methods-to-study-lipid-alterations-neutrophils-subsequent-formation

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

University of Veterinary Medicine Hannover

Lund University

Streptococcus suis is an important cause of different pathologies in pigs and humans, most importantly fibrinosuppurative meningitis. Tissue infected with this pathogen is substantially infiltrated with neutrophils, but the function of neutrophil extracellular traps (NETs) - a more recently discovered antimicrobial strategy of neutrophils - in host defence against Strep. suis has not been investigated. The objective of this work was to investigate the interaction of Strep. suis with NETs in vitro. Strep. suis induced NET formation in porcine neutrophils and was entrapped but not killed by those NETs. As the amount of NETs decreased over time, we hypothesized that a known extracellular DNase of Strep. suis degrades NETs. Though this nuclease was originally designated Strep. suis-secreted nuclease A (SsnA), this work demonstrated surface association in accordance with an LPXTG cell wall anchor motif and partial release into the supernatant. Confirming our hypothesis, an isogenic ssnA mutant was significantly attenuated in NET degradation and in protection against the antimicrobial activity of NETs as determined in assays with phorbol myristate acetate (PMA)-stimulated human neutrophils. Though assays with PMA-stimulated porcine neutrophils suggested that SsnA also degrades porcine NETs, phenotypic differences between wt and the isogenic ssnA mutant were less distinct. As SsnA expression was crucial for neither growth in vitro nor for survival in porcine or human blood, the results indicated that SsnA is the first specific NET evasion factor to be identified in Strep. suis.

Streptococcus suis DNase SsnA contributes to degradation of neutrophil extracellular traps (NETs) and evasion of NET-mediated antimicrobial activity.

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.

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

The formation of neutrophil extracellular traps (NETs) as a host innate immune defence mechanism has been shown to be the result of a novel cell death process called NETosis. The objective of this study was to investigate the role of cholesterol in the formation of NETs. To this end, primary human neutrophils were treated with different concentrations of methy-β-cyclodetxrin (MβCD) to reduce cholesterol level in the cell. The formation of NETs was studied using immunofluorescence microscopy and Picogreen-quantification of released dsDNA. Neutrophils treated with MβCD showed a significant release of NETs in a process that is independent of NADPH-oxidase. The effect of MβCD on the lipid composition of the cells was determined using high performance thin layer chromatography (HPTLC). The identities of lipids separated by HPTLC were confirmed by mass spectrometry. Treatment of neutrophils with MβCD revealed distinct changes in the lipid composition: The percentage of cholesterol in the cell was significantly reduced; other lipids as sphingomyelin were only slightly affected. Interestingly, neutrophils treated with sphingomyelin-degrading sphingomyelinase also showed significant release of NETs. In conclusion, this study shows that lipid alterations facilitate formation of NETs.

Lipid alterations in human blood-derived neutrophils lead to formation of neutrophil extracellular traps.

NETs (neutrophil extracellular traps) have been described as a fundamental innate immune defence mechanism. During formation of NETs, the nuclear membrane is disrupted by an as-yet unknown mechanism. In the present study we investigated the role of human cathelicidin LL-37 in nuclear membrane disruption and formation of NETs. Immunofluorescence microscopy revealed that 5 μM LL-37 significantly facilitated NET formation by primary human blood-derived neutrophils alone, in the presence of the classical chemical NET inducer PMA or in the presence of Staphylococcus aureus. Parallel assays with a random LL-37 fragment library indicated that the NET induction is mediated by the hydrophobic character of the peptide. The trans-localization of LL-37 towards the nucleus and the disruption of the nuclear membrane were visualized using confocal fluorescence microscopy. In conclusion, the present study demonstrates a novel role for LL-37 in the formation of NETs.

The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps.

The porcine and human pathogen Streptococcus suis induces and degrades neutrophil extracellular traps (NETs) in vitro. In this study, we investigated the working hypothesis that NET degradation is mediated not only by the known secreted S. suis nuclease A (SsnA) but also by a so-far undescribed putative endonuclease A of S. suis (designated EndAsuis) homologous to the pneumococcal endonuclease A (EndA). Comparative analysis was conducted to identify differences in localization, expression and function of EndAsuis and SsnA. In contrast to ssnA, endAsuis RNA expression was not substantially different during exponential and stationary growth. Modelling of the 3D structure confirmed a putative DRGH-motif-containing ββα-metal finger catalytic core in EndAsuis. Accordingly, nuclease activity of recombinant EndAsuis with a point-mutated H165 was rescued through imidazol treatment. In accordance with a putative membrane anchor, nuclease activity caused by endAsuis was not detectable in the supernatant. Importantly, endAsuis determined nuclease activity of S. suis prominently during exponential growth. This activity depended on the presence of Mg(2+) but, in contrast to SsnA activity, not on Ca(2+). A pH of 5.4 did not inhibit endAsuis-encoded nuclease activity during exponential growth. NET degradation of S. suis harvested during exponential growth was significantly attenuated in the endAsuis mutant. In contrast to SsnA, mutagenesis of endAsuis did not result in a significantly higher susceptibility against the antimicrobial effect mediated by NETs. As degradation of bacterial DNA caused by S. suis depended on ssnA and endAsuis, further functions of both factors in the host-pathogen interaction might be envisioned.

Identification of a novel DNase of Streptococcus suis (EndAsuis) important for neutrophil extracellular trap degradation during exponential growth.

Neutrophil extracellular traps (NETs) are believed to be essential in controlling several bacterial pathogens. Quantification of NETs in vitro is an important tool in studies aiming to clarify the biological and chemical factors contributing to NET production, stabilization and degradation. This estimation can be performed on the basis of fluorescent microscopy images using appropriate labelings. In this context, it is desirable to automate the analysis to eliminate both the tedious process of manual annotation and possible operator-specific biases.

Automatic determination of NET (neutrophil extracellular traps) coverage in fluorescent microscopy images.

Neutrophil extracellular trap (NET) formation is described as a tool of the innate host defence to fight against invading pathogens. Fibre-like DNA structures associated with proteins such as histones, cell-specific enzymes and antimicrobial peptides are released, thereby entrapping invading pathogens. It has been reported that several bacteria are able to degrade NETs by nucleases and thus evade the NET-mediated entrapment. Here we studied the ability of three different Yersinia serotypes to induce and degrade NETs. We found that the common Yersinia enterocolitica serotypes O:3, O:8 and O:9 were able to induce NETs in human blood-derived neutrophils during the first hour of co-incubation. At later time points, the NET amount was reduced, suggesting that degradation of NETs has occurred. This was confirmed by NET degradation assays with phorbol-myristate-acetate-pre-stimulated neutrophils. In addition, we found that the Yersinia supernatants were able to degrade purified plasmid DNA. The absence of Ca(2+) and Mg(2+) ions, but not that of a protease inhibitor cocktail, completely abolished NET degradation. We therefore postulate that Y. enterocolitica produces Ca(2+)/Mg(2+)-dependent NET-degrading nucleases as shown for some Gram-positive pathogens.

Yersinia enterocolitica-mediated degradation of neutrophil extracellular traps (NETs).

Streptococcus suis is an important meningitis-causing pathogen in pigs and humans. Neutrophil extracellular traps (NETs) have been identified as host defense mechanism against different pathogens. Here, NETs were detected in the cerebrospinal fluid (CSF) of S. suis-infected piglets despite the presence of active nucleases. To study NET-formation and NET-degradation after transmigration of S. suis and neutrophils through the choroid plexus epithelial cell barrier, a previously described model of the human blood-CSF barrier was used. NETs and respective entrapment of streptococci were recorded in the "CSF compartment" despite the presence of active nucleases. Comparative analysis of S. suis wildtype and different S. suis nuclease mutants did not reveal significant differences in NET-formation or bacterial survival. Interestingly, transcript expression of the human cathelicidin LL-37, a NET-stabilizing factor, increased after transmigration of neutrophils through the choroid plexus epithelial cell barrier. In good accordance, the porcine cathelicidin PR-39 was significantly increased in CSF of piglets with meningitis. Furthermore, we confirmed that PR-39 is associated with NETs in infected CSF and inhibits neutrophil DNA degradation by bacterial nucleases. In conclusion, neutrophils form NETs after breaching the infected choroid plexus epithelium, and those NETs may be protected by antimicrobial peptides against bacterial nucleases.

Ariane Neumann

Department of Physiological Chemistry

University of Veterinary Medicine Hannover

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps