The nucleosome (histone-DNA complex) is the TLR9-specific immunostimulatory component of Plasmodium falciparum that activates DCs

Abstract

The systemic clinical symptoms of Plasmodium falciparum infection such as fever and chills correspond to the proinflammatory cytokines produced in response to the parasite components released during the synchronized rupture of schizonts. We recently demonstrated that, among the schizont-released products, merozoites are the predominant components that activate dendritic cells (DCs) by TLR9-specific recognition to induce the maturation of cells and to produce proinflammatory cytokines. We also demonstrated that DNA is the active constituent and that formation of a DNA-protein complex is essential for the entry of parasite DNA into cells for recognition by TLR9. However, the nature of endogenous protein-DNA complex in the parasite is not known. In this study, we show that parasite nucleosome constitute the major protein-DNA complex involved in the activation of DCs by parasite nuclear material. The parasite components were fractionated into the nuclear and non-nuclear materials. The nuclear material was further fractionated into chromatin and the proteins loosely bound to chromatin. Polynucleosomes and oligonucleosomes were prepared from the chromatin. These were tested for their ability to activate DCs obtained by the FLT3 ligand differentiation of bone marrow cells from the wild type, and TLR2(-/-), TLR9(-/-) and MyD88(-/-) mice. DCs stimulated with the nuclear material and polynucleosomes as well as mono- and oligonucleosomes efficiently induced the production of proinflammatory cytokines in a TLR9-dependent manner, demonstrating that nucleosomes (histone-DNA complex) represent the major TLR9-specific DC-immunostimulatory component of the malaria parasite nuclear material. Thus, our data provide a significant insight into the activation of DCs by malaria parasites and have important implications for malaria vaccine development.

Figure 1. Fractionation of P. falciparum nuclear material and preparation of polynucleosomes and histones.

The parasites released from the late trophozoite and schizoint stage-infected erythrocytes were lysed in buffer containing 1% Triton X-100. The insoluble nuclear material was pelleted, extracted with buffer having different salt concentrations to remove proteins loosely bound to the nuclear chromatin material. The chromatin material was sheared to yield polynucleosomes.

Figure 2. The nuclear material and polynucleosomes of P. falciparum efficiently activate DCs to produce inflammatory cytokines.

FL-DCs (panels A and B) or spleen DCs (panels C and D) from WT mice were plated in 96-well plates and stimulated with the indicated doses (based on DNA contents) of nuclear material or polynucleosomes. FL-DCs similarly stimulated with merozoites (MZs, dose indicated by DNA content) or with a standard CpG ODN were analyzed as controls. The levels of TNF-α and IL-12 in the culture medium were measured by ELISA. Data are representatives of three independent experiments, each performed in duplicates. Error bar represents mean values ± SEM.

Figure 3. P. falciparum nuclear material and polynucleosomes induce the maturation of DCs.

FL-DCs prepared from the bone marrow cells of WT mice were plated in 24-well plates and stimulated with merozoites (MZs), parasite nuclear material or polynucleosomes. DCs stimulated with MZs or CpG ODN was used as controls. The upregulated surface expression of costimulatory molecules, CD40, CD80 and CD86, were analyzed by flow cytometry. The percentages of DCs that are positive to each costimulatory molecule are indicated. Data are representatives of two independent experiments.

Figure 4. The analysis of the parasite nuclear material and extracts of nuclear material.

Panel A: The parasite nuclear material, polynucleosomes and buffer/salt extracts of nuclear material were analyzed for proteins by SDS-PAGE (in each lane 15 µg of protein content/well). Lane 1, the supernatant of parasite lysate (see Figure 1 for materials in lanes 1–6); lane 2, 0.3 M KCl extract of the nuclear material; lane 3, 0.6 M KCl extract of the chromatin material; lane 4, 0.4 M NaCl extract of the chromatin material; lane 5, chromatin material after extraction of proteins with buffer A containing various salts; lane 6, polynucleosomes; lane 7, the mixture of standard recombinant histones, H1, H2A, H2B, H3 and H4. The mobility of molecular weight marker proteins is indicated to the right. Panel B: The parasite nuclear material and buffer/salt extracts of nuclear material were electrophoresed on 0.8% agarose gels and the DNA bands were visualized under UV light after ethidium bromide staining. Lane 1, parasite nuclear material before extraction of proteins that loosely bound to chromatin (see Figure 1 for materials in lanes 1–10); lane 2, chromatin material pellet after extraction with buffer/0.3 M KCl; lane 3, chromatin material after extraction with buffer/0.6 M KCl; lane 4, chromatin material after extraction with buffer/0.4 M NaCl; lane 5, soluble polynucleosomes; lane 6, insoluble fibrous material after obtaining polynucleosomes; lane 7, parasite cytoplasmic material plus membrane fragments; lane 8, 0.3 M KCl extract of nuclear material; lane 9, 0.6 M KCl extract; lane 10, 0.4 M NaCl extract. Lanes 1–5, materials having 0.5 µg DNA were analyzed. Lane 6, about 60-fold more material as compared to those in lanes 1–5 based on the total parasite amount used for fractionation. Lanes 7–10, materials equivalent to that in lanes 1–5 based on the total amount of parasite used for fractionation was loaded. The sizes of standard DNA makers are indicated to the right. Panel C: Western blotting of parasite components as shown in panel A was done using anti-H3 histone polyclonal antibodies. Lane descriptions are as outlined in panel A.

Figure 5. Analysis of the immunostimulatory activity and TLR specificity of P. falciparum polynucleosomes.

Panels A and B: FL-DCs prepared from the bone marrow of WT, TLR2^−/−, TLR9^−/− and MyD88^−/− mice were stimulated with the parasite nuclear material or with the indicated doses (based on DNA content) of polynucleosomes. FL-DCs stimulated with Pam₃CSK₄, Poly I∶C, LPS or CpG ODN were used as controls. The levels of TNF-α and IL-12 in the culture supernatants were measured by ELISA. Panels C and D: TNF-α and IL-12 produced by FL-DCs from WT mice stimulated with polynucleosomes, DNase-treated polynucleosomes, DNase-treated polynucleosomes to which parasite genomic DNA (pDNA) was added, trypsin-treated polynucleosomes, trypsin-treated polynucleosomes to which histones (1 µg/ml) were added or mixture of DNase- and trypsin-treated polynucleosomes. The culture supernatants of DCs stimulated with 8 µg/ml parasite genomic DNA (pDNA) and CpG ODN were analyzed as controls. Experiments were repeated three times and each time performed in duplicates. Error bars represent mean values ± SEM.

Figure 6. P. falciparum histone-DNA complex efficiently activate DCs.

Panels A: The parasite chromatin material, 0.8 M NaCl extract of chromatin material, and 0.25 M HCl extracts of chromatin material (Figure 1) were analyzed by SDS-PAGE using 15% gels. Each lane was loaded with 10 µg of protein. Lane 1, polynucleosomes; lane 2, 0.8 M NaCl extract of the chromatin material; lane 3 and 4, 0.25 M HCl extracts (histones). The mobility of molecular weights of marker proteins is indicated to the right. Panels B and C: TNF-α and IL-12 produced by FL-DCs obtained from WT mice stimulated with different doses of isolated parasite histones with or without parasite genomic DNA (pDNA, 8 µg/ml). DCs stimulated with parasite genomic DNA (pDNA, 8 µg/ml), CpG (2 µg/ml) and polynucleosomes (2.5 µg DNA content/ml) were analyzed as controls. Error bars represents mean values ± SEM.

Figure 7. P. falciparum polynucleosomes-activated DCs stimulate NK cells and OT-II T cells to produce IFN-γ.

Panel A: WT FL-DCs were cocultured with NK cells and stimulated with the indicated doses of polynucleosomes (based on DNA content). After 36 h, the IFN-γ produced by NK cells was measured by ELISA. Merozoites (MZs) having the indicated DNA contents and CpG ODN were used as controls. Panel B: WT FL-DCs were stimulated with the indicated doses of polynucleosomes for 6 h and then cocultured with OT-II T cells in the presence of OVA^323–339 peptide. After 72 h, the IFN-γ produced by T cells was measured by ELISA. In parallel NK cells, DCs or T cells were cultured individually or cocultures of DCs and T cells in presence or absence of OVA^323–339 peptide, but not stimulated with polynucleosomes, were used as controls. Data are representative of two independent experiments, each performed in duplicates. Error bars represents mean values ± SEM.

Figure 8. The P. falciparum mononucleosomes and oligonucleosomes efficiently activate DCs to induce the production of proinflammatory cytokines.

Panel A: The parasite polynucleosomes (15 µg DNA content in 200 µl buffer) were treated with the indicated concentrations of micrococcal nuclease and 10 µl aliquots of the enzyme-digestion products (0.75 µg DNA content) were analyzed by electrophoresis using 2% agarose gels. Lane 1, untreated polynucleosomes; lane 2, polynucleosomes incubated with buffer only; lanes 3 to 9, respectively were polynucleosomes treated with 0.5, 1.0, 2.0, 5.0, 10, 20 and 50 units/ml of micrococcal nuclease. The sizes of DNA standards are indicated to the right. Panels B to E: TNF-α and IL-12 produced by WT FL-DCs stimulated with mono- or oligonucleosomes obtained by different doses of micrococcal nuclease treatment of polynucleosomes (panels B and C) or FL-DCs from WT, TLR2^−/−, TLR9^−/− and MyD88^−/− stimulated with parasite oligonucleosomes (panels D and E). FL-DCs stimulated with Pam₃CSK₄, Poly I∶C, LPS or CpG ODN were analyzed as controls. The analysis was performed three times each time in duplicates, and results of a representative experiment are shown. Error bars represent mean values ± SEM. * p<0.05, comparison between 0.5 units/ml of micrococcal nuclease digested and undigested polynucleosomes.

Figure 9. P. falciparum proteins other than histones confer stimulatory activity to the parasite DNA.

Panel A: Fractionation of MZs lysate by sucrose-density gradient centrifugation. The values of absorption at 260 nm (ℓ) and 280 nm (), and DNA contents (O) of the fractions were measured as described in “Materials and Methods”, and plotted. Panel B: SDS-PAGE analysis. The sucrose gradient fractions were dialyzed; aliquots corresponding to 20 µg of proteins were analyzed by SDS-PAGE using 15% gels. Sucrose gradient fraction numbers are indicated at the top. The mobility of molecular weight marker proteins is indicated to the left. Lane S, a mixture of standard H1, H2A, H2B, H3 and H4 histones. Panel C: Western blotting of sucrose gradient fractions using anti-H3 histone polyclonal antibodies. Lane descriptions are same as that in panel B. Panels D and E: TNF-α and IL-12 produced by WT FL-DCs stimulated with the indicated sucrose gradient fractions having 2.5 µg/ml of DNA content or with fractions having 2.5 µg/ml of DNA content to which the purified parasite genomic DNA (pDNA, 8 µg/ml) was added. Cells stimulated with parasite genomic DNA were used as controls. Panels F and G: TNF-α and IL-12 produced by FL-DCs from WT, TLR2^−/−, TLR9^−/− and MyD88^−/− stimulated with sucrose gradient fractions. DCs stimulated with Pam₃CSK₄, Poly I∶C, LPS, CpG ODN or parasite genomic DNA were used as a control. The analysis was repeated three times each time performed in duplicates, and results of a representative are shown. Error bars represent mean values ± SEM. * p<0.05; ** p<0.01 comparison between sucrose gradient fractions with and without added parasite DNA.