Aggregated NETs Sequester and Detoxify Extracellular Histones

Abstract

In response to various infectious and sterile stimuli neutrophils release chromatin decorated with bactericidal proteins, referred to as NETs. Their scaffolds are formed from chromatin fibers which display an apparent diameter of 15-17 nm and mainly consist from DNA (2 nm) and DNA-associated histones (11 nm). The NET-forming strands are thus not naked DNA but higher ordered chromatin structures. The histones may be released from the NET, especially if their tail arginines have been citrullinated. Several studies indicate that extracellular histones are toxic for mammalian epithelia and endothelia and contribute to the microvascular dysfunction observed e.g., in patients suffering from autoimmune diseases or sepsis. NETs formed at sites of very high neutrophil densities tend to clump and form fairly stable enzymatically active aggregates, referred to as aggNETs. The latter are endowed with a bunch of enzymes that cleave, bind, and/or modify autologous as well as foreign macromolecules. The tight binding of the serine proteases to the matrix precludes the spread of these toxic enzymes into the tissue but still allows the access of soluble inflammatory mediators to the enzymatic active internal surfaces of the NETs where they are degraded. Here, we describe that externally added histones are removed from culture supernatants of aggNETs. We will address the fate of the histones and discuss the feature on the background of neutrophil-driven diseases and the resolution of inflammation.

Keywords: NET formation; aggNETs; autoimmunity; histones; proteolysis; sepsis.

Figure 1

aggNETs degrade histones. (A) Histones incubated with aggNETs are degraded as seen in Coomassie and staining and anti-histone H1 Blot. The biotinylationed histones are not bound by the aggNETs. (B) Proteinase3 (PR3) degrades histones. This degradation is inhibited by Elafin as seen in the Coomassie staining. (C) Neutrophil Elastase (NE) degrades histones, specifically inhibited by Sivelestat as shown in the Coomassie staining. (D) SWISS-MODEL of histone H1 (amino acids 39–119) with the cleavage sites for NE as predicted by ExPASy PeptideCutter. (E) NE and aggNETs favor histone over bovine serum albumin (BSA) and human Immunoglobulin G (IgG) for degradation; whereas PR3 can only degrade histones. Degradation of biotinylated histones by aggNETs is not inhibited by Sivelestat or Elafin or a combination of both as seen by the detection of Streptavidin HRP in Western Blot analysis. SDS-PAGE, Western Blot Analysis and Coomassie staining in (A–C) were performed after incubation of the samples for 24 h at 37°C. For (E) the incubation time was 8 h at 37°C. All images shown are representative images of at least three independent experiments. The full-sized images are shown in Figures S1A–D. The successful formation of an aggNET is shown in the macrophotographs in Figure S1E in bright-field and under UV after staining with propidium iodide.

Figure 2

Pre-treatment of histones with aggNETs attenuates histone-mediated cellular cytotoxicity. (A) Light microscope images in 10x magnification of HeLa cells before and after treatment with (1) 500 μg histones, (2) histones pre-treated with aggNETs for 24 h or (3) aggNET supernatant (SN). (B) Overview of different forms of cell death of HeLa cells after treatment assessed by flow cytometry. Pre-treatment of histones with aggNETs increases viability of HeLa cells. (C) Detailed analysis of the different forms of cell death. Viability of HeLa cells incubated with aggNET-treated histones is significantly increased compared to histone treatment due to a reduction in early apoptosis, apoptosis and primary (1°) necrosis. Standard error of mean was calculated from three independent experiments. ***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05 as determined by Two-way ANOVA with Bonferroni post testing. The gating strategy for flow cytometer analysis is depicted in Figure S2.