A proteomic analysis of NETosis in trauma: Emergence of serpinB1 as a key player

Abstract

Background: Release of neutrophil extracellular traps (NETosis) may mediate postinjury organ dysfunction, but mechanisms remain unclear. The intracellular serine protease inhibitor (serpin) B1 is vital to neutrophil function and has been shown to restrict NETosis in inflammatory settings. In this study, we used discovery proteomics to identify the proteomic signature of trauma-induced NETosis. We hypothesized that serpinB1 would be a major component of this NET protein profile and associated with adverse outcomes.

Methods: This was a post hoc analysis of data collected as part of the COMBAT randomized clinical trial. Blood was collected from injured patients at a single Level I Trauma Center. Proteomic analyses were performed through targeted liquid chromatography coupled with mass spectrometry. Abundances of serpinB1 and known NETosis markers were analyzed with patient and injury characteristics, clinical data, and outcomes.

Results: SerpinB1 levels on emergency department (ED) arrival were significantly correlated with proteomic markers of NETosis, including core histones, transketolase, and S100A8/A9 proteins. More severely injured patients had elevated serpinB1 and NETosis markers on ED arrival. Levels of serpinB1 and top NETosis markers were significantly elevated on ED arrival in nonsurvivors and patients with fewer ventilator- and ICU-free days. In proteome-wide receiver operating characteristic analysis, serpinB1 was consistently among the top proteins associated with adverse outcomes. Among NETosis markers, levels of serpinB1 early in the patient's course exhibited the greatest separation between patients with fewer and greater ventilator- and ICU-free days. Gene Ontology analysis of top predictors of adverse outcomes further supports NETosis as a potential mediator of postinjury organ dysfunction.

Conclusion: We have identified a proteomic signature of trauma-induced NETosis, and NETosis is an early process following severe injury that may mediate organ dysfunction. In addition, serpinB1 is a major component of this NET protein profile that may serve as an early marker of excessive NETosis after injury.

Figure 1.

Proteome-wide Spearman’s correlations with serpinB1 for trauma patient plasma samples collected on ED arrival demonstrating significant correlations with NETosis and tissue damage markers. A: U-plot of proteome-wide Spearman’s correlations with serpinB1 with the X-axis displaying the correlation coefficient and the Y-axis displaying the the -log10 transformed P-value for each correlation. Significant (P<0.05) positive correlates are displayed in blue color while insignificant correlates are colored grey. The top significant positive protein correlates were previously identified NETosis and tissue damage markers, which are labeled on the U-plot. B: Table of all included NETosis and tissue damage markers included in this study with most identified on the U-plot in panel A. The table includes the gene name, description, and Uniprot accession number.

Figure 2.

SerpinB1, NETosis markers, and the tissue damage protein myoglobin (MB) are elevated on ED arrival in severely injured patients. Stratification by shock/injury (S/I) phenotypes as defined in Table 1 (Groups 1-4) as well as comparisons with levels in healthy volunteers demonstrate that patients with severe injury (NISS≥25, Groups 3 and 4) have higher serpinB1 and evidence of increased NETosis on ED arrival. In parallel with this pattern of NETosis, MB as a marker of tissue damage was also elevated in more severely injured patients. Lack of significant difference between Groups 3 and 4 may suggest severity of tissue damage is more a driver of post-injury NETosis than hypoperfusion. Displayed proteins in this figure were selected based on having the greatest statistical significance in comparisons between S/I phenotypes and healthy volunteers in Kruskal-Wallis tests; see Panel B of Figure 1 for protein identification. Abundance quantity refers to the label-free quantification of protein level. Plots contain boxes displaying the median and interquartile range (IQR) with whiskers denoting the highest and lowest values within 1.5xIQR; values outside this whisker range were considered outliers and indicated by individual points. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05

Figure 3.

Elevated SerpinB1 and NETosis markers on ED arrival are associated with mortality. A: SerpinB1 and NETosis markers are elevated on ED arrival in non-survivors versus survivors. Displayed proteins were those with the greatest statistical significance in Mann-Whitney tests between non-survivors and survivors; see Panel B of Figure 1 for protein identification. Abundance quantity refers to the label-free quantification of protein level. Plots contain boxes displaying the median and interquartile range (IQR) with whiskers denoting the highest and lowest values within 1.5xIQR; values outside this whisker range were considered outliers and indicated by individual points. B: Receiver operating characteristic (ROC) curves from proteome-wide ROC analysis of top predictors of mortality based on levels measured on ED arrival. The legend shows corresponding areas under the curves (AUC) with 95% confidence intervals. Displayed proteins were NETosis markers with the highest AUC’s among top predictors of mortality. ***P<0.001, ****P<0.0001

Figure 4.

Elevated SerpinB1 and NETosis markers on ED arrival are associated with longer ventilator requirement. A: SerpinB1 and NETosis markers are elevated on ED arrival in patients with fewer than 27 (median) ventilator-free days (VFD) versus those with 27 or more VFD. Displayed proteins were those with the greatest statistical significance in Mann-Whitney tests between groups; see Panel B of Figure 1 for protein identification. Abundance quantity refers to the label-free quantification of protein level. Plots contain boxes displaying the median and interquartile range (IQR) with whiskers denoting the highest and lowest values within 1.5xIQR; values outside this whisker range were considered outliers and indicated by individual points. B: Receiver operating characteristic (ROC) curves from proteome-wide ROC analysis of top predictors of having <27 VFD based on levels measured on ED arrival. The legend shows corresponding areas under the curves (AUC) with 95% confidence intervals. Displayed proteins were NETosis markers with the highest AUC’s among top predictors of having <27 VFD. ****P<0.0001

Figure 5.

Elevated SerpinB1 and NETosis markers on ED arrival are associated with longer ICU stay. A: SerpinB1 and NETosis markers are elevated on ED arrival in patients with fewer than 25 (median) ICU-free days (IFD) versus those with 25 or more IFD. Displayed proteins were those with the greatest statistical significance in Mann-Whitney tests between groups; see Panel B of Figure 1 for protein identification. Abundance quantity refers to the label-free quantification of protein level. Plots contain boxes displaying the median and interquartile range (IQR) with whiskers denoting the highest and lowest values within 1.5xIQR; values outside this whisker range were considered outliers and indicated by individual points. B: Receiver operating characteristic (ROC) curves from proteome-wide ROC analysis of top predictors of having <25 IFD based on levels measured on ED arrival. The legend shows corresponding areas under the curves (AUC) with 95% confidence intervals. Displayed proteins were NETosis markers with the highest AUC’s among top predictors of having <25 IFD. ****P<0.0001