International Immunology

International Immunology Advance Access originally published online on November 29, 2006
International Immunology 2007 19(1):67-79; doi:10.1093/intimm/dxl123

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Pathological role of Toll-like receptor signaling in cerebral malaria

Cevayir Coban^1,2,*, Ken J. Ishii^1,3,*, Satoshi Uematsu¹, Nobuko Arisue⁴, Shintaro Sato^1,3, Masahiro Yamamoto¹, Taro Kawai^1,3, Osamu Takeuchi^1,3, Hajime Hisaeda⁵, Toshihiro Horii⁴ and Shizuo Akira^1,2,3

¹ Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
² 21st Century Center of Excellence, Combined Program on Microbiology and Immunology, Osaka University, Suita, Osaka 565-0871, Japan
³ Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Osaka University, Suita, Osaka 565-0871, Japan
⁴ Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
⁵ Department of Parasitology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan

Correspondence to: S. Akira; E-mail: sakira{at}biken.osaka-u.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
Toll-like receptors (TLRs) recognize malaria parasites or their metabolites; however, their physiological roles in malaria infection in vivo are not fully understood. Here, we show that myeloid differentiation primary response gene 88 (MyD88)-dependent TLR signaling mediates brain pathogenesis of severe malaria infection, namely cerebral malaria (CM). A significant number of MyD88-, but not TIR domain containing adaptor-inducing IFN-beta (TRIF)-deficient or wild-type (WT) mice survived CM caused by Plasmodium berghei ANKA (PbA) infection. Although systemic parasitemia was comparable, sequestration of parasite and hemozoin load in the brain blood vessels was significantly lower in MyD88-deficient mice compared with those in TRIF-deficient or WT mice. Moreover, brain-specific pathological changes were associated with MyD88-dependent infiltration of CD8⁺, CCR5⁺ T cells and CD11c⁺ dendritic cells, including CD11c⁺, NK1.1⁺ and B220⁺ cells, and up-regulation of genes such as Granzyme B, Lipocalin 2, Ccl3 and Ccr5. Further studies using mice lacking various TLRs suggest that TLR2 and TLR9, but not TLR4, 5 and 7, were involved in CM. These results strongly suggest that TLR2- and/or TLR9-mediated, MyD88-dependent brain pathogenesis may play a critical role in CM, the lethal complication during PbA infection.

Keywords: cell trafficking, hemozoin, innate immunity, MyD88, Plasmodium

	Introduction

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
Cerebral malaria (CM) is the lethal complication of malaria caused by Plasmodium falciparum in humans. Besides the high mortality rates, persistent neurocognitive deficits after recovery have become an increasing concern in past decades (1–3). While precise molecular and cellular mechanisms underlying the pathogenesis of CM are not yet fully elucidated, Plasmodium berghei ANKA (PbA) infection in mice provides valuable information as an experimental model of CM (4–6). After infection with PbA, susceptible mice (e.g. C57Bl/6) develop symptoms within a week, such as ataxia, hemi- or paraplegia, seizures and coma, and die within the following 24 h. Profound intravascular changes occur in cerebral vessels, such as endothelial cell damage and sequestration of infected erythrocytes, as well as host immune cells (i.e. platelets and leukocytes).

In recent decades, host immune responses to parasites have been implicated in playing an important role in CM pathogenesis. For example: (i) systemic pro-inflammatory cytokines such as IL-12, IFN and tumor necrosis factor (TNF) , but not IL-1 or IL-18 (7–9); and (ii) specific cell types, such as CD1d-restricted NK T cells and CD8⁺ T cells (10, 11), have been reported as critical mediators in CM development. Immune cell trafficking through CCR5 but not CCR2 has been found to be critical for leukocyte accumulation in the brain of CM-susceptible mice (12). However, the precise mechanism by which innate immune receptors or their signaling triggers such brain as well as systemic inflammation and immune cell trafficking is not known.

Toll-like receptors (TLRs) have been identified as key host molecules in innate immune recognition of and response to microbial products including lipids, proteins and nucleic acids (13). Recent evidence suggests that TLRs are involved in the innate immune responses to Plasmodium species (14). Adachi et al. (15) for the first time has analyzed the involvement of TLRs in vivo using a mouse malaria infection model where myeloid differentiation primary response gene 88 (MyD88), an essential adaptor molecule for most TLRs, is critical for IL-12 induction by P. berghei NK65 parasites, causing liver injury. Recently, glycosyl-phosphatidylinositol (GPI) and hemozoin (a parasite heme metabolite) derived from P. falciparum have been identified as the ligands for TLR2 and TLR9, respectively (16, 17), whereas other heat-labile molecules derived from the malaria parasite are still to be clarified for TLR9-mediated recognition (18).

Based on the above findings, we investigated the role of TLRs and their signaling molecules in the pathogenesis of CM. We used an in vivo experimental model for CM in which various mutant mice lacking TLRs and their adaptor molecules, such as MyD88 and TIR domain containing adaptor-inducing IFN-beta (TRIF), were infected with PbA. Monitoring survival, CM symptoms, parasitemia, hemoglobin level, pathological changes in the brain and host immune responses after infection revealed that TLR2-, TLR9- and MyD88-dependent signaling, but not TLR4-, TLR5-, TLR7- or TRIF-dependent signaling, facilitated CM pathogenesis and its resultant mortality. Analysis of systemic, as well as local inflammatory responses, suggest that the TLR–MyD88-dependent CM pathogenesis was associated with not only systemic inflammatory responses but also with brain sequestration of parasite, as well as hemozoin, and infiltration of particular lymphocytes, such as CCR5⁺, CD8⁺ T cells and CD11c⁺ dendritic cells (DCs) including NK1.1⁺, B220⁺ and CD11c⁺ cells, expressing both DC and NK cell marker.

	Methods

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
Animals
Mice deficient for MyD88, TRIF, TLR2, TLR4, TLR5, TLR7 or TLR9 were generated as described previously (19–25). Except TRIF^–/– mice and their littermate controls which are on a 129/Ola x C57Bl/6 (B6.129) background, all mice used here were backcrossed to C57Bl/6 (B6) background at least for eight generations. Age (6–10 weeks old)- and sex-matched groups of wild-type (WT) (either purchased from CLEA, Japan, or wild-type littermates) and knockout mice were used in the experiments. Animal experiments for infection were approved by the institutional protocol of the Research Institute for Microbial Diseases, Osaka University.

PbA infection and CM assessment
First, donor mice (either B6 or B6.129) were infected with the frozen stock of PbA-infected RBCs (iRBCs), and 6–7 days later, when the parasitemia showed mostly ring stages, and the mice suffered from CM symptoms, blood was drawn and used for infection studies. We chose this method to infect each mouse with similar blood-stage parasites. Then, WT or various mutant mice were infected with 10⁶ iRBCs intra-peritoneally in 200 µl PBS. Parasitemia was assessed every 2 days by microscopy of Giemsa-stained thin blood smears. Survival and signs of disease were monitored daily. Animals that showed neurological signs, such as convulsions, ataxia and paralysis, and died between 6 and 12 days after infection were considered as having CM. Brains were removed and used for histological analysis and reverse transcription (RT)–PCR. Serum was taken for cytokine ELISA and kept at –80°C until use. Blood hemoglobin levels were analyzed by using Drabkin's solution, as described elsewhere (12).

Cell culture, PbA crude extract and hemozoin
The murine microglial cell line BV-2 was cultured in 10% fetal bovine serum (FBS)-containing DMEM medium as described before (26). One million cells ml^–1 were seeded on to a six-well culture plate and stimulated for 24 h as indicated. Then, cells were collected, and total RNA was extracted using TRIzol reagent. RT–PCR was performed as mentioned below. PbA crude extract was prepared from the blood of infected mice, as described elsewhere (27). Hemozoin was prepared from Mycoplasma-free P. falciparum cultures as described before (17). CpG ODN 1555 was used as a control stimulant (26).

Histology
Six days after PbA infection, brains were perfused with PBS and carefully removed and fixed in formaldehyde solution (4% v/v). Tissue sections were prepared and stained with hematoxylin and eosin (HE) as described elsewhere (10). The sections were also stained by Prussian blue in order to visualize and count the hemozoin clusters. After staining with potassium ferrocyanide, counterstaining with nuclear fast red solution allowed us to visualize dark brown hemozoin clusters that were easily counted by light microscopy (Fig. 2B).

View larger version (77K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 PbA-infected MyD88-deficient mouse brain displayed a marked decrease in histopathological changes and brain hemozoin load. MyD88–/– and TRIF–/– mice and their WT controls were infected intra-peritoneally with 106 RBCs infected with PbA. (A) Histological analysis of brain sections of MyD88–/– and TRIF–/– mice and their WT controls 6 days after PbA infection. Brains were removed and fixed for histological examination. Representative sections from areas around the blood vessels of cerebrum with HE staining are shown (n = 3 per group). (a) Normal uninfected mouse brain showing a healthy blood vessel with normal architecture. Endothelial cells (closed triangle, magnification x1000). (b, c and e) Sections of an infected WT (B6) (b), B6.129 (c) and TRIF–/– (e) mice brains showing severe destruction of endothelial cells (open triangles), sequestration of iRBC (closed triangles) and mononuclear cell infiltration (black arrows, magnification x1000). (d) Section of an infected MyD88–/– mouse brain showing mostly intact endothelial cells (closed triangle, magnification x1000) and non-destructed blood vessel. (B) Tissue sections were stained with Prussian blue, and hemozoin clusters were counted by light microscopy. WT brain stained with Prussian blue is shown. Note that shown is a disrupted vessel, with a destruction of endothelial cells (black arrows), and infiltrated mononuclear cells (open triangle) that a counterstaining with nuclear red solution shows very faint staining, but clear visualization of dark brown hemozoin clusters (closed triangle, magnification x1000). (C) Hemozoin clusters in infiltrated brain vessels were counted in 25 microscopic fields. MyD88–/– mouse brains had significantly lower numbers of hemozoin clusters in each area counted (P < 0.001, Mann–Whitney U-test, n = 3 or 4 per group). ND represents not detected in uninfected naive mice.

 
Flow cytometry of brain-infiltrating lymphocytes
On day 6 after PbA infection, lymphocytes from brain were isolated as previously described (12). Briefly, after perfusion with PBS to remove the circulating blood cells, brains were crushed and washed in RPMI 1640 medium supplemented with 10% FBS and penicillin–streptomycin. Brain tissue extracts were then pelleted by centrifugation at 400 x g for 5 min, and were further purified on a 30% Percoll gradient (Amersham) (400 x g for 30 min). Cells were counted, fixed and stained with FITC-, PE-, CyChrome- or allophycocyanin (APC)-labeled antibody in the presence of anti-CD16 antibody for 30 min at room temperature, as previously described (17). Stained cells were washed, re-suspended in PBS/0.1% BSA/0.1% NaN₃ and analyzed by FACSCalibur, followed by analysis using CellQuest software (Becton Dickinson). All antibodies used were purchased from Becton Dickinson. T cell staining was performed using CD8–APC (or –FITC), Thy1.2–PE, TCRß–CyChrome, CD4–APC (or –FITC) and CCR5–PE. NK cells, B cells and DCs were stained with NK1.1–PE, CD45R (B220)–CyChrome and CD11c–APC (28, 29).

DNA microarray analysis
On day 6 of infection with PbA, half of the brain tissues were removed and kept at –80°C. Total RNA was extracted from individual brains with TRIzol and further purified by RNeasy kit (Qiagen, Hilden, Germany), and cDNA was synthesized from total RNA with the SuperScript Choice System (Invitrogen, Carlsbad, CA, USA). These cDNAs were used to prepare biotin-labeled cRNA according to the manufacturer's protocol (Enzo Diagnostics, Farmingdale, NY, USA). Purification of cRNA and hybridization and scanning of the microarray were done according to the manufacturer's instructions (Affymetrix, MG U74A version 2). Data analysis was carried out by using a Suite software version 5.0 (Affymetrix) and GeneSpring software version 6.0 (Silicongenetics, Redwood, CA, USA) (30). To confirm the gene changes, the experiment was performed twice by using two mice per group.

RT–PCR
RT–PCR was carried out as described elsewhere (30). Briefly, brain tissues were homogenized and total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Then, 1 µg total RNA was reverse transcribed with SuperScript II (Invitrogen). PCR amplification was performed using recombinant Taq DNA polymerase (Takara Shuzo). PCR conditions were 30 s denaturation at 94°C, 30 s annealing at 60°C and 1 min elongation at 72°C for 30 cycles. Specific primers used were as follows: Granzyme B, sense 5'-TCGACCCTACATGGCCTTAC-3' and anti-sense 5'-TGGGGAATGCATTTTACCAT-3'; Lipocalin 2, sense 5'-CCAGTTCGCCATGGTATTTT-3' and anti-sense 5'-CACACTCACCACCCATTCAG-3'; MIP-1, sense 5'-ATGAAGGTCTCCACCACTGCCCAAGC-3' and anti-sense 5'-TTAGTCAGGAAAATGACACCTGGCTGGG-3'; IL-6, sense 5'-GACAAAGCCAGAGTCCTTCAGAGAG-3' and anti-sense 5'-CTAGGTTTGCCGAGTAGATCCTC-3' and ß-actin, sense 5'-GACATGGAGAAGATCTGGCACCACA-3' and anti-sense 5'-ATCTCCTGCTCGAAGTCTAGACAA-3'. Density of the PCR products in ethidium bromide-stained gel was measured by NIH image software (http://rsb.info.nih.gov/nih-image/download.html). Quantities of each transcript were compared with the ß-actin reference.

ELISA
Mouse IFN, TNF and IL-12p40 in the serum were measured by ELISA (DuoSet ELISA Kit, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Statistical analysis
Differences between groups were analyzed for statistical significance by using SigmaStat 3.0 software with either Student's t-test or Mann–Whitney U-test. For survival curves, Kaplan–Meier plots and log rank tests were performed. P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
Critical role of MyD88 in CM and its resultant lethality, but not in systemic parasitemia
MyD88 is an essential adaptor molecule for most TLR signaling, whereas TRIF is an indispensable adaptor for TLR3 and TLR4 (31). To examine the possible role of innate immune responses through TLRs in CM pathogenesis, we first infected intra-peritoneally MyD88- or TRIF-deficient mice with a C57Bl/6 (B6) or 129/Ola x C57Bl/6 (B6.129) background, then monitored their survival, parasitemia, percentage hemoglobin in the blood. B6.129 mice were susceptible to CM with 10⁶ PbA and frequency of characteristic CM symptoms were consistent with a previous report (12). After infection, WT mice showed CM symptoms within 6–12 days and died within the following 24 h; however, a significant number of MyD88-deficient mice survived CM and eventually died of extremely high parasitemia, reaching >85% (Fig. 1A, P = 0.003 by log rank survival test). In sharp contrast, infected TRIF-deficient mice died even before the WT controls, during which all of the TRIF-deficient mice suffered from CM (Fig. 1B, P = 0.325 by log rank survival test). Overall survival from CM was significantly higher in MyD88-deficient mice than that in WT or TRIF-deficient mice [Fig. 1C and D, 10% of WT (n = 50) versus 46.15% of MyD88^–/– (n = 39) mice escaped from CM; 9.1% of WT (n = 16) versus none of the TRIF^–/– (n = 16) mice escaped from CM].

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 MyD88- but not TRIF-deficient mice have increased resistance to CM. MyD88–/– and TRIF–/– mice and their WT controls were infected intra-peritoneally with 106 iRBCs of PbA strain. The percentage survival and parasitemia of (A) WT (B6, closed circles) and MyD88-deficient (MyD88–/–, open circles) mice (n = 12 for each group) and (B) WT (B6.129, closed circles) and TRIF-deficient (TRIF–/–, open circles) mice (n = 6 for each group, littermates) after infection. Survival was monitored daily. Parasitemia was assessed by Giemsa-stained blood smears every other day (mean parasitemia ± SE). Data shown here are representative of three (B) to six (A) different experiments (P = 0.003 for the percentage survival curve of WT versus MyD88–/– mice; P = 0.327 for those of WT versus TRIF–/– mice by log rank test). (C) Total escape from CM in MyD88–/– mice until day 12 after infection. Ten percent of WT (B6) mice (n = 50) escaped from CM versus 46.15% of MyD88–/– mice (n = 39). (D) Total escape from CM in TRIF–/– mice until day 12 after infection. In all, 9.1% of WT (B6.129) mice (n = 16) escaped from CM versus 0% of TRIF–/– mice (n = 16). ‘None’ implies that no TRIF–/– mice could survive.

 
Systemic parasitemia and hemoglobin levels were comparable between WT and MyD88- and TRIF-deficient mice during the first week of PbA infection (Fig. 1A and B and data not shown), similar to a previous study on infection with P. berghei NK65 which did not cause CM (15). These data suggest that MyD88, but not TRIF, is involved in CM symptoms and subsequent death, whereas neither MyD88 nor TRIF is involved in controlling parasitemia or the resultant anemia.

MyD88-dependent parasite sequestration and hemozoin load in the brain
We investigated the mechanisms involved in the pathogenesis of MyD88-dependent CM. Since brain-specific immunopathological changes have been shown to play a critical role in CM development (4, 5, 32), we examined the effects of MyD88 and TRIF on immunopathological changes in the brains of mice 6 days after infection, at which most WT mice begin to develop characteristic symptoms of CM. Histological examination of brain sections revealed that WT mice from a B6 or B6.129 background, as well as TRIF-deficient mice, showed typical vascular occlusion with parasitized erythrocytes as well as leukocytes and microvascular destruction including endothelial cell detachment (Fig. 2A, b and c). However, these characteristic pathological changes were absent in MyD88- but not in TRIF-deficient mice, suggesting that a MyD88-dependent immune response may play a role in the brain pathogenesis of CM (Fig. 2A, d and e, respectively).

We have previously demonstrated that hemozoin, a malaria metabolite during the red blood cell stage, activates the innate immune system via TLR9 and MyD88 (17). To investigate whether hemozoin is involved in the pathogenesis of CM, we attempted to visualize hemozoin clusters in HE-stained brain sections. However, we found that when the sections were stained with Prussian blue, visualization was easier due to faint red staining of the background (Fig. 2B). We counted hemozoin clusters in 25 microvessels per group (n = 3 or 4) by light microscopy, and found that hemozoin accumulation was significantly reduced in MyD88-deficient brains compared with that in WT brains at day 6 (Fig. 2C, P < 0.001 by Mann–Whitney U-test), in spite of comparable systemic parasitemia levels (Fig. 1A).

In addition, we noticed that there was hemozoin residue in the tissues, outside the blood vessels (data not shown). It is possible that destruction of the blood–brain barrier might allow parasites or hemozoin itself to enter the brain tissue to activate residual microglia cells. Indeed, microglial cell activation during CM has previously been implicated (33), which prompted us to evaluate whether microglia cells, macrophage-like cells in the brain, can be directly activated by hemozoin or crude extract of iRBCs. We stimulated BV-2 cells (26) (a murine microglia cell line expressing TLR9 mRNA (34)) with PbA crude extract and hemozoin for 24 h (Supplementary Figure 1A and B, available at International Immunology Online). RT–PCR analysis showed activation by hemozoin or PbA crude extract in BV-2 cells to up-regulate mRNA expression of acute inflammatory response genes such as Lipocalin 2, MIP-1 and IL-6. We used CpG ODN 1555 as a control (26). Taken together, these data suggest that microglial cell line can respond to hemozoin and PbA crude extract.

Role of MyD88 and TRIF in the immunopathological changes in the brain during PbA infection
In order to comprehensively analyze and identify the genes involved in MyD88-dependent, brain-specific molecular events during CM, we obtained brains from WT mice or MyD88- or TRIF-deficient mice at day 6 after PbA infection, and examined their mRNA expression profiles by DNA microarray analysis. We compared ‘fold increases’ of mRNA expression in PbA-infected brain tissue over those in uninfected tissue, which were then compared with those of WT mice (B6 or B6.129) and MyD88- and TRIF-deficient mice. A cut-off value was determined as a 5-fold change in infected brains over that of uninfected WT mice. Genes that showed a <2-fold change between WT and mutant mice were determined as ‘independent’, whereas those with a >2-fold change were determined as ‘dependent’. Accordingly, the transcriptional responses to PbA infection were divided into three categories: genes that are regulated solely by MyD88, by either MyD88 or TRIF or solely by TRIF (Table 1, Supplementary Tables 1 and 2, available at International Immunology Online).

View this table: [in this window] [in a new window]   Table 1 Gene expression profiles in brains of MyD88- and TRIF-deficient mice on day 6 after PbA infection

 
Among the genes up-regulated by PbA infection in a MyD88-dependent manner, we noticed that the up-regulation of genes related to TLR-activated microglia cells, such as G-protein-coupled receptor mFpr-2, as well as chemokines such as Ccl3 and Ccl9, were also MyD88 dependent, implying that residual microglia or migrated macrophages may also be involved in MyD88-dependent up-regulation of these genes. We found that the genes related to severe malaria, such as Lipocalin 2 (24p3) (35) and Haptoglobin (Hp), genes associated with cerebral ischemia, such as C4b (complement component 4B), C1qb, Klk7 (Kallikrein 7) and S3-12 (plasma membrane associated protein) and stress response genes, such as Atf3, were up-regulated in the brain in a MyD88-dependent manner.

We also noted that the genes which are (i) associated with cytotoxicity, such as Gzmb (granzyme B) and Pdcd1lg1 (programmed cell death 1 ligand 1, also called B7-H1 or CD274); (ii) related to lymphocyte recruitment, such as Ccl3, Ccl9 and Ccr5; (iii) IFN (type-I and/or -II) inducible, such as Vig1, Oasl2, Tap-1, Ifi44, Ifit3, Ifi35, Isg15, Irf7, Irf9, Usp18, Ifi1 (LRG-47), Stat1, Mx1, Mda5, Ppicap (lectin, also called CyCAP) and Zfp36 and (iv) expressed in T cells and NK cells, such as Ms4a4b (also called Chandra) (36), schlafen 1 and schlafen 2. These data suggest that type-I or -II IFN-producing immune cells such as T cells, DCs and/or NK cells may be involved in the up-regulation of such genes in the brain during PbA infection.

Some of the up-regulated genes that are dependent on either MyD88 or TRIF include type-II IFN-inducible genes such as Lmp7, Ubiquitin D (Ubd), H-2Kd, Ifi205, Gbp2 and Ifi47 and GTPases and chemokines such as Ccl5, Cxcl10, Cxcl9, Cxcl16 and Ccl21a indicate that there is, in fact, TRIF-dependent, possibly TLR3- and/or TLR4-mediated, innate immune recognition during PbA infection (Supplementary Table 1, available at International Immunology Online).

To confirm the results obtained by DNA microarray analysis, some of the gene expression was monitored by RT–PCR in the brain tissues at day 6 after PbA infection. Significant up-regulation of Granzyme B, Lipocalin 2 and Ccl3 mRNA expression was observed in a MyD88-dependent manner (Fig. 3, *P < 0.05 by Student's t-test). The results obtained by DNA microarray analysis revealed novel genes that were up-regulated in a MyD88-dependent manner, suggesting their critical role in CM pathogenesis. Importantly, MyD88-dependent genes, including those coding for chemokines related to lymphocyte recruitment, and lymphocyte-related genes, some of which have been reported as critical for CM pathogenesis (12), prompted us to examine the role of MyD88 in recruitment of lymphocytes during CM pathogenesis.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 MyD88-dependent brain mRNA gene expression after PbA infection. MyD88–/– and TRIF–/– mice and their WT controls were infected with 106 of PbA iRBCs. Brains were removed 6 days after infection and mRNA expression was analyzed by RT–PCR. Density of the PCR products in ethidium bromide-stained gel was measured by NIH image software. Results (mean ± SD) are presented as relative units normalized to ß-actin, which were obtained from three infected mice per group, and experiments were repeated twice. *P < 0.05, infected MyD88–/– or TRIF–/– mice versus infected WT mice (Student's t-test).

 
MyD88-dependent recruitment of CCR5⁺, CD8⁺ T cells and CD11c⁺ DCs, including B220⁺, NK1.1⁺ cells, into PbA-infected mouse brain
It has been shown that recruitment and activation of T cells in the brain during PbA infection play a critical role in CM pathogenesis (37, 38). Based on the brain mRNA expression profile in PbA-infected mice, we examined whether such T cell recruitment occurs and it is controlled by MyD88. Brain cells were isolated from WT or MyD88-deficient mice at 6 days after PbA infection, and analyzed for the number and type of cells by flow cytometry. Similar numbers of mononuclear cells were recovered from all groups of brains (uninfected naive mice contained 14.2 x 10⁶ ± 4.7 x 10⁶ cells per brain, WT mice at day 6 of infection contained 14.9 x 10⁶ ± 8 x 10⁶ cells per brain and MyD88^–/– mice at day 6 of infection contained 10.2 x 10⁶ ± 1 x 10⁶ cells per brain, n = 3–6 mice per group). Large numbers of Thy1.2⁺ and TCRß⁺ T cells were observed among cells isolated from the brain at 6 days after infection, while almost no T cells were stained in naive brains suggesting negligible contamination of the cells from the blood flow (Fig. 4A). More than 70% of these T cells in the infected WT brain were CD8 positive, and the remainder were CD4 positive (Fig. 4A). These CD8⁺ T cells infiltrating the brain also expressed CCR5, whose mRNA was up-regulated in DNA microarray analysis (Fig. 4A and Table 1). Of note, CCL3 (also named MIP-1), the ligand for CCR5, was also highly up-regulated in the infected brain (Fig. 3 and Table 1). In sharp contrast, the brains of MyD88-deficient mice with no CM symptoms had dramatically fewer CCR5⁺, CD8⁺ T cells (Fig. 4A). As CCL3 was highly up-regulated in the brain, these results suggest that CCR5⁺, CD8⁺ T cells may be recruited to the brain via MyD88-dependent up-regulation of CCL3.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 MyD88-dependent recruitment of CCR5+, CD8+ T cells and CD11c+ cells, including CD11c+, B220+ and NK1.1+ cells, into PbA-infected brain. Brain lymphocytes were isolated from infection-naive or PbA-infected WT or MyD88–/– mice, and stained with labeled antibodies as described in the experimental procedure. (A) Thy1.2+ and TCRß+ T cells (left panel) were stained with antibodies against CD4, CD8 and/or CCR5 (middle and right panels). (B) Cells were stained with anti-CD11c, NK1.1 and/or B220. Numbers represent the percentage of gated cell population in isolated brain lymphocytes. Results are representative of one mouse from each group of three mice. Experiments were repeated three times with similar results.

 
We also found that CD11c⁺ DCs, but not B220⁺ B cells, were increased in number in infected brains in a MyD88-dependent manner (Fig. 4B). Among those CD11c⁺ cells increased, we found that CD11c⁺ and NK1.1⁺ cells were also increased in a MyD88-dependent manner, all of which were CD11c⁺ and B220 dim (Fig. 4B), which displayed identical staining pattern to those identified as hybrid type cells for DCs and NK cells namely IFN-producing killer dendritic cells (IKDCs) (type-I and -II) (28, 29) and certain NK cell subsets as previously described (39, 40). These results suggest that MyD88-mediated signaling triggers expression of genes, such as chemokines, including CCL3, resulting in recruitment of CCR5⁺, CD8⁺ T cells, as well as CD11c⁺ DCs, including CD11c⁺, B220⁺ and NK1.1⁺ cells into the brain. It was also shown by DNA microarray analysis and RT–PCR that genes such as Ms4a4b (also called Chandra), granzyme B and Ifng, which are expressed in CD4⁺ and CD8⁺ T cells and NK1.1⁺ cells, and possibly in CD11c⁺, B220⁺ and NK1.1⁺ cells (36), were also highly up-regulated in WT mice brains, but not in those from infected MyD88-deficient mice or uninfected mice (Table 1).

Role of TLRs in the pathogenesis of CM
MyD88 is an essential adaptor molecule for intracellular signaling mediated by most TLRs, as well as IL-1R/IL-18R. Previous reports suggest that IL-18 is up-regulated and involved in protective immunity, rather than lethal complications such as CM in PbA infection (9). Plasmodium spp. have been shown to contain ligands for TLR2 and TLR9, such as GPI anchor and hemozoin, respectively (16, 17), which prompted us to examine whether these TLRs are, in fact, involved in the pathogenesis of CM. We infected mice deficient for TLR2, TLR4, TLR5, TLR7 or TLR9 with PbA, and then monitored incidence of CM and survival. Each TLR-deficient mouse was backcrossed to C57BL/6 background at least for eight generations. A significant number of mice lacking TLR2 or TLR9, but not TLR4, TLR5 or TLR7, survived CM and early death between 6 and 12 days after infection (Fig. 5A–E) (P < 0.001 for WT versus TLR2^–/– mice; P = 0.027 for WT versus TLR9^–/– mice; P > 0.05 for WT versus TLR4^–/–, TLR5^–/– or TLR7^-/Y mice by log rank survival analysis). Overall escape from CM in TLR2- and TLR9-deficient mice until day 12 after infection was significantly higher [10.55% of WT (B6) mice (n = 28) versus 55% of TLR2^–/– mice (n = 21) escaped from CM; 14.7% of WT (B6) mice (n = 27) versus 45.8% of TLR9^–/– mice (n = 19) escaped from CM]. However, TLR4-, TLR5- or TLR7-deficient mice could not escape from CM [10.55% of WT (B6) mice (n = 28) versus 0% 16.75% of TLR4^–/– mice (n = 20) escaped from CM; 14.28% of WT (B6) mice (n = 14) versus 0% of TLR5^–/– mice (n = 11) escaped from CM; 0% of WT (B6) mice (n = 5) versus 0% of TLR7^-/Y mice (n = 5) escaped from CM].

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 TLR2–/– and TLR9–/– mice have decreased susceptibility to CM. WT (B6) or mice lacking (A) TLR2, (B) TLR9, (C) TLR4, (D) TLR5 or (E) TLR7 (n = 28 for WT and n = 21 for TLR2–/–, n = 27 for WT and n = 19 for TLR9–/–, n = 28 for WT and n = 20 for TLR4–/–, n = 5 for WT and n = 5 for TLR5–/– and n = 5 for WT and n = 5 for TLR7–/Y) were infected with 106 iRBCs of PbA strain. Survival was monitored daily. Parasitemia was examined from Giemsa-stained blood smears every 2 days (mean parasitemia ± SE). P < 0.001 for the percentage survival curve of WT versus TLR2–/– mice, and P = 0.027 for that of WT versus TLR9–/– mice by log rank test. (F) Total escape from CM in TLR-deficient mice until day 12 after infection. In all, 10.55% of WT (B6) mice (n = 28) escaped from CM versus 55% of TLR2–/– mice (n = 21), 14.7% of WT (B6) mice (n = 27) escaped from CM versus 45.8% of TLR9–/– mice (n = 19), 10.55% of WT (B6) mice (n = 28) escaped from CM versus 16.75% of TLR4–/– mice (n = 20), 14.28% of WT (B6) mice (n = 14) escaped from CM versus none of TLR5–/– mice (n = 11) and none of WT (B6) mice (n = 5) escaped from CM versus none of TLR7-/Y mice (n = 5). ‘None’ implies that no infected mice could survive.

 
Of note, parasitemia was comparable between the WT and each strain of TLR-deficient mice (Fig. 5A and B), which survived CM, while that of MyD88^–/– was slightly higher (Fig. 1A). It may be due to compensated innate and adaptive immune responses in TLR2^–/– or TLR9^–/– mice compared with MyD88^–/– mice which lack combined effects of each TLR. While we did not formally exclude the less likely involvement of IL-1 and IL-18, which also require MyD88 for their subsequent functions, these results strongly suggest that TLR2 and TLR9, but not TLR4, TLR5 or TLR7, are involved in the pathogenesis of CM, but not in controlling parasitemia during PbA infection.

Systemic responses and hemozoin load in the brain
Based on the evidence that pro-inflammatory cytokines such as IFN, IL-12 and TNF are associated with the severity of malaria infection including CM (7–9), we investigated the role of MyD88/TLR2/TLR9 pathways on the systemic productions of such cytokines in serum at day 6 after infection. Productions of serum cytokines IFN, TNF and IL-12p40 were significantly dependent on MyD88 (P < 0.05 by Mann–Whitney U-test) (Fig. 6A). The up-regulation of serum IFN, TNF and IL-12p40 was significantly reduced in MyD88-deficient mice, but not in TRIF- or TLR2-deficient mice. On the other hand, in TLR9^–/– mice only TNF production was TLR9 dependently secreted after PbA infection (Fig. 6A, P < 0.05, Mann–Whitney U-test). These results suggested that PbA infection causes systemic cytokine productions via MyD88-dependent signaling; however, systemic cytokine productions are not critical for CM development.

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Systemic cytokine responses and brain hemozoin load after PbA infection. (A) MyD88–/–, TRIF–/–, TLR2–/– and TLR9–/– mice and their WT controls were infected with 106 of PbA iRBCs. Serum levels of IFN, TNF and IL-12p40 at day 6 after infection with PbA were measured by ELISA. Results (mean ± SE) are from three different experiments that cytokine levels were measured at the same time (n = 10–25 mice per group). *P = 0.02 for IFN of MyD88–/– versus WT mice, *P = 0.005 for TNF of MyD88–/– versus WT mice *P = 0.002 for TLR9–/– versus WT mice and *P < 0.001 for IL-12p40 of MyD88–/– versus WT mice (Mann–Whitney U-test). (B) Brain tissue sections were stained with Prussian blue, and hemozoin clusters in infiltrated brain vessels were counted in 20–25 microscopic fields by light microscopy. TLR2–/– and TLR9–/–mouse brains had significantly lower numbers of hemozoin clusters in each area counted (P < 0.038 and P < 0.019, respectively, Mann–Whitney U-test, n = 2 or 3 per group). ND represents not detected in uninfected naive mice.

 
We also counted hemozoin clusters in brains of TLR2^–/– and TLR9^–/– mice after Prussian blue staining and found that hemozoin accumulation was significantly reduced in TLR2- and TLR9-deficient mice brains compared with that in WT brains at day 6 (Fig. 6B, P < 0.038 and P < 0.019, respectively, by Mann–Whitney U-test).

	Discussion

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
In this study, we showed, for the first time, that TLRs and their adaptor molecules play distinct roles in CM pathogenesis. Mice deficient in MyD88, but not in TRIF, displayed significantly less CM-characteristic neurological symptoms, which resulted in significantly reduced mortality. This was supported by histological analysis in which the brains from infected MyD88-deficient mice displayed reduced endothelial cell damage and sequestration of infected erythrocytes. We could detect hemozoin in infected WT brain blood vessels, as well as in the other brain tissues by microscopy, which was significantly less in MyD88-deficient mice. Moreover, flow cytometry of the recovered lymphocytes from the infected brain suggested that infiltration of T cells, most of which are CCR5⁺, CD8⁺ T cells and CD11c⁺ DCs, including CD11c⁺, B220⁺ and NK1.1 cells, is controlled at least in part by MyD88-dependent signaling. Although not all MyD88-deficient mice survived CM caused by PbA infection, our results strongly suggest that MyD88, but not TRIF, is a key signaling molecule in CM pathogenesis, at least in currently available experimental CM models.

The effect of MyD88 on CM was more obvious in local brain where CM symptom attribute to, rather than systemic control of parasitemia as well as inflammation. Damage of brain blood vessels is a characteristic feature of CM pathogenesis, such as destruction of the endothelial cell layer, sequestration of iRBCs and infiltration of lymphocytes, all of which are dramatically reduced in MyD88-deficient mice compared with either WT or TRIF-deficient mice. We observed parasite sequestration and hemozoin load in these damaged blood vessels in WT infected mice, which was almost diminished in MyD88-deficient mice. Together with the other observation that hemozoin was also detected in brain tissue, and that the microglia cell line was activated by both crude extracts of the iRBCs and hemozoin, but not uninfected RBCs, this suggests that residual microglial cells may be activated by iRBCs that are known to contain TLR ligands, such as GPI and/or hemozoin (16, 17), which have escaped or leaked through the damaged blood–brain barrier.

Comprehensive analysis of MyD88- and/or TRIF-dependent genes in response to PbA infection revealed several important genes that could be involved in CM pathogenesis. MyD88-depedent genes up-regulated by PbA infection included not only those up-regulated by the microglial cell line, but also type-I or -II IFN-inducible genes, T or NK cell-related genes and stress response genes. A recent report identified several genes such as Gzmb, Samhd1, Fkbp5, Ifit3, Igf2r, Ctla2a and C1qb which were up-regulated specifically in CM-susceptible mice strains infected with PbA (32). Our results confirmed and further extended these findings; Gzmb and Ifit3 were up-regulated during PbA infection, which was exclusively dependent on MyD88. In addition, we found that Isg15, Mx1, Cxcl10 (IP-10), Ccl3 (MIP-1), Ccr5 and serum amyloid A were also up-regulated by PbA infection in a MyD88-dependent manner. Of note, MIP-1 was reported to be up-regulated by hemozoin in macrophages (41). Moreover, Lipocalin 2 (24p3), a gene important for iron metabolism and for resistance against certain bacteria (42) and presumably important marker for severe malaria in humans (35), was highly up-regulated in the brain of PbA-infected mice in a MyD88-dependent manner (Table 1 and Fig. 3). Our preliminary data, however, suggest that mice deficient for Lipocalin 2 (24p3) suffered from and died from CM, as did WT mice (C. Coban, K. J. Ishii, S. Sato and S. Akira, unpublished results).

Among the MyD88-dependent CM-related genes, we noted some that may be involved in innate immune responses (especially type-I and -II IFN-inducible genes) and the recruitment of lymphocytes, such as T cells and/or NK cells. We were initially interested in type-I IFN-inducible genes; however, infection of mice lacking IFNßR or TBK1 (and/or TNF, which are deficient for TRIF- or TLR-independent type-I IFN induction) suffer from CM and died comparably to those of WT mice (C. Coban, K. J. Ishii and S. Akira, unpublished results). Rather, type-II IFN may play a more critical role in MyD88-dependent IFN-inducible genes up-regulated during PbA infection, since systemic as well as brain IFN but not IFN were up-regulated in a MyD88-dependent manner (Fig. 6A, and data not shown). In fact, systemic production of IFN, TNF and IL-12p40 was MyD88 dependent, but not TRIF dependent (Fig. 6A). In case of mice lacking TLR2 or TLR9, however, systemic cytokines were not altered except TNF in TLR9-deficient mice (Fig. 6A), suggesting that systemic cytokines such as IFN were compensated between TLR2 and TLR9, although mice lacking TLR2 or TLR9 survived CM significantly better than WT (Fig. 5). It is also conceivable that the systemic cytokine production such as IFN may be regulated by TLR-independent, MyD88-dependent signaling pathway, in which IL-1R and IL-18R utilize. We then focused on genes related to T and NK cells which may be the result of their migration into brain controlled by chemokine genes such as Ccl3, Ccl9 and Ccr5 which were up-regulated in a MyD88-dependent manner (Table 1 and Fig. 3). In fact, we found that both CD4⁺ and CCR5⁺, CD8⁺ T cells and CD11c⁺ DCs including CD11c⁺, B220⁺ and NK1.1⁺ cells infiltrated into the brain after PbA infection, which was clearly MyD88 dependent. The cells positive for CD11c, B220 and NK1.1 were with identical staining pattern of newly described IKDCs (28, 29) and certain subsets of NK cells expressing either CD11c or B220 (39, 40), suggesting that they may possess cytotoxic ability via granzyme. In addition, we found that certain chemokines were highly up-regulated in a MyD88-dependent manner; CCL3 and CCL4 are known to recruit CCR5-positive cells, including T cells, macrophages and DCs. In agreement with previous reports showing that perforin-deficient C57Bl/6 mice (11, 43, 44) and CCR5-deficient mice (12) which lack their killing and recruiting functions, respectively, display increased resistance to CM, we conclude that the MyD88-dependent recruitment of T cells, DCs and cells expressing both DC and NK markers, possibly via chemokine productions in the brain, may play a critical role in CM pathogenesis.

Results obtained in similar experiments using various TLR-deficient mice suggest that TLR2- and/or TLR9-mediated, MyD88-dependent innate immune cascades may play a critical role in the pathogenesis of CM. This coincides with recent evidence that GPI and hemozoin are found to be agonists for TLR2 and TLR9, respectively (16, 17). TLR2 and TLR9 have been shown to co-operate for protective immune responses against various infectious organisms including Mycobacterium tuberculosis (45), while TLR9 and TLR2 have been shown to play a reciprocal role in protective immunity and pathology during Herpes simplex virus infection, respectively (46). In the case of PbA infection, an absence of TLR2 or TLR9 increases resistance to CM-related mortality but not parasitemia, suggesting that these two TLRs play a critical role in the pathogenesis, and not in protective immunity. A similar strategy was observed for West Nile Virus to utilize TLR3 to facilitate infection in the brain (47). However, further investigation may be needed to clarify whether these TLRs play similar roles in the case of the other Plasmodium species such as Plasmodium yoelii or chabaudi.

In contrast to TLR2 and TLR9, the other TLRs utilizing MyD88 as an essential adaptor, such as TLR5 and TLR7, as well as TLR4, which utilize both MyD88 and TRIF, were not involved in CM pathogenesis and its resultant mortality (Fig. 5). It is of note that, although TRIF-deficient mice die in the same way as WT mice, some genes up-regulated by PbA infection were TRIF dependent (Supplementary Table 2, available at International Immunology Online). These results suggest that there are TRIF-dependent innate immune responses during PbA infection, and therefore, it is interesting to investigate the role of TRIF-dependent recognition in the immune response to PbA infection and to identify as yet unknown ligands. Receptors for IL-1 and IL-18 are also known to utilize MyD88 for their sequential signaling; however, previous reports have suggested that both IL-1 and IL-18 are not involved in CM pathogenesis (8, 9). While specific cell types, by which TLR2 and/or TLR9 mediate MyD88-dependent innate and adaptive immune cascades leading to CM, have not been formally identified, the present study will hopefully help in discerning the complex role of innate immunity in CM pathogenesis.

Overall, the results presented in the current work suggest that innate immune responses via TLR2-, TLR9- and MyD88-dependent pathway are critically involved in the pathogenesis of CM, in which local rather than systemic pro-inflammatory responses plus adaptive immune responses particularly in brain tissue lead to infiltration of CD8 T cells, DCs including those with NK cell marker into brain, and up-regulation of variety of genes related to CM, resulting in the pathogenic changes as we described above. A few information are available whether TLRs or their signaling pathways are involved in human malaria infection; TLR4 frequent single-nucleotide polymorphism (SNP), Asp299Gly, is associated with severe malaria and risk of maternal malaria, whereas TLR9 SNP, T-1486C increased the risk of maternal malaria, but was not associated with severe malaria (48, 49). Although relevance of murine experimental CM models to human CM is under debate; however, our findings with the best available model of CM with PbA infection strongly suggest that host innate immune system against malaria and its exploitation by parasites hold a key to further understanding of pathogenesis of human CM.

	Supplementary data

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
Supplementary figure 1 and tables 1 and 2 are available at International Immunology Online.

	Acknowledgements

 
We thank Yukiko Fujita for her excellent technical assistance. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan and from the 21st Century Center of Excellence Program of Japan.

	Abbreviations

 

APC, allophycocyanin

CM, cerebral malaria

DC, dendritic cell

FBS, fetal bovine serum

GPI, glycosyl-phosphatidylinositol

HE, hematoxylin and eosin

IKDC, IFN-producing killer dendritic cell

iRBC, infected RBC

MyD88, myeloid differentiation primary response gene 88

PbA, Plasmodium berghei ANKA

RT, reverse transcription

SNP, single-nucleotide polymorphism

TLR, Toll-like receptor

TNF, tumor necrosis factor

TRIF, TIR domain containing adaptor-inducing IFN-beta

WT, wild type

	Notes

 
^* These authors contributed equally to this study.

Transmitting editor: K. Inaba

Received 23 August 2006, accepted 24 October 2006.

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