Objective To investigate the clinical value of peripheral serum cell-free DNA/neutrophil extracellular traps (cf-DNA/NETs) level in diagnosis and severity assessment of sepsis patients. Methods Forty patients with sepsis and 40 patients with non-infectious systemic inflammatory response syndrome (nf-SIRS) were enrolled in this study. The cf-DNA/NETs level in serum of all subjects were measured. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic ability of the cf-DNA/NETs, white blood cell count (WBC), procalcitonin (PCT) and interleukin-6 (IL-6). The sepsis patients were stratified into a survival group and a death group according to the prognosis. Sequential organ failure (SOFA) score were recorded in the sepsis patients, and the correlations between SOFA and cf-DNA/NETs, PCT, WBC, IL-6 were analyzed. Results Compared with the nf-SIRS group, cf-DNA/NETs and PCT levels were significantly higher in the sepsis group (both P<0.05). WBC and IL-6 showed no significant differences between the two groups (bothP>0.05). The area under the ROC curve (AUC) of cf-DNA/NETs was 0.884 for diagnosis of sepsis, and it was higher than the AUC of PCT (0.803). The cf-DNA/NETs showed better sensitivity (81.2% and 79.2%) and specificity (81.0% and 82.4%) than PCT. cf-DNA/NETs and PCT were significantly higher in the death group than those in the survival group. Bivariate collection analysis revealed positive correlations between SOFA score and the two biomarkers of cf-DNA/NETs and PCT (r1=0.573, r2=0.518; both P<0.01). Conclusions cf-DNA/NETs and PCT have certain value in early diagnosis of sepsis, and cf-DNA/NETs shows better diagnostic value in distinguishing sepsis from nf-SIRS than PCT. cf-DNA/NETs can be used as a routine monitoring index to help assess disease severity in sepsis.
Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.
The effect of neutrophil extracellular traps (NETs) on promoting intravascular microthrombi formation and exacerbating the severity of sepsis in patients has gained extensive attention. However, in sepsis, the mechanisms and key signaling molecules mediating NET formation during direct interactions of endothelial cells and neutrophils still need further explored. Herein, we utilized lipoteichoic acid (LTA), a component shared by Gram-positive bacteria, to induce NET extrusion from neutrophils firmly adhered to the glass slides coated with intercellular adhesion molecule-1(ICAM-1). We also used Sytox green to label NET-DNA and Flou-4 AM as the intracellular Ca2+ signaling indicator to observe the NET formation and fluctuation of Ca2+ signaling. Our results illustrated that LTA was able to induce NET release from neutrophils firmly attached to ICAM-1-coated glass slides, and the process was time-dependent. In addition, our study indicated that LTA-induced NET release by neutrophils stably adhered to ICAM-1 depended on Ca2+ signaling but not intracellular reactive oxygen species (ROS). This study reveals NET formation mediated by direct interactions between endothelial ICAM-1 and neutrophils under LTA stimulation and key signaling molecules involved, providing the theoretical basis for medicine development and clinical treatment for related diseases.