Neutrophil extracellular traps have active DNAzymes that promote bactericidal activity

Abstract

The mechanisms of bacterial killing by neutrophil extracellular traps (NETs) are unclear. DNA, the largest component of NETs was believed to merely be a scaffold with antimicrobial activity only through the charge of the backbone. Here, we demonstrate for the first time that NETs DNA is beyond a mere scaffold to trap bacteria and it produces hydroxyl free radicals through the spatially concentrated G-quadruplex/hemin DNAzyme complexes, driving bactericidal effects. Immunofluorescence staining showed potential colocalization of G-quadruplex and hemin in extruded NETs DNA, and Amplex UltraRed assay portrayed its peroxidase activity. Proximity labeling of bacteria revealed localized concentration of radicals resulting from NETs bacterial trapping. Ex vivo bactericidal assays revealed that G-quadruplex/hemin DNAzyme is the primary driver of bactericidal activity in NETs. NETs are DNAzymes that may have important biological consequences.

Graphical Abstract

Figure 1.

Immunofluorescence stain of putative G4/H colocalization on NETs. (A–G) Unstimulated neutrophils (PMN). (A) Bright field. (B) Condensed chromatin within nuclei, Hoechst 33342 stain. (C) G4 structures within nuclei, BG4 antibody staining. (D) 1D3 antibody staining of hemin. (E) Anti-citrullinated histone H3 antibody staining of histone H3. (F) Merge of panels (C) and (D), showing possible colocalization of G4 structures and hemin. (G) Merge of panels (B)–(E) portraying defined cellular boundaries with intracellular labeling. (H–N) Escherichia coli stimulated neutrophils. (H) Bright field. (I) Extruded NETs DNA, Hoechst 33342 stain. (J) BG4 antibody staining of G4 structures within NETs. (K) 1D3 antibody staining of hemin with NETs. (L) Anti-citrullinated histone H3 antibody staining of histone H3 within NETs confirming the formation of NETs after E. coli stimulation. (M) Merge of panels (J) and (K), showing the possible colocalization of G4 structures and hemin. (N) Merge of panels (H)–(N) showing potentially extensive colocalization of G4 and hemin on NETs, evidenced by the Manders’ coefficient of 0.998 for G4 overlapping hemin and 0.843 for hemin overlapping G4. (O) Intact cell numbers under microscopy field of view. (P–S) Fluorescent area % under field of view. Scale bar = 25 μm. **** indicates P-value < 0.0001.

Figure 2.

Peroxidase activity of G4/H DNAzyme in purified NETs DNA using AR. Peroxidase activity using equal masses of Telo G4 and purified NETs DNA were assayed with various combinations of individual components, G4 specific binding inhibitors (BRACO19 and NMM) and antioxidant (vitamin C). Telo G4 + hemin + H₂O₂ and HRP + H₂O₂ used have comparable activity. NETs + hemin + H₂O₂ had greater activity than individual components and 7.6x higher than hemin alone. Peroxidase activity was abrogated by G4 inhibitors and antioxidant. All experiments were performed in triplicate with mean and standard deviation plotted. One-way ANOVA test with Tukey’s honestly significant difference test performed to compare the different combinations. **** indicates P-value < 0.0001.

Figure 3.

NETs DNA-based proximity labeling assay. (A) Schematic of proximity labeling assay. Labeling of bacteria trapped in purified NETs DNA, versus untrapped bacteria in nuclease-digested NETs DNA, with biotin-phenoxyl radicals by the DNAzyme activity of G4/H complexes. (B) Purified NETs DNA from PMA-stimulated neutrophils incubated with EC, biotin-phenol, hemin, H₂O₂ and fluorescein–streptavidin to allow for bacterial surface labeling. Upon digestion of NETs DNA with EcoR I and DNAse I on the same bacteria, the resulting fluorescent intensity showed a reduced labeling effect comparable to that of free-floating Telo G4 (∼54%) compared with intact NETs. (n = 3 biologically independent experiments; bars represent mean signal, and error bars denote s.e.m.; one-way ANOVA performed; **** indicates P-value < 0.0001).

Figure 4.

Ex vivo bactericidal activity of the G4/H DNAzyme against EC. (A) Schematic of the ex vivo bactericidal assay. Neutrophils from whole blood were stimulated with IL-8 and treated with various combinations of antiphagocytic cytochalasin D, G4 specific inhibitor BRACO19, G4 binding hemin analog NMM and DNase I with no additional hemin or H₂O₂. (B) Bactericidal activity of isolated neutrophils through NETs against EC. Approximately 80% of inoculated EC was killed by IL-8-stimulated NETs. Phagocytosis accounts for an additional ∼10% of killing. Abrogation of NETs killing by G4-specific inhibitors like BRACO19, NMM or antioxidant vitamin C (<20%). DNase I treatment only reduced killing by ∼20%. (n = 3 biologically independent experiments; bars represent mean signal, and error bars denote s.e.m.; one-way ANOVA performed; **** indicates P-value < 0.0001).