Endogenous pioneer neutrophils release NETs during the swarming response in 1 zebrafish 2

Neutrophils are rapidly recruited to inflammatory sites where they coordinate their migration 25 to form clusters, a process termed neutrophil swarming. The factors which modulate neutrophil 26 swarming during its early stages are not fully understood, requiring the development of new 27 in vivo models. Using transgenic zebrafish larvae to study endogenous neutrophil migration in 28 a tissue damage model, we demonstrate that neutrophil swarming is a conserved process in 29 zebrafish immunity, sharing essential features with mammalian systems. We show that 30 neutrophil swarms initially develop around a pioneer neutrophil, in a three-phase sequence of 31 events. By adopting a high-resolution confocal microscopy approach, we observed the release 32 of cell fragments by early swarming neutrophils. We developed a neutrophil specific histone 33 H2A transgenic reporter line TgBAC(mpx:GFP)i114;Tg(lyz:H2A-mCherry)sh530 to study 34 neutrophil extracellular traps (NETs), and found that endogenous neutrophils recruited to sites 35 of tissue damage released NETs at the start of the swarming process. The optical 36 transparency achieved using the zebrafish model has provided some of the highest resolution 37 imaging of NET release in vivo to date. Using a combination of transgenic reporter lines and 38 DNA intercalating agents, we demonstrate that pioneer neutrophils release extracellular traps 39 during the swarming response, suggesting that cell death signalling via NETosis might be 40 important in driving the swarming response. 41 prior


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A robust inflammatory response against invading pathogens or endogenous danger signals 45 requires the coordination of multiple cellular and humoral components. Neutrophils are one of 46 the first responders to tissue inflammation and rapidly home to inflamed tissue within hours of 47 injury. Within inflamed tissue, neutrophils destroy pathogens 1 and clear wound debris 2 , 48 ultimately leading to the restoration of tissue homeostasis. The anti-microbial repertoire of 49 neutrophils can cause substantial secondary tissue damage, thus neutrophil recruitment to, 50 and removal from, inflammatory sites must be tightly controlled.

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Neutrophils are recruited to sites of inflammation through a series of well-defined molecular 52 events 3-5 . During their recruitment, neutrophils are primed by pro-inflammatory signals 53 including growth factors, inflammatory cytokines and chemoattractants. Neutrophils are 54 capable of integrating host-and pathogen-derived environmental signals, resulting in their 55 polarisation and migration towards the initiating inflammatory stimulus 6 . Within the 56 interstitium, neutrophils coordinate their migration patterns to form clusters in several models 57 of sterile-inflammation and infection [7][8][9][10][11][12][13] . The parallels between these cellular behaviours and 58 migration patterns seen in insects has led to use of the term "swarming".

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A series of sequential phases leading to neutrophil swarming have been described in murine 60 models; the initial migration of 'pioneer' or neutrophils proximal to the wound site (scouting) is 61 followed by large scale synchronised migration of neutrophils from distant regions 62 (amplification) leading to neutrophil clustering (stabilisation) and eventual resolution 7-10 . The 63 initial arrest and death of early recruited pioneer neutrophils correlates with the onset of 64 neutrophil swarming 8,9,13 , which is mediated by many chemoattractants including lipid and 4 cytoplasmic proteins, which are able to capture and kill pathogens extracellularly 17 .

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Neutrophils release NETs following a series of intracellular changes resulting in chromatin 79 decondensation, breakdown of the nuclear envelope and mixing of DNA with granular and 80 cytoplasmic proteins 18

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The zebrafish (Danio rerio) is a powerful model organism in which to study neutrophil function 86 that has been used extensively to study neutrophil migration towards and away from sites of 87 sterile inflammation [19][20][21] . The optical transparency of zebrafish embryos allows for the tracking 88 of endogenous neutrophils at wound sites within minutes following injury in transgenic reporter 89 lines 22 . In this study, we use zebrafish larvae to study pioneer neutrophil behaviour prior to 90 the onset of swarming. We use both inflammation and infection assays to demonstrate that 91 neutrophil swarming is conserved in zebrafish immunity, indicating importance of this 92 neutrophil behaviour across evolution. We define a three-stage sequence of migration events 93 which leads to the swarming of endogenous neutrophils within the inflamed tissue and verify 94 that the neutrophil relay signal, LTB4, is required for amplification of neutrophil recruitment.

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Importantly, we show that a single pioneer neutrophil is sufficient to induce a swarming 96 response and that this neutrophil adopts a rounded, non-motile morphology distinct from other 97 neutrophils within the inflamed tissue. We develop a transgenic reporter for NETs and perform 98 live imaging of neutrophil extracellular trap release. We study pioneer neutrophil cell death 99 using cell viability assays and transgenic reporters for cell death, and identify that pioneer 100 neutrophils are viable prior to the onset of swarming, but die following the release extracellular 101 traps from within the swarm centre. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/521450 doi: bioRxiv preprint

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Neutrophil swarming is conserved in zebrafish immunity 106 Neutrophil swarming is characterised by the highly directed and coordinated movement of 107 neutrophils to sites of infection or injury followed by accumulation and clustering 23

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Persistent swarming was defined as the formation of clusters which grew by the coordinated 119 migration of individual neutrophils ( Figure 1C). Persistent swarms were observed from 40 120 minutes post injury (Supplemental Figure 3A) and remained stable for, on average, 2.17 hours 121 ± 0.32 (Supplemental Figure 3B). Persistent neutrophil swarms were observed in 50% of 122 larvae, transient swarms (persisting for <1 hour) were seen in 14% of larvae, and 36% of 123 larvae showed no evidence of swarming behaviour within the imaging period ( Figure 1D).

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In mammalian neutrophil swarming, biphasic neutrophil responses are modulated by the lipid 125 LTB4 8 . During the imaging period, two waves of neutrophil recruitment were observed: the 126 early migration of neutrophils proximal to the wound site between 0.5-2hpi, followed by a later 127 influx of neutrophils from more distant sites ( Figure 1E). We investigated the requirement for 128 LTB4 in neutrophil chemotaxis towards the wound site in zebrafish using the CRISPR/Cas9 129 system. Biosynthesis of LTB4 in zebrafish occurs through fatty acid metabolism of arachidonic 130 acid via common intermediates, resulting in the production of LTB4 by the enzyme leukotriene 6 larvae was significantly lower than control (tyr) crRNA injected larvae ( Figure 1F). These 140 results are in agreement with data from mouse 8 and human neutrophils 7 , supporting a 141 requirement for LTB4 signalling in neutrophil recruitment at the later stages.   The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/521450 doi: bioRxiv preprint a distinct morphology at the wound site prior to swarm formation, which is not seen in scouting 175 neutrophils responding to chemoattractants produced at the wound edge.

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A series of sequential phases leading to neutrophil swarming has been described in 178 mammalian systems 8,9 , so we next determined the stages leading to swarming in zebrafish.

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Although there was temporal variation from fish-to-fish, all swarms formed by: 1) the early

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To study these cell fragments, a high-resolution confocal microscopy approach was adopted   The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/521450 doi: bioRxiv preprint 9 To confirm these structures contained histones, a transgenic reporter line for neutrophil 248 histone H2A was generated to provide a cell-autonomous, intrinsically-expressed reporter of 249 NET release in vivo. A genetic construct containing histone H2A with a C-terminal fusion of 250 the fluorescent protein mCherry (H2A-mCherry), driven by the neutrophil specific lyz promoter 251 32,33 was generated using gateway cloning ( Figure 8A). The construct was introduced into the 252 genome of mpx:GFP larvae by Tol2 mediated transgenesis, and a stable was line generated:

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The H2A transgene was expressed by neutrophils ( Figure 8C), colocalising with the DNA stain 255 DAPI within neutrophil nuclei ( Figure 8D). The construct did not affect neutrophil migration to

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After determining that NETs were released by early swarming neutrophils, we next determined 266 whether pioneer neutrophils were amongst the NET releasing cells. The fate of pioneer 267 neutrophils was studied within developing swarms using a photo conversion approach.  injected into the mouse ear, we propose that just one pioneer neutrophil is sufficient to drive 300 a swarming response in our model.

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The single-cell resolution achieved in our study enabled us to study pioneer neutrophils with 303 optical clarity prior to the onset of swarming. Other groups have found that within inflamed or 304 infected interstitial tissue, the initial arrest of a small number of 'pioneer' or 'scouting'  The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/521450 doi: bioRxiv preprint 11 neutrophils call to other neutrophils for help as well as capturing pathogens by NETosis.

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Interestingly caspase-3 was intact during the swarm initiation phase, indicating that swarm 323 initiating pioneer neutrophils were not undergoing neutrophil apoptosis prior to swarming. Due 324 to the requirement for live imaging to study pioneer neutrophils prior to swarming, it was not 325 technically possible to confirm our apoptosis results using staining assays such as TUNEL.

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However, other studies have found that results using the mpx:FRET transgenic line 327 recapitulate TUNEL staining 29 , demonstrating this is a reliable way to read out neutrophil 328 apoptosis. These observations suggest that signals actively released from pioneer neutrophils 329 initiate swarming, rather than the bursting of neutrophils and release of DAMPs into the tissue.  The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/521450 doi: bioRxiv preprint recruitment to tail fin inflammation is bi-phasic; neutrophils proximal to the wound edge are 357 recruited within minutes following injury, whilst neutrophils from further away r ecruited 358 between 2-6 hours following injury. Using CRISPR/Cas9 to knock down lta4h and blt1, we 359 found that neutrophil responses were impaired only in the later stages of recruitment (3-6hpi).

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These findings are in keeping with data from human and murine neutrophils 7,8 . Neutrophil

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All procedures were performed on embryos less than 5.2 dpf which were therefore outside of 382 the Animals (Scientific Procedures) Act, to standards set by the UK Home Office.

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To induce an inflammatory response, zebrafish larvae at 2 or 3dpf were anaesthetised in

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The initiation stage is observed 58 minutes prior to swarming (rounded pioneer neutrophil).          The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/521450 doi: bioRxiv preprint 26 Tail fin nick was performed on 3dpf mpx:GFP larvae which were imaged using a 40X objective 881 lense on a perkin elmer spinning disk confocal microscope.  The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/521450 doi: bioRxiv preprint      The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/521450 doi: bioRxiv preprint