Nucleosomes and neutrophil extracellular traps in septic and burn patients

Highlights

• Nucleosomes and HNE-DNA complexes are elevated in septic and burn patients.

• Septics' nucleosomes correlate with SOFA at 1 dpa and were associated with mortality.

• Patient's neutrophils exhibit spontaneous NETosis and are unresponsive to stimulation.

• Nucleosomes could be organ damage indicators and mortality predictors in septics.

• Platelets, VWF and cytokines do not seem to mediate NETosis in these patients.

Abstract

NETosis is a host defense mechanism associated with inflammation and tissue damage. Experimental models show that platelets and von Willebrand factor (VWF) are key elements for intravascular NETosis.

We determined NETosis in septic and burn patients at 1 and 4 days post-admission (dpa).

Nucleosomes were elevated in patients. In septics, they correlated with Human Neutrophil Elastase (HNE)-DNA complexes and SOFA score at 1 dpa, and were associated with mortality.

Patient's neutrophils had spontaneous NETosis and were unresponsive to stimulation. Although platelet P-selectin and TNF-α were increased in both groups, higher platelet TLR-4 expression, VWF levels and IL-6 were found in septics at 1 dpa. Neither platelet activation markers nor cytokines correlated with nucleosomes or HNE-DNA.

Nucleosomes could be indicators of organ damage and predictors of mortality in septic but not in burn patients. Platelet activation, VWF and cytokines do not appear to be key mediators of NETosis in these patient groups.

Introduction

The formation of neutrophil extracellular traps (NETs) is a novel antimicrobial process named NETosis through which neutrophils can trap and kill microbes [1]. Despite NETosis being initially described as a beneficial host response against pathogens, it is being increasingly recognised that the uncontrolled formation of these traps switches NETosis from a beneficial response into a major cause of tissue damage and organ failure [2], [3], [4]. Besides pathogens, cytokines or danger signals can trigger NET formation [5]. Thus, NETs can be formed not only in infectious conditions but also under inflammatory sterile states. In addition, NETs can be found in tissues as well as inside blood vessels [6]. Experimental models show that platelets, von Willebrand factor (VWF) and endothelial cells are key elements for the intravascular formation of NETs [7], [8], [9]. The role of these factors in NETosis occurring under clinical conditions is yet to be investigated.

Sepsis and severe burns are two acute inflammatory conditions, one triggered by an infectious insult and the other by tissue injury. Even though they share many aspects, and multiorgan failure is frequent in both pathologies, the evolution of these two entities is not exactly the same. Although early mortality in burn patients is mostly due to hypovolemic shock and inhalation injuries, in septic patients, multiorgan damage is the most frequent cause of death, being respiratory and renal failure the most common among them [10]. Following the initial 5–7 days period, the evolution of the burn patient becomes similar to that of the septic, existing three possible states, early death associated to a highly pro-inflammatory profile, resolution of the infection and progressive improvement of the organ function due to an adequate balance between pro- and anti-inflammatory states, or torpid and lengthy evolution due to a tendency to immunosuppression from the onset of the disease. It is noteworthy that multiorgan failure prevalence is higher in burn patients when they present acute inhalation injury in a similar way to what happens during sepsis-associated respiratory distress [11]. In this context, several studies in animal models have proven that the lung would be a key organ in the NET formation process [6], [12], [13] and that it is there where a critical interaction between platelets and neutrophils takes place, leading to a systemic release of NETs and inflammatory cytokines.

Despite multiple technological breakthroughs in the support and treatment of the etiology, septic and burn patient's morbi-mortality remains high. Clinical management of these patients is challenging and largely limited to supportive therapies, which is in part related to a limited understanding of the underlying pathophysiology. After the discovery of the NETosis process, some studies in septic and a few in burn patients have analysed the role of cell free DNA (cfDNA) or nucleosomes in these inflammatory diseases. However, these biomarkers can be released upon several conditions including necrosis, apoptosis or NETosis, [14] and therefore, the real involvement of NETs in clinical inflammatory conditions is still not clear. In order to further understand the role of NETs in the inflammatory response, herein we aimed to determine the presence of NETs in septic and burn patients and correlate them with organ damage, patient evolution, platelet activation, VWF and pro-inflammatory cytokines.