ARTICLE
Auteur(s) : Matthew BB McCall1, Bart
Ferwerda2, Joost Hopman1, Ivo
Ploemen1, Boubacar Maiga3, Modibo
Daou3, Amagana Dolo3, Cornelus C
Hermsen1, Ogobara K Doumbo3, George
Bedu-Addo4, Jos W
van der Meer2, Marita
Troye-Blomberg5, André JAM
van der Ven2, Ralf R
Schumann6, Robert W Sauerwein1,
Frank P Mockenhaupt7, Mihai G Netea2
1Department of Medical Microbiology, Radboud
University Nijmegen Medical Centre, Nijmegen,
The Netherlands
2Department of General Internal Medicine, 463,
Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB
Nijmegen, The Netherlands
3Malaria Research and Training Centre, Faculty
of Medicine, University of Bamako, Bamako, Mali
4School of Medical Sciences, Kwame Nkrumah
University of Science and Technology, Kumasi, Ghana
5Wenner-Grenn Institute of Immunology, Stockholm
University, Stockholm, Sweden
6Institute of Microbiology and Hygiene,
Charité – University Medical Center, Berlin, Germany
7Institute of Tropical Medicine
and International Health, Charité – University Medical Center,
Berlin, Germany
accepté le 31 Mars 2010
Caspase-12 is a member of the large family of cysteine proteases
involved in apoptotic and inflammatory pathways [1], although it
has limited proteolytic activity of its own [2]. Recently,
caspase-12 has been implicated in the regulation of the
pro-inflammatory cytokine IL-1β, through the inhibition of
caspase-1-mediated cleavage of pro-IL-1β to IL-1β [3] and through
the inhibition of NOD/RIP signalling [4]. Mice lacking the
caspase-12 gene generated a stronger pro-inflammatory response than
wild-type animals [5]. In man, two allelic forms of the caspase-12
locus exist, the long (L-) and short (S-) variants. The former
encodes for a full-length protein, whereas the latter encodes for a
product that is prematurely terminated during translation. The
emergence of the S-variant represents a relatively recent event in
human evolution, since this variant is not found in chimpanzee
populations [6]. Clinical studies have found an association between
the L-variant of caspase-12 and an increased risk of sepsis [7].
Whereas in Caucasian populations exclusively the S-variant of
caspase-12 is found, in African populations the L-variant is still
common [8].
The distinct global distribution of caspase-12 alleles on the
one hand suggests selection of the inactive S-variant outside of
Africa, presumably due to its protective role in sepsis [9]. On the
other hand, maintenance of the functional L-variant in sub-Saharan
Africa implies a comparative advantage of this allele against some
major selective force specific to this continent.
Malaria remains responsible for high morbidity and mortality in
large parts of the developing world, but nowhere more so than in
sub-Saharan Africa, that suffers, by far, the highest transmission
intensities. This burden, combined with its target of particularly
young, pre-pubescent children, means that the disease has exerted a
strong selective pressure on human populations for at least the
last 10,000 years. Such selective pressure is visible today in the
genetic make-up of humans from malaria-endemic areas, more
specifically in the presence of allelic variants or other genetic
polymorphisms that confer some measure of protection against
malaria [10]. Indeed some of the oldest and best-studied human
genetic variations, those of the haemoglobin genes, including the
HbS and HbC variants and α-and β-thalassaemias, are found in
African populations as a result of selection by malaria [11]. In
addition to red blood cell phenotypes, malaria has also exerted
strong selective pressure on genes involved in both the adaptive
[12, 13] and innate immune response [14-18].
The strong selective pressure malaria is known to have exerted
on other genes, particularly in African populations, suggests that
it may also have played a role in the maintenance of full-length
caspase-12 on this continent. In the present paper, we have
combined caspase-12 genotype data from three separate studies
investigating susceptibility to malaria in different African
populations, and assessed associations with immunological, clinical
and obstetrical data.
DONORS AND METHODS
Mali study
Blood samples for functional assays were collected in the Koro
district of Mali as part of investigational studies into
inter-ethnic differences in susceptibility to malaria. This study
site has been described in detail elsewhere [19]. Briefly, it is a
rural Sahelian area with intense malaria transmission exclusively
during the rainy season (June-September). Samples for this study
were collected both during the 2006 rainy season (September) and
the 2007 dry season (April). The study included healthy Dogon or
Fulani adult male volunteers and patients with
symptoms of uncomplicated malaria. A thick blood film was
made from each participant; slide-negative patients were excluded
from the study. No positive blood films were recorded during the
April 2007 inclusion. Approval for the study was provided by the
institutional review boards of the University of Stockholm
(National Ethical Committee no. 03 536) and the University of
Bamako (N°0527/FMPOS).
Cameroon study
This was a hospital-based study, set up to investigate inflammatory
parameters in relation to clinical severity of malaria, details of
which are published elsewhere [20]. Briefly, the samples for this
study were collected from children aged between eight months and
14 years (mean age 5.8) presenting at the outpatient clinic or
admitted to the Central Hospital of Yaounde, Cameroon, with
symptoms of malaria. Based on a positive thick blood smear for
Plasmodium falciparum and a complete physical examination, patients
were classified by clinical presentation according to the 2000
World Health Organization (WHO) standard [21] as uncomplicated or
complicated/severe malaria. The number of patients categorised as
suffering from mild and complicated/severe disease was 100 and
38, respectively. Clinical parameters at admission, including
temperature, heart rate and respiratory rate were recorded, and
venous whole blood was collected for a complete blood count.
Informed consent was provided in all cases by a parent or guardian.
Ghana study
This study on the diagnosis and manifestation of placental malaria
in pregnancy has been described in detail [22]. The study area is
characterized by perennial and hyper- to holoendemic malaria
transmission. Briefly, 889 women attending the Presbyterian
Mission Hospital in Agogo, Ghana for delivery, were recruited after
informed, written consent was obtained. Peripheral venous blood
and, following delivery, placental intervillous blood were
collected and examined for P. falciparum by microscopy,
immuno-chromatographic test (ICT Malaria Pf/Pv,
Becton Dickinson, Germany) and nested PCR assays [23].
Haemoglobin (Hb) was measured using a HemoCue photometer
(Ångelholm, Sweden), and anaemia defined as Hb < 11 g/dL. Crude
birth weight and gestational age were assessed within 24 hours
of delivery. Low birth weight (LBW) was defined as < 2,500 g,
and preterm delivery (PD) as gestational age < 37 weeks applying
the Finnström score [24]. The study protocol was reviewed and
approved by the Committee on Human Research Publications and
Ethics, School of Medical Sciences, University of Science and
Technology, Kumasi.
Cytokine stimulation assay
In the Mali study, venous blood was collected from both healthy
volunteers and patients into lithium heparin vacutainers. Further
manipulations took place in Bandiagara, approximately three hours
from the study sites; all samples were worked-up on the day of
collection. Peripheral blood mononuclear cells (PBMCs) were
isolated by density gradient centrifugation on Ficoll, washed
3 x in cold RPMI, counted and resuspended in complete culture
medium [RPMI 1640 containing 2 mM glutamine, 1 mM
pyruvate, 50 μg/mL gentamicine and 10% pooled human AB+
serum (Sanquin, Nijmegen, NL)], for a final concentration of 2.5 x
106/mL. PBMCs were transferred into 96-well,
round-bottom plates and were immediately stimulated in duplo with
cryopreserved NF54 strain P. falciparum-infected erythrocytes
(PfRBC), uninfected erythrocytes (uRBC) at 5 x
106/mL final, or RPMI only. Cells were incubated for
21 hours at 37°C, following which cell supernatants were
collected and stored at - 80°C for subsequent cytokine
measurement by ELISA. Cell supernatants were analysed for cytokine
production using commercially available ELISA kits according to the
manufacturers’ instructions: IFN-γ, IL-10 (Sanquin, Nijmegen, NL)
and IL-1β (R&D, Abingdon, UK).
Caspase-12 genotyping
DNA was extracted from whole blood using either the Puregene
isolation kit (Gentra Systems, Minneapolis, MN, USA) or the QIAmp
blood mini-kit (Qiagen, Hilden, Germany). Primers flanking the
T125C polymorphism region and the methodology were adapted as
described by Saleh et al. [7]. Sequencing was performed on
either a 48-capillary 3730 sequencer (Applied Biosystems) or a
Lightcycler 480 (Roche). Genotypes were analysed using the software
4Peaks by A. Griekspoor and Tom Groothuis, (mekentosj.com).
Statistics
Data were analysed in SPSS; differences in genotype distributions
between populations were assessed using the χ2-test or
Fisher's exact test. Differences in clinical parameters between
groups were analysed using the Kruskal-Wallis test. Differences in
cytokine responses to PfRBC between groups were analysed using the
Mann-Whitney test after subtracting the response to uRBC; negative
values were set to zero. P-values of < 0.05 were considered
statistically significant in all analyses.
Results
Distribution of caspase-12 alleles in African
populations
Table 1 presents the varying caspase-12
genotype- and allele-frequencies amongst the African populations
studied. Frequencies of the L-allele as high as 34% were observed.
None of the populations studied showed evidence of violation of the
Hardy-Weinberg equilibrium. Caspase-12 genotype-frequencies
differed significantly (p < 0.05) between all populations
studied except those from Cameroon and Dogon (p = 0.13); similarly,
allele-frequencies differed between all populations except those
from Ghana and Dogon (p = 0.12).
Table 1 Distribution of caspase-12 genotypes and
allele-frequencies in the populations studied
|
Population
|
N
|
Caspase-12 genotypea
|
Allele frequency
|
|
|
S/Sb
|
S/Lb
|
L/Lb
|
S
|
L
|
|
Cameroon
|
138
|
81 (58.7%)
|
53 (38.4%)
|
4 (2.9%)
|
0.78
|
0.22
|
|
Ghana
|
885
|
699 (79.0%)
|
172 (19.4%)
|
14 (1.6%)
|
0.89
|
0.11
|
|
Mali Dogon
|
81
|
56 (69.1%)
|
25 (30.9%)
|
0 (0%)
|
0.85
|
0.15
|
|
Mali Fulani
|
91
|
42 (46.2%)
|
37 (40.7%)
|
12 (13.2%)
|
0.66
|
0.34
|
Mali study: cytokine profile amongst caspase-12 genotypes
During the malaria transmission season, parasite prevalence
differed between caspase-12 genotypes within the Fulani ethnic
group [S/S, 13/31 (41.9%); S/L, 11/26 (42.3%); L/L, 10/11 (90.9%);
p = 0.012], but not within the sympatric Dogon [S/S, 20/42 (47.6%);
S/L, 9/15 (60.0%); p = 0.41]. Irrespective of ethnicity, no effect
of the caspase-12 genotype was found on clinical parameters,
including parasite density or haemoglobin (Hb) levels (data not
shown).
Ex vivo stimulation assays were performed with PBMCs from
33 uninfected and 31 P. falciparum-infected donors in
September 2006, and a further 38 uninfected volunteers in
April 2007. Cytokine production by volunteer peripheral blood
mononuclear cells (PBMCs) stimulated ex vivo with P.
falciparum-infected erythrocytes (PfRBC) is presented in figure 1.
Interferon-γ (IFN-γ) and interleukin-10 (IL-10) responses to
uninfected-red blood cells (uRBC) or to medium alone were generally
below the detection limit of the assay (IFN-γ, 3.1 pg/mL; IL-10,
4.7 pg/mL). IFN-γ, but not IL-10, responses to PfRBC were
significantly stronger in uninfected than in infected volunteers (p
< 0.001 and p = 0.29, respectively), and therefore cytokine
responses were analysed separately by infection status; cytokine
responses did not differ between uninfected volunteers in the rainy
and dry season. IFN-γ responses to PfRBC were stronger in
caspase-12 S/L carriers than in S/S carriers for the infected group
(p = 0.011), but not for the uninfected group, although the pattern
remained similar (figure 1). IL-10
responses were higher in S/L than in S/S carriers among uninfected
donors (p = 0.023) but not among infected donors, although again
the pattern remained similar. Furthermore, these patterns were
observed individually in both Fulani and Dogon populations and
during both the transmission- and non-transmission seasons (data
not shown). Cytokine data were only available for three infected
L/L carriers; both IFN-γ and IL-10 appeared slightly lower than in
S/L carriers. We also analysed the IFN-γ /IL-10 ratio as an
estimate of the overall pro/anti-inflammatory balance. This ratio
was lower in S/L than in S/S carriers for both uninfected and
infected donors, although not significantly so. Interleukin-1β
(IL-1β) production was also assessed, but responses to PfRBC were
barely measurable above the detection limit of the assay (40 pg/mL
IL-1β) (data not shown).
Cameroon study: clinical presentation amongst caspase-12
genotypes
Table 2 presents the distribution of
caspase-12 genotypes amongst Cameroonian children with
uncomplicated or severe malaria. Amongst the 38 patients with
severe malaria, most had symptoms of cerebral malaria [31 were in
coma (Blantyre score < 3) and 10 suffered repeated
convulsions], nine were in severe respiratory distress and one had
severe anaemia (Hb < 5g/dL); not unexpectedly, a number of
patients presented with two or more symptoms of severe malaria.
However, no association was found between the caspase-12 genotype
and disease severity overall (p = 0.86), or between caspase-12
genotype and individual manifestations of severe malaria (table 2). Furthermore, there was no
obvious effect of the caspase-12 genotype on individual clinical
parameters among either uncomplicated or severe cases (table 2).
Table 2 Cameroon study: clinical presentation amongst
caspase-12 genotypes
|
N
|
Caspase-12 genotype
|
P-value
|
|
|
S/S
|
S/L
|
L/L
|
|
|
Overall genotype frequency, n (%)
|
138
|
81 (58.7%)
|
53 (38.4%)
|
4 (2.9%)
|
|
|
Clinical presentation
|
|
|
|
|
|
|
Uncomplicated malaria, n (%)
|
100
|
60 (74.1%)
|
37 (69.8%)
|
3 (75.0%)
|
|
|
Severe malaria (all forms), n (%)
|
38
|
21 (25.9%)
|
16 (30.2%)
|
1 (25.0%)
|
0.86a
|
|
Individual forms of severe malariab
|
|
|
|
|
|
|
- Coma (Blantyre score < 3), n (%)
|
31
|
16 (76.2%)
|
14 (87.5%)
|
1 (100%)
|
0.66c
|
|
- Repeated convulsions, n (%)
|
10
|
6 (28.6%)
|
4 (25.0%)
|
0 (0.0%)
|
0.80c
|
|
- Respiratory distress, n (%)
|
9
|
6 (28.6%)
|
3 (18.8%)
|
0 (0.0%)
|
0.80c
|
|
- Severe anaemia (haemoglobin < 5 g/dL), n (%)
|
1
|
1 (4.8%)
|
0 (0.0%)
|
0 (0.0%)
|
0.70c
|
|
Clinical parameters- uncomplicated cases (n = 100)
|
|
|
|
|
|
|
Gender, maled
|
|
36 (60%)
|
19 (53%)
|
0 (0%)
|
0.11
|
|
Age, yearse
|
|
4.8 ± 3.5
|
5.2 ± 3.8
|
6.3 ± 5.0
|
0.79
|
|
Temperature, oCe
|
|
39.2 ± 1.1
|
38.3 ± 6.7
|
39.2 ± 0.6
|
0.89
|
|
Parasitaemia, log10/μLe
|
|
4.4 ± 0.9
|
4.5 ± 1.1
|
4.7 ± 1.8
|
0.61
|
|
Haemoglobin, g/dLe
|
|
8.9 ± 2.0
|
9.0 ± 1.7
|
7.3 ± 3.4
|
0.45
|
|
Leukocytes, x 106/mLe
|
|
8.7 ± 3.9
|
9.8 ± 5.2
|
8.4 ± 1.2
|
0.60
|
|
Heart rate, /mine
|
|
126 ± 22
|
127 ± 23
|
127 ± 11
|
0.92
|
|
Respiratory rate, /mine
|
|
37 ± 9
|
39 ± 14
|
36 ± 7
|
0.99
|
|
Clinical parameters - severe cases (n = 38)
|
|
|
|
|
|
|
Gender, maled
|
|
11 (55%)
|
7 (47%)
|
1 (100%)
|
0.56
|
|
Age, yearse
|
|
3.3 ± 2.2
|
3.3 ± 2.2
|
7.8
|
0.31
|
|
Temperature, oCe
|
|
39.1 ± 1.2
|
38.7 ± 1.2
|
39.0
|
0.57
|
|
Parasitaemia, log10/μLe
|
|
4.4 ± 1.3
|
4.6 ± 1.0
|
5.1
|
0.89
|
|
Haemoglobin, g/dLe
|
|
8.7 ± 1.9
|
9.3 ± 1.5
|
9.4
|
0.52
|
|
Leukocytes, x 106/mLe
|
|
11.2 ± 5.2
|
14.3 ± 7.7
|
11.9
|
0.37
|
|
Heart rate, /mine
|
|
137 ± 27
|
130 ± 44
|
160
|
0.57
|
|
Respiratory rate, /mine
|
|
43 ± 13
|
45 ± 18
|
32
|
0.69
|
Ghana study: pregnancy outcome amongst caspase-12
genotypes
Table 3 shows the distribution of
caspase-12 genotypes amongst 885 women attending antenatal
clinics at the Agogo District Hospital, Ghana for whom genetic data
was available. Overall, neither the prevalence of P. falciparum by
PCR nor parasite density was found to vary significantly between
caspase-12 genotypes (table 3). In
a sub-group analysis of primigravidae (n = 315), the prevalence of
peripheral (but not placental) parasitaemia by PCR was slightly
higher in S/L than S/S mothers (70.8 versus 56.9%, p = 0.04), and
this association remained stable adjusting for age, presence of
pyrimethamine in plasma, and wet season (adjusted odds ratio, 1.89;
95%CI, 1.03-3.46; p = 0.04). Peripheral parasite densities in these
S/L mothers were, if anything, lower (2.6 versus 3.1 log10/μL, p =
0.09).
A mother's caspase-12 genotype status was not associated with
fever, preterm delivery, or LBW (table 3). In L/L mothers, anaemia
appeared to occur at a reduced rate, but this marginal association
(p = 0.04) was lost after stratification for malaria. Also,
stratification into infected and non-infected women did not change
the observed absence of an effect of the caspase-12 genotype on
maternal and foetal morbidity (data not shown).
Interestingly, the incidence of perinatal mortality was lower in
infants of S/L mothers (0/172) than in either S/S mothers (19/699,
p = 0.02) or L/L mothers (2/14, p = 0.005). This effect could not
be ascribed to malaria however, since the majority of all such
cases occurred in mothers without placental parasitaemia (table 3). Although the precise cause of
death could not always be defined, LBW appeared to contribute in a
majority (58%) of recorded cases. Furthermore, perinatal mortality
was associated with a history of complicated labour (47% of cases),
often requiring caesarean section (47% of cases). Finally, 52% of
cases involved either still-birth or poor delivery outcome
(10-minute Apgar score < 7).
Table 3 Ghana study: pregnancy outcome amongst
caspase-12 genotypes
|
Caspase-12 genotype
|
P-value
|
|
|
|
|
S/S
|
S/L
|
L/L
|
Overalla
|
S/S vs S/Lb
|
S/S vs L/Lb
|
S/L vs L/Lb
|
|
Overall genotype frequency, n (%)
|
699 (79.0%)
|
172 (19.4%)
|
14 (1.6%)
|
|
|
|
|
|
Data for all 885 women
|
|
|
|
|
|
|
|
|
Age of mother in years (mean)
|
26.3 ± 6.2
|
26.4 ± 6.8
|
27.8 ± 4.9
|
0.57
|
|
|
|
|
Primigravidae, n (%)
|
246 (35.5%)
|
65 (38.2%)
|
4 (28.6%)
|
0.68
|
|
|
|
|
Maternal fever, n (%)
|
22 (3.2%)
|
6 (3.5%)
|
0 (0.0%)
|
0.77
|
|
|
|
|
Maternal anaemia, n (%)
|
262 (37.5%)
|
58 (33.7%)
|
1 (7.1%)
|
0.048
|
0.36
|
0.020
|
0.04
|
|
Perinatal mortalityc, n (%)
|
19 (2.7%)
|
0 (0.0%)
|
2 (14.3%)
|
0.001
|
0.020
|
0.06
|
0.005
|
|
- amongst infectedd (n = 516), n (%)
|
9 (2.2%)
|
0 (0.0%)
|
0 (0.0%)
|
0.28
|
0.22
|
1.0
|
-
|
|
- amongst uninfected (n = 368), n (%)
|
9 (3.1%)
|
0 (0.0%)
|
2 (33.3%)
|
< 0.001
|
0.22
|
0.017
|
0.006
|
|
Parasite prevalence (by PCR)
|
|
|
|
|
|
|
|
|
- peripheral venous blood, n (%)
|
363 (51.9%)
|
92 (53.5%)
|
6 (42.9%)
|
0.73
|
|
|
|
|
- placental intervillous blood, n (%)
|
405 (57.9%)
|
104 (60.5%)
|
8 (57.1%)
|
0.82
|
|
|
|
|
Geometric mean parasite density
|
|
|
|
|
|
|
|
|
- peripheral blood, log10/μL
|
2.79
|
2.69
|
2.24
|
0.58
|
|
|
|
|
- placental blood, log10/100 fields
|
1.91
|
1.81
|
2.43
|
0.36
|
|
|
|
|
Data of live, singleton deliveries
|
|
|
|
|
|
|
|
|
Preterm deliverye, n (%)
|
119/647 (18.4%)
|
31/168 (18.5%)
|
4/12 (33.3%)
|
0.42
|
|
|
|
|
Low birth weightf, n (%)
|
105/653 (16.1%)
|
27/169 (16.0%)
|
1/12 (8.3%)
|
0.77
|
|
|
|
Discussion
The striking global distribution of caspase-12 alleles begs the
question of what evolutionary pressures have shaped this pattern.
Humans are the only mammals, apart from rabbits and cows, to have
lost a functional caspase-12 gene, indicating relatively recent and
species-specific selective pressure [6]. The proposed evolutionary
benefit of an untranslated caspase-12 gene lies in protection
against sepsis [7], although to our knowledge no further studies
have yet confirmed this. In any case, estimates of the genetic age
of the caspase-12 S-variant suggest fixating pressure starting as
late as 60 thousand years ago [9].
In line with this previous report, we confirm the presence of
the L-variant caspase-12 in African populations, at allele
frequencies of up to 34%. Whatever the selective force that has
eliminated the full-length L-variant outside Africa, within Africa,
it must have been counter-balanced by a factor acting with an
opposite effect, in order to explain the continued high frequencies
of this allele in populations on this continent. Malaria, given its
strong selective effect, specifically in Africa, would appear to be
a prime candidate for this role.
However, in none of the three studies did we find any convincing
evidence for an effect of the full-length caspase-12 L-allele on
either the prevalence or density of parasitaemia or the clinical
severity of malaria. Furthermore, caspase-12 genotypes did not
directly affect most individual obstetric parameters, including
prevalence or density of placental parasitaemia, fever, preterm
delivery or LBW. Nor was there any robust effect on Hb levels or
anaemia in delivering women (or either of the other two study
populations).
One limitation that must be borne in mind when considering these
findings is the low number of data on L/L homozygotes, limiting the
characterisation of their phenotype. However, although L/L
homozygotes may arguably show a more pronounced phenotype than
heterozygous individuals, as for any recessive genetic trait this
difference would not be expected to be greater than the observed
(lack of) difference between S/L heterozygotes and S/S homozygotes,
since both L/L homozygotes and S/L heterozygotes express some
full-length protein whereas S/S homozygotes do not express any
caspase-12 protein at all.
The full-length caspase-12 protein has been proposed to reduce
pro-inflammatory cytokine production through inhibition of
caspase-1 mediated activation of IL-1β [3]. To test this
hypothesis, we measured cytokine responses to P.
falciparum-infected erythrocytes by PBMCs from healthy and
parasitaemic African volunteers. Although IL-1β production was too
weak to draw meaningful conclusions, we were able to measure robust
IFN-γ responses. IFN-γ is induced downstream of IL-18, which itself
requires activation by caspase-1 in a manner similar to IL-1β [25,
26]. Remarkably, IFN-γ production in caspase-12 S/L-heterozygotes
tended to be stronger than in S/S individuals. However, S/L donors
also elicited stronger anti-inflammatory IL-10 responses than S/S
homozygotes and, overall, the IFN-γ/IL-10 ratio was lower in
heterozygotes. For diseases in which inflammation contributes
significantly to pathology, as is the case for various
presentations of severe malaria [27], a relatively stronger
anti-inflammatory balance may actually provide a survival
advantage. IL-10 production has been shown to reduce susceptibility
to severe malaria in animal models [28, 29], but in humans the
role of IL-10 is less well established. Clinical and post-mortem
studies have shown elevated levels of both pro-inflammatory
cytokines and IL-10 in most forms of severe malaria [30-32].
However, the ratios of pro-inflammatory cytokines (IFN-γ, TNF-α,
IL-6) to IL-10 are usually increased in severe malaria patients
[30, 31, 33, 34], suggesting that enhanced anti-inflammatory
responses protect against severe manifestations of malaria. In
particular, high IL-10 levels appear to attenuate severe anaemia
[33, 35]. It remains unclear however, by what pathway a full-length
caspase-12 might lead to stronger IL-10 production.
Finally, the association observed between maternal caspase-12
genotype and infant perinatal mortality risk, although potentially
coincidental, remains intriguing. This effect could not be ascribed
to malaria, or to other infections specifically. Rather, perinatal
death was associated with LBW and complicated labour requiring
caesarean section, possibly implying a role of caspase-12 in
the physiology of pregnancy. Nevertheless, this finding may
represent a balanced evolutionary force acting on caspase-12
alleles. Bearing in mind sampling error, the observed relative
selection against both homozygote states predicts an equilibrium
frequency of the L-allele of 2.7/(2.7 + 14.3) = 0.16 by standard
population genetics theory, which remarkably similar to the
observed frequency of 0.11 in this population. This alone, of
course, could not explain the disappearance of the L-allele in
Europe and Asia, which would then presumably be due to the
superimposed effects of other infections on those continents (e.g.
plague, influenza).
In summary, we aimed to investigate the evolutionary pressures
that maintain a full-length caspase-12 allele on the African
continent, with an emphasis on malaria infection. No major effects
of the caspase-12 genotype on clinical or parasitological
parameters were apparent, although functional studies assessing
cytokine stimulation by P. falciparum-infected erythrocytes showed
a slightly more anti-inflammatory profile in the presence of
functional caspase-12. These data, obtained in cross-sectional
settings, should be followed by larger prospective studies, in
order to fully understand the impact of caspase-12 polymorphisms on
malaria. Coincidentally, we found indications of an effect of the
caspase-12 genotype on perinatal mortality, which could itself
represent an important selective pressure. Alternatively, genetic
drift or other (parasitic) infections that could have led to the
balanced evolution of caspase-12 in Africa should be
considered.
Acknowledgments
We are grateful to the participants in all three studies in
Cameroon, Ghana and Mali for their time, cooperation and
enthusiasm. We thank the members of the three study teams involved
in sample collecting, for their collaboration.
Disclosure. None of the authors has any conflict of
interest to disclose.
Financial support. This work was supported by an FP6
European Network of Excellence (BioMalPar) fellowship (to MBBM) and
a Vici grant from the Netherlands Organisation for Scientific
Research (to MGN).
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