Prehospital immune responses and development of multiple organ dysfunction syndrome following traumatic injury: A prospective cohort study.

BACKGROUND: Almost all studies that have investigated the immune response to trauma have analysed blood samples acquired post-hospital admission. Thus, we know little of the immune status of patients in the immediate postinjury phase and how this might influence patient outcomes. The objective of this study was therefore to comprehensively assess the ultra-early, within 1-hour, immune response to trauma and perform an exploratory analysis of its relationship with the development of multiple organ dysfunction syndrome (MODS). METHODS AND FINDINGS: The immune and inflammatory response to trauma was analysed in 89 adult trauma patients (mean age 41 years, range 18-90 years, 75 males) with a mean injury severity score (ISS) of 24 (range 9-66), from whom blood samples were acquired within 1 hour of injury (mean time to sample 42 minutes, range 17-60 minutes). Within minutes of trauma, a comprehensive leukocytosis, elevated serum pro- and anti-inflammatory cytokines, and evidence of innate cell activation that included neutrophil extracellular trap generation and elevated surface expression of toll-like receptor 2 and CD11b on monocytes and neutrophils, respectively, were observed. Features consistent with immune compromise were also detected, notably elevated numbers of immune suppressive CD16BRIGHT CD62LDIM neutrophils (82.07 x 106/l ± 18.94 control versus 1,092 x 106/l ± 165 trauma, p < 0.0005) and CD14+HLA-DRlow/- monocytes (34.96 x 106/l ± 4.48 control versus 95.72 x 106/l ± 8.0 trauma, p < 0.05) and reduced leukocyte cytokine secretion in response to lipopolysaccharide stimulation. Exploratory analysis via binary logistic regression found a potential association between absolute natural killer T (NKT) cell numbers and the subsequent development of MODS. Study limitations include the relatively small sample size and the absence of data relating to adaptive immune cell function. CONCLUSIONS: Our study highlighted the dynamic and complex nature of the immune response to trauma, with immune alterations consistent with both activation and suppression evident within 1 hour of injury. The relationship of these changes, especially in NKT cell numbers, to patient outcomes such as MODS warrants further investigation.

Neutrophil Dysfunction, Immature Granulocytes, and Cell-free DNA are Early Biomarkers of Sepsis in Burn-injured Patients: A Prospective Observational Cohort Study.

OBJECTIVE: The aim of this study was to measure neutrophil function longitudinally following burn injury and to examine the relationship between neutrophil dysfunction and sepsis. BACKGROUND: Sepsis prevalence and its associated mortality is high following burn injury, and sepsis diagnosis is complicated by the ongoing inflammatory response. Previous studies have suggested that neutrophil dysfunction may underlie high infection rates and sepsis postburn; however, neutrophil dysfunction has not been thoroughly characterized over time in burns patients. METHODS: Neutrophil phagocytosis, oxidative burst capacity, and neutrophil extracellular trap (NET) generation (NETosis) were measured from 1 day to up to 1 year postburn injury in 63 patients with major burns (≥15% total body surface area). In addition, immature granulocyte (IG) count, plasma cell-free DNA (cfDNA), and plasma citrullinated histone H3 (Cit H3) levels were measured. RESULTS: Neutrophil function was reduced for 28 days postburn injury and to a greater degree in patients who developed sepsis, which was also characterized by elevated IG counts. Plasma cfDNA and Cit-H3, a specific marker of NETosis, were elevated during septic episodes. The combination of neutrophil phagocytic capacity, plasma cfDNA levels, and IG count at day 1 postinjury gave good discriminatory power for the identification of septic patients. CONCLUSION: Neutrophil function, IG count, and plasma cfDNA levels show potential as biomarkers for the prediction/early diagnosis of sepsis postburn injury and neutrophil dysfunction may actively contribute to the development of sepsis. Targeting neutrophil dysfunction and IG release may be a viable therapeutic intervention to help reduce the incidence of nosocomial infections and sepsis postburn.

Traumatic injury is associated with reduced deoxyribonuclease activity and dysregulation of the actin scavenging system.

BACKGROUND: Traumatic injury is associated with increased concentrations of cell-free DNA (cfDNA) in the circulation, which contribute to post-injury complications. The endonuclease deoxyribonuclease 1 (DNase-1) is responsible for removing 90% of circulating cfDNA. Recently, DNase activity was reported to be significantly reduced following major non-traumatic brain injury (TBI), but the processes responsible were not investigated. Moreover, it is not known how quickly following injury DNase activity is reduced and whether this also occurs after TBI. METHODS: At 3 post-injury time points (≤1, 4-12 and 48-72 hours), blood samples were obtained from 155 adult trauma patients that had sustained an isolated TBI (n = 21), TBI with accompanying extracranial injury (TBI+) (n = 53) or an extracranial injury only (ECI) (n = 81). In addition to measuring cfDNA levels and the activity and expression of DNase, circulating concentrations of monomeric globular action (G-actin), an inhibitor of DNase-1, and the actin scavenging proteins gelsolin (GSN) and vitamin D binding protein (VDBP) were determined and values compared to a cohort of healthy controls. RESULTS: Significantly elevated concentrations of plasma cfDNA were seen in TBI, TBI+ and ECI patients at all study time points when compared to healthy controls. cfDNA levels were significantly higher at ≤1 hour post-injury in ECI patients who subsequently developed multiple organ dysfunction syndrome when compared to those who did not. Plasma DNase-1 protein was significantly elevated in all patient groups at all sampling time points. In contrast, DNase enzyme activity was significantly reduced, with this impaired function evident in TBI+ patients within minutes of injury. Circulating concentrations of G-actin were elevated in all patient cohorts in the immediate aftermath of injury and this was accompanied by a significant reduction in the levels of GSN and VDBP. CONCLUSIONS: The post-traumatic increase in circulating cfDNA that occurs following extracranial trauma and TBI is accompanied by reduced DNase activity. We propose that, secondary to reduced GSN and VDBP levels, elevated circulating concentrations of G-actin underlie the post-injury reduction in DNase activity. Reducing circulating cfDNA levels via therapeutic restoration of DNase-1 activity may improve clinical outcomes post-injury.

Traumatic Injury and Exposure to Mitochondrial-Derived Damage Associated Molecular Patterns Suppresses Neutrophil Extracellular Trap Formation.

Major traumatic injury induces significant remodeling of the circulating neutrophil pool and loss of bactericidal function. Although a well-described phenomenon, research to date has only analyzed blood samples acquired post-hospital admission, and the mechanisms that initiate compromised neutrophil function post-injury are therefore poorly understood. Here, we analyzed pre-hospital blood samples acquired from 62 adult trauma patients (mean age 44 years, range 19-95 years) within 1 h of injury (mean time to sample 39 min, range 13-59 min). We found an immediate impairment in neutrophil extracellular trap (NET) generation in response to phorbol 12-myristate 13-acetate (PMA) stimulation, which persisted into the acute post-injury phase (4-72 h). Reduced NET generation was accompanied by reduced reactive oxygen species production, impaired activation of mitogen-activated protein kinases, and a reduction in neutrophil glucose uptake and metabolism to lactate. Pre-treating neutrophils from healthy subjects with mitochondrial-derived damage-associated molecular patterns (mtDAMPs), whose circulating levels were significantly increased in our trauma patients, reduced NET generation. This mtDAMP-induced impairment in NET formation was associated with an N-formyl peptide mediated activation of AMP-activated protein kinase (AMPK), a negative regulator of aerobic glycolysis and NET formation. Indeed, activation of AMPK via treatment with the AMP-mimetic AICAR significantly reduced neutrophil lactate production in response to PMA stimulation, a phenomenon that we also observed for neutrophils pre-treated with mtDAMPs. Furthermore, the impairment in NET generation induced by mtDAMPs was partially ameliorated by pre-treating neutrophils with the AMPK inhibitor compound C. Taken together, our data demonstrate an immediate trauma-induced impairment in neutrophil anti-microbial function and identify mtDAMP release as a potential initiator of acute post-injury neutrophil dysfunction.

Soluble GPVI is elevated in injured patients: shedding is mediated by fibrin activation of GPVI.

Soluble glycoprotein VI (sGPVI) is shed from the platelet surface and is a marker of platelet activation in thrombotic conditions. We assessed sGPVI levels together with patient and clinical parameters in acute and chronic inflammatory conditions, including patients with thermal injury and inflammatory bowel disease and patients admitted to the intensive care unit (ICU) for elective cardiac surgery, trauma, acute brain injury, or prolonged ventilation. Plasma sGPVI was measured by enzyme-linked immunosorbent assay and was elevated on day 14 after thermal injury, and was higher in patients who developed sepsis. sGPVI levels were associated with sepsis, and the value for predicting sepsis was increased in combination with platelet count and Abbreviated Burn Severity Index. sGPVI levels positively correlated with levels of D-dimer (a fibrin degradation product) in ICU patients and patients with thermal injury. sGPVI levels in ICU patients at admission were significantly associated with 28- and 90-day mortality independent of platelet count. sGPVI levels in patients with thermal injury were associated with 28-day mortality at days 1, 14, and 21 when adjusting for platelet count. In both cohorts, sGPVI associations with mortality were stronger than D-dimer levels. Mechanistically, release of GPVI was triggered by exposure of platelets to polymerized fibrin, but not by engagement of G protein-coupled receptors by thrombin, adenosine 5'-diphosphate, or thromboxane mimetics. Enhanced fibrin production in these patients may therefore contribute to the observed elevated sGPVI levels. sGPVI is an important platelet-specific marker for platelet activation that predicts sepsis progression and mortality in injured patients.

Endotheliopathy is associated with higher levels of cell-free DNA following major trauma: A prospective observational study.

BACKGROUND: Cell free deoxyribonucleic acid (cfDNA) has been proposed as a biomarker of secondary complications following trauma. Raised thrombomodulin and syndecan-1 levels have been used to indicate endotheliopathy, and are associated with inflammation, coagulopathy, and mortality. The current study aimed to analyse the association between cfDNA and biomarkers of endotheliopathy in a cohort of trauma patients, and whether raised levels of cfDNA were associated with poorer clinical outcomes. METHODS: Serum thrombomodulin and syndecan-1 were used as biomarkers of endotheliopathy and compared to plasma cfDNA in trauma patients from two prospective longitudinal observational studies. Cohort A (n = 105) had a predicted injury severity score (ISS) >8, and had blood sampled within 1h of injury and at 4-12h. Cohort B (n = 17) had evidence of haemorrhagic shock, and had blood sampled at a median time of 3.5h after injury. Relationships between biomarkers were tested using multivariable linear regression models that included the covariates of gender, age, ISS, Glasgow Coma Scale, lactate, systolic blood pressure, and heart rate. A model was fitted to investigate whether changes in cfDNA were associated with similar changes in endothelial biomarkers. RESULTS: The mean age was 41 (SD 19), and the median ISS was 25 (IQR 12-34). There was a significant association between cfDNA levels and both syndecan-1 and thrombomodulin levels (both p<0.001). This was independent of all covariates except for ISS, which significantly correlated with cfDNA levels. 50 ng/ml change in syndecan-1 and 1 ng/ml change in thrombomodulin corresponded to 15% and 20% increases in cfDNA levels respectively (both p<0.001). Patients who died had significantly higher prehospital and in-hospital cfDNA levels (both p<0.05). CONCLUSIONS: Raised cfDNA levels are associated with markers of endotheliopathy following trauma, and are associated with mortality. This relationship is present within the first hour of injury, and a change in one biomarker level is reflected by similar changes in the others. These findings are in keeping with the hypothesis that circulating DNA and endothelial injury share a common pathway following trauma.