Histones: Better In than Out?

Extracellular histones are major mediators of death in sepsis.

Xu, et. al. Nature Medicine 15: 1318-1321 (2009).

Sepsis, a life-threatening systemic inflammatory condition caused by bacterial, viral, or fungal infection, is one of the leading causes of death in the United States and a common problem in hospitals. A surprising report by Xu, et. al. in the November 2009 issue of Nature Medicine presents evidence that death due to sepsis is largely caused by the abnormal presence of histones circulating outside of cells. That's right, histones - the abundant, seemingly inert component of chromatin around which the DNA of every eukaryotic cell is wound.

Currently, one of the few treatment options for severe sepsis is activated protein C (APC), a serine protease. Although the therapuetic benefit of APC is believed to be related to an anti-inflammatory function, the exact mechanism of action is not completely understood. Here, Xu, et. al. tested the hypothesis that proteolytic degradation of target proteins by APC occurs during sepsis (using an in vitro sepsis model of murine macrophages with an activated inflammatory response) by comparing the proteins present in the presence or absence of APC via SDS-PAGE. They found that the APC-conditioned cells had three specific new bands, which sequencing and Western blotting revealed to be fragments of the core histones H2A, H3, and H4. Surprisingly, the authors found that histones, especially histones H3 and H4, were cytotoxic to human endothelial cells and that APC could alleviate this effect. Furthermore, in a purified system, APC could cleave H3 and H4 both in a dose-dependent manner and in the presence of an inflammatory agent. Seredipitously, the authors had access to archival plasma samples of baboons that had been treated with sepsis-inducing doses of E. coli before rescue with APC, and examined the baboons' extracellular H3 levels both after E. coli and APC treatment. As predicted by their in vitro results, in two baboons (it is not mentioned out of how many) extracellular H3 levels increased in response to challenge with E. coli and the amount of cleaved H3 increased in response to APC treatment. The same was also true in archival samples of human sepsis patients, although again, it was unclear how many, if any, patient samples lacked elevated extracellular H3 or increased H3 cleavage following APC treatment.

Regardless, these results suggest that histones - the same histones that are essential for packaging our genomes - are involved in the pathology of a devastating medical condition. The shocking toxicity of extracellular histones is then demonstrated in vivo, as injection of 75mg/kg purified calf thymus histones killed 5/5 mice in less than an hour, with 4/5 mice dying in less than 10 minutes, and 3/5 mice dying almost instantly. To prove this was a rapid, specific induction of sepsis and not just some horribly botched injection, the authors show that the pathological changes in the mouse tissues were consistent with what occurs during sepsis and, impressively, that coinjection of histones with APC completely rescued their rapid lethality. The authors then provide evidence that targeting histones may be effective in treating sepsis, as an H4 antibody can almost completely block sepsis induced by any of three ways.

The idea that the abnormal presence of histones in the bloodstream can kill cells and trigger a systemic condition like sepsis is fascinating yet hard to grasp. At first glance, one might dismiss the presence of extracelullar histones during sepsis as merely an indicator of widespread cell lysis caused by an offending infectious agent. However, other studies have shown that histones may have anti-microbial properties, either individually like histone H2A, which can be cleaved into a potent anti-microbial peptide, or as part of neutrophil extracelluar traps (NETs), web-like structures formed from apoptotic neutrophils during inflammatory responses that are composed of chromatin and neutrophilic proteins. Likewise, the data of Xu, et. al. indicates that the histones themselves are functional during systemic infection, acting to galvanize a cytotoxic inflammatory response. Their work emphasizes that, despite the great strides that have been made in the last few years in understanding the roles of histones in regulating gene expression, we know shockingly little about their activities in the world outside the nucleus.

Other references:

Brinkmann, V. and Zychlinsky A. Beneficial suicide: why neutrophils die to make NETs. Nature Reviews Microbiology 5: 577-582 (2007).