Abstract
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.
Highlights
Major injury induces significant phenotypic and functional remodeling of the peripheral neutrophil pool, attributable in part to the emergence into circulation of immature granulocytes (IGs) and highly mature neutrophil subsets [1,2,3,4,5]
Compared to neutrophils isolated from healthy controls (HCs), neutrophils acquired from trauma patients within 1 h of injury exhibited significantly enhanced basal neutrophil extracellular trap (NET) generation (Figure 1A), a hyperactivity that was accompanied by significantly elevated serum concentrations of high mobility group box-1 (HMGB-1) (Figure 1B) and IL-33 (Figure 1C)
Due to the significant lymphocytosis that occurs within minutes of traumatic injury [3], and the small blood volume collected from patients at the scene of injury, we were unable to isolate a sufficient number of neutrophils from prehospital blood samples to examine MAPK signaling
Summary
Major injury induces significant phenotypic and functional remodeling of the peripheral neutrophil pool, attributable in part to the emergence into circulation of immature granulocytes (IGs) and highly mature neutrophil subsets [1,2,3,4,5]. Whilst understanding of trauma-induced changes in neutrophil intracellular bactericidal function is well-developed, few studies have investigated the impact of injury on the extracellular defensive mechanisms of neutrophils and how soon after injury, any compromise occurs. In terms of stimulus-induced NET generation, comparable [13], or reduced [1, 10] NET production in response to stimulation with phorbol 12-myristate 13-acetate (PMA) has been reported post- trauma. Of these studies, only one performed quantitative analysis [1], and neither study that reported a post-injury reduction in NET formation investigated the mechanism(s) responsible [1, 10]
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