High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Podsumowanie

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Streszczenie

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation.

Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

Wprowadzenie

NET formation is a novel mechanism by which neutrophils fight pathogens.¹ The core of NETs is nuclear DNA.¹ This DNA network is associated with neutrophil granule proteins and histones.¹ The main form of NET formation requires the death of neutrophils characterized by chromatin decondensation, disappearance of granular and nuclear membranes, translocation of neutrophils elastase to the nucleus, citrullination of histones and finally the spill of DNA-based NETs.² NETs entrap and kill a wide variety of microbes and are an essential part of the innate immune weapon repertoire. Uncontrolled NET formation has, however, been linked to numerous autoinflammatory diseases.^3,4 Despite their increasingly established relevance, little is known about the mechanism and regulation of NET release.

Neutrophils dying by releasing NETs are different from apoptotic or necrotic neutrophils.^3,5 NET-releasing neutrophils show several features that are characteristic for NET formation. Granule components are associated with DNA in NETs.¹ Myeloperoxidase (MPO) and human neutrophil elastase (HNE) are both found in primary granules in resting cells but are translocated to the nucleus to bind to DNA in NETs.¹ MPO-DNA and HNE-DNA complexes are specific for NETs, do not occur in apoptotic or necrotic neutrophils.^1,3,5 Chromatin decondensation is another feature typical for NETosis.² NET release also requires citrullination of histones by peptidyl aminidase 4 (PAD4).⁶ Citrullinated histones are hallmarks of neutrophils that underwent NET release.⁶

Here three methods are introduced that in combination provide excellent tools to quantitate NETs on a high-throughput scale. The first assay has been used on the field with different changes and quantitates extracellular DNA release in a microplate format. The second and third assays provide confirmation of NETs by measuring NET-specific MPO-DNA and HNE-DNA complexes.

Protokół

The Institutional Review Board of the University of Georgia approved the human subject study to collect peripheral blood from healthy volunteers (UGA# 2012-10769-06).^5,7,8 Volunteers signed the required informed consent form before blood draw. The research performed in this article is in compliance with the ethical guidelines for medical research involving human subjects of the Declaration of Helsinki.

1. Isolation of Neutrophils from Peripheral Human Blood (Figure 1)

Note: There are several ways to isolate neutrophils from peripheral blood. The following protocol provides one possibility. This protocol yields large numbers of non-activated human neutrophils capable of releasing NETs upon external stimulation. Use 40 ml whole blood obtained from healthy volunteers by venipuncture.^7,9

1. Aliquot 20 ml blood into two 50 ml conical tubes. Add 10 ml 6% Dextran. Mix gently.
2. After 20 min transfer the leukocyte-rich upper phase without taking any pelleted red blood cells into clean 50 ml conical tubes, and fill it up with sterile PBS.
3. Centrifuge at 400 x g 10 min. Resuspend pellet in 4 ml sterile PBS.
4. Prepare a 5-step Percoll gradient of 85%-80%-75%-70%-65% in two 15 ml conical tubes and layer 2 ml leukocyte suspension on top of each gradient.
5. Centrifuge 800 x g for 30 min with brakes off.
6. Collect all the cells (neutrophils) in the layers at the 75%/80% and 70%/75% interface.
7. Wash cells twice in PBS (350 x g, 10 min, RT) and count cells using a hemocytometer.

2. Measurement of Kinetics of Extracellular DNA Release Using a Microplate Fluorimeter

Note: This method enables measurement of changes in fluorescence of a membrane-impermeable DNA-binding dye indicative of DNA release on a 96-well microplate format (Figure 2).

1. Prepare a suspension of neutrophils in assay medium (HBSS + 1% (v/v) autologous serum + 5 mM glucose) at a concentration of 2 x 10⁶ cells/ml.
2. Add 5 µmol/L of the membrane impermeable DNA-binding dye. Mix gently.
3. Aliquot human neutrophils (50 µl/well) onto 96-well black, transparent bottom microplates. Make sure the assay medium covers the entire bottom of the well. If not, tap the plate gently on the side.
4. Warm up the plate containing neutrophils for 10 min at 37 °C.
5. In the meantime prepare solutions of stimuli at double of their final concentrations to be used (in 37 °C warm assay medium). As a positive NET control use human neutrophils with 100 nM phorbol-myristate-acetate (PMA) for 4 hr to trigger maximal NET release.^5,8 PMA stimulation for 4 hr ensures maximal NET release while spontaneous NET formation remains low^5,8.
6. Prepare the microplate fluorimeter for the measurement (4 hr, 37 °C, 530 nm for excitation and 590 nm for emission).
7. Stimulate neutrophils by adding 50 µl stimulus solution to 50 µl neutrophil suspensions. Use 0.5 µg/ml saponin in one well to measure maximal DNA release signal.
8. Place plate in reader and measure changes in fluorescence for 4 hr at every 2 min with no shaking.
9. For data analysis, calculate normalized increases in fluorescence over time.
   1. Subtract baseline fluorescence from endpoint values for all wells including saponin (Figure 2A, B). Rise in saponin fluorescence should be the highest signal. It is referred to as "maximal DNA release" (Figure 2A).
   2. Calculate percentages of DNA release in unknown samples by dividing increases in fluorescence of unknown samples by maximal DNA release.
   3. Average results of replicates and present them as "% of maximal DNA release" (Figure 2C).

3. Quantitation of NET Formation by MPO-DNA and HNE-DNA ELISA Assays

Note: These assays modified (MPO-DNA) or established (HNE-DNA) in our laboratory quantitate NET formation by measuring levels of MPO-DNA and HNE-DNA complexes.⁵

1. Coat 96-well high-binding capacity ELISA plates overnight with capture antibodies (50 μl/well): anti-MPO (1:2,000 dilution) or anti-HNE (1:2,000).
2. Wash ELISA plates three times with PBS (200 μl/well) and block them with 5% BSA and 0.1% human serum albumin for 2 hr at RT (200 μl/well). Wash three times with PBS.
3. Seed neutrophils on 96-well microplates at a density of 100,000 cells/well in 100 µl assay medium, and stimulate NET formation. Incubate cells: 4 hr for 37 °C.
4. Perform a limited DNase digestion by adding 2 U/ml DNase to neutrophil supernatants, mix them well. Keep them at RT.
5. Stop the DNase after 15 min by adding 11 µl 25 mM EGTA (final concentration 2.5 mM). Mix well. Collect supernatants in clean microfuge tubes. Spin samples to get rid of cell debris at 300 x g for 5 min at RT. Transfer their supernatants into clean microfuge tubes. Keep them on ice until ready.
6. Use an aliquot of a previously prepared "NET standard".
   Note: The NET-standard is a mix of DNase-digested supernatants of PMA-stimulated neutrophils obtained from at least 5 different, independent human donors.
   1. To use the same standard for a long time, collect and aliquot a large volume of neutrophil supernatants from each individual donor. Collecting for example 2 ml of DNase-digested supernatants of PMA-stimulated neutrophils will result in 200 aliquots (10 µl each) enough for 200 ELISA plates. Data characterizing the NET standard are found in Figure 5.
   2. Stimulate human neutrophils with 100 nM PMA for 4 hr and apply DNase digest to their supernatants according steps 3.4-3.5.
   3. Collect, pool, aliquot (10 µl) and freeze (-20 °C) neutrophil supernatants.
   4. To prepare the "NET-standard", thaw one aliquot/donor obtained from at least five separate donors, mix them and keep them on ice.
   5. Discard thawed samples and use fresh ones for the next experiment.
   6. Prepare a 1:2 serial dilution of the NET-standard in PBS+EGTA.
7. Dilute DNase1-digested samples (standard and unknown supernatants) 20-fold in PBS+EGTA and aliquot them on ELISA plates coated with capture antibodies.
8. Incubate ELISA plates over night at 4 °C and wash them three times with PBS.
9. Apply detection antibody solution (100 µl/well): horse radish peroxidase conjugated anti-DNA antibody (1:500, mouse). Incubate for 1 hr in dark.
10. After four washes with PBS add TMB peroxidase substrate: 100 µl/well, 30 min. Blue coloration is an indication for peroxidase activity (NETs present).
11. Stop reaction by adding 100 μl/well 1 M HCl. The blue solutions will turn yellow (Figure 5A).
12. Read absorbance at 450 nm using a microplate photometer.
13. For data analysis, calculate the amount of MPO-DNA or HNE-DNA complexes compared to the NET standard.
    1. Subtract the background optical density value of the assay medium from all samples.
    2. Plot the absorbance values (X-axis) of the diluted standard samples against their relative NET content (Y-axis) (Figure 5A).
    3. Using the nonsaturated range of this standard curve establish a trend line (use best exponential fit from the software used) (Figure 5A). The equation of this trend line determines the conversion of OD values to quantitative amounts of NETs.
    4. Insert the measured OD values of the unknowns into the trend-line equation from 3.13.3 that will give the amount of NETs in the unknown sample as percentage of the NET-standard (Figure 5A).
    5. Calculate averages of replicates (triplicates) and present the final data as "amount of MPO-DNA or HNE-DNA complexes (% of standard)" (Figure 5C).

Wyniki

The figures in this manuscript describe the method of neutrophil isolation, experimental procedures, and present representative results with explanation of data analysis. Figure 1 shows the sequential steps of human neutrophil preparation. This protocol represents only one possible way of neutrophil isolation. It yields large amounts of resting neutrophils capable of releasing NETs upon stimulation. Figure 2 shows how the fluorescence-based DNA release as...

Dyskusje

NETs represent a fascinating novel mechanism by which neutrophils kill pathogens.¹ Although the literature of NETs has been continuously expanding over the last ten years since their discovery, several important questions related to their role in biology, mechanism and regulation remain unclear. Appropriate methodology has to be developed to measure NETs, this very unique antimicrobial mechanism. This article describes methods that can be used to quantitate NETs in a high throughput manner. The first assay fol...

Materiały

Name	Company	Catalog Number	Comments
Anti-Human Neutrophil Elastase  Rabbit Ab	Calbiochem	481001	1:2,000x coated
Anti-Myeloperoxidase Ab (Rabbit)	Millipore	07-496	1:2,000x coated
DNase-1	Roche	10-104-159-001	1 µg/ml used for digestion
20 mM EGTA/ PBS	Sigma-Aldrich	E3889-25G
2.5 mM EGTA/PBS	Sigma-Aldrich	E3889-25G
Cell death detection ELISA Anti-DNA POD	Roche	11544675001	1:500x
Eon Microplate Spectrophotometer	Biotek
Gen5 All-in-One microplate software	Biotek		analytical tool (ELISA)
Sytox orange	Life Technology	S11368	0.2% final concentration/volume
1 M Hepes	Cellgro	25-060-Cl	Use 10 mM final concentration.
1 M glucose	Sigma		Use 5 mM final concentration.
HBSS	Corning	21-023-CM
Varioskan Flash Ver.2.4.3	Thermoscientific
PMA	Sigma	P 8139	100 nM final used
ELISA Plate	Greiner bio-one	655061
Conical tubes 15 ml	Thermoscientific	339650
Conical tubes 50 ml	Thermoscientific	339652
Percoll (pH 8.5-9.5)	Sigma	P 1644	Sodium Chloride, Sigma, S7653-250G
Dextran	Spectrum	D1004
RPMI 1640 media	Corning Cellgro	17-105-CV
96 well assay plate black plate clear bottom	Costar	3603