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Neutrophils are rapidly recruited to inflammatory sites where their coordinated migration forms clusters, a process termed neutrophil swarming. The factors that modulate early stages of neutrophil swarming are not fully understood, requiring the development of new in vivo models. Using transgenic zebrafish larvae to study endogenous neutrophil migration in a tissue damage model, we demonstrate that neutrophil swarming is a conserved process in zebrafish immunity, sharing essential features with mammalian systems. We show that neutrophil swarms initially develop around an individual pioneer neutrophil. We observed the violent release of extracellular cytoplasmic and nuclear fragments by the pioneer and early swarming neutrophils. By combining in vitro and in vivo approaches to study essential components of neutrophil extracellular traps (NETs), we provide in-depth characterisation and high-resolution imaging of the composition and morphology of these release events. Using a photoconversion approach to track neutrophils within developing swarms, we identify that the fate of swarm-initiating pioneer neutrophils involves extracellular chromatin release and that the key NET components gasdermin, neutrophil elastase, and myeloperoxidase are required for the swarming process. Together our findings demonstrate that release of cellular components by pioneer neutrophils is an initial step in neutrophil swarming at sites of tissue injury.

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

Introduction and objectives Exposure to respiratory pathogens is a leading cause of exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). We have shown that Pellino-1, an E3 ubiquitin ligase, is involved in viral-induced TLR3 signalling; thus we sought to describe the role of Pellino-1 in the host response to viral stimulation of the airway. Methods Pellino-1 expression was examined in bronchial sections from patients with GOLD stage 2 COPD (n=7) and healthy controls (n=8). Primary bronchial epithelial cells (PBECs, n=5) in which Pellino-1 expression had been knocked down using siRNA, were extracellularly challenged with the TLR3 agonist poly(I:C). C57BL/6 Peli1-/- mice and wild type littermates were subjected to intranasal infection with the clinically-relevant respiratory viruses rhinovirus (RV1B) and influenza A, and responses monitored up to 8 days later. Results We show that Pellino-1 is expressed in the airways of normal subjects and those with COPD. In the absence of Pellino-1, PBECs showed significant reduction in proinflammatory cytokines, including CXCL8 and IL-6, upon TLR3 activation. Surprisingly however, knockout of Peli1 in the murine lung resulted in increased production of the proinflammatory cytokines IL-6 and TNFα with viral infection. This was accompanied by an enhanced recruitment of immune cells to the airways in these animals, including a population of innate B cells, without loss of viral replication. Conclusions We conclude that Pellino-1 functions as a positive regulator for antiviral responses in isolated bronchial epithelial cells and in systemic responses to TLR3 activation, but it has the opposite effect in the lung. Here, Pellino-1 has a distinct function in the down-regulation of antiviral inflammation; a role dissociated from viral replication. Our data therefore suggest that Pellino-1 may offer therapeutic potential to limit viral inflammation in COPD and asthma.

Between 2007 and 2014, 337 free-living red squirrels (