Dysregulation of the actin scavenging system and inhibition of DNase activity following severe thermal injury

R J Dinsdale, J Hazeldine, K Al Tarrah, P Hampson, A Devi, C Ermogenous, A L Bamford, J Bishop, S Watts, E Kirkman, J J Dalle Lucca, M Midwinter, T Woolley, M Foster, J M Lord, N Moiemen, P Harrison, R J Dinsdale, J Hazeldine, K Al Tarrah, P Hampson, A Devi, C Ermogenous, A L Bamford, J Bishop, S Watts, E Kirkman, J J Dalle Lucca, M Midwinter, T Woolley, M Foster, J M Lord, N Moiemen, P Harrison

Abstract

Background: Circulating cell-free DNA (cfDNA) is not found in healthy subjects, but is readily detected after thermal injury and may contribute to the risk of multiple organ failure. The hypothesis was that a postburn reduction in DNase protein/enzyme activity could contribute to the increase in cfDNA following thermal injury.

Methods: Patients with severe burns covering at least 15 per cent of total body surface area were recruited to a prospective cohort study within 24 h of injury. Blood samples were collected from the day of injury for 12 months.

Results: Analysis of blood samples from 64 patients revealed a significant reduction in DNase activity on days 1-28 after injury, compared with healthy controls. DNase protein levels were not affected, suggesting the presence of an enzyme inhibitor. Further analysis revealed that actin (an inhibitor of DNase) was present in serum samples from patients but not those from controls, and concentrations of the actin scavenging proteins gelsolin and vitamin D-binding protein were significantly reduced after burn injury. In a pilot study of ten military patients with polytrauma, administration of blood products resulted in an increase in DNase activity and gelsolin levels.

Conclusion: The results of this study suggest a novel biological mechanism for the accumulation of cfDNA following thermal injury by which high levels of actin released by damaged tissue cause a reduction in DNase activity. Restoration of the actin scavenging system could therefore restore DNase activity, and reduce the risk of cfDNA-induced host tissue damage and thrombosis.

© 2019 The Authors. BJS published by John Wiley & Sons Ltd on behalf of BJS Society Ltd.

Figures

Figure 1
Figure 1
Thermal injury results in reduced DNase activity in the presence of stable or increased antigen levelsa DNase activity in serum taken from 64 patients on day (D) 1 to month (M) 12 after thermal injury, and from healthy controls (HC). Median (horizontal bars) and individual values are shown. *P < 0·001 versus HC (1‐way ANOVA followed by Kruskal–Wallis test). b DNase 1 antigen levels in serum taken from 24 patients on D1 to D28 after thermal injury, and from HC. Median (horizontal bars) and individual values are shown. *P < 0·010 versus HC (1‐way ANOVA followed by Bonferroni's multiple comparison test). c Fluorescence microscopic imaging of neutrophil extracellular trap (NET) degradation with phorbol myristate acetate (PMA)‐induced NET DNA stained with SYTOX® Green for untreated control (N = 3), and NETS treated with serum from patients with thermal injuries (N = 3), serum from healthy controls (N = 3) and DNAse 1 positive controls (N = 3) (original magnification ×20).
Figure 2
Figure 2
Thermal injury causes release of actin into the blooda Western blot of actin in plasma samples from nine patients (P1–P9) taken within 24 h of injury. Actin was not detected in plasma from healthy controls (HC). Data are from two independent western blots. b Western blot for a patient (P10) with a burn affecting 66 per cent of the total body surface area who developed multiple organ failure (MOF). Actin was detected within 24 h and up to day (D) 3 after thermal injury. Actin was detectable at later time points, day 20–23, corresponding to the later development of MOF. A second western blot for four independent patients (P11–14) shows actin at later time points.
Figure 3
Figure 3
Levels of vitamin D‐binding protein and gelsolin are reduced after thermal injury Levels of a vitamin D‐binding protein (VDBP) in 50 patients and b gelsolin in 64 patients from day (D) 1 to month (M) 12 after thermal injury, and in healthy controls (HC). Median (horizontal bars) and individual values are shown. *P < 0·010 versus HC (1‐way ANOVA followed by Bonferroni's multiple comparison test).
Figure 4
Figure 4
Blood products increase circulating gelsolin levels and protect against inhibition of DNase activity in patients with polytrauma Comparison of a DNase activity, b gelsolin, c vitamin D‐binding protein (VDBP) and d cell‐free (cf) DNA levels at hospital admission between five patients who had previously received blood products (fresh frozen plasma) and five who had not. Median (horizontal bars) and individual values are shown. *P = 0·028, †P = 0·032 (Mann–Whitney U test).
Figure 5
Figure 5
Hypothesis model suggesting how severe thermal injury results in disruption of the actin scavenging system which predisposes to DNase activity inhibition and accumulation of cell‐free DNA Following severe thermal injury, polymerized and monomeric actin is released, which immediately reduces vitamin D‐binding protein and gelsolin levels. The polymerized actin can then bind to DNase and inhibit its activity. Injury‐ and immune cell‐derived DNA can accumulate and potentially cause activation of the intrinsic clotting cascade, impaired fibrinolysis, bind to platelets, and cause cellular damage and/or inflammation. NET, neutrophil extracellular trap.

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