SUPPORTING INFORMATION
(References added in separate list)
Supporting results
Reduced increase of IgM, IgG2c and IgG2b levels after PcAS infection in
B6.TLR9-/-(OR) mice
The data presented in figure 1 suggested that B6.TLR9-/-(OR) have a less efficient
anti-malarial immunity compared to B6.WT(Jv). Antibody-dependent immunity is
considered essential during blood-stage malaria infection, particularly for the
resolution of the chronic stage of infection [1-4]. Hence, we tried to explain
differences in antiparasite immunity by effects on antibody responses [5,6]. Total
IgM, IgG and IgG subtype (IgG2c, IgG2b and IgG1) antibody responses in serum
from naive and infected B6.WT(Jv) and B6.TLR9-/-(OR) mice (day 21 and day 42 pi)
were analyzed by ELISA. IgG2c was measured since C57Bl/6 mice lack the gene for
IgG2a and instead express IgG2c. Titers of IgM antibodies were significantly raised
during the course of infection only in the commercial B6.WT(Jv) mice on day 21 (3.5
fold, p < 0.001) and day 42 (4 fold, p < 0.001) (Figure S3A). In contrast, in the
B6.TLR9-/-(OR) mice, the IgM increase was absent. The total IgG response was
significantly elevated during blood stage PcAS infection and no differences in titers
were observed between B6.WT(Jv) and B6.TLR9-/-(OR) mice (Figure S3B).
However, B6.TLR9-/-(OR) mice displayed an altered IgG subclass profile during a
malaria infection. Overall significant increases of both IgG2c and IgG2b antibody
titers were observed in B6.WT(Jv) and TLR9-/-(OR) mice after infection with PcAS
(Figures S3C, S3D respectively). Both titers of IgG2c and IgG2b subtypes were
more increased in infected B6.WT(Jv) mice versus infected B6.TLR9 -/-(OR) mice. In
particular, a significantly increased amount of IgG2c (1.3 fold, p < 0.001) and IgG2b
(1.1 fold, p < 0.05) antibodies was detected in plasma from B6.WT(Jv) mice on day
21 pi compared to B6.TLR9-/-(OR) mice. This pattern is reversed when considering
the IgG1 response (Figure S3E). The IgG1 response was elevated upon infection
only in B6.TLR9-/-(OR) mice and they produced significantly higher IgG1 antibody
titers on day 21 (2.2 fold, p < 0.001) and day 42 (2.1 fold, p < 0.01) pi in comparison
with B6.WT (Jv) mice. In B6.WT(Jv) mice, only on day 42 pi, a slight but significant
increase in IgG1 was observed compared to day 0. Altogether, these data suggest
that IgM, IgG2c and IgG2b antibodies may play a role in the protective immunity to
control acute and chronic stage PcAS infection, whereas IgG1 is inversely correlated
with parasite control.
Differential antimalarial effect of passive transfer of B6.WT(Jv) and B6.TLR9 -/(OR) immune serum
To investigate whether the altered antibody profile in B6.TLR9 -/-(OR) mice during
PcAS blood stage infection was associated with reduced parasite control, serum
transfer experiments were performed to test the protective ability of immune serum
from B6.WT(Jv) and B6.TLR9-/-(OR) mice. Naive B6.WT(Jv) mice were either shamtreated or treated ip with 200 µl of immune serum (day 21 pi) from B6.WT(Jv) or
B6.TLR9-/-(OR) mice. Immediately thereafter, mice were infected with 10 4 PcAS
pRBCs and parasitemia was monitored in Giemsa-stained blood smears during 44
days. As illustrated in figure S4, mice transferred by B6.WT(Jv) immune serum
followed by infection showed significantly reduced peak parasitemia values
compared to sham-treated mice (2.4 fold reduction, p < 0.001). This corresponded
with previous transfer studies which have also reported a protective role of
antibodies during PcAS infection [7,8]. In contrast, transfer of B6.TLR9-/-(OR)
immune serum into B6.WT(Jv) mice had no effect on acute parasitemia levels, which
were comparable to the parasitemia in sham-treated mice. In conclusion, the
difference in the course of PcAS infection between commercial B6.WT(Jv) and
B6.TLR9-/-(OR) mice was associated with alterations in IgM and IgG subclasses and
the production of antiparasite immune sera was shown to be protective only when
derived from commercial B6.WT(Jv) mice and not from B6.TLR9-/-(OR) mice.
Supporting Materials and Methods
Genotyping
Genomic DNA was obtained from mouse tail samples using the EZNA tissue DNA
purification kit (Omega Bio-Tek, Inc., Norcross, GA, USA). The TLR9 KO versus WT
genetic status of animals was detected by PCR as described previously [9] with two
sets of primers. One set was specific for the wild type TLR9 transcript (upstream 5’CAGTGCTGGCTGAGACTCTG-3’; downstream 5’-TAGCTCAGGTTCAGCTCCTC3’), the other set was specific for the TLR9-neomycin transcript (upstream 5’CAGTGCTGGCTGAGACTCTG-3’; downstream 5’-CGCCTTCTTGACGAGTTCTT3’). Denaturation was attained with 5 min at 95°C, followed by 50 amplification cycles
(1 min at 95 °C, 30 s at 59°C, 1 min at 72°C), and a final extension step of 10 min at
72°C. The resulting PCR products were separated and visualized on a 1.2 %
agarose FlashGel DNA cassette (57029, Lonza Rockland, USA). FlashGel loading
dye was added to all samples prior to loading (50462, Lonza Rockland). The
FlashGel DNA marker was used to provide accurate size estimation of DNA
fragments (100bp-4kb, sizes 100/200/300/500/800/1250/2000/4000 bp bands)
(50473, Lonza Rockland).
Measurement of hemoglobin concentration
Anemia during infection was assessed by colorimetric analysis of hemoglobin
concentration using the ‘SDS-haemichrome method’ as described by Theodorsen
[10]. Since hemoglobin in blood exists under various forms with each their specific
absorption, dilutions were made in buffer containing 3.5 mM SDS and 154 mM NaCl
to create denatured globin hemichrome with the absorption maximum at 536 nm.
Hemoglobin concentrations were calculated with the use of a serial dilution of human
hemoglobin (Sigma-Aldrich, St. Louis, MO, USA) as a standard.
Measurement of antibody isotype and subclass levels in plasma
Heparinized plasma was obtained from orbital punctures at the indicated time
intervals. Circulating plasma levels of IgM, IgG and IgG subtypes (IgG2c, IgG2b and
IgG1) were measured by ELISA (Enzyme Linked Immunosorbent Assay). Although
commercial anti-IgG2a sera are often used in isotyping and measuring the
concentration of Ig subclasses in mice, it is of note that C57Bl/6 mice lack the gene
for IgG2a and instead express the IgG2c isotype [11]. ELISA plates (96-well, Nalge
Nunc Inc., Roskilde, Denmark) were coated overnight at 4°C with 3 µg/ml of the
appropriate polyclonal anti-mouse isotype/subclass specific antibody (Jackson
ImmunoResearch Laboratories, Suffolk, United Kingdom) in coating buffer (100 mM
NaHCO3, pH 9.6). After washing the wells several times with PBS pH 7.4 containing
0.1% Tween 20 (wash buffer), the wells were blocked for 1 h at 37°C with 1% bovine
serum albumin (BSA, Cohn fraction V, Sigma-Aldrich) and 0.05% Tween 20 in PBS
(also used as diluent for samples, standard and antibodies) to avoid aspecific
binding. Wells were washed and 100 µl of serial dilutions of individual plasma
samples were added in the coated plate, incubated for 1h at 37°C and washed
extensively. For detection, horseradish peroxidase (HRP)-conjugated polyclonal antimouse isotype/subclass specific antibody (Jackson ImmunoResearch Laboratories)
was used. After 1h incubation at 37°C followed by extensively washing the wells,
HRP activity was visualized by adding 0.55 µM of the substrate tetramethylbenzidine
(TMB, Sigma-Aldrich) and 0.004% hydrogen peroxide (H2O2, Merck, Darmstadt,
Germany) in 0.1 M citrated/acetate buffer pH 4.3. The reaction was stopped with
H2SO4 (1 M) and optical densities were determined at 450 nm on a microplate
spectrophotometer (Power Wave XS, Biotek, Winooski, VT, USA). A laboratory
standard was prepared by pooling various plasma samples and was given an
arbitrary value of 100 units. Using the equation generated after plotting dilutions of
the laboratory standard versus the optical densities, the antibody levels in the
individual plasma samples were determined and expressed in units.
Passive transfer of immune serum
Immune sera were collected by cardiac puncture from B6.WT(Jv) and B6.TLR9 -/(OR) mice 21 days after infection with 104 PcAS pRBCs and pooled for each group.
Naive B6.WT(Jv) mice were transferred ip with 200 µl of either PBS, B6.WT(Jv)
immune serum or B6.TLR9-/-(OR) immune serum, followed immediately by ip
infection with 104 PcAS pRBCs. Parasitemia was monitored for a period of 44 days
by microscopic examination of Giemsa-stained blood smears.
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