Results of this study provide new insights into the mechanisms of HMGB1 release and show that DNA, while unable to stimulate HMGB1 when incubated directly with RAW 264.7 cells, can nevertheless stimulate this release when transfected into cells. This release depends on the action of IFN-β which, like IFN-α, can also induce HMGB1 release. The transformation of the immune activity of DNA by transfection is consistent with previous results indicating that mammalian DNA acquires immune activity in the context of transfection reagents (Shirota, Ishii et al. 2006
; Zhu, Reich et al. 2003
). Together, these results suggest that transfection allows access of DNA to other, non-TLR receptors, which do not require the presence of CpG motifs.
The entry of DNA into cells and the trafficking among compartment are poorly understood, although synthetic ODN are increasingly used as immunomodulatory agents and are currently under trial for a number of clinical indications. Unlike other TLR ligands, both natural and synthetic DNA must enter cells for activity. Since TLR9 is functional in a lysosomal compartment, free DNA, when incubated with macrophages, accesses the cytoplasmic space for uptake into lysosomes where activation by TLR9 occurs (Latz, Schoenemeyer et al. 2004
). Ultimately, activation depends on the content of immunostimulatory CpG motifs since even high concentrations of mammalian DNA or an non-CpG ODN fail to activate cells (Pisetsky and Reich 2000
In contrast to the situation with free DNA, transfected DNA displays distinct structure-function relationships for stimulation, with both CpG and non-CpG DNA similarly active. Furthermore, as shown in previous studies as well as results presented herein, transfected DNA can activate certain signaling pathways (e.g., JNK) much more effectively than free DNA (Jiang, Reich and Pisetsky 2004
). Transfected DNA also leads to the presence of DNA in the cytoplasm although it appears to differ from that free DNA since it leads to macrophage activation even in the absence of immunostimulatory motifs.
The activation of macrophages by transfected DNA may reflect interaction with non-TLR internal receptors that are usually inaccessible to free DNA. As shown in recent studies, cytoplasmic nucleic acids can activate a number of receptor systems which may be important in triggering innate immunity (Meylan, Tschopp et al. 2006
). Such cytoplasmic nucleic acid, which may be mimicked by transfected DNA, may result from either viral or bacterial infection as well as entrance of DNA into cells in the form of immune complexes as may occur in SLE. Thus, cytoplasmic RNA can activate cells through a retinoic acid-inducible gene I/melanoma differentiation-associated gene 5 (RIG-I/MDA5) signaling pathway (Andrejeva, Childs et al. 2004
; Yoneyama, Kikuchi et al. 2004
). This pathway involves signaling through the adapter caspase recruitment domain adapter inducing interferon-beta (Cardif) followed by the activation of NF-κB and IRFs through the recruitment of IKK and TBK1 (Meylan, Tschopp, and Karin 2006
). In addition, cytoplasmic DNA can activate immune cells to produce IFN-β through an IRF3-dependent pathway, however, the cytoplasmic receptor for this pathway is yet to be identified (Stetson and Medzhitov 2006
Our study extends these findings by showing that transfected DNA can also induce macrophages to release HMGB1, an endogenous molecule with alarmin activity. Similar to activation by LPS or poly (I:C), this release appears to be dependent on IFN-β, a cytokine which can be induced by transfected but not free DNA. The role of IFN-β was established by showing that neutralizing IFN-β by an antibody can inhibit HMGB1 release induced by transfected DNA. It is of interest, in this regard, that a previous study showed that transfected Po ODNs can induce IFN-β production if the length of these ODNs is greater than 25 bases (Stetson and Medzhitov 2006
). In our studies, Po ODNs with the same sequence and length (20 bases) as the Ps ODNs did not induce HMGB1 release even when transfected. With the length of these Po ODNs increased to 45 bases, these compounds also induced HMGB1 release when complexed with lipofectamine. These findings point to a difference in the size requirement for stimulation by transfected DNA.
In a previous study, we showed that the HMGB1 release induced by LPS and poly (I:C) depends on JNK activation (Jiang and Pisetsky 2006
). JNK activation may be important to this release process since JNK activation can lead to the activation of AP-1 (Derijard, Hibi et al. 1994
) and AP-1 mediates IFN-β transcription (Maniatis, Falvo et al. 1998
). In our studies, while we found that transfected DNA activates JNK, the role of JNK in HMGB1 release differ depending on DNA backbone structure. As such, HMGB1 release induced by transfected Ps ODNs appeared dependent on JNK. On the other hand, inhibition of JNK did not prevent HMGB1 release induced by a natural DNA. These findings are consistent with a large body of data indicating that Po and Ps DNA may utilize different signaling transduction pathways despite a common interaction with TLR9.
While these studies involve transfected DNA, the mechanism we postulate may nevertheless occur during infection or inflammatory disease. During infection, internal nucleic acids most likely arise from intracellular viruses or microorganisms. During inflammation, the internal nucleic acid may result from immune complexes or complexes of DNA with DNA binding molecules that functionally “transfect” DNA into cells. Among these molecules, HMGB1 itself has transfecting properties and can promote DNA entry into cells (Bottger, Vogel et al. 1988
; Kato, Nakanishi et al. 1991
). In the setting of cell death or inflammation, extracellular DNA-HMGB1 complexes may form from nuclear material extruded or released from dead and dying cells; activated cells may also release these complexes. In addition, such complexes may form because of a high concentration of extracellular DNA and HMGB1.
Whatever their origin, such complexes may allow access of DNA to internal nucleic acid receptors which lead to a cycle of cytokine production and HMGB1 release that further intensifies inflammation by expanding the array of pro-inflammatory mediators released. As shown in studies on immune complexes in SLE, HMGB1 may also affect activation because of triggering of other receptors (i.e. RAGE) as well as its to interact with TLR9 in the lysosomal compartment in way that determine stimulation by CpG DNA (Ivanov, Dragoi et al. 2007
; Tian, Avalos et al. 2007
). The interaction of DNA and HMGB1 in inflammation may therefore be multifaceted. Future studies are in progress to determine whether DNA-HMGB1 complexes can act similarly as transfected DNA and to identify agents that can block this distinct pathway of DNA stimulation.