5-LOX in brain tissue
and there was PPAR- cytoplasm-to-nucleus translocation, which may have adequately modulated
the response against Plasmodium. The inflammatory response was more regulated in cerebral
malaria-resistant BALB/c mice than in the -susceptible CBA and C57BL/6 models, showing that
the deregulated response may contribute to cerebral disease development.

Conflict of interest
This study was exclusively designed and conducted by the authors that have no conflict of interest.

Acknowledgements
Tatiana Karla Borges and rica Alessandra Rocha Alves were supported by CAPES (Molecular
Pathology, University of Braslia, Brazil). Maria Imaculada Muniz-Junqueira is an investigator
27

supported by the Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq), Brazil

(process number 308985/2013-3). This research was partially supported by a grant from Fundao
de Apoio Pesquisa do Distrito Federal (FAPDF), Brazil (193.000.021/2012). The authors used a
copy-editing service for language revision. We thank Dr. David Lima Duarte for reviewing the
statistical analyses. We thank Andria Cristina Gonalves Cascaes, Danilo Corazza, Marcelo
Henrique de Nvoa Netto, Simone Schmil, Daniela Aquino, Mayara G C de Oliveira and Luciana
Magalhes, as well as all technical staff, for their assistance with the experiments and animal care.
We thank Mrs. Fernanda Muniz Junqueira Ottoni and Mrs. Jackelline Cavalcante do Nascimento
Borges for the excellent work in image processing.

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Legends to Figures

Fig. 1. Survival rates of Plasmodium berghei ANKA-infected mice and parasitemia curve. In A.
Groups of BALB/c (n = 18), CBA (n = 10) and C57BL/6 (n = 15) mice were infected with 106 P.
berghei ANKA-parasitized erythrocytes, and survival was observed twice daily. C57BL/6 mice
began to die on the sixth day post-infection, and the last animal died on the sixteenth day. CBA and
BALB/c mice began to die on the eighth day post-infection. All mice in the CBA group had died by
the eighteenth day, while the last BALB/c mouse died on the thirty-fourth day after infection.
Survival was plotted according to the Kaplan-Meier method, and significant differences were
evaluated with the Mantel-Cox log-rank test (8.3; P = 0.016) with the p value adjusted by the
Bonferroni method, considering three groups. In B: Parasitemia was assessed in the tail blood from
cerebral malaria-susceptible CBA (n = 14) and C57BL/6 (n = 25) mice and resistant BALB/c (n =
21) mice after Plasmodium berghei ANKA infection. C57BL/6 mice had higher parasitemia levels
than did CBA and BALB/c mice. Regarding parasitemia levels in the mouse groups, on day 3, iC57
> iBAL (P = 0.0005, Kruskal-Wallis followed by Dunns comparison test); on days 5 and 8, iC57 >
35

iBAL and iCBA (P < 0.0001, ANOVA followed by Student-Newman-Keuls test). The curve is
expressed in median.
Fig. 2. Reactive species production by macrophages from P. berghei ANKA-infected and noninfected mice. Nitric oxide (A) and hydrogen peroxide (B) production in vitro by macrophages from
P. berghei ANKA-infected CBA, C57BL/6 and BALB/c mice and controls (n = 10 per group). The
data are shown as medians, quartiles and extremes. In A: basal NO and H2O2 production were
evaluated in the culture supernatants of macrophages from non-infected (BAL, CBA and C57) and
infected animals (iBAL, iCBA and iC57). After infection, NO production by macrophages from the
infected C57BL/6 mice was lower than that of macrophages from infected BALB/c mice (P =
0.007, ANOVA followed by Student-Newman-Keuls test, iC57 < iBAL). NO production by
macrophages from infected C57BL/6 mice was lower than that of macrophages from non-infected
animals (P = 0.011, Mann-Whitney test). In B: macrophages from non-infected C57BL/6 mice
produced lower levels of H2O2 than did cells from CBA mice (P = 0.007, Kruskal-Wallis test
followed by Dunns multiple comparison test, C57 < CBA). After infection, C57BL/6 macrophages
also produced lower levels of H2O2 than did CBA macrophages (P = 0.02, Kruskal-Wallis test
followed by Dunn multiple comparison test). In response to infection, C57BL/6 macrophages
produced higher levels of H2O2 (P = 0.004, t test).

Fig. 3. Representative image of lipid body staining in peritoneal macrophages from P. berghei
ANKA-infected and non-infected mice from a pool of 5 mice, twice repeated.

The green color

indicates BODIPY staining, which marks neutral lipids, while the blue color represents DAPIstained nuclei. Macrophages were obtained on the sixth day post-infection. The first column shows
macrophages from non-infected-mice (BAL, C57 and CBA). Note that after infection, macrophages
from all strains (iBAL, iC57 and iCBA) contained increased amounts of lipid bodies. The images
were captured with a Leica SP5 confocal microscope and analyzed with ImageJ software. Pinhole:
1.26 Airy; 1 frame average; 3 lines average; zoom: 5 X. Scale bars: 7.5 m.
36

Fig. 4. Lipid body indices in macrophages from non-infected and P. berghei ANKA-infected mice.
Macrophages were stained with oil red and Mayers hematoxylin (n = 9 per group). The cells were
analyzed with a light microscope by determining the percentage of macrophages with lipid bodies
and the number of lipid bodies within the macrophages. The images (A and B) were captured with
an Axiophot Leica TCS SP5 microscope (620X). (A) Peritoneal macrophages from a non-infected
group. (B) Macrophages from an infected group with red-stained lipid bodies in the cytoplasm of
the cells (representative image). (C) Lipid body index; infection increased the LBI in all mouse
groups. (D) Number of lipid bodies per macrophage. (E) Percentage of macrophages with lipid
bodies. Data were plotted as the means and SEM.

Fig. 5. Image representative of COX-2 and 5-LOX expression in the brain tissue of experimental
malaria mouse models (n = 5 per group). COX-2 and 5-LOX expression in brain tissues of BALB/c,
CBA and C57BL/6 infected (iBAL, iCBA and iC57) and non-infected (BAL, CBA, C57) mice were
determined by immunohistochemistry on the sixth day post-infection with Plasmodium berghei
ANKA. Brown staining indicates positive cells. All sections were incubated in the same conditions
and simultaneously. The images were captured with an Aperio ScanScope slide scanner. Scale
bar: 50 m. Original magnification: 400 X. In A (COX-2) and D (5-LOX): Plasmodium berghei
ANKA-infected BALB/c mice. Pattern 0 and 1 vessels predominated. There was no congestion,
sequestration or vasodilatation. The yellow arrow indicates vessel pattern 0. In B (COX-2) and E
(5-LOX): Plasmodium berghei ANKA-infected CBA mice. Yellow arrows indicate pattern 3
staining for COX-2 and show a congested vessel with adherent erythrocytes. Note the perivascular
region hemorrhage (red arrow). In C (COX-2) and F (5-LOX): Plasmodium berghei ANKAinfected C57BL/6 mice. In C, the yellow arrow indicates vessel pattern 2 staining; only the
capillaries are stained. In F, the red arrow indicates pattern 3 staining.

37

Fig. 6. COX-2 or 5-LOX staining in brain cells on the sixth day post-infection. Coronal brain
sections were taken from Plasmodium berghei-ANKA-infected (iBAL, iCBA, iC57) or noninfected BALB/c, CBA and C57BL/6 (BAL, CBA, C57) mice. A total of 500 cells were analyzed in
the cortex, hippocampus and thalamus from 5 animals per group. (A) Percentage of COX-2expressing cells; (B) percentage of 5-LOX-expressing cells. The data are shown as medians,
quartiles and extremes. Mann-Whitney test was used.

Fig. 7. Representative histograms of COX-2 or 5-LOX expression. Histograms of COX-2 or 5-LOX

expression in macrophages (A and C, respectively) and microglia (B and E, respectively) from noninfected (BAL, CBA and C57) and infected (iBAL, iCBA and iC57) mice by flow cytometry (n = 5
per group). Cells were harvested on the sixth day post-infection with Plasmodium berghei-ANKAinfected or non-infected BALB/c, CBA and C57BL/6, and stained for COX-2 and 5-LOX.

Fig. 8. PPAR- nuclear translocation in peritoneal macrophages from Plasmodium berghei ANKAinfected (iBAL, iCBA and iC57) and non-infected (BAL, CBA and C57) mice. Representative
image from a pool of 5 mice. Experiments were twice repeated. The macrophages were assessed on
the sixth day post-infection after labeling with anti-PPAR- primary antibodies (1:200), Alexa Fluor
546-conjugated secondary antibodies (in red; 1:3000) and DAPI (in blue; 1:5000). The first column
shows macrophages from non-infected mice, while the second column shows macrophages from
Plasmodium berghei ANKA-infected mice. Pinhole: 120.5, line average: 3, zoom: 2.5 X.

Fig. 9. PPAR- expression and localization in peritoneal macrophages from Plasmodium berghei
ANKA-infected (iBAL, iCBA and iC57) and non-infected mice (BAL, CBA and C57). (A) Nuclear
PPAR-. (B) Cytoplasmic PPAR-. (C) Total PPAR-. (D) Nuclear/cytoplasmic PPAR- ratio.
PPAR- translocation from the cytoplasm to the nuclei of macrophages was only evident in the
cerebral malaria-resistant BALB/c mice. The data are shown as medians, quartiles and extremes
38

from an analysis of 5 separate high-power field images. Experiments were twice repeated (n = 5 per
group. Mann-Whitney and Kruskal-Wallis tests).

Fig. 10. Responses to Plasmodium berghei ANKA infection in cerebral malaria-susceptible and
resistant mice. (A) The cerebral malaria-susceptible C57BL/6 mice had more severe disease.
These mice died on sixty day post-infection with high parasitemia and low production of
plasmodicidal NO and H2O2 molecules, which contributed to the lower survival time. Both
eicosanoid-forming-enzymes COX-2 and 5-LOX shown an enhanced expression in brain tissue
cells and vessels. There was no translocation of PPAR- from cytoplasm to nucleus in
macrophages.

The lack of PPAR-, NO and H2O2 regulatory molecules may had enhanced

expression of eicosanoid-forming-enzymes in both pathways, and this imbalanced response

associated to the lack of plasmodicidal NO e H2O2 molecules increased the severity of the disease in
the C57BL/6 mice. (B) The cerebral malaria-susceptible CBA mice showed a less severe disease
than C57BL/6 mice. They died two days later and shown lower parasitemia than C57BL/6 mice.
They had mainly enhanced COX-2 expression in brain tissue cells and vessels and lacked PPAR-
cytoplasm-to-nucleus translocation. The less severe imbalance in COX-2 pathway activation may
had caused a less severe evolution in these mice. (C) The cerebral malaria-resistant BALB/c mice
presented higher survival time, lower parasitemia and higher NO and H2O2 production on the sixth
day post-infection than -susceptible C57BL/6 and CBA mice. BALB/c mice did not express either
COX-2 or 5-LOX in brain tissue cells and vessels. There was post-infection translocation of PPAR from the cytoplasm to the nucleus. The translocation of the regulatory PPAR- molecules may had
modulate the eicosanoid pathway, and together a higher production of plasmodicidal molecules may
had contributed to a more controlled initial response to Plasmodium berghei ANKA. It can be
concluded that besides the high parasite burden and lack of microbicidal molecules, an imbalance
with high COX-2 and 5-LOX eicosanoid expression and a lack of regulatory PPAR- cytoplasm-tonucleus translocation in macrophages were observed in mice that developed cerebral malaria.
39

Table 1. COX-2 or 5-LOX expression in the brain vessels of non-infected and infected mice
according to immunohistochemistry.
Cyclooxygenase-2
Pattern
0
Median
(quartiles)
70.0
(68.381.7)
60.0
(42.368.3)

1
Median
(quartiles)
13.3
(10.021.6)
23.3
(15.031.7)

2
Median
(quartiles)
0.0
(0.011.7)
11.2
(4.915.6)

3
Median
(quartiles)
0.0
(0.05.0)
3.3
(0.014.2)

0.062

0.211

0.211

0.589

63.3
(42.581.6)
30.0
(17.532.5)

28.3
(12.541.7)
40.0
(24.248.3)

6.7
(3.315.0)
15.0
(10.821.7)

1.7
(0.003.4)
21.7
(9.229.2)

0.029

0.378

0.186

0.028

60.0
(31.785.0)
19.1
(4.655.0)

13.3
(13.340.0)
40.8
(27.553.6)

6.7
(1.711.7)
18.3
(11.726.7)

0.0
(0.00.0)
8.3
(0.015.8)

**Test (P)

0.091

0.090

0.047

0.095

Non-infected

0.048

0.523

0.539

0.499

Infected

0.119

0.128

0.271

0.178

Mice strain (groups)

BALB/c

Non-infected
(n = 5)
Infected
(n = 4)
**Test (P)

CBA

Non-infected
(n = 4)
Infected
(n = 4)
**Test (P)

C57BL/6

***Test
(P)

Non-infected
(n = 5)
Infected
(n = 8)

5-Lipoxygenase
Pattern
0
Median
(quartiles)
69.1
(48.990.6)
43.3
(31.770.0)

1
Median
(quartiles)
18.3
(4.932.5)
38,3
(21.742.5)

2
Median
(quartiles)
9.9
(2.215.3)
15,0
(8,326,7)

3
Median
(quartiles)
1.8
(0.04.1)
0.0
(0.02.5)

0.200

0.114

0.486

0.408

61.6
(39.880.8)
41.7
(30.043.33

22.0
(14.129.3)
36.7
(23.340.0)

11.6
(4.923.8)
20.0
(15.025.0)

3.3
(0.08.5)
6.7
(0.817.5)

0.200

0.114

0.343

0.459

66.7
(57.578.3)
43.3
(27.363.3)

33.3
(15.039.2)
22.2
(16.727.3)

3.30
(0.08.3)
11.1
(6.719.0)

0.0
(0.00.0)
19.0
(0.030.0)

**Test (P)

0.073

0.268

0.073

0.005

Non-infected

0.819

0.418

0.179

0.376

Infected

0.807

0.111

0.348

0.075

Mouse strain (groups)

BALB/c

Non-infected
(n = 4)
Infected
(n = 4)
**Test (P)

CBA

Non-infected
(n = 4)
Infected
(n = 4)
**Test (P)

C57BL/6

***Test
(P)

Non-infected
(n = 5)
Infected
(n = 6)

Expressed as a percentage and counted per 500 cells per mouse.

** t test or Mann-Whitney test; *** ANOVA or Kruskal-Wallis test.

40

Table 2. COX-2 or 5-LOX expression in macrophages and microglia from non-infected and
infected mice.
Cyclooxygenase-2
Mouse
strain
(groups)

Peritoneal macrophages
median
(lower and upper quartiles)
BALB/c
CBA
C57BL/6
(n = 5)
(n = 5)
(n = 5)

Microglia
median
(lower and upper quartiles)
BALB/c
CBA
C57BL/6
(n = 5)
(n = 5)
(n = 5)

Noninfected

1461
(881 2445)

767
(652 1217)

1644
(723 3994)

32.0
(28.9 45.7)

39.9
(38.4 47.15)

45.8
(30.0 53.6)

Infected

2308
(978 2361)

621
(449 1794)

1389
(908 2406)

25.0
(19.1 30.7)

27.7
(23.4 36.1)

28.7
(21.5 30.6)

*Test (P)

0.786

0.556

0.548

0.056

0.056

0.082

**Noninfected
**Infected

P = 0.221

P = 0.365

P = 0.155

P = 0.941
5-Lipoxygenase

Mouse
strain
(groups)

Peritoneal macrophages
median
(lower and upper quartiles)
BALB/c
CBA
C57BL/6
(n = 5)
(n = 5)
(n = 4)

Microglia
median
(lower and upper quartiles)
BALB/c
CBA
C57BL/6
(n = 4)
(n = 5)
(n = 4)

Noninfected

505
(327 2369)

1619
(1162 1961)

2542
(2469 2836)

34,2
(17.7 54.0)

45.3
(24.8 55.3)

42.9
(19.8 63.1)

Infected

2371
(1852 2642)

1773
(1422 2004)

1060
(595 1589)

24,3
(15.8 37.4)

22,8
(18.2 28.7)

18.2
(11.4 27.9)

*Test (P)

0.250

0.556

0.029*

0.571

0.111

0.343

**Noninfected
**Infected

P = 0.038

P = 0.695

P = 0.031

P = 0.582

Expressed as fluorescence intensity.

* Mann-Whitney test; ** Kruskal-Wallis test.

41