Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses.

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Neutrophil Swarming in Damaged Tissue Is Orchestrated by Connexins and Cooperative Calcium Alarm Signals.

Neutrophils are major inflammatory cells that rapidly infiltrate wounds to provide antimicrobial functions. Within the damaged tissue, neutrophil migration behavior often switches from exploratory patrolling to coordinated swarming, giving rise to dense clusters that further disrupt tissue architecture. This aggregation response is self-organized by neutrophil paracrine chemoattractant signaling (most notably of the inflammatory mediator leukotriene B4 [LTB4]). The coordination mechanism and possible evolutionary benefits of neutrophil swarms are elusive. Here, we show that neutrophil swarms require mutual reinforcement of damage signaling at the wound core. New biosensors and live imaging in zebrafish revealed that neutrophil chemoattractant synthesis is triggered by a sustained calcium flux upon contact with necrotic tissue that requires sensing of the damage signal ATP. This "calcium alarm" signal rapidly propagates in the nascent neutrophil cluster in a contact-dependent manner via connexin-43 (Cx43) hemichannels, which are mediators of active ATP release. This enhances chemoattractant biosynthesis in the growing cluster, which is instrumental for coordinated motion and swarming. Inhibition of neutrophil Cx43 compromises clearance of wound-colonizing P. aeruginosa bacteria and exacerbates infection-induced morbidity. Thus, cooperative production of alarm signals among pioneer clustering neutrophils fuels the growth of dense antimicrobial cell masses that effectively seal off breached tissue barriers from opportunistic pathogens.

Author Correction: Chemokine receptor trafficking coordinates neutrophil clustering and dispersal at wounds in zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chemokine receptor trafficking coordinates neutrophil clustering and dispersal at wounds in zebrafish.

Immune cells congregate at specific loci to fight infections during inflammatory responses, a process that must be transient and self-resolving. Cell dispersal promotes resolution, but it remains unclear how transition from clustering to dispersal is regulated. Here we show, using quantitative live imaging in zebrafish, that differential ligand-induced trafficking of chemokine receptors such as Cxcr1 and Cxcr2 orchestrates the state of neutrophil congregation at sites of tissue damage. Through receptor mutagenesis and biosensors, we show that Cxcr1 promotes clustering at wound sites, but is promptly desensitized and internalized, which prevents excess congregation. By contrast, Cxcr2 promotes bidirectional motility and is sustained at the plasma membrane. Persistent plasma membrane residence of Cxcr2 prolongs downstream signaling and is required for sustained exploratory motion conducive to dispersal. Thus, differential trafficking of two chemokine receptors allows coordination of antagonistic cell behaviors, promoting a self-resolving migratory response.