As all organisms are defined by their nucleic acids, it should not be surprising that nucleic acids have emerged as key triggers of innate immunity. In this report, we have identified a novel DNA sensing pathway important in innate immunity to Malaria. This pathway does not involve any of the known DNA receptors described to date but otherwise follows a canonical core STING-TBK1-IRF signaling pathway. Plasmodial DNA activates this novel AT-r stem-loop DNA sensing pathway either as single stranded or double stranded in a sequence specific manner.
It is probable that AT-r stem-loop DNA only activates this STING-dependent pathway when it finds its way into the cytosol. Experimentally, this was achieved using a transfection reagent like lipofectamine. Our previously published findings, that plasmodial
DNA signals in DCs via TLR9, were explored using DOTAP (Parroche et al., 2007
), a transfection reagent that preferentially targets DNA to the endolysosomal compartment. In the absence of transfection reagents, a key question is how DNA could gain access to cytosolic sensors during an actual infection. We propose that hemozoin acts as a vehicle to deliver DNA to a subcellular compartment where it can have biological effects. This may be particularly relevant as during Plasmodium
infections, concentrations achieved by free hemozoin in the blood approach 0.2–1mg/ml (Hanscheid et al., 2007
). Upon uptake into an immune cell, hemozoin traffics to the phagolysosomal compartment, and TLR9 appears to gain access there to the CpG motifs on the crystalline surface (Parroche et al., 2007
). The hemozoin crystal itself activates the cytosolic NLRP3 inflammasome(Dostert et al., 2009
; Griffith et al., 2009
; Shio et al., 2009
). One of the mechanisms by which NLRP3 is activated by crystals like hemozoin, silica and uric acid is a result of their ability to destabilize the phagolysosome, which allows the delivery of phagosomal contents, including cathepsins, to the cytosol (Halle et al., 2008
; Hornung et al., 2008
). Phagosomal destabilization likely delivers the AT-r stem-loop DNA into the cytosol, where it could then interact with its cytosolic sensor. Indeed, we have observed that hemozoin causes phagolysosomal instability and allows the contents of this compartment, such as the hemozoin crystal and potentially P. falciparum
DNA on its crystalline surface, access to the cytosol. These observations suggest that after the uptake of DNA-coated hemozoin, sequential activation of a TLR9-dependent response in the phagolysosome and a TLR9-independent response in the cytosol are likely to occur. This observation is supported by the fact that components of both the TLR9 pathway (namely MyD88) as well as those of the STING-dependent DNA response contribute to the IFN response to parasite laden RBCs. While the defect in Sting
-deficient cells was incomplete we found a complete defect in type I IFN induction in Tbk1−/− 1
and Irf3−/− Irf7−/−
macrophages. Moreover, in line with its previous role in conferring TLR-independent sensitivity to DNA vaccines (Ishii et al., 2008
), TBK1 is critical in the development of ECM, likely through the induction of TLR-independent pathways. Thus, our study suggests that two innate pathways are initiated during malaria: a TLR9- and a STING-driven pathway, both of which converge on the IRFs to regulate IFN gene transcription.
A number of reports have revealed the ability of Plasmodium spp
to induce type I IFNs and a type I IFN gene signature ((Aucan et al., 2003
; Pichyangkul et al., 2004
; Vigario et al., 2007
; Voisine et al.). Previous reports have shown that exogenous recombinant IFNα can even inhibit ECM and reduce parasite burden in mice infected with P. berghei
) (Vigario et al., 2007
), although this effect requires pretreatment and is reminiscent of the ability of LPS, for example, to prevent Gram-negative sepsis in mice.
Although, pDCs are typically considered major sources of IFNα during microbial infections, recent studies indicate that pDCs are not important in regulating immunity to P. chaubadi
infections (deWalick et al., 2007
; Voisine et al.). In the case of P. chaubadi
, this observation is not surprising because these mice also do not benefit from the loss of the type I IFN receptor (Voisine et al.). But in mice infected with PbA
, this is a surprise, given the central role pDCs have in generating type I IFNs. Thus, alternative sources of IFNβ, likely mediated by cells other than pDCs via the cytosolic AT-r ODN pathway may account for the apparent pathology mediated by the type I IFNs in cerebral malaria thus explaining the lack of importance of pDCs in ECM models and perhaps in human cerebral malaria.
The importance of DNA recognition and type I interferon production is only now beginning to be appreciated in a large spectrum of infectious illnesses well beyond the traditional role of IFNα/β in anti-viral immunity. The data in this paper are suggestive that plasmodial DNA has the capability of generating type I IFN (as well as pro-inflammatory cytokines), which in turn determine the outcome of disease. This view of the pathophysiology of malaria is in agreement with the effect of polymorphisms in the human IFN receptors on clinical infections (Aucan et al., 2003
; Koch et al., 2002
; Naka et al., 2009
) as well as by our observation that patients with malaria express a panoply of interferon-stimulated genes. A further observation is that by our analysis, P. vivax
, although seldom fatal, has a preponderance of both AT-motifs (~5500) as well as CpG motifs (~3000) (data not shown). In contrast, Pf
, most often associated with fatal malaria, has a rather large number of AT-motifs (~6000) but much smaller numbers of CpG motifs (~300) (data not shown). The importance of this distribution is not immediately clear and may further shift our perception of DNA as a pathogen associated molecular pattern (PAMP) useful in resolving disease.
In view of these results, as well as the emerging literature on DNA sensing, we predict that immune recognition of DNA will be an increasingly common theme in pathogenesis. Microbes as diverse as Listeria monocytogenes
(O’Connell et al., 2004
; Rayamajhi et al.), Staphylococcus aureus
(Martin et al., 2009
), Group B streptococcus (Streptococcus agalactiae
) (Charrel-Dennis M, 2008
) (Xiao et al., 2009
) and Trypanosoma cruzi
(Chessler et al., 2008
) all seem to induce significant amounts of type I IFN through nucleotide recognition. This then suggests that any immunomodulatory therapy contemplated for use as an adjunct for any infectious disease will need to take into account the role of DNA-induced type I IFN in a specific disease and whether or not type I IFNs promote a healthy outcome or enhance pathogenesis.