Novel role of the antimicrobial peptide LL-37 in the protection of neutrophil extracellular traps against degradation by bacterial nucleases

Abstract

Neutrophil extracellular traps (NETs) have been described as a fundamental innate immune defence mechanism. They consist of a nuclear DNA backbone associated with different antimicrobial peptides (AMPs) which are able to engulf and kill pathogens. The AMP LL-37, a member of the cathelicidin family, is highly present in NETs. However, the function of LL-37 within NETs is still unknown because it loses its antimicrobial activity when bound to DNA in the NETs. Using immunofluorescence microscopy, we demonstrate that NETs treated with LL-37 are distinctly more resistant to S. aureus nuclease degradation than nontreated NETs. Biochemical assays utilising a random LL-37-fragment library indicated that the blocking effect of LL-37 on nuclease activity is based on the cationic character of the AMP, which facilitates the binding to neutrophil DNA, thus protecting it from degradation by the nuclease. In good correlation to these data, the cationic AMPs human beta defensin-3 and human neutrophil peptide-1 showed similar protection of neutrophil-derived DNA against nuclease degradation. In conclusion, this study demonstrates a novel role of AMPs in host immune defence: beside its direct antimicrobial activity against various pathogens, cationic AMPs can stabilise neutrophil-derived DNA or NETs against bacterial nuclease degradation.

Fig. 1

LL-37 protects NETs against degradation by S. aureus nuclease. Confocal immunofluorescence (a) and electron micrograph (b) of structures, e.g. NETs and nanoparticles released by PMA-activated neutrophils which are associated with a high amount of LL-37. Bar: 8 µm (a), 100 nm (b). c Human blood-derived neutrophils were stimulated with 25 nM PMA for 4 h to induce 100% NET formation. Aprotinin (40 µg/ml) was used to block formation of endogenous active LL-37. The NETs were then treated with 0.01 U/ml S. aureus nuclease (MN) in the presence or absence of externally added LL-37 (5 µM). d Representative fluorescent micrographs displaying the results of the column bar graph (c). Bars: 50 µm. NETs were visualized using an Alexa 488-labelled antibody against H2A-H2B-DNA complexes (green) in combination with DAPI to stain the nuclei in blue. The area covered with NETs was quantified using Image J. The graph represents the mean ± SEM of a minimum of 18 images derived from 3 independent experiments. * p < 0.05, *** p < 0.001 by t test.

Fig. 2

LL-37 protects host DNA against degradation by S. aureus nuclease. Quantification of DNA and its degradation using different DNA-intercalating dyes: PicoGreen (a, b, d), SYTOX Green (c) and ethidium bromide (e) in the presence or absence of 5 µM LL-37 ± 100 U/ml S. aureus nuclease (MN). All graphs represent the mean ± SEM of minimum 3 independent experiments. * p < 0.05, ** p < 0.005, *** p < 0.001 by t test.

Fig. 3

DAPI and immunostaining of NETs. Confocal immunofluorescent micrograph of NETs. NETs were visualized using an Alexa 488-labelled antibody against H2A-H2B-DNA complexes (green) and with DAPI to stain the extra- and intracellular DNA in blue.

Fig. 4

LL-37 protects host DNA against degradation by nucleases derived from different Gram-positive bacteria: DNA degradation in the presence or absence of 5 µM LL-37 by purified EndA (a), supernatants of three different GAS strains (b) and S. aureus USA300 LAC (c) using PicoGreen as a marker. c A panel of nuclease (nuc)-deficient mutants and nuclease-producing control strains of S. aureus USA300 LAC strain was used: S. aureus LAC WT empty vector control (WT + pCM28), nuc-mutant empty vector control (nuc + pCM28) or complemented mutant strain (nuc + pCM28nuc). Electron micrograph of NETs after degradation with purified MN: only small left-over NET-structures that are decorated with gold-labelled LL-37 are visible. All NETs that are not decorated with LL-37 (partially seen in fig. 1b) have been enzymatically degraded and are not detectable anymore. Bar: 100 nm (d). Entrapment of S. aureus USA300 LAC strain by NET-releasing neutrophils in the presence or absence of 5 µM LL-37: percentage of entrapment was calculated compared to total amount of surviving CFU under the respective conditions in the presence or absence of LL-37 (e). All graphs represent the mean ± SEM of a minimum of 3 independent experiments. * p < 0.05, ** p < 0.005 by t test.

Fig. 5

Cationicity is involved in the protection of host DNA against bacterial nuclease-mediated degradation. a Sequences and biochemical characteristics of LL-37, LL-37 fragments, scrambled LL-37 (Scr. LL-37), hBD-3 and HNP-1. Quantification of DNA-degradation by 100 U/ml S. aureus nuclease (MN) in the presence or absence of 5 µM LL-37, LL-37 fragments, scrambled LL-37 (b) or 5 µM HNP-1 and hBD-3 (c, d). All graphs represent the mean ± SEM of minimum 3 independent experiments. * p < 0.05, ** p < 0.005 by t test.