Background
Malaria remains one of the most devastating diseases worldwide, with ~40% of the population at risk, and 200-300 million new cases each year, resulting in about one million deaths annually [
1]. The causative agents of malaria are parasitic protozoa belonging to the genus
Plasmodium. Except for the virulence of infected malaria parasite, presentation of clinical malaria is mainly dependent on the balance between pro- and anti-inflammatory responses against these parasites. Individuals who exhibit particularly weak immune responses often lead to uncontrolled parasitaemia. Thus, understanding the regulation mechanism of the immune response brought on by infection with
Plasmodium parasites will provide us with potential therapeutic approaches for treating infected individuals.
Although adaptive immune effectors, such as
Plasmodium-specific CD4
+ αβ T cells and antibody [
2], are mandatory for effective clearance of parasitized red blood cells (pRBCs) after infection, the control of parasite growth during the early stage of infection is largely dependent on the innate immune response. Previous study has shown that the primary peak of parasitaemia in T-cell-deficient mice tends to be comparable to that of wild-type mice [
3]. It has been reported that NK cell-derived IFN-γ that contributes to the early control of
Plasmodium chabaudi and
Plasmodium yoelii infections [
4,
5]. However, a recent study showed that the macrophage-mediated innate immune response, but not IFN-γ, has a significant role in controlling the primary wave of
P. yoelii infection [
6]. Another study recently reported that a population of CD11b
highLy6C
+ monocyte migration from bone marrow to the spleen was important for killing of
P. chabaudi blood stage at early stage [
7].
It is well established that macrophages are activated by malaria parasites to release pro-inflammatory cytokines, mainly through toll-like receptors (TLRs). For example,
Plasmodium falciparum-derived glycosylphosphatidylinositols (GPI) moieties are known to induce potent TNF responses in macrophages by TLR2, and to a lesser extent TLR4[
8,
9]. It has also been reported that
P. falciparum haemozoin, a crystalline by-product of haemoglobin metabolism by malaria parasites, is recognized by TLR9 on macrophages[
10], although more recently it has been suggested that instead haemozoin-bound nucleic acids are the true ligand for this receptor [
11,
12].
TLR signalling is subject to modulation by microorganisms. For instance, it is well known that lipopolysaccharide (LPS), the toxic wall component of Gram-negative bacteria, induces macrophages into a tolerance status by down-regulating TLR4 receptor expression [
13], or its association with MyD88, and by IRAK-1 activation [
14]. In contrast, infection of
P. falciparum primes peripheral blood mononuclear cells (PBMC) for TLR signalling by enhancing mitogen-activated protein kinase (MAPK) activation during the early stage [
15‐
17]. However, the infection of
P. yoelii non-lethal strain 17XNL was reported to induce dendritic cell (DC) TLR tolerance during the late stage [
18].
Little is known about the regulation of macrophages TLR signalling by the infection of lethal or non-lethal strain of rodent malaria parasites at the early stage. In the present study, both P. yoelii 17XL and 17XNL strains were found to be able to enhance the response of peritoneal macrophages to pRBC lysate and TLR agonists, through up-regulating the expression of TLR2 and intracellular signalling molecules MyD88, IRAK-1, and TRAF-6.
Methods
Mice and plasmodium
BALB/c mice (specific pathogen free, ~6-8 week old females) were purchased from the Animal Institute of Third Military Medical University (Chongqing, China). These studies have been reviewed and approved by the Third Military Medical University Institute of Medical Research Animal Ethics Committee.
P. yoelii 17XNL is a non-lethal strain, originally isolated in 1965 from the blood of a wild thicket rat,
Thamnomys rutilans [
19]
, and P. yoelii 17XL is a lethal strain cloned from
P. yoelii 17XNL, which was suddenly virulent in the laboratory of J. Finerty [
20]. Cohorts of 10 mice were infected intraperitoneally with 2 × 10
5 non-lethal strain
P. yoelii 17XNL-infected pRBCs or 2 × 10
5 lethal strain
P. yoelii 17XL. For control purposes, 10 mice were also infected with 2 × 10
5 RBCs taken from normal mice (nRBCs).
Reagents and antibody
Biotin anti-mouse TLR4 (MTS510), TLR2 (6 C2 clone), TLR9 (M9.D6 clone), Biotin rat IgG2a and IgG2b isotype control antibodies, anti-mouse CD16/32 (93 clone), FITC-F4/80 and PE-streptavidin were all purchased from eBioscience (San Diego, CA). TLR2 agonist Pam3CSK4, TLR4 agonist LPS (Escherichia.coli 0111:B4), TLR9 agonist CpG(ODN 1826), and its control CpG were purchased from InvivoGen (San Diego, CA).
Isolation of peritoneal macrophage
All the mice infected with P. yoelii 17XL or 17XNL, or injected with nRBCs, were administrated with 1 mg/ml thioglycollate (Sigma, St. Louis, MO, USA) via intraperitoneally three days before killing. Peritoneal exudate cells (PECs) were then extracted and allowed to adhere on tissue culture dishes for two hours, and non-adherent cells were removed. The adherent cells were collected as peritoneal macrophages, and F4/80 expression was analysed using a FACSCalibur flow cytometer with Cell Quest software (Becton Dickinson, Lincoln Park, NJ).
Cytokine detection by ELISA
Peritoneal macrophage from mice infected with P. yoelii 17XL, 17XNL, or nRBCs were cultured in the presence of 1 × 107 nRBC or pRBC lysate, or LPS (10 μg/ml), Pam3CSK4 (10 μg/ml), or CpG (10 μg/ml). Lysates from 1 × 107 red blood cells (RBCs) or pRBCs were prepared by twice freeze-thaw. After 24 hours, supernatants were collected and analysed by ELISA (eBioscience) according to manufacturer's instructions to detect IL-6 and TNF.
Flow cytometry assay
Peritoneal macrophage were isolated from mice infected with P. yoelii 17XL or 17XNL, or from mice injected with nRBCs, at one, three and five days after infection. 1 × 106 peritoneal macrophages were blocked with anti-mouse CD16/32 by incubation on ice for 30 min, then labelled with biotin-conjugated anti-mouse TLR4 and TLR2 for 30 min, followed by incubation with PE-streptavidin for 30 min. To examine TLR9 expression, peritoneal macrophages were permeabilized with fixation/permeabilization (eBioscience) prior to labelling with biotin-conjugated anti-mouse TLR9 and PE-streptavidin. Cells were finally analysed using a FACSCalibur flow cytometre using Cell Quest software (Becton Dickinson).
Semi-quantitive reverse-transcriptase PCR
Total RNA was isolated from 1 × 106 peritoneal macrophages from mice injected with P. yoelii 17XL or 17XNL strain, or with nRBCs, one, three and five days after infection using Trizol (Boehringer Mannheim, Germany), and reversely transcribed with MMLV (Promega, Madison, WI, USA). Initial cDNAs amount for PCR amplification were normalized with GAPDH signal, which was optimized to be visible, but not saturated. Changes in the expression of intracellular signalling molecules TRAF-6, IRAK-1, and MyD88 were investigated in peritoneal macrophages from mice infected with or without malaria parasites. Specific primers used were as follows: IRAK-1781: 5'- GAACAGCTATCAAGGTTTCGTCA-3'; IRAK-11260: 5'-ACCAGCAAGGGTCTCCAGTA-3'; MyD2341: 5'-CCCACTCGCAGTTTGTTG-3'; MyD2569: 5'-CTCCCAGTTCCTTTGTTTG-3'; TRAF162: 5'-GGGCTACGATGTGGAGTT-3'; TRAF396: 5'-TACCGTCAGGGAAAGAAT-3'.
Statistic analysis
Statistical significance was determined with SPSS software (version 13.0). Differences between two experimental groups (P.yoelii 17XNL-infection vs naive or P.yoelii 17XL-infection vs naive), or two time points were analysed for statistical significance by means of a nonparametric Mann-Whitney U test, and for multiple groups (TLR2 expression among P.yoelii 17XNL-infected, P.yoelii 17XL-infected and naive mice) using Kruskal-Wallis test. *P < 0.05 or **P < 0.01 were considered statistically significant.
Discussion
Macrophage-mediated innate immune response is critical for controlling the
P. yoelii parasitaemia at its early stage, so it tends to be modulated by exposure to malaria parasites. It was previously reported that
Plasmodium berghei infection inhibits IL-12 p40 production by peritoneal macrophages at the transcriptional level [
22]. However, infection with lethal strain
P. yoelii 17XL or non-lethal strain
P. yoelii 17XNL was found to be able to prime the response of macrophages through up-regulating TLR2 expression and signalling intracellular molecules, although infection of the two strains resulted in dramatically different disease outcome.
Pre-exposure to a variety of TLR agonists, including LPS, Pam3CSK4, and CpG, often induces macrophages into a tolerant status to prevent over-activation [
23‐
25]. It is well known that pre-administration of a low dose (sub-lethal dose) of LPS induces macrophages into endotoxin tolerance to protect the host from the challenge of lethal-dose of LPS. However, tissue injury[
26] and
Propionibacterium infection [
21] have been demonstrated to prime the TLR response. In this study, the lethal
P. yoelii 17XL strain was also found to enhance the response of murine peritoneal macrophages to pRBC at one, there and five days post-infection (Figure
3B-D), which is consistent with recent reports of priming the TLR response with
P. f at the early stage [
15‐
17]. Interestingly, infection with the non-lethal
P. yoelii 17XNL strain could also prime the response of macrophages to pRBC lysate (Figure
3A-C). Like the response of macrophages to pRBC lysate, macrophages from
P. yoelii 17XL- or 17XNL-infected mice responded to TLR2, TLR4, and TLR9 agonists much more strongly than macrophages from nRBCs-injected mice at one and there days post-infection (Figure
4). Thus, the increased response of macrophages to TLR2, TLR4, and TLR9 agonists resulted in their hypersensitivity to pRBC lysate.
In a previous study, McCall
et al attempted to correlate TLR2/4 expression with the priming response, however the investigators did not observe enhanced expression of TLR2/4 on PBMCs [
17]. In contrast, Flanklin
et al found that TLR2, TLR4 and TLR9 expression was significantly augmented in PBMCs from patients with relatively high parasitaemia [
15]. Here, the infection with
P. yoelii 17XL or 17XNL induced the expression of TLR2, but not TLR4 and TLR9, on murine macrophages in the present study (Figure
5). The disparity could be interpreted as
P. yoelii used in this study, but
P.f was used in their research. Interestingly, the transcription levels of intracellular molecules of the MyD88-dependent pathway were also found to be augmented in macrophages from
P. yoelii 17XL- or 17XNL-infected mice (Figure
5). Therefore, hypersensitivity of macrophages to TLR agonists was contributed to up-regulation of intracellular signalling molecules by malaria parasite infection. However, it remains to be determined whether up-regulation of MyD88, IRAK-1, and TRAF-6 would result in enhancement of MAPK activation, which was previously contributed to priming the TLR response on PBMCs from
P. f- infected patients [
16].
It was recently reported that malaria-induced priming of the TLR response was TLR9-, MyD88-, and IFN-γ-dependent[
15]. Hence, it is reasonable to find that lethal and non-lethal strains can prime the macrophage response in this study, as a relative high level of IFN-γ is induced in the spleen of either strain-infected mice during the early stage [
27]. It is well known that IFN-γ was mainly secreted by NK and T cells after infection with lethal strain
P. yoelii 17XNL and nonlethal strain
P. yoelii 17XL [
4], but the production of IFN-γ by NK cells required the help of IL-12 of DC activated by rodent malaria parasite [
28].
Single amino acid substitution of erythrocytic binding ligand (EBL) was reported to determine the erythrocyte invasion preference and virulence of
P. yoelii 17XL and
P. yoelii 17XNL [
29], but the early induction of TGF-β[
30] and activation of CD4
+CD25
+T cells could suppress the host immune response, and result in the overgrowth of
P. yoelii 17XL in mice. Although the level of macrophage response between the two strains could not be compared in this study, as their parasitaemia were significantly different at one, there and five days post infection (Figure
1), the duration time of primed TLRs response of macrophage from
P. yoelii 17XNL-infected mice was much longer than that from
P. yoelii 17XL-infected mice. This is consistent with a relative higher level of IFN-γ in the spleen of
P. yoelii 17XNL-infected mice than that of
P. yoelii 17XL-infected mice [
27], and might be associated with more efficiently controlling of
P. yoelii 17XNL growth than
P. yoelii 17XL in mice at the early stage.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
WYX designed research; YF, YD, TLZ, XLF performed research; WYX analysed data and wrote paper. All authors have read and approved the final manuscript.