Impact of plasma histones in human sepsis and their contribution to cellular injury and inflammation

Sepsis is a multifactorial life-threatening syndrome arising from the immune response to invading microorganisms, resulting in excessive cell activation and tissue damage [1],[2]. Based on current understanding of the pathophysiology of the host response, endothelial activation and dysfunction [3], as well as culminating multiple organ failure, are known hallmarks of the clinical course, which determines the prognosis of patients [4]-[6]. Multiple immune activators during systemic inflammation known as pathogen-associated molecular pattern (PAMPs) [7] and damage-associated molecular patterns (DAMPs) [8] have been reported to be involved in the aggravation of host response.

Recently reported is a concept implicating extracellular histone isoforms, mainly histone H4 (H4), as a result of host response, which aggravates maladaptive mechanisms through a direct action on endothelial cells [9] and platelets [10]. Furthermore, free histones in circulation mediate extensive cellular damage, hemostatic imbalance and amplification of the inflammatory response by inducing cytokine production as reported in an animal model of sepsis. Intravenous injection of purified histones in mice elicited thrombocytopenia, neutrophil migration, and organ failure mimicking the pathophysiological signature of sepsis [9]. Interestingly, co-infusion of histones with APC [9] or intravenous injection of histones in toll-like receptor (TLR)4-null mice abrogates histone induced effects [11]. Additionally, histones have been reported to mediate fatal liver injury in mice [11], and have recently been identified as essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury [12] and in trauma-associated lung injury in humans, with concentrations ranging between 10 and 280 μg/ml as measured using immunoblotting [13].

The study demonstrated for the first time and in two independent cohorts of septic patients that plasma concentrations of extracellular histones are elevated during human sepsis as compared to ICU controls. Interestingly, the observed concentrations are similar in both cohorts with low inter-assay variation. Histones were also significantly increased during the course of disease progression until day 5 of sepsis. This is in line with previous reports of an increase in histone concentration in baboons after endotoxin challenge [9]. Interestingly, histone levels were higher in septic patients compared to patients with multiple organ failure without infection, potentially indicating that the source of histones in circulation could be strongly associated with the underlying infection and the release of neutrophil extracellular traps by neutrophils as demonstrated by others [15],[16]. In addition, we found lower levels of histones in patients with minor trauma. This might be related to the limited tissue damage representing a lower release of histones into circulation, possible clearance of small quantities of histones in circulation by macrophages or complete eradication through active cleavage by proteases in plasma such as endogenous activated protein C. Most interestingly, we observed that in our cohort of patients with multiple trauma, histone concentrations were higher than levels found in septic patients. This supports previous reports of elevated levels of histones found in patients with blunt traumatic lung injury. Nevertheless, there was a clear disparity in the concentrations of histones measured in our multiple trauma patient cohort in the nanogram range compared to the levels reported in traumatic lung injury in the microgram range [13]. However, we could not confirm histone concentrations by mass spectrometry due to high abundance plasma proteins, which interfere with the detection of low abundance proteins despite the existence of high abundance protein depletion protocols. Our efforts to quantify histones by immunoblotting also failed because levels of histones found in patients were far below the lower limit of detection for immunoblotting (>500 ng/ml). Nevertheless, our investigation using two independent cohorts of sepsis patients demonstrates and justifies consistency and reproducibility of plasma histone measurements in septic patients. In addition, the inclusion of a second cohort enabled us to determine plasma histones not only at onset of sepsis but also during disease progression.

A hallmark of sepsis is the occurrence of renal failure requiring renal replacement therapy [17]. Consistently, our data suggested that higher levels of histones correlated with the need for RRT. In addition, histones have been shown to bind to platelets recruiting plasma adhesion molecules, thereby promoting platelet aggregation [10]. In our study, increased histone levels were associated with a decrease in platelet count corroborating this concept. Interestingly, the occurrence of renal failure in sepsis and thrombocytopenia is often associated with high mortality [18],[19]. The observed increase of plasma histone levels in patients undergoing RRT also implies that this increase might be a consequence of impaired renal excretion of the protein. However, to the best of our knowledge, there is no evidence that histones are excreted and become present in urine, either in healthy or pathophysiological conditions. On the other hand, microvascular permeability is increased when renal microvasculature is exposed to extracellular histones, released from dying tubular epithelial cells [20]. In lipopolysaccharide (LPS)-induced endotoxemia, neutralization of released histones reduced tubular injury and an improved renal function as measured by creatinine levels was obvious [20], identifying histones as mediators of acute kidney injury. Strikingly, higher histone levels correlated with ICU-, 28- and 90-day mortality. Interestingly, thrombocytopenia and organ dysfunction following histone administration have also been demonstrated in mice [8],[10]. Based on this concept, it could be hypothesized that the interaction of histones with the endothelium will result in endothelial impairment. This damage leads to endothelial dysfunction promoting platelet activation and aggregation, which impairs microcirculation and results in organ dysfunction, which is associated with an increased risk of death [21]. In line with this, our ex-vivo findings further demonstrate that histone mediated cytotoxicity on endothelium cells and underline the results of Fuchs et al. demonstrating direct platelet activation and aggregation [10]. Besides the direct effects on platelets and cytotoxicity, we were able to show an activation of immune cells which led to an increase in sepsis prototypic cytokines in both low and high concentrations of histone stimulation. From a pathomechanistic perspective, histone mediated effects might be initiated by TLR4 signaling as neutralization or blocking of the TLR4 receptor in our study completely abrogated histone-mediated cytotoxicity in our in vitro model. This underlines a report by Xu et al. who showed that TLR4 knock-out mice are protected from the fatal effects of histone infusion [11] and also, at least in part, is in line with results from Abrams et al. showing that TLR4 blocking results in a decrease in cytokine production but not cytotoxicity.

We report lower concentrations of histones in the nanogram range during sepsis which contradicts reports on the levels of histones measured in various species and diseases (baboons after gram-negative challenge, approximately 15 μg/ml [9], in patients with blunt traumatic lung injury, approximately 10 to 280 μg/ml [13], 200 μg/ml in mice models of acute lung injury [12]) and reported concentration used in all histone stimulation studies so far (approximately 10 to 1000 μg/ml) [9],[11]-[13],[22]. It could also be speculated that the discrepancy in histone levels released in animal (mice and baboons) sepsis and the actual concentration measured in plasma of our septic patients might be due to the treatment with heparin in our septic ICU patients. Heparin is highly negatively charged and was recently shown to bind to positively charged histones reducing their cytotoxic effect [22]. In line with other reports, the observed histone levels in multiple trauma patients might be related to a higher degree of tissue damage as represented by the injury severity score [13]. In our cohort stratified according to need for RRT on day 5 (cohort II), all of the patients underwent anticoagulation either with heparin (2/3 of all our ICU patients) or citrate (1/3 of all our ICU patients). However, measurements of histones to associate the level with the clinical endpoint of need for RRT were performed prior to initiation of anticoagulation for supportive treatment. From the low number of samples with different anticoagulants, from which one might interact with the readout, no comparison between these was performed.

We demonstrate also for the first time the ability of plasma to degrade histones. As plasma was used for these experiments, a potential binding of positively charged histones to negatively charged membranes of erythrocytes could be excluded. The observed fast initial degradation of histones might be related to free proteases within the plasma such as endogenous APC and might explain why co-injection of histones with APC abrogated the lethal effects of histone injection in mice [9]. This might also support our finding of a negative correlation between histone levels and APC in our septic patients. Unclear, however, is the decline to a steady state of histones in plasma after 30 minutes. However, we speculate that the biological half-life of histones in plasma could be affected by active metabolites, binding of fragments to other proteins as well as receptor interactions and therefore will not follow the first order kinetics with a fixed rate constant, because the generated fragments were also detected by the cited polyclonal antibodies. Nevertheless, this finding indicates the need for early centrifugation and measuring or freezing of the samples. Finally, further studies should elucidate the origin and sources of circulating histones in plasma during sepsis.

In conclusion, our data provides evidence that plasma histones are not only elevated in human sepsis but are associated with sepsis-related organ dysfunction, mediating cellular damage and severe inflammation responses. The observed effects might be mediated via the TLR4 receptor.