Ex Vivo Human Neutrophil Swarming Against Live Microbial Targets

Abstract

Neutrophils often communicate with each other and coordinate their actions to seal off large sites of injury and infection that individual neutrophils could not cover. The concerted actions of neutrophils are essential for the expeditious protection of healthy tissues from wounds and microbes. These processes, collectively known as swarming, are typically studied in vivo in mice. However, these studies are low throughput and their relevance to human disease is limited. Recently, new tools have been developed for the study of human neutrophil swarming ex vivo. The emergent microscale swarming assays are providing significant insights into the molecular mediators of swarming. By enabling the direct study of human cells, these assays also shed new light on human diseases and host responses against infections. Here, we describe a robust technique for manufacturing microscale swarming arrays with live microbial targets (e.g., clusters of Candida albicans). These arrays allow for the direct, precise, and efficient interrogation of the antimicrobial functions of human swarming against a variety of targets.

1. Introduction

Recent characterization of neutrophil behavior has revealed that neutrophils communicate with each other, coordinating their actions to accomplish functions that independent neutrophils could not achieve [1–4]. This process, termed “neutrophil swarming,” helps seal off sites of injury and infections, separating these from healthy tissues. The process also allows neutrophils to restrict and contain microbes and microbe clusters that would be too big to phagocytose by individual neutrophils. Swarming is characterized by exponential neutrophil recruitment and driven by feedback loops that the neutrophils themselves perpetuate [2–4]. Swarming is distinct from phagocytosis and is only triggered against targets above a certain size threshold [4]. While important insights have been provided by in vivo mouse models, these systems present numerous challenges including low throughput, poor access to molecular signals for analysis and difficulty in translating protocols for the study of human neutrophil swarming [2, 3]. Recently, microscale and microfluidic technologies have been used to probe swarming biology. These technologies are particularly beneficial in elucidating the constellation of signaling molecules coordinating neutrophil swarming and interactions with other immune and nonimmune cells. The microscale neutrophil swarming arrays enable the monitoring of hundreds of swarms at once. The arrays are particularly useful for enriching the supernatant in the molecules mediating neutrophil communication through the synchronized release from hundreds of swarms [4].

Here we describe recent work that has extended the swarming array technology to directly interrogate the ability of human neutrophils to restrict the growth of live fungi. We print microarrays of poly-l-lysine/ZETAG directly onto glass slides in predetermined patterns. We use commercially-available multiwell attachment to create individual wells above each printed array. We then add a solution of live microbes to each well, allow them to adhere to the arrays, and then wash off the excess. This creates patterned arrays of the desired microbial targets, ready for use. As each array is isolated from one another in individual wells, many different microbial targets can be tested at once. Alternatively, the same target can be run in multiple wells with neutrophils from multiple sources or in multiple treatment conditions. Neutrophil swarming responses can be directly observed against live microbes by time-lapse microscopy. As this is an open system, the supernatants or cells can be readily recovered and subject to molecular analysis.

2. Materials

2.1. Array Printing

1. Poly-l-lysine solution: 0.1% poly-l-lysine (w/v) in sterile H₂O.

2. ZETAG solution: 1.6 mg/mL ZETAG 8185 (BASF) in sterile H₂O.

3. Printing Solution: Mix 1 mL of the poly-l-lysine solution with 12 μL of the ZETAG solution. Add 0.1 mg/mL FITC or another fluorophore to allow for visualization of the printed arrays. Vortex vigorously for 20 s.

4. PolyPico microspotter.

5. PolyPico cartridges.

6. Ultraclean glass slides.

7. Heat block or 37 °C incubator.

2.2. Microorganism Patterning

1. Appropriate media to culture desired microorganism (Yeast Extract-Peptone-Dextrose (YPD) broth for C. albicans).

2. Proplate multiarray 16 well attachment.

3. Spray bottle with sterile phosphate-buffered saline (PBS) solution.

2.3. Isolation of Human Neutrophils from Blood

1. EasySep Direct Human Neutrophil Isolation kit and EasySep Buffer.

2. Appropriate magnet for the selected blood volume and isolation kit.

3. Iscove’s Modified Dulbecco’s Medium (IMDM) + 20% Fetal bovine serum (FBS).

2.4. Neutrophil Swarming Assay

1. Microscope with fluorescent imaging capabilities and temperature control chamber (e.g., Nikon BioStation IM-Q or higher throughput), automated Nikon Eclipse Ti2 series.

3. Methods

Carry out all procedures at room temperature unless otherwise noted.

3.1. Printing of Arrays

1. Open a disposable PolyPico cartridge and place the suction cup attachment on the end of a P200 pipet. Set the pipet to 100 μL. Aliquot printing solution in the well of a 24-well plate. Align the holding rack over the well and place your cartridge in the well. Place the pipet with the suction cup over the top of cartridge and press down to create a seal. Draw up on the pipet to load solution into the cartridge (see Note 1).

2. Transfer the loaded cartridge into the head of the PolyPico microspotting machine (see Note 2). Place an ultraclean glass slide on the platform of the machine, with its edge aligned at the “A1” corner.

3. Open the PolyPico Software and turn on the PolyPico microspotting machine. Select the appropriate array design in the software. Go to the “Calibrate” panel and select “find tip.” The machine will automatically find the tip of the loaded cartridge. Next, click the “dispense on” box to view the droplet being produced. Adjust the amplitude, width, and frequency settings as necessary to see a well formed (spherical) droplet. Move to the “Dispense” panel. Select the “use current parameters” option and then hit the play button to dispense droplets in the selected array pattern (see Note 3).

4. Screen the arrays by microscopy to ensure accuracy. For small volume arrays, the addition of a fluorophore like FITC in the printing solution is very helpful to visualize the arrays (see Fig. 1).

5. Dry the slides on a heat block at 37–40 °C for 2 h. Slides can then be left at room temperature until use.

Fig. 1.

Printing of microscale arrays. Swarming arrays were printed on glass slides using a PolyPico microspotter and a solution of poly-l-lysine/ZETAG. FITC was spiked in the solution to allow visualization of the spots in the arrays (shown on the right). The printed spots shown are 100 μm in diameter

3.2. Patterning of Microorganisms

1. Grow the desired target microorganism according to standard culture methods. Methods described here will be for C. albicans. Spin down an overnight culture in a centrifuge at max speed for 30 s. Remove the media and discard it. Resuspend the pellet in an equivalent amount of sterile water (see Note 4).

2. Place the 16-well attachment and gasket system onto the glass slide with the printed arrays. Slide on the clips on each side to hold the attachment in place (Fig. 2a).

3. Load 50 μL of the microorganism solution into each well. Incubate on a rocker for 5–10 min.

4. Wash the wells clean with PBS, using a spray bottle. Vigorous and repeated washing is required to ensure nonbound microorganism is removed from the glass. Check periodically with microscopy to determine when arrays are clean (Fig. 2b).

5. Dispense 60 μL of PBS into each well to ensure that the microorganism arrays do not dry out before running the assay.

Fig. 2.

Assembly of swarming chambers and microbe patterning. After printing and drying, slides are ready for use. Grace Bio-labs multiwell chambers are attached on top of the glass slide. It is secured in place using the slide on clips on each side (a). A solution containing the desired target is added to each well and incubated with rocking. Following incubation, each well is thoroughly washed to remove excess target, leaving behind only those adhered to the printed arrays. An image of an array of the live fungus C. albicans is shown as an example (b)

3.3. Isolation of Neutrophils from Human Blood

1. Many methods are available for isolating neutrophils from human peripheral blood including density gradient centrifugation and both positive and negative magnetic selection (e.g., see Chapters 3 and 4 of this volume). For our swarming assays, we isolate neutrophils from peripheral blood using negative selection with Direct EasySep Neutrophil Isolation kits. Isolations are done according to the manufacturer’s protocols.

2. Validating the functionality of neutrophils could be performed by checking their migration signatures in response to traditional chemoattractants (fMLF, LTB₄, etc.) [5]. If migration defects are observed, the neutrophil separation protocol can be validated by comparing the migration of isolated neutrophils with that of neutrophils in whole blood, using microfluidic devices designed to accommodate both whole blood and isolated neutrophils.

3.4. Neutrophil Swarming Assay

1. Place the slides with patterned microorganism arrays on an appropriate microscope for imaging. If multipoint functionality is available, select all points prior to the loading of neutrophils into the wells (see Note 5).

2. Add 5 × 10⁵ neutrophils per well. Typically, this is delivered as 200 μL of a cell suspension at a concentration of 2.5 × 10⁶ cells/mL.

3. Start the time-lapse imaging within seconds after adding the neutrophil suspension. Incubate the arrays at 37 °C for the duration of time-lapse imaging.

4. To track the dynamics of swarming over time, the area of individual swarms can be determined using the analysis software appropriate for the microscope used, or by using ImageJ. The addition of certain stains, such as Hoechst or SYTOX, also enables measurements of fluorescent intensity to be useful in examining swarming dynamics and function. We use Nikon TiE microscopes and therefore NIS-elements or FIJI for our area and fluorescent intensity analysis.

3.5. Swarming Area Quantification

For analysis of area, there are two main outputs: “area of the swarm” and “area of microbial growth.” For area of microbial growth, you will want to outline the area covered by the microbe as it grows throughout the time course (Fig. 3a–c). Identifying and quantifying the area can be done automatically or manually in ImageJ (see below). The irregular nature of microbial components such as fungal hyphae can make automated analysis inaccurate, so analysis of microbial growth can be done manually. For area of the swarm, you want to define the outlines of the swarm (Fig. 4). If neutrophils are stained with Hoechst, this fluorescent channel can be used to identify and outline the boundaries of area covered by neutrophils in the swarm (Fig. 4a, b). This can often be done automatically or manually in ImageJ.

Fig. 3.

Analysis of microbial growth. Individual spots in the arrays can be followed over time to determine the dynamics of microbial growth. Panels from a time course show the germination and growth of live C. albicans over time (a). To quantify this growth, the area can be determined (b, c). For filamentous fungi like C. albicans, irregular growths like hyphae should be included in the area of growth quantitation (b, right panels). Quantitation of C. albicans growth over time is shown. This quantitation follows 16 individual C. albicans spots over a 12 h time course. Error bars represent standard deviation

Fig. 4.

Analysis of swarming area. By adding neutrophils to the arrays of live microbes, the impact of swarming on microbial growth can be examined. Human neutrophils isolated from peripheral blood were added to arrays of live C. albicans to look at swarming. A representative sequences of images showing neutrophils (stained with Hoechst) swarming to C. albicans is shown (a). The dynamics of swarming can be tracked by determining the area of the swarm, using Hoechst fluorescence to identify just the neutrophils in the swarm (b, left panels). By examining microbial growth in the context of swarming and comparing to microbial growth without neutrophils, you can determine the impact of swarming on the microbe. To determine microbial growth, use the brightfield channel to identify any elements of microbial growth that have escaped beyond the area of the swarm and determine area as outlined previously (Fig. 3, Fig. 4b left panels). If the microbe expresses fluorescence, this can be combined with the brightfield images during quantitation to provide greater accuracy (see also Fig. 5). Quantitation showing the ability of neutrophils to restrict C. albicans growth is shown. The area of fungal growth seen during neutrophil swarming is overlaid on the data of C. albicans growing alone to illustrate the significant restriction mediated by neutrophils (c). N > 16 individual swarms per time point. Error bars represent standard deviation

1. Automated area analysis:

   1. Open your image file in ImageJ. Convert the image to 8-bit by opening the Image tab, going to “Type” and selecting 8-bit.
   2. Create a thresholded binary image. Go to the “Process” tab, go to “Binary” and select “Make Binary.”
   3. Analyze Area. Open the “Analysis” tab and select “Analyze Particles.” Enter the upper and lower size limits for what should be considered a swarm in your image. Check the “show outlines” and “display results” boxes before clicking OK. Data measurements will be in a new window that you can export as an Excel file and will include the area. A copy of your image with the swarms outlined will also appear. Be sure to check this image closely to ensure that swarms were accurately outlined.

The automated method outlined above works well for getting just the area of the swarm, but if swarms are irregular in shape or if you want to capture irregular microbial growth that is escaping the swarm, you may need to use manual outlining for analysis (Fig. 3).

1. Manual area analysis:

   1. Open your image in FIJI. Zoom in on the swarms to an appropriate level so you can accurately see all the details you wish to include in the analysis.
   2. Select the “Freehand Tool.” Click and hold to manually outline the area to be analyzed. Release once finished.
   3. Analyze area. Use the “Control” and “M” keys on the keyboard to measure the outlined area. Results will appear in a new window and can be exported or copy/pasted into excel.

2. Fluorescent Intensity Analysis

By including different fluorescent probes, you can measure many aspects of neutrophil function within a swarm (Fig. 5). Inclusion of SYTOX Green in the assay, for example, can allow the measurement of the release of neutrophil extracellular traps over time within the swarm (Fig. 5a, b). Furthermore, the use of microbial strains which constitutively express fluorescent proteins can allow you to use fluorescent intensity as a measure of microbial viability and growth (or killing) within the swarm (Fig. 5c, d).

Fig. 5.

Analysis of fluorescent intensity during swarming. In addition to area measurements, examination of fluorescent intensity can provide useful information of events during swarming. By incorporating fluorescent probes into the assay, such as SYTOX Green, you can use fluorescent intensity measurements to quantify dynamics within the swarm. In the case of SYTOX Green, you can monitor the release of DNA and NETs within the swarm. The fluorescent intensity and a selected panel of images of SYTOX Green staining throughout a time course are shown for a representative swarm (a). Using microbes that express fluorescent proteins allows the use of fluorescent intensity to track microbial growth (or killing) during swarming. Using C. albicans that expresses a far red fluorescent protein, fluorescent intensity was followed over time to examine microbial growth [6]. Fluorescent intensity of C. albicans was also followed after the addition of neutrophils to the swarming array, showing the restriction of fungal growth (b)

1. Open your image in ImageJ. Select the appropriate fluorescent channel. You will now need to define the area over which you want to measure the fluorescent intensity. This can be done using the “rectangle,” “oval,” “polygon,” or “freehand” tools (see Note 6).

2. Use the “Control” and “M” on the keyboard to measure the intensity in the defined area. Results will appear in a new window and can be exported to Excel.

4. Conclusion

Fully understanding neutrophil behaviors during infection represents a key barrier to harnessing these critical cells in the design of novel therapies against infectious disease. Neutrophil swarming represents a novel neutrophil function that is in the early stages of being characterized [2–4]. Neutrophil swarming may play a critical role in how neutrophils attack and contain the invasion of large pathogens like fungal hyphae and microbe clusters [1,3]. Microscale arrays present an important tool by which to interrogate neutrophil swarming in a high-throughput format, in molecular detail, in human cells [4]. Here, we provide a detailed protocol for the creation and use of microscale arrays of microbial targets, for use in neutrophil swarming experiments with human cells. While described here in the context of examining swarming in restricting infection, these arrays are well positioned to be leveraged to study neutrophil swarming in other situations, particularly in the context of inflammatory diseases where exuberant neutrophil responses could contribute to tissue damage and disease symptoms. Taken together, this technique can be utilized to provide significant insight into the process of neutrophil swarming in the context of infection and inflammation.