Plasma NETs are associated with disease severity and outcomes in H1N1 infection
The characteristics of all subjects in the present study including 30 individuals with severe H1N1 infection and 88 individuals with mild H1N1 infection are presented in Table 1 and Table 2. Consistent with our previous findings, the severely infected patients had higher levels of NETs (cfDNA, histone-DNA, MPO-DNA and LTF-DNA) in the plasma than the patients with mild infection and healthy controls (all p < 0.001; Figure 1a).
Among the 30 severely infected patients, the levels of both plasma cfDNA and histone-bound DNA were negatively correlated with the oxygen index (Figure 1b). The patients who received extracorporeal membrane oxygenation (ECMO) support (n=14) displayed significantly higher levels of cfDNA and histone-bound DNA than those without undergoing ECMO support (cfDNA: median: 501 ng/ml [IQR: 348-711 ng/ml] vs 310 ng/ml [240-403 ng/ml], p = 0.005; histone-bound DNA: 25.9 ng/ml [21.1-39.3 ng/ml] vs 17.2 ng/ml [12.0-23.5 ng/ml], p = 0.012; Figure 1c).
In addition, the levels of both cfDNA and histone-bound DNA were positively correlated with sequential organ failure assessment (SOFA) scores (Figure 1d). Accordingly, the patients with MODS had greater levels of NETs than those without MODS (cfDNA: 572 ng/ml [405- 676 ng/ml] vs 306 ng/ml [248-354 ng/ml], p < 0.001; histone-bound DNA: 31.5 ng/ml [23.4-35.9 ng/ml] vs 17.1 ng/ml [11.6-23.0 ng/ml], p < 0.001; Figure 1e). Furthermore, higher levels of NETs were also observed in the plasma of the 17 patients who died within 60 days than in that of the 13 patients who survived (cfDNA: 400 ng/ml [333-622 ng/ml] vs 298 ng/ml [244-461 ng/ml], p = 0.020; histone-bound DNA: 24.1 ng/ml [19.1-34.9 ng/ml] vs 16.6 ng/ml [11.2-23.0 ng/ml], p = 0.012; Figure 1f). Thus, the data from the present cohort confirmed our previous finding that plasma NET levels correlated with disease severity and outcomes in H1N1 infection.
Enhanced levels of BALF NETs are not associated with disease severity or outcomes in patients with severe H1N1 infection
Next, we evaluated NET levels in BALF samples from the patients with severe H1N1 infection. Significant increases in the levels of cfDNA, histone-DNA, MPO-DNA and LTF-DNA were observed in the BALF from the severe H1N1 patients compared with those from the healthy controls (all p < 0.001; Figure 2a). Although the NET content in alveoli was diluted during the lavage (120 ml saline per person), the concentrations of cfDNA, histone-DNA, MPO-DNA and LTF-DNA in BALF samples were still 13.3-, 9.5-, 2- and 9.7-fold higher, respectively, than those in plasma samples from the same patients (all p < 0.001; Figure 2b). Further analysis showed that the cfDNA concentrations in the BALF were not correlated with those in the plasma (Figure 2c). Additionally, the cfDNA levels in the BALF were not correlated with the proportions of neutrophils in the BALF or the levels of the cytokines IL-6, IL-8, MIP-1α and MIP-1β in the BALF (Figure 2c, Table 3) and additional tables show these in more detail (see Additional files 2 and 3). Notably, we found distinct increases in permeability of alveolar epithelial cells when co-cultured with the supernatant of BALF from severe patients, compared with those from HCs. (Figure 2d).
Next, we investigated whether the cfDNA levels in the BALF correlate with disease severity. Surprisingly, we observed no correlation between the BALF cfDNA levels and the oxygen index (Figure 3a). Among the severely infected patients, those who received ECMO support had levels of cfDNA and histone-bound DNA in the BALF comparable to the levels in the patients who did not receive ECMO support (Figure 3b). Furthermore, the BALF cfDNA levels were not correlated with SOFA scores (Figure 3c).
The patients with MODS and those who did not progress to MODS had similar levels of BALF NETs (Figure 3d). Accordingly, the NET levels in the BALF were comparable between the 17 patients who died within 60 days and the 13 survived patients (Figure 3e).
The levels of cfDNA and histone-bound DNA in the plasma predict disease severity and clinical outcomes in H1N1 infection
Next, we plotted a ROC curve for plasma levels of cfDNA to predict severe illness of H1N1 influenza. With an optimal cut-off value of 286.6 ng/ml, the AUC of cfDNA was 0.922 (95% CI, 0.872-0.972; p < 0.001), and the sensitivity and specificity of cfDNA were 80.0% (95% CI, 61.4%-92.3%) and 89.8% (95% CI, 81.5%-95.2%), respectively (Figure 4a). The cfDNA levels in the plasma were measured in the H1N1 cohort 2009 in our previous study. Using the cut-off value of cfDNA obtained from cohort 2017 (286.6 ng/ml), we performed a separate validation study with cohort 2009. The sensitivity and specificity were 87.5% (95% CI, 71.0%-96.5%) and 76.5% (95% CI, 58.8%-89.3%), respectively (Figure 4a).
In addition to cfDNA, another marker of NETs, the histone-bound DNA levels in the plasma, also accurately predicted severe illness with an AUC of 0.996 (95% CI, 0.987-1.005; p < 0.0001) in cohort 2017. With a cut-off value of 9.4 ng/ml, the sensitivity and specificity were 100.0% (95% CI, 88.4%-100.0%) and 98.0% (95% CI, 89.2%-100.0%), respectively (Figure 4b).
We further performed ROC curve analysis to predict influenza mortality in cohort 2017. As shown in Table 4 and an additional figure (see Additional file 4), the AUC of plasma cfDNA was 0.751. With a cut-off value of 306.3 ng/ml, the sensitivity and specificity of cfDNA were 94.1% and 61.6%, respectively. Consistently, plasma histone-bound DNA had an AUC of 0.769 in predicting mortality. In addition to NETs, three other parameters that were closely related to a poor prognosis, the oxygen index, neutrophil proportion and plasma IL-8 level, were also included[26, 31, 32]. The AUCs of the oxygen index, neutrophil proportion and IL-8 were 0.679, 0.586 and 0.728, respectively, all of which were lower than the AUC of cfDNA or histone-bound DNA. Furthermore, a validation study with cohort 2009 showed that the AUC of plasma cfDNA (0.711) was comparable to that determined for cohort 2017. Taken together, these data suggest that the plasma levels of NETs on the day of admission could predict severe illness and a fatal outcome in H1N1 influenza.