Early Cytokine Production Is Associated with Protection from Murine Cerebral Malaria

Abstract

Cerebral malaria (CM) is an infrequent but serious complication of Plasmodium falciparum infection in humans. Animal and human studies suggest that the pathogenesis of CM is immune mediated, but the precise mechanisms
leading to cerebral pathology are unclear. In mice, infection with Plasmodium berghei ANKA results in CM on day 6 postinoculation (p.i.), while infection with the closely related strain P. berghei K173 does not result in CM. Infection with P. berghei K173 was associated with increased plasma gamma interferon (IFN-γ) at 24 h p.i. and with increased splenic and hepatic mRNAs
for a range of cytokines (IFN-γ, interleukin-10 [IL-10], and IL-12) as well as the immunoregulatory enzyme indoleamine 2,3-dioxygenase.
In contrast, P. berghei ANKA infection was associated with an absence of cytokine production at 24 h p.i. but a surge of IFN-γ production at 3 to
4 days p.i. When mice were coinfected with both ANKA and K173, they produced an early cytokine response, including a burst
of IFN-γ at 24 h p.i., in a manner similar to animals infected with P. berghei K173 alone. These coinfected mice failed to develop CM. In addition, in a low-dose P. berghei K173 infection model, protection from CM was associated with early production of IFN-γ. Early IFN-γ production was present
in NK-cell-depleted, γδ-cell-depleted, and Jα281−/− (NKT-cell-deficient) mice but absent from β2-microglobulin mice that had been infected with P. berghei K173. Taken together, the results suggest that the absence of a regulatory pathway involving IFN-γ and CD8+ T cells in P. berghei ANKA infection allows the development of cerebral immunopathology.

Full text

INFECTION AND IMMUNITY, Sept. 2005, p. 5645–5653 Vol. 73, No. 9
0019-9567/05/$08.00⫹0 doi:10.1128/IAI.73.9.5645–5653.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Early Cytokine Production Is Associated with Protection from Murine
Cerebral Malaria
Andrew J. Mitchell,
1
† Anna M. Hansen,
1,2
† Leia Hee,
1
Helen J. Ball,
1
Sarah M. Potter,
1
‡
John C. Walker,
2
and Nicholas H. Hunt
1
*
Departments of Pathology
1
and Medicine,
2
University of Sydney, Sydney, Australia
Received 21 December 2004/Returned for modification 18 February 2005/Accepted 9 May 2005
Cerebral malaria (CM) is an infrequent but serious complication of Plasmodium falciparum infection in
humans. Animal and human studies suggest that the pathogenesis of CM is immune mediated, but the precise
mechanisms leading to cerebral pathology are unclear. In mice, infection with Plasmodium berghei ANKA
results in CM on day 6 postinoculation (p.i.), while infection with the closely related strain P. berghei K173 does not
result in CM. Infection with P. berghei K173 was associated with increased plasma gamma interferon (IFN-␥)at
24 h p.i. and with increased splenic and hepatic mRNAs for a range of cytokines (IFN-␥, interleukin-10 [IL-10], and
IL-12) as well as the immunoregulatory enzyme indoleamine 2,3-dioxygenase. In contrast, P. berghei ANKA infection
was associated with an absence of cytokine production at 24 h p.i. but a surge of IFN-␥production at 3 to 4 days
p.i. When mice were coinfected with both ANKA and K173, they produced an early cytokine response, including a
burst of IFN-␥at 24 h p.i., in a manner similar to animals infected with P. berghei K173 alone. These coinfected mice
failed to develop CM. In addition, in a low-dose P. berghei K173 infection model, protection from CM was associated
with early production of IFN-␥. Early IFN-␥production was present in NK-cell-depleted, ␥␦-cell-depleted, and
J␣281
ⴚ/ⴚ
(NKT-cell-deficient) mice but absent from ␤2-microglobulin mice that had been infected with P. berghei
K173. Taken together, the results suggest that the absence of a regulatory pathway involving IFN-␥and CD8
ⴙ
T
cells in P. berghei ANKA infection allows the development of cerebral immunopathology.
Despite the best efforts of public health authorities, malaria
remains a major global health concern, with estimates ranging
from 300 to 500 million people infected every year (10, 48).
The majority of deaths attributed to malaria occur in sub-
Saharan Africa as a result of infection by Plasmodium falcipa-
rum. One of the major life-threatening complications of P.
falciparum infection is cerebral malaria (CM), which is char-
acterized by convulsions and unarousable coma. Although only
a small percentage of infected individuals develop CM, the
mortality rate once it has developed is around 20% (47).
While there are competing hypotheses about the etiology of
CM, there is strong evidence that it is an immunopathological
process (9, 26). In particular, animal studies using the rodent
malaria strain Plasmodium berghei ANKA have been revealing.
For P. berghei ANKA infections of susceptible mouse strains, the
development of CM is dependent upon a variety of immunolog-
ical processes. For example, the production during the course of
infection of cytokines such as gamma interferon (IFN-␥) (21, 39),
as well as the presence of both CD4
⫹
and CD8
⫹
T cells (4, 24, 35,
49), has been shown to be essential for CM pathology to occur.
Investigations of CM pathogenesis have examined almost
exclusively the immune processes immediately preceding the
development of cerebral manifestations, that is, the end-stage
pathological processes. Thus, there is little understanding of
the very early immune system responses to malarial parasites.
In many infection models, such early innate immune processes
have been shown to critically influence the later development
of adaptive immune responses (reviewed in references 27 and
37), so it seems possible that early responses may also influence
the immunopathology of CM.
We therefore investigated the early immune responses to
two closely related Plasmodium strains and correlated these
with the pathological outcome. Susceptible mice infected with
Plasmodium berghei ANKA succumb to CM 6 to 7 days after
infection. In contrast, mice infected with Plasmodium berghei
K173 develop high parasitemia levels and low hematocrits and
die without cerebral symptoms 2 to 3 weeks after infection. We
found that 24 h after parasite inoculation with P. berghei K173,
but not P. berghei ANKA, there was a transient production of
a range of cytokines in the spleen, most notably of IFN-␥.
Upon simultaneous infection of mice with both P. berghei
ANKA and P. berghei K173, a similar pattern of cytokine pro-
duction to that seen with K173 alone was seen, and this cor-
related with the absence of later CM. This suggested that an
active suppression of immunopathological processes was oc-
curring in P. berghei K173 infection and that this dominated over
the processes usually occurring in P. berghei ANKA infection.
Since IFN-␥may be involved in immunosuppression through the
induction of indoleamine 2,3-dioxygenase (IDO), the cellular pro-
cesses occurring during early IFN-␥production were investigated
for P. berghei K173 infection, and production of the cytokine was
found to be ␤2-microglobulin (␤2-M) dependent.
MATERIALS AND METHODS
Animal procedures. C57BL/6 mice were purchased from the Animal Re-
sources Centre (Canning Vale, Western Australia). Breeding pairs of CD1d
⫺/⫺
* Corresponding author. Mailing address: University of Sydney, De-
partment of Pathology, Medical Foundation Building (K25), 92-94
Parramatta Rd., Camperdown NSW 2042, Australia. Phone: 61-2-9036
3242. Fax: 61-2-9036 3286. E-mail: nhunt@med.usyd.edu.au.
‡ Present address: De´partement d’Immunologie, Institut Cochin,
Institut National de la Sante´ et da la Recherche Me´dicale Unite´ 567,
Paris, France.
† A.J.M. and A.M.H. contributed equally to the work in this paper.
5645

and J␣281
⫺/⫺
mice were obtained from M. Smyth (Peter McCallum Institute,
Melbourne, Australia), and IFN-␥
⫺/⫺
mice were obtained from G. Karupiah
(John Curtin School of Medical Research). ␤2-Microglobulin
⫺/⫺
(␤2-M
⫺/⫺
)
mice were obtained from the Australian National University (John Curtin School
of Medical Research). All animals were housed in the Blackburn Animal House,
University of Sydney, under a 12-hour light/dark cycle and were given food and
water ad libitum. For some experiments, selected cell populations were depleted
using antibodies. Natural killer cells were depleted (approximately 90% efficient)
by the administration of 25 ␮l anti-asialo GM-1 antibody (Wako Chemical
Company). Gamma delta T cells were depleted by intraperitoneal administration
of 400 ␮g of hamster anti-␥␦-T-cell receptor (␥␦TCR, clone GL-3; Walter and
Elisa Hall Monoclonal Antibody Facility, Victoria, Australia). Antibodies were
administered 24 h prior to infection, and cell depletion was verified by flow
cytometry. NK cells were identified as NK1.1
⫹
(clone 145-2C11) CD3
⫺
cells
(clone KT3-1-1). Gamma delta cells were identified as CD3
⫹
␥␦
⫹
(GL-3) B220
⫺
(RA-6B2) ␣␤TCR
⫺
(H57-597) CD11b
⫺
(CBL-131P) cells. All antibodies used
for flow cytometry were from Pharmingen. All animal procedures were approved
by the University of Sydney Animal Ethics Committee.
Parasite inoculation. Mice were intraperitoneally inoculated with 2 ⫻10
6
parasitized red blood cells (pRBC) from an ANKA- or K173-infected mouse. For
coinfection experiments, animals were either infected with 2 ⫻10
6
ANKA, 2 ⫻
10
6
K173, or a mixture of 2 ⫻10
6
ANKA plus 2 ⫻10
6
K173 cells. It has been
established that P. berghei ANKA causes CM across a very wide range of inoc-
ulum sizes (44). The P. berghei ANKA strain was obtained from G. Grau (Uni-
versite de la Me´diterrane´e, Marseille, France), and the P. berghei K173 strain was
obtained from I. Clark (Australian National University, Canberra, Australia).
The strains were independently isolated and have not been cloned subsequently.
Molecular biology. Gene expression in tissues was measured by real-time
quantitative reverse transcription-PCR (RT-qPCR). A mouse spleen or liver was
placed in 1 ml of Tri reagent (Sigma) and homogenized using 1-mm-diameter
zirconium beads in a Fastprep homogenizer (Qbiogene). Chloroform (0.2 ml)
was added, and the lysate was mixed well. After centrifugation at 12,000 ⫻gfor
15 min, the aqueous layer was transferred to a new tube. RNAs were precipitated
with 500 ␮l of isopropanol and pelleted at 12,000 ⫻gfor 15 min. The pellet was
washed with 70% (vol/vol) ethanol and resuspended in water. Any contaminating
genomic DNA was removed by DNase treatment using a DNAfree kit (Ambion).
cDNAs were synthesized from up to 2 ␮g of total RNA in a reaction mixture
containing 0.1 ␮g of oligo(dT)
18
, 0.6 mmol/liter each nucleotide,5UofPrime
RNase inhibitor (Eppendorf), and Moloney murine leukemia virus reverse tran-
scriptase (Invitrogen). Approximately 20 ng of cDNA was used for each 20-␮l
PCR mixture, and PCRs were performed in an ABI7700 PCR machine (Applied
Biosystems) by use of Platinum Quantitative PCR SuperMix-UDG with added
ROX reference dye (Invitrogen), 0.3⫻SYBR green nucleic acid stain (Molec-
ular Probes), and 100 nmol/liter each primer (listed below). After a 10-min
incubation at 95°C, amplification was achieved by 40 cycles of a 15-s incubation
at 95°C followed by a 60-s incubation at 60°C. The identity and purity of the PCR
products were confirmed by melting-curve analysis. Expression levels in infected
mice were compared with those in uninfected controls after adjustment accord-
ing to the levels of the reference housekeeping gene hypoxanthine guanine
phosphoribosyltransferase, using the 2
⫺⌬⌬Ct
method (31). The primers used
were as follows: for hypoxanthine guanine phosphoribosyltransferase, 5⬘-GCTT
TCCCTGGTTAAGCAGTACA-3⬘and 5⬘-CAAACTTGTCTGGAATTTCAAA
TC-3⬘; for IFN-␥,5⬘-CAGCAACAGCAAGGCGAAA-3⬘and 5⬘-GCTGGATT
CCGGCAACAG-3⬘; for interleukin-10 (IL-10), 5⬘-GCCCTTTGCTATGGTGT
CCTTT-3⬘and 5⬘-TGAGCTGCTGCAGGAATGATC-3⬘; for IL-12, 5⬘-CTCAC
ATCTGCTGCTCCACA-3⬘and 5⬘-AATTTGGTGCTTCACACTTCAGG-3⬘;
for IL-18, 5⬘-TTCCATGCTTTCTGGACTCCTG-3⬘and 5⬘-TGCTGGAGGTT
GCAGAAGATG-3⬘; and for IDO, 5⬘-GGCTTCTTCCTCGTCTCTCTATT
G-3⬘and 5⬘-TGACGCTCTACTGCACTGGATAC-3⬘.
IFN-␥in plasma was quantified by an enzyme-linked immunosorbent assay
(ELISA) using either an OptEIA antibody set (catalog no. 555138; Becton
Dickinson-Pharmingen, CA) for low-dose P. berghei K173 CM studies or the
AN-18 antibody set no. 555138 for all other studies, according to the manufac-
turer’s instructions.
Statistical analysis. For time-series comparisons, one- and two-way analyses of
variance with Tukey’s posttest were performed using GraphPad Prism, version
3.00, for Macintosh (GraphPad Software, San Diego, California). Survival curves
were compared using a log-rank test (GraphPad Prism).
RESULTS
Early cytokine production occurs in P. berghei K173 infec-
tion. A number of in vitro studies using human peripheral
blood mononuclear cells (PBMC) have suggested that IFN-␥
may be produced rapidly by the innate immune system in
response to Plasmodium-parasitized red blood cells (pRBC)
(2, 3, 23). Since IFN-␥is centrally involved in a range of
immunological processes, IFN-␥protein levels were quantified
in the plasmas of both P. berghei ANKA- and K173-inoculated
mice over the course of infection (Fig. 1a). There was a clear
peak of IFN-␥protein in the blood on days 3 and 4 in ANKA-
infected mice, and slightly lower but more sustained levels of
IFN-␥protein were also seen for K173 infection from day 3
onwards. However, the most striking difference in IFN-␥pro-
files between the two infections was a large, transient burst of
plasma IFN-␥protein 24 h following K173 inoculation that was
absent for mice given ANKA. The general pattern of IFN-␥
protein production was mirrored when mRNA levels in the
spleen (Fig. 1b) and liver (Fig. 1c) were examined. By this
method, IFN-␥mRNA levels were significantly increased on
FIG. 1. Early production of IFN-␥during K173 infection. C57BL/6
mice were infected with either P. berghei ANKA (squares) or P. berghei
K173 (triangles), plasma IFN-␥protein was quantified by ELISA (a),
and changes in IFN-␥mRNA in the spleen (b) and liver (c) were
determined by RT-qPCR. There was a significantly increased produc-
tion of IFN-␥(*, P⬍0.05; **, P⬍0.01; and ***, P⬍0.001) at 24 h
post-K173 infection compared to that in ANKA-infected mice at the
same time point. Data are means and standard errors of the means
(SEM) for groups (n⫽5).
5646 MITCHELL ET AL. INFECT.IMMUN.

days 3 and 4 in the spleens of ANKA-infected mice, with a
lower and more prolonged increase in IFN-␥expression seen
for K173-infected animals. Again, however, the most obvious
difference between the two parasite strains was the large burst
of IFN-␥mRNA produced at 24 h postinfection in both the
spleens and livers of K173-infected mice (Fig. 1b and c), but
not in those of ANKA-infected animals.
In addition to IFN-␥mRNA, the mRNAs for a number of
other cytokines and the immunomodulatory enzyme IDO were
also upregulated 24 h after P.berghei K173, but not ANKA,
infection. Interleukins-10 and -12, but not -18, were signifi-
cantly induced in the spleens (Fig. 2) and livers (data not
shown) of K173-infected mice. Since the early production of
IDO by dendritic cells (DC) has been implicated in the sup-
pression of T-cell proliferation (18, 33) and since the produc-
tion of IDO is typically induced by IFN-␥(42), changes in the
level of IDO mRNA were examined in IFN-␥
⫺/⫺
mice follow-
ing K173 infection. These mice failed to upregulate IDO at
24 h post-K173 infection (Fig. 3), while none of the other
cytokines tested (IL-10, -12, and -18) were found to be depen-
dent on IFN-␥(data not shown).
Coinfection with P.berghei K173 inhibits the development of
P.berghei ANKA-induced CM. It was hypothesized that im-
mune processes that are activated at 24 h post-K173 infection
would be able divert the immune response to ANKA away
from the development of CM. Therefore, coinfection studies
were undertaken to investigate the potential influence of K173
infection on the disease course of ANKA infection. Groups of
mice were infected with either ANKA or K173 alone or with a
mixture of K173 and ANKA, and the hematocrit and survival
were determined over the course of infection (Fig. 4). In ad-
dition, the early cytokine response was investigated (Fig. 5). As
FIG. 2. Early expression of IL-10, IL-12, and IL-18 mRNA during K173 infection. Mice were infected with either strain ANKA (squares) or
strain K173 (triangles), and changes in cytokine or enzyme mRNA in the spleen were determined by RT-qPCR. There was a significant
upregulation of mRNAs for IL-10, IL-12, and IDO (*, P⬍0.05; **, P⬍0.01; and ***, P⬍0.001) at 24 h post-K173 infection compared to those
for ANKA-infected mice at the same time point. Data are means and SEM for groups (n⫽5).
FIG. 3. Induction of early IDO expression is IFN-␥dependent.
Wild-type and IFN-␥
⫺/⫺
mice were infected with strain K173 (PbK),
and changes in the levels of IDO mRNA were examined by RT-qPCR.
IDO mRNA was significantly upregulated (*, P⬍0.05) in wild-type
mice but not in IFN-␥
⫺/⫺
mice in both the spleen (a) and liver (b) at
24 h postinfection. Data are means and SEM for groups (n⫽5).
VOL. 73, 2005 EARLY CYTOKINE PRODUCTION IN MURINE CEREBRAL MALARIA 5647

expected, mice infected with P.berghei ANKA alone suc-
cumbed to CM on days 6 and 7 (median survival time, 6 days),
while in contrast, the majority of P.berghei K173-infected mice
survived until the second week of infection (median survival
time, 15.5 days), when they became moribund, with high par-
asitemia levels and low hematocrits. Saliently, mice that were
inoculated with both ANKA and K173 followed a similar dis-
ease course to those infected with K173 alone. These mice also
became moribund, with low hematocrits and high parasitemia
levels, in the second week postinfection (median survival time,
17 days) and showed no signs of CM. Also significantly, the
pattern of cytokine expression seen 24 h after infection with a
mixed K173-ANKA inoculum was essentially identical to that
of mice infected with K173 alone. IFN-␥protein levels in the
plasma, as well as mRNA levels in the spleen, were signifi-
cantly raised in both K173-infected and K173-ANKA-coin-
fected animals (Fig. 5a). Furthermore, the mRNA levels of
interleukin-10 and -12 as well as those of IDO were also raised
to levels similar to those seen with K173 infection alone (Fig.
5b).
Protection from CM correlates with early IFN-␥production
in a low-dose K173 model. To further investigate the signifi-
cance of the IFN-␥produced at 24 h postinoculation during P.
berghei K173 infection, a low-dose K173 model of CM was
used. In contrast to inoculation with high doses of K173 (⬃2⫻
10
6
), which typically follows a non-CM disease course in
C57BL/6 mice, lower doses of the parasite lead to an increased
incidence of early mortality associated with cerebral pathology
(12). Therefore, mice were inoculated with 10
6
K173 parasites,
blood samples were collected at 24 h for plasma IFN-␥deter-
mination, and the disease course was then followed in individ-
ual mice. Animals that became moribund in the second week
following inoculation (8/29 mice) showed signs of CM, while
those that continued beyond 14 days (21/29 mice) developed
high parasitemia levels and severe anemia (data not shown).
The presence or absence of CM was confirmed by brain his-
topathology. Plotting the values for the concentration of
plasma IFN-␥at 24 h against the disease outcome (CM or
non-CM) revealed that the group of animals that did not de-
velop CM had significantly higher levels of plasma IFN-␥at
24 h than those that did ultimately develop CM (Fig. 6a).
To extend this finding, a further experiment was performed
with groups of mice receiving either high or very low doses of
K173. Groups of animals (n⫽5/group) were inoculated with
either 2 ⫻10
6
,2⫻10
4
,or5⫻10
3
K173 parasites, and the
disease course was followed. A cohort of animals (n⫽5/group)
that received corresponding parasite inocula were killed at 24 h
for determination of plasma IFN-␥levels. Animals in groups
receiving low doses of K173 (2 ⫻10
4
or 5 ⫻10
3
parasites)
produced low levels of IFN-␥at 24 h, while those that received
a high dose of K173 (2 ⫻10
6
parasites) had significantly higher
levels of plasma IFN-␥at that time. Importantly, CM only
developed in mice receiving low doses of parasite, while those
receiving high doses were protected (Fig. 6b).
Early IFN-␥production during K173 infection is ␤2-micro-
globulin dependent. Since the early cytokine response, in par-
ticular the production of IFN-␥, appeared to influence the
development of cerebral immunopathology, the cellular source
of IFN-␥was investigated. Although NK cells and ␥␦ T cells
were the most likely candidates as the source of early IFN-␥in
response to pRBC, a number of other cell types, in particular
NKT and CD8
⫹
T cells, also may rapidly produce IFN-␥in
response to an antigen. Therefore, a systematic series of ex-
periments was performed using either gene-deficient animals
or animals depleted of cell types by antibody treatment to
investigate the cellular source of IFN-␥. The depletion of ei-
ther NK cells (Fig. 7a and b) or ␥␦ T cells (Fig. 7c and d) did
not lead to any significant changes in either plasma IFN-␥or
splenic IFN-␥mRNA in response to K173 infection. Similarly,
when the production of IFN-␥in K173-infected mice deficient
in NKT cells was examined using J␣281
⫺/⫺
(Fig. 8a and b) and
CD1d
⫺/⫺
(Fig. 8c and d) mice, these animals also produced
FIG. 4. Coinfection with K173 and ANKA inhibits the develop-
ment of CM. Mice were infected with either 2 ⫻10
6
K173 (triangles)
or 2 ⫻10
6
ANKA (squares) parasites alone or with an inoculum
containing both 2 ⫻10
6
ANKA and 2 ⫻10
6
K173 parasites (circles),
and survival was determined (n⫽15 per group). (a) Mice infected with
a mixed inoculum of ANKA and K173 survived significantly longer
than those infected with ANKA alone (P⬍0.001). The median sur-
vival times of groups inoculated with K173 or ANKA-K173 were not
significantly different (P⬎0.05). Mice infected with ANKA developed
classical signs of CM, while both K173- and ANKA-K173-infected
mice developed high parasitemia levels (b) and low hematocrits (c).
Note that for clarity, the parasitemia and hematocrit values for the
single surviving ANKA-infected mouse are not shown after day 6.
5648 MITCHELL ET AL. INFECT.IMMUN.

IFN-␥at similar levels to those of controls. The contribution of
CD8
⫹
T cells to early IFN-␥production was examined in
␤2-M
⫺/⫺
mice. In these animals, in contrast to other deficient
animals, the 24-hour IFN-␥response was completely abro-
gated (Fig. 8e and f).
DISCUSSION
From studies with many infectious disease models, it has
become clear that events occurring in the early immune re-
sponse can ultimately lead to dramatically divergent immune
effector responses. The classical example is polarization of an
undifferentiated immune response into either a Th1 or Th2
response, which depends in large part upon the early cytokine
milieu to which naı¨ve T cells are exposed when they are acti-
vated. While the Th1/Th2 dichotomy is highly stereotypic (29),
the coordination of immune effector mechanisms by appropri-
ate Th1 or Th2 responses typically leads to clearance or control
of infection (1, 27). In contrast to the majority of immune
responses, the activation of inappropriate effector responses
conceivably could lead to the development of immune-medi-
ated pathology. Inappropriate immune activation has been im-
plicated in the development of CM following P. berghei ANKA
infection, where it has been argued that the pathology shows
features of an exaggerated Th1 response (reviewed in refer-
ence 26). Therefore, we compared two mouse models of Plas-
modium infection to investigate the early phases of CM devel-
opment. We hypothesized that immune deviation caused by
early activation could not only influence the clearance or per-
sistence of a pathogenic organism but also influence the de-
velopment of immune pathology.
When the early production of cytokines was examined for
ANKA and K173 infections, striking differences were seen. For
ANKA infections, there was no detectable cytokine production
within the first 24 h. In contrast, for K173 infections, there was
a transient increase in the hepatic and splenic mRNA levels of
a variety of cytokines, including IFN-␥, IL-10, and IL-12, as
well as the immunomodulatory enzyme IDO. For Plasmodium
yoelii infection, it also has been reported that IFN-␥is pro-
duced at 24 h p.i. and that this IFN-␥production correlates
with protection from lethality (14). Such early production of
IFN-␥may be particularly relevant to human infection, since a
rapid induction of IFN-␥in human PBMC cultures following
P. falciparum challenge has been demonstrated previously and
since the kinetics of this production were broadly similar to
those noted in the present study (2, 3, 23). Furthermore, rapid
IFN-␥production by a variety of cells has been shown to be
dependent upon IL-12 (19, 20, 30, 36, 44), and IL-12 also is
transiently produced during K173 infection. Teasing apart the
involvement of early IFN-␥in CM is complicated by the role
that this cytokine plays late in the course of murine CM. It
appears that IFN-␥plays divergent roles at different stages of
P. berghei infection. While the results presented in this study
show that the IFN-␥produced at 24 h correlates with protec-
tion from CM, previous studies have shown that a later pro-
duction of IFN-␥is essential for the development of CM (21).
It was therefore impossible to use IFN-␥
⫺/⫺
mice or to inhibit
IFN-␥with antibodies to examine the direct contribution of the
early production of this cytokine to the disease course, because
any later production of IFN-␥would also be inhibited/absent
and these animals therefore would also lack the later IFN-␥
production involved in immunopathology.
A dual approach was used to further investigate the signif-
icance of the early IFN-␥production. Firstly, the production of
early IFN-␥was examined in both a coinfection model and a
low-dose P. berghei K173 infection model. In the coinfection
FIG. 5. Early cytokine production in mice coinfected with K173 and ANKA. Mice were infected with either K173 (PbK) or ANKA (PbA) alone
or with an inoculum containing both ANKA and K173, and the changes in cytokine expression were determined by ELISA and RT-qPCR. (a)
Changes in plasma IFN-␥were determined by ELISA, and those in splenic IFN-␥mRNA were determined by RT-qPCR. (b) Changes in splenic
IL-10, IL-12, and IDO mRNA were determined by RT-qPCR. For ANKA-K173-coinfected animals, the levels of all cytokines were significantly
upregulated 24 h after infection compared with those for uninfected controls (P⬍0.05), but they were not significantly different from those for
animals infected with K173 alone (ns, not significant). Data represent means and SEM for groups (n⫽5).
VOL. 73, 2005 EARLY CYTOKINE PRODUCTION IN MURINE CEREBRAL MALARIA 5649

model, the disease course was followed in mice infected with
both ANKA and K173. The rationale behind this was the
assumption that the early response to strain K173 might influ-
ence the response to the closely related ANKA strain. This
indeed appeared to be the case, since when animals were
simultaneously infected with ANKA and K173, a similar pat-
tern of cytokine production to that observed for K173 infection
alone was seen. Importantly, ANKA-K173-coinfected mice did
not develop CM, but followed a disease course similar to that
of K173 infection. One potential drawback of this coinfection
study is that the population dynamics of the individual strains
could not be determined. It is conceivable that in animals
receiving a mixed inoculum of both ANKA and K173, the K173
parasites outcompeted ANKA parasites, thereby inhibiting the
development of CM by a non-immune-mediated mechanism.
However, the correlation between increased circulating IFN-␥
and a lack of development of CM still held. To address this
issue, an alternative model using low-dose K173 inocula was
used to assess the functional significance of the early IFN-␥
peak. As reported by others, C57BL/6 mice infected with low
doses of K173 typically develop CM, while those that receive
high doses do not (12). In this model, protection from CM in
animals receiving a high dose of K173 correlated with high
levels of plasma IFN-␥at 24 h postinoculation. Furthermore,
when an intermediate inoculum size was given, which led to a
variable disease outcome, the production of high levels of
IFN-␥at 24 h correlated with protection from CM. Taken
together, the results from these two models strongly indicate
that the early production of IFN-␥during K173 infection is at
the very least associated with protection from CM and may
play an active role.
Since it appeared that IFN-␥production could be an impor-
tant modulator of the developing immune response, the cellu-
lar source of the early IFN-␥seen for K173 infection was
investigated with mice deficient in or depleted of various cell
types. The rapid production of IFN-␥is consistent with its
cellular source being a component of the innate immune sys-
tem. Natural killer cells (2, 3) or ␥␦ T cells (23) have been
argued to be the cellular source of IFN-␥in human PBMC
exposed to pRBC in vitro. Consistent with these in vitro stud-
ies, NK cells and ␥␦ T cells have been argued to produce IFN-␥
rapidly following P. yoelii infection in mice (8). However, the
authors of those studies were unable to definitively rule out the
involvement of ␣␤ T cells in IFN-␥production, as anti-Thy 1.1
was used to deplete ␥␦ T cells. Surprisingly, in the present
studies neither NK cells nor ␥␦ T cells appeared to be respon-
sible for early IFN-␥production in our experimental system, as
mice treated with either an anti-asialoglycoprotein receptor
antibody (NK depleted) or anti-␥␦TCR (␥␦ T-cell depleted)
still produced substantial IFN-␥peaks 24 h after K173 infec-
tion. Although not directly tested in the present studies, NKT
cells have been argued to be at least partially responsible for
the IFN-␥production seen on day 5 of ANKA infection of
C57BL/6 mice (22), which is associated with the pathogenesis
of CM, and therefore they were also a candidate for the cel-
lular source of early IFN-␥in the present model. However,
NKT cells did not appear to be responsible for early IFN-␥
production during K173 infection, as both CD1d
⫺/⫺
and
J␣281
⫺/⫺
mice, which are deficient in the major NKT-cell
subpopulations (7, 11, 32), showed normal IFN-␥production
at 24 h. Further light was shed on the source of IFN-␥pro-
duction when ␤2-M
⫺/⫺
mice were examined. In these animals,
the production of early IFN-␥was completely absent.
The most obvious deficit in ␤2-M
⫺/⫺
mice is that they lack
CD8
⫹
T cells, as ␤2-M is a component of major histocompat-
ibility complex class I (50). However, ␤2-M is also a component
of nonclassical major histocompatibility complex molecules,
most notably CD1. Consequently, an absence of ␤2-M also
results in an absence of NKT cells. Since J␣281
⫺/⫺
and
Cd1d
⫺/⫺
mice, which lack the major NKT-cell populations,
showed an early induction of IFN-␥in response to K173 in-
fection, it is unlikely that NKT cells are involved. Therefore,
although the production of IFN-␥by minor cell populations
cannot be excluded, it is possible that CD8
⫹
T cells are directly
involved in IFN-␥production during the early phase of K173
infection.
Although best characterized for their role in adaptive im-
munity, subpopulations of CD8
⫹
T cells have been reported to
rapidly produce IFN-␥in a nonclonally restricted manner un-
FIG. 6. Plasma IFN-␥levels at 24 h correlate with disease outcome
of K173 (PbK) infection. (a) Mice (n⫽29) were inoculated with 10
6
K173 parasites, and 24 h later, blood was collected for determinations
of plasma IFN-␥levels. The disease course was then followed in indi-
vidual mice. Animals were categorized into CM and non-CM out-
comes based on clinical signs and postmortem histopathology, and the
24-h IFN-␥levels were plotted based on the disease outcome. Points
represent individual animals and group means. **, P⬍0.01 versus the
CM group. (b) Groups of mice (n⫽5/group) were inoculated with
either a high dose (2 ⫻10
6
) or a low dose (2 ⫻10
4
or 5 ⫻10
3
) of K173
parasites, and the disease course was followed. Cohort groups (n⫽
5/group) were infected with corresponding parasite inocula, and the
levels of IFN-␥protein in the plasma of these animals at 24 h postin-
oculation were determined by ELISA. The data represent means and
SEM of plasma IFN-␥levels, and numbers above the bars indicate the
number of animals per group that developed CM. Note that a different
matched pair of antibodies was used for the IFN-␥ELISA in this
experiment than that used for time-course and depletion studies, which
gave higher determinations of plasma IFN-␥.
5650 MITCHELL ET AL. INFECT.IMMUN.

der a range of conditions. In particular, memory CD8
⫹
T cells
have been argued to contribute significantly to early IFN-␥
production in response to bacterial (5, 6, 30) or lipopolysac-
charide (28) challenge in an IL-12- and/or IL-18-dependent
process. Since the production of IL-12 was noted in the present
study, it is possible that an indirect mechanism such as this
could account for the CD8
⫹
T-cell stimulation seen here. Al-
ternatively, it has been argued that the majority of early IFN-␥
produced in response to an anti-CD3 antibody is from a nu-
merically minor subpopulation of CD8
⫹
cells with nonclassical
ontogeny that may also express markers that are present on
memory CD8
⫹
cells (13, 41). The reason for the discordance of
the present study with previous in vitro studies that suggested
that NK or ␥␦ T cells rapidly produce IFN-␥in response to P.
falciparum is unclear, but it may reflect study differences (hu-
man versus mouse model, spleen cells versus PBMC, or P.
berghei versus P. falciparum) or the occurrence of idiosyncratic
processes in vitro.
The downstream immune mechanisms for the inhibition of
CM pathology are unclear. Both IL-10 and IDO were seen as
likely candidates for modulating the developing immune re-
sponse to P. berghei and the consequent pathology, since den-
dritic cell production of these factors is capable of influencing
the developing T-cell response. Such an inhibition of T-cell
activation could conceivably account for the absence of CM in
P. berghei K173 infection, as both CD4
⫹
(24, 49) and CD8
⫹
(4,
35, 38) T cells have been shown to be involved in the later
stages of CM immunopathology during ANKA infection. A
possibly relevant observation is that antigen-specific T cells are
depleted during ANKA infection of BALB/c mice, a mouse
strain in which, unlike the case for C57BL/6 mice, CM does not
develop in response to ANKA infection (25), perhaps provid-
ing circumstantial evidence that similar processes of T-cell
inhibition may be at work in this model. The early production
of IL-10 acts predominantly in an autocrine manner by down-
regulating the activation of antigen-presenting cells, which
then has the downstream effect of inhibition of T-cell activa-
tion and expansion (15–17). Indeed, some cloned lines of P.
falciparum have been shown to downregulate DC activation in
vitro, a phenomenon that correlated with IL-10 production and
the binding of parasitized RBC to CD36 on DC (45, 46). It also
has been argued that high-affinity binding to CD36 may inhibit
the development of severe malaria (46). In addition to IL-10
production, the induction of IDO in dendritic cells has been
argued to be a critical immunoregulatory mechanism. IDO
breaks down the essential amino acid tryptophan, which is
FIG. 7. NK and ␥␦ cells are not responsible for early IFN-␥production. Mice that were either untreated or depleted of specific cell types by
prior antibody treatment were infected with K173 (PbK), and 24 h later, both plasma IFN-␥(by ELISA) and splenic IFN-␥mRNA (by RT-qPCR)
levels were determined. The levels of IFN-␥protein and mRNA expression in K173-infected NK-depleted (a and b) or ␥␦-T-cell-depleted (c and
d) mice were not significantly different from those in either non-antibody-treated or control antibody-treated, K173-infected animals (ns, not
significant). Data represent means and SEM (n⫽5 or 6/group).
VOL. 73, 2005 EARLY CYTOKINE PRODUCTION IN MURINE CEREBRAL MALARIA 5651

required for cellular division, and its early production by DC is
capable of mediating T-cell apoptosis and consequently of
regulating the T-cell response (18, 33, 34). Although IDO
expression in the brain has been implicated in the late-stage
immunopathology of CM (40), its role in early responses to
malarial infection is as yet unclear. However, in the present
study, the dependence of early IDO production on IFN-␥,as
well as the correlation of early IDO production with an ab-
sence of later immunopathology, provides indirect evidence
that it may be involved in immune modulation.
The results of the present study suggest that the initial
strain-dependent interaction of the immune system with ma-
larial parasites can critically influence the development of later
immunopathology. Moreover, not only may infection with dif-
ferent isolates of malaria parasites lead to differing patholog-
ical outcomes, but the immune response to one strain may
modulate the development of immunopathology caused by an-
other parasite strain.
ACKNOWLEDGMENTS
This study was funded by an Australian National Health and Med-
ical Research grant.
We thank G. Grau for stimulating discussions.
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