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Treatment with DNases rescues hidden neutrophil elastase from aggregated NETs

The release of neutrophil extracellular traps (NETs) is one of the weapons neutrophils have in their armory. NETs consist of extracellular chromatin fibers decorated with a plethora of cytoplasmic and granular proteins, such as the antimicrobial serine protease neutrophil elastase (NE). Because the first description of NETs as beneficial to the host, reports on their double‐faced role in health and disease have considerably increased recently. On one hand, NETs reportedly trap and kill bacteria and also participate in the resolution of the acute inflammation associated with infection and with tissue damage. On the other hand, numerous negative aspects of NETs contribute to the etiopathogenesis of autoimmune disorders. Employing soluble and solid fluorescent substrates, we demonstrate the interaction of NE with aggregated NETs (aggNETs), the limitation of its enzymatic activity and the containment of the enzyme from surrounding tissues. These events prevent the spread of inflammation and tissue damage. The detection of DNase 1‐dependent elevation of NE activity attests the continuous presence of patrolling neutrophils forming NETs and aggNETs even under conditions physiologic conditions.


INTRODUCTION
Neutrophils, the most abundant leukocytes, are the first cells attracted to the sites of inflammation and play an essential role in the initiation and modulation of inflammatory responses. Recruited from the blood vessels to the site of infection or sterile tissue damage, they phagocytose pathogens, generate reactive oxygen species, release microbicidal molecules (degranulation), and secrete proinflammatory cytokines. 1 After fulfilling their mission, neutrophils undergo apoptosis and are ingested by tissue-resident macrophages. 2 to initiation, perpetuation, and chronification of autoimmunity. 11 Much effort has been put forward to investigate the role of NETs in autoimmune diseases. A potentially pathogenic role of NETs has been shown for antineutrophil cytoplasmic antibodies-associated small-vessel vasculitis, 12,13 rheumatoid arthritis, 14 and systemic lupus erythematosus. [15][16][17] Another intriguing deleterious effect of NET formation in the body is the accumulation of macroscopically visible aggregates of NETs that conditions pancreatitis initiated by ductal occlusion. 18 Contrastingly, aggregated NETs (aggNETs) have been shown to contribute to the resolution of sterile inflammation in gout, where they trap and degrade proinflammatory cytokines and chemokines. This stops subsequent chemotaxis and further infiltration and activation of neutrophils. 6 A better understanding of the presence of NETs in various tissues and body fluids in health and disease is required to elucidate the contribution of NETs and aggNETs to the etiopathogenesis of several diseases.
The common method to detect NETs is the measurement of extracellular DNA [19][20][21] and the visualization of DNA strains by immunocytochemistry. 12,[22][23][24] The sole detection of extracellular DNA with fibrillary pattern is not enough to prove the presence of NETs. It is widely accepted that the detection of specific NET-markers is necessary to confirm the presence of NETs. This refers to DNA, the main component of NETs, as well as to one of the proteins that are associated with decondensed chromatin, for example, NE, citrullinated histone H3, or myeloperoxidase. In some cases, detection by immunocytochemistry of protein markers is impeded by the intricate structure of aggNETs.
Endogenous macromolecular inhibitors, including 1-antitrypsin, 2-macroglobulin in the circulation, and the secretory leukocyte protease inhibitor in various epithelia, contain the lytic activity of NE released to extracellular space. 1 The presence of NE in the interstitium has been demonstrated in severe conditions such as the emphysematous lung and the acute respiratory distress syndrome. 25,26 Because NETs and aggNETs are heavily decorated with NE, we analyzed the interaction of the latter with DNA, the limitation of its enzymatic activity, and its containment from surrounding tissues. These events prevent the spread of inflammation and tissue damage. We argue for a regulatory role of aggNETs during neutrophil-driven inflammation, lowering the activity of NE and sequestering the enzyme from large molecular weight substrates. Furthermore, we show the presence of NETs and aggNETs in various body fluids attesting the continuous presence of patrolling neutrophils forming NETs even in health.

Generation of NETs and aggNETs for enzymatic assays
NETs and aggNETs were generated in V-bottom tubes with 5 × 10 6 PMN/ml that had been settled to the bottom of the tube.

Analysis by microscopy of NETs/aggNETs
NETs and aggNETs were generated in chamber slides with 5 × 10 6 PMN/ml that had been settled to the bottom of the chamber slide. These cells were stimulated with 10 ng/ml of PMA (Sigma

NE activity
All experiments were performed in reaction buffer consisting of PBS

Digestion of gelatin-coated slides
Rounded cover slips were sterilized in 70% ethanol and placed in 24well plates. Next, they were coated with 50 µg/ml of poly-L-lysine

Murine samples
BALB/c mice were purchased from The Janvier Labortories (Roubaix, France) and kept in the local animal facility with free access to food and water. We euthanized the mice with isoflurane before blood was drawn by cardiac puncture. We collected serum, gall fluid, and feces and stored them at −20 • C until use.

Enzymatic kinetics of NE
The kinetic model used to analyze the experimental results was the Michaelis-Menten equation: where S is the concentration of the substrate; V max represented the maximum reaction rate; and K m was the substrate concentration at the half-maximal rate. The V max and K m values were obtained from a nonlinear regression between substrate concentrations and reaction velocities using the fit equation. Dataset used for analyses was obtained from at least 2 donors.

Statistics
Statistics were calculated employing GraphPad Prism 5.03 software.

NETs form aggregates and tightly trap sterile particulate matter
We induced NETs by PMA (Fig. 1A) or MSU crystals (Fig. 1B) as reported before. 3,6 Fluorescence staining revealed decondensed chromatin (PI, red) decorated by NE (green). Analysis of the colocalization of these markers is often employed for the detection of NETs.
In high PMN densities, NETs reportedly aggregate and form so called aggNETs. 6 In order to show differences between the aggregation in the presence and in the absence of particulate matter, we display the macroscopic appearance and PI epifluorescence of aggNETs induced by PMA (Fig. 1C) and MSU (Fig. 1D), both prone to DNase 1 digestion (Fig. 1E, F). PMA-induced aggNETs appear as a slack mass of extracellular DNA (PI positive, Fig. 1C). When compared to MSU-induced aggNETs the extracellular DNA appears more compacted (Fig. 1D).
This suggests that, during aggNET formation, MSU crystals serve as a scaffold for the decondensed chromatin fibers. 6

Digestion with DNase 1 of NETs and aggNETs increased the apparent activity of NE
NE activity was measured in aggNETs (closed symbols) induced by PMA ( Fig. 2A) or MSU crystals (Fig. 2B) in the absence and presence of DNase 1. The specificity of the reaction was confirmed by blocking substrate conversion with sivelestat, a specific NE inhibitor (open symbols). As shown in Fig. 2A and B, digestion with DNase 1 (squares) increased the NE activity in both kinds of aggNETs.
PMA-induced aggNETs ( Fig. 2A) show higher NE activity in comparison to MSU crystals-induced tophus-like aggNETs (Fig. 2B). This suggests that more NE is available to convert the low molecular weight substrate in the slack mesh of PMA-induced aggNETs (Fig. 1C) and supports the hypothesis that MSU crystals trigger more tight aggNETs (Fig. 1D). The accessibility of the enzymes contained in aggNETs to the external milieu is, therefore, regulated by the tightness of the chromatin mesh. In order to test whether the activity of the NE is affected To show that digestion of the NET-DNA and not a putative interaction with NE of DNase 1 is responsible for the increased NE activity after treatment with the endonuclease DNase 1, we employed the endo-exo nuclease MNase to test its influence on the NE activity. Nevertheless, in the case of neutrophil-driven conditions affecting the pulmonar parenquima, an increased activity of NE results clearly disadvantageous. [28][29][30][31][32][33][34] Based on these and our observations, we conclude that dsDNA in aggNETs physically interacts and interferes with the enzymatic activity of NE and thus plays a regulatory role at the sites of neutrophil-driven inflammation.

Digestion of DNA increased NET-associated NE activity in samples of body fluids
The formation of aggNETs reportedly causes pathologic ductal occlusion and pancreatitis due to patrolling neutrophils that enter the bicarbonate rich pancreatic ducts and execute NET formation 40 and aggregation. 18 Neutrophils patrol the ocular surface and form aggNETs to wipe out dust and microorganisms. 41 NETs participate in the initiation and growth of gallstones. 42 We assessed the NE activity in samples from murine gall fluid (Fig. 4A), feces (Fig. 4B), and serum ( Fig. 4C), as well as eye wash ( Fig. 4D and E) and gall fluid (Fig. 4E) samples from normal healthy donors. Inhibition by sivelestat of NE (open symbols) confirmed the specificity of the assay. We detected robust NE activity in murine gall fluid (Fig. 4A), feces (Fig. 4B), and human eye wash samples (Fig. 4D) only after digestion of the samples by DNase 1.
For serum samples, no essential conversion of the substrate by NE was to be detected (Fig. 4C). This can easily be explained by the presence in sera of anti-proteases like 1-antitrypsin, a natural agent blocking the activities of various serine proteases. 43 Many reports have shown the presence of patrolling neutrophils in various body compartments under healthy conditions, such as the eye 44 or sputum. 45