Using the Lionheart™ FX to Image and Analyze Stimulated dHL60 Cells

Abstract

Neutrophils play a key role in the body’s defense against infections. As part of the innate immune system, they employ multiple strategies to degrade and kill microbes, including the release of neutrophil extracellular traps (NETs). NETs are a web-like structure composed of antimicrobial proteins and DNA that are released during a distinct form of programmed neutrophil cell death known as NETosis. In general, NETs allow neutrophils to kill extracellular pathogens while minimizing damage to the host cells, but can also result in an autoimmune response. This process in vivo is not well understood, so in vitro models are important to help clarify the mechanisms and results of this phenomenon. Here we describe the use of the Lionheart FX to image and analyze differentiated HL60 cells stimulated to undergo NETosis in vitro.

Introduction

Neutrophil extracellular traps (NETs) are networks of extracellular fibers, primarily composed of DNA from neutrophils, which bind pathogens^[1]. Neutrophils, which are the most abundant white blood cell and the immune system’s first line of defense against infection, have conventionally been thought to kill invading pathogens, either by engulfing microbes or by secreting antimicrobials^[2]. In 2004, a third function; the formation of NETs, was identified by Brinkmann et al^[1]. High-resolution scanning electron microscopy has shown that NETs consist of stretches of DNA and globular protein domains with diameters of 15-17 nm and 25 nm, respectively, which can aggregate into larger threads with a diameter of 50 nm^[1]. Under the flow conditions present in the vasculature in vivo, much larger structures can form^[3].

Figure 1. NETosis process.

Following PMA stimulation, NETosis is activated through NADPH oxidase stimulation. NADPH oxidase assists in the translocation of neutrophil elastase (NE) from cytosolic granules into the nucleus where it aids in chromatin breakdown via histone cleavage. Myeloperoxidase (MPO) is required for chromatin and nuclear envelope breakdown and granular mixing within the NET vacuole. Following intracellular NET formation, the neutrophil outer membrane ruptures, and the mature NET is extruded (Figure 1).

Immunofluorescence has shown that NETs contain proteins from several different neutrophil granule types, as well as the nucleus, but many common cytoplasmic proteins, such as actin and tubulin were lacking^[1,4]. NETs provide for a high local concentration of antimicrobial components that bind, disarm, and kill microbes extracellularly and independent of phagocytic uptake. Delivery of the granule proteins into NETs may also keep potentially injurious proteins like proteases from diffusing away and inducing damage in tissue adjacent to the site of inflammation.

The complete NETosis activation pathway is still under investigation but a few key proteins have been identified. The NETosis pathway can be initiated through activation of Toll-like Receptors (TLRs), Fc receptors, and complement receptors with various ligands such as antibodies, PMA, and IL-8^{[5, 6]}. Following stimulation by PMA through activation of protein kinase C, raf–mitogenactivated protein kinase and (MEK)–extracellular signal-regulated kinase (ERK) pathways, downstream signaling results in the release ofcalcium from the endoplasmic reticulum. This intracellular influx of calcium, in turn, activates NADPH oxidase, causing the activation of protein-arginine deiminase 4 (PAD4) via reactive-oxygen species (ROS) intermediaries. PAD4 is responsible for the citrullination of histones in the neutrophil, resulting in decondensation of the chromatin. Azurophilic granule proteins such as myeloperoxidase (MPO) and neutrophil elastase (NE) enter the nucleus and further the decondensation process. Eventually, the nuclear envelope ruptures and the uncondensed chromatin enters the cytoplasm where additional granule and cytoplasmic proteins are added to the early-stage NET. The end result of the process then depends on whether the suicidal or vital NETosis pathway is activated (Figure 1)^[7]. Suicidal NETosis, first described in 2007, demonstrated that the release of NETs resulted in neutrophil-death through a different pathway than apoptosis or necrosis^[5]. In suicidal NETosis, the intracellular NET formation is followed by the rupture of the plasma membrane, releasing it into the extracellular space. Vital NETosis, on the other hand, results in the blebbing of the nucleus, producing a DNA-filled vesicle that is exocytosed leaving the plasma membrane intact. While not viable, neutrophils can continue to phagocytose and kill microbes after vital NETosis^[6]. Of note, suicidal NETosis can take hours, while vital NETosis can be completed in a matter of minutes, which suggests different functions.

In this application note, we demonstrate an in vitro live cell assay to monitor NETosis activity using differentiated HL-60 cells as a model. The basis of this assay is to kinetically follow cell membrane integrity using both cell membrane permeable and impermeable nuclear stains using fluorescence microscopy. This allows for the determination of inhibitor potency of NETosis activity.

Materials and Methods

Reagents

RMPI-1640 tissue culture media, fetal bovine serum (FBS), glutamine, and penicillin/streptomycin were from Life Technologies. HL-60 human promyeloblast cells were obtained from ATCC (Manassas, VA). Nuclear Red™ and Nuclear Green™ stains were obtained from Cayman Chemical (Ann Arbor, MI). DMSO and ATRA were procured from Sigma-Aldrich (St. Louis, MO). Stimulant and inhibitor small molecule compounds, PMA, Nigericin, A23187, DPI, Go 6976, nocodazole, apocynin, ML 171, VAS 2870, and NoxA 1ds were purchased from Tocris Bioscience (Minneapolis, MN).

Cell Culture

HL-60 cells were grown in RPMI-1640 plus 10% FBS supplemented with 2 mM glutamine, penicillin and streptomycin. The suspension cells were split 1:5 with fresh media every 2-3 days. HL-60 cells were diluted to 1 x 105 cells/mL and differentiated into neutrophils by exposure to 1.25% DMSO and 0.1 μM ATRA for 4-5 days (Figure 2).

Figure 2. Differentiation of HL60 promyelocytic leukemia cells. Cultured HL60 cells are induced to differentiate to neutrophils by exposure to 1.25% DMSO and 100 nM ATTRA for 5 days. Differentiated HL 60 cells (dHL60) are harvested and stained with Nuclear Red™ for 30 minutes at 37 °C. The cells are then pelleted and Nuclear Green™ stain added immediately before plating.

Staining

Differentiated HL-60 cells (dHL-60) were counted and 1.2 x 106 cells were pelleted by centrifugation in a 15 mL conical tube. Cells were resuspended in 6 mL of PBS and 6 μL of Nuclear Red™ stain added. Cells were incubated for 30 minutes at 37 °C, then an additional 6 mL of PBS added and the cells were again pelleted by centrifugation. Cells were resuspended in 12 mL of NETosis imaging buffer (PBS, 0.5% w/v BSA, 0.1% w/v glucose) to which 12 μL of Nuclear green™ stain has been added and immediately aliquoted (100 μL, 20,000 cells/well) into the wells of a Corning (cat # 3904) 96-well, black sided, clear bottom microplate (Figure 3).

Figure 3. NETosis stimulation and inhibition assay process. Differentiated HL60 (dHL60) cells, pre-stained with Nuclear red™ and Nuclear Green™ dyes are added to different concentrations of NETosis inhibitors prior to the addition of a NETosis stimulant such as PMA. After the addition of stimulant, NETosis is monitored by imaging in the GFP and CY5 channels.

Several different compounds were used to stimulate dHL-60 cells to undergo NETosis. Compound dilutions in NETosis imaging buffer were added to the wells in 100 μL at 2x of the desired concentration. Cells were then added to the wells in 100 μL and the microplate centrifuged at 200g for 60 seconds to seat the nonadherent cells on the bottom of the well. Imaging was initiated immediately after centrifugation.

Several potential inhibitors were studied for their ability to reduce or eliminate compound induced NETosis stimulation. Inhibitor compounds were diluted in NETosis imaging buffer and aliquoted (100 μL) into wells of a microplate at 3x the final intended concentration. dHL-60 cells (100 μL), stained as described previously, were added to the inhibitor dilutions and incubated for 15 minutes at RT, after which 100 μL of a 3x final concentration NETosis stimulate was added. The plate was centrifuged and imaged as described previously.  

Imaging

Cultures were imaged using a Lionheart™ FX Automated Microscope (BioTek Instruments, Winooski, VT) configured with brightfield, as well as GFP and CY5 LED cubes. The imager uses a combination of LED light sources and bandpass filters with dichroic mirrors to provide appropriate wavelength light. Images were taken kinetically every 15 minutes for 4 hours using a 20x objective. Laser autofocus (LAF) was used to focus the image for each well automatically. A LAF reference scan was obtained using focused CY5 fluorescent images (Figure 4).  

Table 1. Image-capture and image-analysis parameters for NETosis.

Figure 4. Cell staining. Cells were stained using a NETosis Imaging Assay Kit (Cayman Chemical, Ann Arbor MI), which consist of a combination of Nuclear Red™ and Nuclear Green ™ stains. The cell permeable red stain was used to set initial Laser Autofocus (LAF) parameters, while impermeable green stain was used to monitor NETosis.

Analysis

After imaging, cellular analysis identified GFP objects using a threshold value of 5000 (Figure 5). The object masks were not split into individual objects, but were limited to greater than 10 μm in size in order to exclude debris. Holes in the mask were filled and objects along the imaging-edge were also included in the object area metric determination (Table 1).  

Figure 5. Object area analysis. NETs were identified using the Cellular Analysis Data reduction option in Gen5 ™ software (BioTek Instruments). The total object area was determined for each 20x image.

Figure 6. Integral of Total Green Area. The total object area of green fluorescence is determined for each time point in every well of the kinetic experiments. The area under the curve (AUC) or integral of the kinetic plots for each well are automatically calculated and plotted against compound (stimulant or inhibitor) concentration depending on the experiment. The green and red striping represents the AUC for a positive and negative control, respectively.

The total GFP object area was automatically calculated for each well at each time point and plotted using Gen5™ Microplate Reader and Imager Software. The integral or area under the curve (AUC) of these plots determined automatically by Gen5 (Figure 6). The resultant AUCs were graphed against compound (stimulant or inhibitor) concentration using GraphPad Prism 7.

Results

Different compounds were tested for their ability to stimulate NETosis in dHL60 cells. By plotting the AUC of the kinetic curves as a function of compound concentration one can ascertain the potency of various stimulators or inhibitors of NETosis. The compound phorbol 12-myristate 13-acetate (PMA) was found to be a potent stimulator of NETosis. PMA acts in a concentration-dependent manner with an EC₅₀ of 5 nM (Figure 7). PMA mimics diacylglycerol and binds to protein Kinase C (PKC), activating the kinase. Downstream effects include the promotion of cell growth and inflammation^[8].

The ionophore compounds A23187 and Nigericin were also demonstrated to induce NETosis, as measured by GFP fluorescence, in a concentrationdependent manner. However, these compounds are significantly less potent than PMA (Figure 8). A23187 is a calcium ionophore that besides directly facilitating extracellular calcium movement into the cell also activates phospholipase C-dependent mobilization of intracellular Ca2+ stores^[9]. Nigericin, is an antibiotic derived from Streptomyces hygroscopicus and is reported to act as a potassium ionophore, which can activate the NLRP3 inflammasome^[10].

Figure 7. Effect of PMA Concentration on NETosis. dHL60 cells were treated with PMA at various concentrations in the presence of Nuclear Green™ stain. Analysis of kinetic images determined the total NETosis area of green fluorescence for each well as a function of time. AUC for each kinetic curve was and plotted as a function of PMA concentration. Each data point represents the mean of 8 determinations.

Figure 8. Comparison of the potency of different NETosis stimulant. dHL60 cells were treated with PMA, A23187 or Nigericin at various concentrations in the presence of Nuclear Green™ stain. Analysis of kinetic images determined the total NETosis area of green fluorescence for each well as a function of time. AUC for each kinetic curve was calculated and plotted as a percentage of the AUC at maximum compound concentration. Each data point represents the mean of 8 determinations.

NETosis in vivo has been reported to act through the action of NADPH oxidase (NOX). As such it would be expected that NOX inhibitors would ameliorate the effect of NETosis stimulants. As demonstrated in figure 9, diphenylene iodonium (DPI) is capable of inhibiting NETosis stimulated by PMA (100 nM) in a concentration dependent manner. Interestingly, the compound was ineffective against the ionophores A23187 or Nigericin. This suggests that these compounds act primarily through their ionophore activity rather than true stimulation of NETosis.

Figure 9. Inhibition of NETosis stimulation of dHL60 Cells by diphenylene iodonium (DPI). Differentiated HL60 cells were pretreated with DPI at various concentrations prior to stimulating cells by the addition of 100 nM PMA, 25 μM A 23187, or 25 μM Nigericin in the presence of Nuclear Green™ stain. The integral (AUC) of the kinetic plots for total area of fluorescence for each well was calculated by Gen5 Microplate Reader and Imager Software. The data was expressed as the % total AUC of untreated positive control and plotted as a function of DPI concentration. Each data point represents the mean of 8 determinations.

Several compounds were tested for their ability inhibit the effects of PMA in regards to NETosis. As seen in Figure 10, DPI is significantly more potent and effective than the PKC inhibitor Go 6976 at negating the NETosis produced by PMA. The EC₅₀ of Go 6976 is approximately 20-fold higher concentration than that observed with DPI. In addition, the effective inhibition was about half that observed with DPI. Nocodazole, which interferes with microtubule polymerization and a known cell cycle inhibitor was ineffective at preventing NETosis (Figure 9).

Figure 10. Effect of Cellular inhibitors on PMA stimulation of dHL60 Cells. Differentiated HL60 cells were pretreated with either DPI, Go 6976 or Nocodazole at various concentrations prior to stimulating cells by the addition of 100 nM PMA in the presence of Nuclear Green™ stain. The integral (AUC) of the kinetic plots for the total area of fluorescence for each well were calculated. Data was expressed as the % total AUC of untreated positive control and plotted as a function of inhibitor concentration. Each data point represents the mean of 8 determinations.

The effectiveness of the NOX inhibitor DPI at inhibiting PMA induced NETosis led us to test the efficacy of other known NOX inhibitors. Of the inhibitors tested, DPI had the greatest potency (Figure 11). Interestingly nonspecific NOX inhibitors such as DPI, Apocynin and VAS 2870 all had some degree of efficacy, while NOX 1 specific inhibitors had little effect (Figure 10).

Figure 11. Effect of NADPH oxidase inhibitors on PMA stimulation of dHL60 Cells. Differentiated HL60 cells were pretreated with either DPI, VAS 2870, Apocynin, NoxA 1ds, and ML 171 at various concentrations prior to stimulating cells by the addition of 100 nM PMA in the presence of Nuclear Green™ stain. The integral (AUC) of the kinetic plots for the total area of fluorescence for each well were calculated, expressed as the % total AUC of untreated positive control, and plotted as a function of inhibitor concentration. Each data point represents the mean of 8 determinations.  

Discussion

We have used differentiated HL-60 (dHL60) cells as an in vitro model for human neutrophils. dHL60 cells exhibit the same NETosis response as freshly isolated human cells and serve as a good model to better understand this phenomenon. NETosis was examined using imaging after staining with a combination of Nuclear Red and Nuclear Green (Cayman Chemical). While both stains bind nucleic acids and become fluorescent, Nuclear Red stain is a cell permeable stain that can stain live cell nuclei and Nuclear Green is impermeable and will only fluorescence when cell membranes have been disrupted. Initial imaging and focus are accomplished using a CY5 LED cube that detects the nuclei of all cells stained with Nuclear Red. With NETosis, cell membranes become permeable to Nuclear Green™ dye. Entry of the dye allows an increase in green fluorescence as a result of binding to nuclear DNA. With time, the nuclear membrane breaks down and karyoplasm and cytoplasm contents mix and the nuclear contents are released. The extracellular DNA is also accessible to the Nuclear Green™ Reagent, resulting in a greater area of green fluorescence.

The phorbol ester PMA was demonstrated to stimulate NETosis in HL60 cells that had been differentiated into neutrophils in a dose-dependent manner. The effect could also be inhibited with NADPH oxidase inhibitors such as DPI. Ionophores such as Nigericin and A23187 were shown to have a similar effect, but could not be inhibited with DPI which suggest that their effect was not pharmacologic of NETosis, but rather the result of the ionophores allowing access of the membrane impermanent dye to the cellular nucleic acids. The NOX inhibitor data suggests, that while NADPH oxidase protein activity is required for NETosis, NOX1 is not the isoform directly responsible for this activity.

These data demonstrate that the Lionheart™ FX Automated Microscope in conjunction with Gen5 ™ Microplate Reader and Imager Software can be used to quantitate NETosis of neutrophils in vitro. The Lionheart FX is an ideal platform for these studies. The imager maintains the physiologic temperature within the read chamber, as well as providing humidity with use of the humidity plate carrier during the kinetic imaging. Gen5 Microplate Reader and Imager Software controls the imager in regards to timing of kinetic imaging as well as automatically identifying and quantifying areas of fluorescent staining. These data are then plotted as individual well kinetic graphs and the integral (AUC) calculated. Subsequent AUC determinations are available for concentration dependent graphs.

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