Neutrophils, the first-responder cells of innate immunity, converge on sites of tissue injury and infection through swarming, a self-amplifying collective migration in which early-arriving cells release signals that recruit hundreds to thousands of followers into a cluster around the insult. This problem asks for the complete control circuit of swarming in living tissue: the molecular signals that initiate swarm formation, the relay signal that amplifies it, the signals that terminate it, and the rule by which swarm size is scaled to the magnitude of the insult. The relay amplifier (leukotriene B4) is established, and several partial start and stop mechanisms are known - phospholipase cPLA2-derived arachidonic acid for initiation and scaling, GRK2-mediated receptor desensitization and an NADPH-oxidase negative feedback for termination, and lipoxin A4 and resolvin E3 as candidate but unconfirmed stop signals - but no integrated, in-vivo-validated account exists. Closing this gap would establish the feedback-control logic by which the innate immune system sizes local inflammatory responses to match the threat while limiting collateral tissue damage.

Background

Neutrophils are the most abundant circulating white blood cells in mammals and the first cells recruited into injured or infected tissue. Beyond individual chemotaxis (directed movement along a chemical gradient), neutrophils in tissue engage in swarming: a collective migration behavior in which one or a few pioneer cells that contact a damage or microbial target release intercellular signals that recruit, amplify, and coordinate hundreds to thousands of additional neutrophils into a dense cluster around the insult. Swarming has been observed by intravital microscopy (direct imaging of cells inside living tissue) in many organs, across sterile injury and infection with bacteria, fungi, and viruses; it improves microbial containment but risks collateral tissue damage, so both its amplification and its shutdown must be actively controlled.

The relay signal is the best-understood circuit element. Leukotriene B4 (LTB4LTB_4), a lipid chemoattractant released by activated neutrophils and sensed by the G-protein-coupled receptor BLT1 on neighboring cells, is required for swarm amplification in vivo (Lämmermann et al. 2013, mouse); notably, swarm initiation at sites of cell death was LTB4LTB_4-independent, indicating a separate start signal. Profiling of synchronized human swarms on microscale arrays (Reátegui et al. 2017) confirmed LTB4LTB_4-driven amplification and revealed temporally ordered production of other lipid mediators, among which lipoxin A4 (LXA4LXA_4) and resolvin E3 (RvE3RvE_3), members of the specialized pro-resolving mediator (SPM) family of inflammation-terminating lipids, emerged as candidate stop signals: exogenous LXA4LXA_4 slows recruitment and shrinks swarms in vitro, but an endogenous stop-signal role has never been confirmed.

Termination is partially dissected into two programs that operate on different axes. In vivo (mouse lymph node), desensitization of the chemoattractant G-protein-coupled receptors (GPCRs) by GPCR kinase 2 (GRK2) shuts swarm signaling off in time; neutrophils lacking GRK2 fail to self-limit swarming and clear bacteria less well (Kienle et al. 2021). Ex vivo (human blood on defined microscale targets), swarming proceeds through pulsatile LTB4LTB_4 relay waves that are self-extinguishing: an NADPH-oxidase (NOX2, the phagocyte oxidase) negative feedback limits calcium influx and LTB4LTB_4 production, controlling the spatial range of recruitment, and patients with chronic granulomatous disease, who lack NOX2, show defective wave termination (Strickland et al. 2024). How these two programs relate to each other and to the candidate SPM stop signals is unknown.

Initiation and scaling have recent but incomplete answers. In the ex vivo human system, swarm initiation requires activation of the mechanosensitive phospholipase cPLA2 by two coincident inputs, calcium influx upon pathogen contact and nuclear deformation as the cell spreads along fungal hyphae, and its product arachidonic acid (AA, the 20-carbon fatty-acid substrate of all eicosanoid lipid mediators) is necessary and sufficient for swarming, with AA availability regulating swarm magnitude; Strickland et al. (2026, preprint) propose that neutrophils share AA across pathogen-engaged cells to collectively assess infection magnitude. Whether this substrate-availability logic scales swarm size to insult size in living tissue, and how it composes with the near-constant total recruitment observed across starting cell densities (a homeostatic property of the relay-wave system), is unresolved. Initiation in vivo also involves tissue-side licensing signals, for example epithelial thrombospondin-1 activating latent TGF-β1, which engages leukotriene synthesis by neutrophil 5-lipoxygenase (the enzyme that converts arachidonic acid into leukotrienes such as LTB4LTB_4) and clustering in a urinary tract infection model (Widera et al. 2025), and swarms can nucleate on a single dying neutrophil in infected airway epithelial cultures (Robinson et al. 2023).

Problem Statement

Identify and integrate the molecular control circuit that governs neutrophil swarming in living tissue. A complete answer must: (i) establish the molecular identity and regulation of the initiation signal or signals, including the proposed cPLA2/arachidonic-acid substrate-availability logic, tissue-derived licensing cues such as epithelial thrombospondin-1/TGF-β1, and the danger-signal nucleation of swarms at dying cells; (ii) account for the established LTB4LTB_4 relay amplification and how it composes with the initiation and termination elements of the circuit; (iii) identify the termination signal or signals and determine how the known and candidate stop programs, namely GRK2-mediated GPCR desensitization, the NADPH-oxidase negative feedback, and specialized pro-resolving mediators such as LXA4LXA_4 and RvE3RvE_3, relate to one another and jointly extinguish a swarm, including whether the candidate SPM stop signals act as endogenous terminators in vivo; and (iv) establish how swarm size (radius and recruited cell number) is scaled to the magnitude and nature of the insult, including whether arachidonic-acid availability and its sharing across insult-engaged cells implement this scaling and how this composes with the near-constant (homeostatic) total recruitment observed across starting cell densities. The deliverable is a coherent account of start, relay, stop, and scaling. As in the cited sources, the question is posed at species- and context-generality (human and mouse; sterile injury and fungal, bacterial, and viral infection), and evidence from in vivo, ex vivo, and in vitro systems alike bears on it. A complete answer is one coherent integrated account of start, relay, stop, and scaling assembled from that evidence, and is not required to include new primary in vivo validation: for each element it must state what is established, what is proposed, and the decisive open test, and the mechanisms named above must all be addressed without being an exhaustive list of what the general circuit may contain.

References

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