Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9

Abstract

Hemozoin (HZ) is an insoluble crystal formed in the food vacuole of malaria parasites. HZ has been reported to induce inflammation by directly engaging Toll-like receptor (TLR) 9, an endosomal receptor. "Synthetic" HZ (beta-hematin), typically generated from partially purified extracts of bovine hemin, is structurally identical to natural HZ. When HPLC-purified hemin was used to synthesize the crystal, beta-hematin had no inflammatory activity. In contrast, natural HZ from Plasmodium falciparum cultures was a potent TLR9 inducer. Natural HZ bound recombinant TLR9 ectodomain, but not TLR2. Both TLR9 stimulation and TLR9 binding of HZ were abolished by nuclease treatment. PCR analysis demonstrated that natural HZ is coated with malarial but not human DNA. Purified malarial DNA activated TLR9 but only when DNA was targeted directly to the endosome with a transfection reagent. Stimulatory quantities of natural HZ contain <1 microg of malarial DNA; its potency in activating immune responses was even greater than transfecting malarial DNA. Thus, although the malarial genome is extremely AT-rich, its DNA is highly proinflammatory, with the potential to induce cytokinemia and fever during disease. However, its activity depends on being bound to HZ, which we propose amplifies the biological responses to malaria DNA by targeting it to a TLR9(+) intracellular compartment.

Fig. 1.

Highly pure β-hematin has no stimulatory activity. (A) FL-DCs (2 × 10⁵ cells per well) were exposed for 18 h to medium alone or 150 μM β-hematin (“synthetic” HZ) prepared from two different sources of hemin chloride. IL12p40 (data not shown) and TNFα were measured in the culture supernatant by ELISA. (B) Bone marrow-derived FL-DCs from WT and knockout mice were tested for their response by overnight stimulation with 50 μM natural HZ. The production of IL12p40 and TNFα (data not shown) was assessed by ELISA. Results are the mean level of triplicate determinations of released cytokines (±SD). (C) WT FL-DCs were stimulated for 24 h with medium alone or medium plus the indicated ligand. IFNα release into the medium was then measured by ELISA.

Fig. 2.

Natural HZ stimulates FL-DCs in a DNase-sensitive fashion. WT FL-DCs were treated with 50 μM natural HZ for 24 h with HZ before (black bars) and after (red bars) treatment with DNase I, S. aureus micrococcal nuclease S7 (blue bars), EDTA-inactivated S7 nuclease (gray bars), or RNase (white bars). The supernatants were collected and analyzed for Rantes and IL12p40 by ELISA. CpG oligonucleotide 2336 and poly[IC] were treated in the same way as the HZ. Medium controls represent tissue culture medium to which the final concentration of each nuclease was added. Each condition was assayed in triplicate; the results represent mean level of Rantes (±SD). Nearly identical results were observed when the release of IL12p40 was measured.

Fig. 3.

The ectodomain of TLR9 but not TLR2 binds to the DNA coating of natural HZ. Chimeric proteins composed of the extracellular domain of TLR9 or TLR2 and the Fc portion of mouse IgG2a were used to assess ligand binding. Titrated amounts (0–150 μM) of HZ or S7-nuclease treated-HZ (Hz plus nuclease) were absorbed to the plastic bottoms of 96-well black plates. The respective TLR2- or TLR9-Fc fusion proteins were added at 2 μg/ml. Bound Fc proteins were detected by using an anti-mouse IgG Alexa Fluor 488 pAb; fluorescent emissions were read at 535 nm. The binding of TLR2:Fc to PAM2CK4 (Pam2) and TLR9:Fc to CpG oligonucleotide 2006 (ODN 2006) was assessed as positive controls. Results representing triplicate determinations are shown as mean fluorescence emitted (±SD).

Fig. 4.

Malarial DNA is coated with HZ; the activity of HZ as an inducer of TLR9 is due to malaria DNA. (A) PCR was performed by using 3.5 nmol of natural HZ or 25 ng of genomic DNA as template. Plasmodium-specific primers (P. falciparum, P. spp), human-specific primers (TLR7, TRAM), or mouse-specific primers (CD14) were tested in the absence or presence of template as indicated in the table. Note that the presentation of the data required reorganization of digital images; the original gels can be seen as SI Fig. 5. (B) FL-DCs from WT mice were stimulated with 32, 75, and 150 μM natural HZ (red X symbols) or purified genomic DNA (black squares) mixed with DOTAP. The x axis of the HZ data has been aligned to also indicate its content of genomic DNA. After 24 h, supernatants were analyzed by ELISA.