Neutrophils are innate immune cells and the first to be recruited to sites of injury, where they are essential for host defence. Defective neutrophil recruitment can inhibit pathogen clearance, while impaired neutrophil resolution can lead to chronic inflammation. Therefore, it is essential that neutrophil migration is tightly controlled and self-resolving. It was originally thought that neutrophilic inflammation was resolved through apoptosis. However, more recent studies have revealed that neutrophils can actively depart (reverse migrate) from target sites, which is believed to support resolution of the response.

Immune cell migration is regulated by chemoattractants, including the chemokine family. Chemokines bind to cognate G protein-coupled receptors to induce cell migration and are upregulated in many chronic inflammatory diseases characterised by aberrant immune cell migration. While chemokines represent an attractive therapeutic target, the development of drugs targeting them in chronic inflammatory diseases has been hampered by the complexity of the chemokine system. It is thought that a greater understanding of the context-specific roles of chemokines will aid identification of suitable targets for the treatment of chronic inflammatory diseases.

Here, using a zebrafish model of inflammation, I investigated the ligand preferences and subcellular trafficking of two chemokine receptors, Cxcr1 and Cxcr2, which were previously shown to mediate clustering and dispersal of neutrophils, respectively. I demonstrate that Cxcr1 recognises Cxcl8a and is rapidly internalised, whereas Cxcr2 recognises Cxcl8b and traffics in a manner consistent with recycling. These differences explain, in part, how Cxcr1 and Cxcr2 mediate opposing neutrophil behaviours, ensuring a balanced response. To investigate whether different spatial or temporal expression of the chemokines Cxcl8a and Cxcl8b also contributes to this balanced response, I explored CRISPR/Cas9 technology to generate a fluorescent reporter line of endogenous Cxcl8a expression. While I introduced mutations in the target gene, the knock-in approach was ultimately not successful, and I discuss possible approaches which could be applied in the future to achieve this technically challenging goal.

The observation that neutrophils can reverse migrate provided a potential new mechanism for the resolution of sterile inflammation. However, the role of reverse migration in the resolution of infected wounds remained unclear. By generating a neutrophil-specific photoconvertible line, I determined the fate of neutrophils recruited to sterile and infected wounds, finding that reverse migration plays an important role in the resolution of infected wounds. A potential regulator of neutrophil reverse migration is desensitisation of chemokine receptors. Previous work in our lab showed that desensitisation of Cxcr1 is required for neutrophils to stop and cluster. Combining a Cxcr1-desensitisation mutant with the photoconvertible line, I showed that receptor desensitisation is required for reverse migration. Finally, I sought to investigate the functional impacts of Cxcr1 desensitisation on wound healing and responses to secondary wounds and infections. I generated new protocols to study such impacts, which will form the basis of future studies with Cxcr1-desensitisation mutants.