Elevation of neutrophil‐derived factors in patients after multiple trauma

Abstract Trauma represents one of the leading causes of death worldwide. Traumatic injuries elicit a dynamic inflammatory response with systemic release of inflammatory cytokines. Disbalance of this response can lead to systemic inflammatory response syndrome or compensatory anti‐inflammatory response syndrome. As neutrophils play a major role in innate immune defence and are crucial in the injury‐induced immunological response, we aimed to investigate systemic neutrophil‐derived immunomodulators in trauma patients. Therefore, serum levels of neutrophil elastase (NE), myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) were quantified in patients with injury severity scores above 15. Additionally, leukocyte, platelet, fibrinogen and CRP levels were assessed. Lastly, we analysed the association of neutrophil‐derived factors with clinical severity scoring systems. Although the release of MPO, NE and CitH3 was not predictive of mortality, we found a remarkable increase in MPO and NE in trauma patients as compared with healthy controls. We also found significantly increased levels of MPO and NE on Days 1 and 5 after initial trauma in critically injured patients. Taken together, our data suggest a role for neutrophil activation in trauma. Targeting exacerbated neutrophil activation might represent a new therapeutic option for critically injured patients.


| INTRODUC TI ON
Unintentional and violence-related injuries are responsible for 4.4 million deaths every year, accounting for 8% of all deaths worldwide. Road traffic accidents, homicide and suicide constitute three of the top five causes of death between the ages of 5 and 29. 1 Although Van Breugel et al. 2 reported a decrease of approximately 1.8% per year in all-cause mortality in polytrauma patients admitted to the intensive care unit (ICU) over the last 35 years, in-hospital mortality remains high, at approximately 15%. 3 After initial trauma, around 32% of patients develop multipleorgan failure (MOF) during hospitalisation. Also, acute respiratory distress syndrome (ARDS) is one of the most common complications observed in polytraumatized patients, with reported rates of up to 50%. 3 The endogenous response to trauma includes systemic release of pro-and anti-inflammatory cytokines. Disbalance of this response can lead to systemic inflammatory response syndrome (SIRS) or compensatory anti-inflammatory response syndrome (CARS), potentially leading to sepsis and multi organ failure. [4][5][6][7] While SIRS is caused by a systemic pro-inflammatory state, CARS is a consequence of complex anti-inflammatory signalling leading to immunosuppression. However, their underlying pathways and mechanisms remain to be completely understood, 4,5,8 These distinct immunological states occur predominantly simultaneously and can lead to MOF and infections. 8,9 Neutrophils play a major role as the first line of innate immune defence and are the most abundant leukocyte in humans, constituting 60%-70% of circulating leukocytes, 8,10,11 Neutrophils exert several anti-microbial mechanisms, including phagocytosis, release of effector molecules, and formation of neutrophil extracellular traps (NETs). 12,13 NETs were first described by Brinkman et al. in 2004. 14 Accumulations of NET-forming activated neutrophils in damaged tissue following injury have been described. 8,15 NET formation occurs by the formation of citrullinated histones due to the activation of protein-arginine deiminase 4 (PAD4), resulting in chromatin decondensation. Following neutrophil plasma membrane rupture, granule proteins such as myeloperoxidase (MPO) and neutrophil elastase (NE) together with intracellular DNA are released. MPO and NE additionally promote DNA decompaction. 16 Previously, we were able to demonstrate systemic neutrophil activation after burn injury. 12 This is in accord with other studies reporting a role for neutrophils and NETs in burn injury, 17 critically ill patients, 11 sepsis 18 and lung injury. 19 Whereas NET formation represents a protective process that captures and sequesters microbes, thereby preventing the spread of infection, a dysregulation of NET formation with increased concentration of extracellular DNA may contribute to the perpetuation of inflammation and severe tissue injury. 15,20 Although, NET formation has been reported following trauma and subsequent surgery, 21 systemic neutrophil-derived factors indicating NET formation such as MPO and NE have so far not been comprehensively studied in severely injured patients.
We therefore aimed to investigate systemic neutrophil-derived immunomodulators in patients suffering from multiple injuries.

| Study population
In total, 106 patients were enrolled prospectively, meeting the following inclusion criteria: age over 18, admission at our urban level I trauma centre with severe injuries (injury severity score, ISS above 15) within 1 h following trauma, and primary treatment at the inten-

| Serum samples
We obtained study-specific serum samples within the first 2 h after admission ('day 0', initial assessment) and then on Day 1, 3, 5, 7 and 10 after admission. Following blood withdrawal and an interval of 15-30 min to allow thorough coagulation, samples were centrifuged at 3000g for 15 min at room temperature. Serum samples were then aliquoted and stored at −80°C until analysed.

| Quantification of serum NE, CitH3 and MPO
Serum NE, CitH3 and MPO were quantified by enzyme-linked immunosorbent assay (ELISA) using commercially available kits and following the manufacturers' protocols (human neutrophil elastase, human MPO: all R&D Systems, Bio-Techne; human CitH3, clone 11D3, Cayman Chemical). Colorimetric measurements were performed using a Tecan F50 infinite microplate reader (Tecan Group) with Magellan software (version 7.2, Tecan). Analytes were quantified according to external standard curves.

| Statistical analysis
Statistical analyses and visualisation were performed using IBM SPSS Statistics 26.0 (IBM) and GraphPad Prism (version 9, GraphPad Software Inc.). The Mann-Whitney U-test was used for comparison of metric and ordinal values between the two independent groups.
The Kruskal-Wallis test with multiple-comparison post hoc tests with Bonferroni correction was calculated to compare metric and ordinal values between three or more independent groups. A p-value <0.05 was considered statistically significant.

| Serum concentrations of CitH3, MPO and NE in survivors versus nonsurvivors
CitH3 levels were slightly decreased on Days 0-10 after injury in nonsurvivors when compared to survivors. Measurements of MPO and NE showed slightly elevated serum levels of MPO and NE in nonsurvivors on Day 7 post injury (see Figure 2), but these differences were not significant.

| Routine laboratory values in survivors versus nonsurvivors
The course of routine laboratory values from Day 0 to Day 10 after injury of survivors and nonsurvivors is displayed in Figure 3. On the day of admission (Day 0) and on Day 10 after injury, nonsurvivors of polytrauma showed significantly reduced thrombocytes (Day 0: TA B L E 1 Demographic details of study population.

| NE and MPO are markedly increased in severely injured patients with ISS over 50 as compared with moderately injured patients
We found increased levels of the neutrophil-derived factors NE and MPO in patients with higher ISS. On Days 1 and 5 after   Figure 4).

| DISCUSS ION
In severely injured patients, two distinct pathomechanisms with partially overlapping occurrence are known to be involved in the endogenous immunological response. The initial pro-inflammatory state with increased secretion of pro-inflammatory cytokines is later accompanied by an anti-inflammatory response with increased immunological tolerance, leading to increased risk for secondary infections and late sepsis. 8,9,22 Whereas many previously published studies have shown a potential role for neutrophils in patients after injury, 8  were able to detect CitH3-positive cells using immunofluorescence in the bloodstream of critically ill patients. CitH3 was also identified as reliable blood biomarker for the diagnosis and treatment of endotoxic shock 24 in a mouse model. They also indicated that CitH3 was mainly circulating in mice suffering from lipopolysaccharide shock syndrome, whereas they could rarely detect any CitH3 in mice with haemorrhagic shock. This might also serve as an explanation for the lack of a significant difference in CitH3 serum levels between severely injured patients and healthy controls.
Interestingly, CitH3 levels were reduced in surviving patients on Days 1, 5 and 10 after trauma. However, we were not able to detect significant differences in the MPO and NE serum levels of survivors versus nonsurvivors. We could only demonstrate slight increases in MPO and NE on Day 0 in nonsurviving patients, as depicted in

ACK N O WLE D G E M ENTS
We thank Dr H. P. Haselsteiner and the CRISCAR Familienstifung for their belief in this private-public partnership to augment basic and translational clinical research.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Raw data are available from the corresponding authors upon request.