Synthetic neutrophil extracellular traps dissect bactericidal contribution of NETs under regulation of α-1-antitrypsin

Abstract

Deciphering the complex interplay of neutrophil extracellular traps (NETs) with the surrounding environment is a challenge with notable clinical implications. To bridge the gap in knowledge, we report our findings on the antibacterial activity against Pseudomonas aeruginosa of synthetic NET-mimetic materials composed of nanofibrillated DNA-protein complexes. Our synthetic system makes component-by-component bottom-up analysis of NET protein effects possible. When the antimicrobial enzyme neutrophil elastase (NE) is incorporated into the bactericidal DNA-histone complexes, the resulting synthetic NET-like structure exhibits an unexpected reduction in antimicrobial activity. This critical immune function is rescued upon treatment with alpha-1-antitrypsin (AAT), a physiological tissue-protective protease inhibitor. This suggests a direct causal link between AAT inhibition of NE and preservation of histone-mediated antimicrobial activity. These results help better understand the complex and, at times, contradictory observations of in vivo antimicrobial effects of NETs and AAT by excluding neutrophil, cytokine, and chemoattractant contributions.

Engineering in vitro models of cystic fibrosis lung disease using neutrophil extracellular trap inspired biomaterials

“…1A). 26,30,31 Fluorescence microscopy of the sNETs revealed successful recreation of a porous DNA scaffold structure, similar in appearance to endogenous NETs (Fig. 1B).…”

“…antimicrobial peptides) which could influence growth behavior of P. aeruginosa . It should be noted sNETs prepared with other NET-associated proteins has been recently reported 31 where we plan to adapt these procedures for future studies.…”

“…Synthesis of sNETs is based on a previously published protocol, with some minor adaptations. 26,30,31 DNA (Sigma-Aldrich; sodium salt from salmon testes) was stirred for 2 hours at a concentration of 6 mg ml −1 in a physiological buffer consisting of 154 mM NaCl, 3 mM CaCl 2 , and 15 mM NaH 2 PO 4 at pH 7.4. Histones from calf thymus (Sigma-Aldrich; Type II-A) were dissolved separately in the same buffer at a concentration of 6 mg ml −1 .…”

“…To do this, we used a synthetic NET (sNET) biomaterial based on previous work to mimic the chromatin scaffold of endogenous NETs. 26,30,31 This cell-free protocol can be used to generate NET-like biomaterials without the difficulties associated with in vitro generation and isolation of endogenous NETs or co-culture of neutrophils within the HAE model. To understand the impact of NETs on the viscoelastic properties of mucus, we incorporated the sNETs into a synthetic mucus hydrogel as described in our prior work 32–34 and mucus produced by HAE cells.…”

Mechanisms of Neutrophil Extracellular Trap Formation and Regulation in Cancers

“…Exogenous replenishment of these substances restores NETs formation [ 8 ]. The results of a study of the antibacterial activity of synthetic NETs simulated materials against Pseudomonas aeruginosa show a decrease in antibacterial activity when NE was bound to the antibacterial DNA-histone complexes [ 9 ]. These results suggest that protein components play an indispensable role in the formation and functioning of NETs.…”