Neutrophils collectively migrate to sites of injury and infection. How these swarms are coordinated to ensure the proper level of recruitment is unknown. Using an ex vivo model of infection, we show that human neutrophil swarming is organized by multiple pulsatile chemoattractant waves. These waves propagate through active relay in which stimulated neutrophils trigger their neighbors to release additional swarming cues. Unlike canonical active relays, we find these waves to be self-terminating, limiting the spatial range of cell recruitment. We identify an NADPH-oxidase-based negative feedback loop that is needed for this self-terminating behavior. We observe near-constant levels of neutrophil recruitment over a wide range of starting conditions, revealing surprising robustness in the swarming process. This homeostatic control is achieved by larger and more numerous swarming waves at lower cell densities. We link defective wave termination to a broken recruitment homeostat in the context of human chronic granulomatous disease.

Neutrophil Isolation Protocol:

Imaging media was first prepared with RPMI (w/o Phenol Red, 25mM HEPES, L-Glutamate) and 0.4% Human Serum Albumin (HSA) (Sigma Cat: A5843). HSA was added directly to RPMI and then centrifuged at 500xg for 5 min until fully dissolved. The mix was then filtered with a .22 μm Steriflip filter (Millipore Sigma: SE1M179M6) before further use. Imaging media was always prepared fresh on the same day of imaging.

Blood specimens from patients were obtained with informed consent according to the institutional review board-approved study protocol at the University of California - San Francisco (Study #21-35147). Fresh samples of peripheral blood (2 tubes, 7 mL each) from healthy adult volunteers were collected via a BD 23-gauge butterfly needle collection set (SKU: 23-021-022) into 10 ml BD Vacutainer EDTA tubes (SKU:366643). Volunteers were informed not to take ibuprofen, acetaminophen, or more than one drink of alcohol within 24 hrs, class 1 or 2 antihistamines within 5 days, or aspirin within 7 days of blood draw. Blood was kept on a shaker at minimum setting and utilized within 2 hours of the draw. Neutrophils were isolated using the Stemcell EasySep Direct Human Neutrophil Isolation Kit (#19666) with the BigEasy magnet (#18001) according to the manufacturer’s protocol.

Isolated neutrophils were spun down at 200xg for 5 min and then resuspended in a dye media consisting of imaging media plus 5 μg/ml Hoechst 3334 (Invitrogen, Cat:H3570), and 1 μM CalBryte 520 AM (AAT Bioquest, Cat: 20650). This cell suspension was incubated at room temperature in the dark for 15 min, and then spun down at 200xg for 5 min. The dye medium was aspirated and replaced with an amount of cell culture media of RPMI (Gibco) with 10% heat inactivated FBS (Gibco) needed to achieve a final cell density at or below 1x10⁶ cells/mL. Purified neutrophils were then kept in polystyrene T25 flasks at 37°C in a 5% CO2 environment until imaging. Cells were allowed to incubate for at least one hour before imaging began and not more than 5 hours after isolation. Allowing the cells to rest in culture before imaging helped ensure that Ca²⁺ signaling was more consistent and less noisy across volunteers.

CGD Cell Isolation:

For CGD cells use in this work, the following changes to the above protocol were made. First, blood specimens were drawn from patients at the NIH campus for either control healthy donors or donors verified to have CGD. Samples were collected into 10ml BD Vacutainer EDTA tubes, and blood was shipped overnight in a temperature-controlled box using Phase 22 room temperature packs. Both CGD and healthy control blood were shipped together with each run to control for effects of shipping blood overnight to MGH. Blood was isolated in the same method as above as soon as it arrived in lab at around 10:30 am each day experiments were performed. Cells were not used more than 5 hours after isolation, and all accumulation experiments took place within 2 hours post isolation.

Neutrophil Swarming Chip Protocol:

Swarming arrays of C. albicans were prepared as described previously, with modifications (6, 7). Briefly, we used a microprinting platform (Picospotter PolyPico Galway, Ireland) to print a solution of 0.1% poly-l-lysine (Sigma-Aldrich) with ZETAG 8185. For experiments, we printed arrays with either 1.0 mm or 1.5 mm spacing as indicated in an 8 well format on full sized No. 1.5H glass coverslips (Ibidi, Cat # 10812). Coverslips were dried and then left at room temperature until required. To attach an infection-like material to these arrays, 8-well sticky-Slide attachments (Ibidi, Cat # 80828) were overlaid on the printed arrays. An overnight culture of live C. albicans yeast was heat killed at 90°C for 20 minutes before being washed and re-suspended in dH2O, then 750 µL of this suspension was added to each well and incubated for 5 minutes. Following incubation, wells were thoroughly washed out with dH₂O to remove unbound targets from the glass surface. Wells were screened to ensure appropriate patterning of targets onto the spots with minimal non-specific binding before use. The well attachment was then removed, and the coverslips were stored at 4°C until ready for use.

When preparing to use a patterned coverslip, the coverslip was first re-inspected for coating and target integrity. An Ibidi 8 well sticky-slide (Cat: 80828) was pressed firmly on to the slide, and a pipette tip was run along the bottom to ensure a proper seal was formed. The coverslip-well combo was then incubated in a 37°C oven overnight. Next a 200 µL mixture of imaging media plus 15 µg/ml Fibronectin from human plasma (Sigma, Cat: F0895) was pipetted into each well that was to be used that day. The slide was then incubated for 30 min at 37°C and washed 3x with 200 µL/well of PBS (-/- Ca/Mg). The final wash of PBS was left on the well until imaging.

Neutrophil Swarming Imaging Protocol:

Cells were imaged as follows except when using CGD cells (See below). Neutrophils in culture were taken and placed into Fisherbrand LowRetention 1.5mL microcentrifuge tubes (Cat: 02681320) and spun down at 200xg for 5 min. Cells were resuspended at variable concentrations ranging from 3x10⁶ - 10x10⁶ cells/mL in a freshly made solution of imaging media. These cell solutions were then allowed to rest at room temperature for 15 min. When the wait time had elapsed, the wash PBS was taken off the well to be imaged and 200 µL of the neutrophil solution pipetted into the well. Imaging began as soon as imaging conditions could be verified after placing the cells into the well. Most confocal microscopy data was collected using a Nikon Ti2-E body scope configured with a CrestOptics X-Light V2 confocal spinning disk system, a Lumencor Celesta light engine, Nikon 10x CFI Plan Apo Lambda objective, an Okobox temperature and CO₂ controlled environment, and a Photometrics Prime 95B sCMOS camera. Revision confocal microscopy data was collected using a Nikon Ti2-E body scope configured with a CrestOptics X-Light V3 confocal spinning disk system, a Lumencor Celesta light engine, Nikon 10x CFI Plan Apo Lambda objective, an Okobox temperature and CO₂ controlled environment, and a Photometrics Kinetix sCMOS camera. All data was taken using the same levels of laser power from the 405 nm and 488 nm. The camera was run in a 2x2 binning mode with a set exposure time of 200 ms for all channels. All movies are taken with a frame interval of 5 seconds between exposures unless otherwise indicated. All movies were taken at 37°C with 5% CO2 for the duration of imaging.

Where indicated, this protocol was modified as follows to add the inhibitors used in this study. For the LTB4 inhibitor BIIL315 (Boehringer Ingelheim via opnMe), the drug first resuspended in DMSO at a stock concentration of 10 mM. The stock was then diluted in resuspension media (RPMI+0.4%HSA) to a final concentration of 1 µM before resuspending cells in the final step for imaging sample preparation. Cells were incubated in this drug-media solution for 15 min before starting the experiment. The drug was kept in solution for the duration of imaging. For Diphenyleneiodonium chloride, or DPI (MedChemExpress, Cat: HY-100965), the drug was bought in a premixed solution of DMSO at a stock concentration of 10 mM. The drug was diluted to a final concentration of 50 µM in RPMI + 0.4% HSA before resuspending cells in the final step for imaging sample preparation. Cells were incubated in this drug-media solution for 15 min before starting the experiment. The drug was kept in solution for the duration of imaging. For SOD + Catalase, each lyophilized protein was separately suspended in imaging media (RPMI + 0.4% HSA) and allowed to dissolve. Aliquots were made and stored at -80C. Before imaging, aliquots were thawed on ice and diluted in imaging media to a final concentration of 200 U/mL for SOD (Sigma Cat #9636-1KU), >200U/mL for Catalase (Sigma Cat #219008-1MG). Since Catalase was provided with only a range of specific activity (>50,000 U/mL), the final concentration in the swarming assay was 4.27ug/mL to give an activity range of >213U/mL. Cells were incubated in this protein containing media solution for 15 min before starting the experiment. The proteins were kept in solution for the duration of imaging.

CGD Donor Cell Accumulation Imaging:

Neutrophils in culture were taken and placed into 1.5 mL microcentrifuge tubes and spun down at 200xg for 5 min. Cells were resuspended at variable concentrations ranging from 3x10⁶ - 10x10⁶ cells/mL in a freshly made solution of imaging media. These cell solutions were then allowed to rest at room temperature for 15 min. When the wait time had elapsed, the wash PBS was taken off the well to be imaged and 200 µL of the neutrophil solution placed in the well. End point accumulation imaging was performed 60 min after the wells were seeded with cells. Images were collected using a widefield florescence Nikon Ti-E body scope configured with a Nikon 10x Plan Flour objective equipped with a 37°C temperature and 5% CO2 environmental chamber.

CGD Donor Calcium Imaging:

Neutrophils in culture were taken and placed into 1.5 mL microcentrifuge tubes and spun down at 200xg for 5 min. To avoid cell activation, cells were resuspended in the same media they were cultured in, RPMI + 10% FBS. These cells were left to rest for 15 min at room temperature, and then added to the well and imaged immediately. Timelapse imaging was performed on a Nikon Ti-E body widefield florescence scope, with a 10x Nikon objective, and wells were inside a stage top 37°C incubation unit.

Image Analysis:

All image analysis presented in this work was achieved via the use of both Fiji (ImageJ) and Python. Images were pre-processed when needed to split one field of view into 4 quadrant ROIs to simplify the downstream Python analysis. All python analysis for each graph is provided as open-source code in the form of both scripts and Jupyter notebooks on Github (https://github.com/strickland-ev/swarming-self-extinguishing-relay-publication). A brief description of each data analysis tool used is provided in plain text below. The raw data for all graphs generated will be provided as a zip file on Box or the publisher website. We also intend to publish our raw data, code, and all files that went into making these figures and movies on Zenodo or comparable platform at the time of publication.

ARCOS Wave Tracking:

See code posted on Github for a more direct explanation of the methods used and the code used for each figure. Broadly, to track cells trackpy was used to link frames where nuclei were identified via a custom StarDist model. The ARCOS algorithm was used to group like calcium events. Further analysis of cell tracks was done via custom python code included in the Github jupyter notebooks.