By purifying the various components of P. falciparum
schizont rupture to near homogeneity, we dissected the immunostimulatory part of the parasite responsible for innate immune responses by DCs. Notably, the results show that, among the components released from schizonts, MZs are the inducers of innate immune responses in DCs; parasite FVs, HZ, parasite membrane fragments formed during the differentiation of matured trophozoites into MZs, and parasite cytoplasmic material are non-stimulatory. The activation of DCs by malarial MZs occurs exclusively through the engagement of TLR9-MyD88 signaling pathway. Thus, DCs deficient in TLR9 or MyD88 were completely nonresponsive to malarial MZs or whole schizonts, whereas DCs lacking TLR2 or TLR4 were as efficient as wild type DCs in producing cytokine responses and cell maturation. The TLR9-dependent recognition of P. falciparum
by DCs is consistent with the previous finding that the IL-12-mediated liver injury in P. berghei
-mouse malaria model and the activation of human and mouse DCs are mediated by TLR9-MyD88 signaling pathway (35
). Also, TLR9-mediated recognition of the parasite has recently been shown to be involved in the acquisition of malaria-specific memory B cells (36
In malaria infected individuals, the synchronous burst of schizonts releases huge amounts of MZs within a short window of the parasite's 48 h life cycle. The MZs are short lived and as such, they must quickly invade erythrocytes for the efficient survival and parasite propagation. However, in reality, a substantial portion of the released MZs cannot attach and invade erythrocytes. Thus, massive amounts of MZs become the target of DCs, leading to DC maturation, cytokine production, and eventually the development of adaptive immunity.
In this study, we conclusively dissected, for the first time, the stimulatory activity of the malaria parasite. Importantly, our results show that protein-DNA complexes are the immunostimulatory component that is responsible for the inflammatory cytokine production by DCs in response to P. falciparum
. The parasite proteins are essential for stimulatory activity of an otherwise non-stimulatory parasite DNA. Further, notably, the data show that parasite HZ pigment, which is widely believed to be either a potent immunostimulatory molecule by directly interacting with TLR9 or a carrier of parasite DNA for TLR9 recognition to induce pro-inflammatory responses (21
), is neither stimulatory by itself for activation of DCs nor is able to confer activity to parasite DNA. Several lines of evidence support our conclusions. These include: (i) the efficient activation of DCs by MZs devoid of HZ; (ii) complete loss of activity upon treatment of MZs or schizont stage IRBCs with DNase, despite the latter containing HZ; (iii) gain of activity of DNase-treated MZs upon the addition of parasite DNA; (iv) total loss of activity when MZs or IRBCs were treated with trypsin, (v) gain of full activity when trypsin-treated MZs was combined with DNase-treated MZ lysates; (vi) the inability of parasite or synthetic HZ to confer activity to parasite DNA. Together these data conclusively show that parasite proteins but not HZ plays a critical role in the TLR9-mediated activation of DCs by malarial DNA.
The ability to confer immunostimulatory activity to P. falciparum
DNA is not restricted to parasite proteins. PLL, a polycationic protein, imparted a potent stimulatory activity to parasite DNA. Chitosan, a nonprotein polycationic molecule, was also able to impart a significant activity to the parasite DNA. In contrast, polyanionic molecules, including PLG, DS, PS, and heparin were unable to impart activity to the parasite DNA. Thus, it is clear that a complex formation by the ionic interaction with polycationic molecules is essential for the TLR9-dependent activity of parasite DNA. Recently, a cationic antimicrobial peptide LL37, produced during human skin psoriasis, has been shown to complex with DNA released by the tissue, thereby was able to convert the non-stimulatory human DNA into a potent immunostimulatory molecule by facilitating the endosomal entry of DNA (29
). The activity was highly specific to the amino acid sequence of LL37 as the peptide with a scrambled sequence was unable to impart activity to human DNA. Thus, it appears that, in addition to aiding endosomal entry of DNA, LL37 converts self-DNA into a structure distinctively different from endogenous components for recognition as a foreign molecule for cell activation. This is in contrast to malarial DNA, which does not require specific polycationic molecule as evident by its conversion into an active immunostimulatory molecule by a diverse type of polycationic molecules, including polypeptides and carbohydrates. By being a molecule of pathogenic origin, malarial DNA is recognized as a foreign molecule by human and mouse DCs. Our findings that polycationic proteins and carbohydrate polymers can convert DNA of pathogenic origin into potent immunostimulatory molecule represent novel findings.
The malaria parasite genome has very high (76%) AT content and CpG motifs are minor constituents (22
). It is unlikely that CpG motifs of the parasite are the main trigger of stimulatory activity in DCs, although they may contribute to a certain extent (22
). A recent study found that the AT-rich motifs of DNA, particularly those containing repeats of several T residues that abundantly occur in parasite DNA, activate cells by engaging TLR9 (38
). These findings when considered with our observations that human and mouse genomic DNA, which contain high levels of CpG motifs, are unable to exhibit stimulatory activity in the presence of PLL or malarial MZ proteins, strongly suggest that the high AT content of parasite DNA is distinctively recognized by TLR9.
Our results further demonstrate that while TLR9 and MyD88 are critically required for activation of DCs, these receptors are not required for the NK cells to produce IFN-γ in response to parasite-stimulated DCs. Thus, TLR9-/-
DCs stimulated with P. falciparum
MZs/schizont lysates were unable to activate NK cells, whereas WT DCs stimulated with MZs or IRBCs efficiently activated NK cells to produce IFN-γ. A recent study reported that TLR9-dependent stimulation of DCs was required for the activation of regulatory T cells by malaria parasite (39
). This process has been suggested to be involved in the malaria parasite immune evasion. Therefore, TLR9-mediated recognition of malaria parasites by DCs is involved in both immune activation and immune suppression by distinct mechanisms. These findings highlight the complexities involved in malaria immunity.
The results of this study also demonstrate that contact-dependent interactions between mDCs, pDCs and NK cells are essential for the efficient production of IFN-γ by NK cells in response to malarial protein-DNA complex. Although mDC population of mouse FL-DCs efficiently produced IL-12, mDCs are inefficient in activating NK cells in co-cultures to secrete IFN-γ in response to MZs. pDC population was unable to activate NK cells to produce IFN-γ completely. NK cells were also unable to produce IFN-γ when DCs were cultured in separate compartments of transwells. In contrast, when both pDCs and mDCs were cocultured with NK cells, efficient production of IFN-γ was observed, demonstrating that interactions between all three cell types is essential for the optimal production of IFN-γ by NK cells.
Previous studies have shown GPIs purified from P. falciparum
can activate macrophages through mainly TLR2- and to some extent TLR4-mediated recognition, leading to inflammatory responses. In this study, although merozoites and parasite membrane fragments of schizont rupture contain the membrane-bound GPIs, both parasite components unable to exhibit TLR2-dependent activation of DCs. A minimum of 20 ng/ml of purified GPIs are required to observe a detectable level of cytokine responses by macrophages primed with IFN-γ, and saturated level of cytokine response requires at least 200 ng/ml of GPI concentration (40
). The dose of merozoites required for the saturated level of cytokine responses contain an estimated amount of ~5 ng/ml of GPIs (41
). Thus, it is evident that the potency of TLR9-dependent activity of DNA-protein complex in merozoites is markedly higher than the ability of parasite membrane-bound GPIs to activate DCs and it appears that the contribution of GPIs toward activation of DCs in vivo
Our data have important considerations for the development of an effective vaccine for malaria. Several MZ proteins, including MSP1, MSP2, MSP3, AMA1, and EBA-175 have been extensively studied as vaccine candidates, either as a single protein or as multi-subunit structures (42
). Although these candidate proteins are in various stages of clinical trials, so far none has been proved to be useful as an efficacious vaccine. Because of low immunogenicity of malarial antigens, adjuvants are essential for efficient immune responses. Several adjuvants, including alum, gels, and oil-based components have been studied but all exhibit various levels of toxicity (44
). In this regard, MZs or whole IRBCs may prove to be very effective for malaria vaccination as they contain repertoire of antigens involved in MZ invasion of erythrocytes and immune-boosting protein-DNA complex for strong adjuvant activity. Since MZs or IRBCs can effectively stimulate DCs through TLR9 recognition, this process will likely to induce an effective adaptive immunity. Recently, MZs have been proposed for malaria vaccination (46
). Our data strongly support this notion. Furthermore, our results provide a conceptual framework for strategies aimed at designing DNA-protein antigen complexes as a malaria vaccine.
In summary, this study clearly demonstrates that protein-DNA complex is the exclusive component that activates DCs and HZ has no role in the DNA-mediated activation of DCs by malaria parasite, clarifying the hitherto widely held belief that HZ is either directly or indirectly immunostimulatory. The results set a stage for refocusing of studies that aimed at understanding of the initiation and regulatory mechanisms of malaria immunity by DCs. More importantly, the involvement of the parasite protein-DNA complex-mediated immune responses may have potential for the development of immune-based therapeutics or a vaccine for malaria.