Extracellular histones, cell-free DNA, or nucleosomes: differences in immunostimulation

Abstract

In inflammation, extensive cell death may occur, which results in the release of chromatin components into the extracellular environment. Individually, the purified chromatin components double stranded (ds)DNA and histones have been demonstrated, both in vitro and in vivo, to display various immunostimulatory effects, for example, histones induce cytotoxicity and proinflammatory signaling through toll-like receptor (TLR)2 and 4, while DNA induces signaling through TLR9 and intracellular nucleic acid sensing mechanisms. However, DNA and histones are organized in nucleosomes in the nucleus, and evidence suggests that nucleosomes are released as such in inflammation. The cytotoxicity and proinflammatory signaling induced by nucleosomes have not been studied as extensively as the separate effects brought about by histones and dsDNA, and there appear to be some marked differences. Remarkably, little distinction between the different forms in which histones circulate has been made throughout literature. This is partly due to the limitations of existing techniques to differentiate between histones in their free or DNA-bound form. Here we review the current understanding of immunostimulation induced by extracellular histones, dsDNA and nucleosomes, and discuss the importance of techniques that in their detection differentiate between these different chromatin components.

Figure 1

The immunostimulatory effects of histones. Purified histones disturb plasma membrane integrity, which induces a calcium flux, resulting in cellular lysis. In addition, histones have also been shown to signal via TLR2 and 4. It is unclear whether TLR binding of histones induces their uptake and translocation into early endosomes

Figure 2

The immunostimulatory effects of dsDNA. Purified DNA is endocytosed and signals via TLR9 or activates cytoplasmic DNA sensing mechanisms. Purified DNA is not easily endocytosed. Several proteins such as C1q, anti-dsDNA antibodies, and histones appear to enhance dsDNA endocytosis. The constraints for TLR9 signaling by dsDNA, including CpG content, the phosphodiester backbone, and DNA curvature, are discussed in the text

Figure 3

The immunostimulatory effects of nucleosomes. In contrast to purified histones, dsDNA, or mixed preparations, nucleosomes appear to follow additional and different routes of immunostimulation, also depending on the cell type it encounters. Similar to dsDNA, anti-dsDNA antibodies increase the uptake of nucleosomes by phagocytic cells. Moreover, purified nucleosomes with bound HMGB1 mediate immunostimulation of human macrophages via TLR2. In contrast, purified nucleosomes lacking HMGB1 are stimulatory to neutrophils and dendritic cells in a MyD88-independent manner, indicating that stimulation by nucleosomes is also cell-type specific. In contrast to histones, nucleosomes do not appear cytotoxic. Given that nucleosomes were repeatedly found to bind to the plasma membrane, the existence of a nucleosome-specific receptor has been proposed, but this receptor has thus far not been identified. Finally, it is unclear whether nucleosomes that have been taken up by cells are able to stimulate intracellular DNA sensing mechanisms

Figure 4

The origin of histones, dsDNA, and nucleosomes. Upon insufficient clearance of dead cells, or the induction of (neutrophil) extracellular traps, chromatin components are released into the extracellular environment. This release may occur passively, but several plasma proteins are known to regulate the release of chromatin (components) from dead cells