Tails of twists and turns: Using zebrafish to unravel neutrophil chromatin remodelling

Neutrophils are the most abundant leucocytes and the primary effector cells in innate inflammation. Because neutrophils play an important role in returning the body to homeostasis following immune challenge, neutrophil dysfunction is linked to chronic inflammatory diseases and age-related immune decline. Understanding mechanisms that control neutrophils can therefore help develop therapies for inflammatory dysfunction. The role of epigenetic modifications and specifically chromatin remodelling in neutrophils is only beginning to be explored. Recent work has shown that neutrophil response is regulated by chromatin changes, and further work has shown neutrophil function alters with ageing. We therefore, hypothesised that changes to chromatin conformation drive neutrophil dysfunction in ageing and disease. I aimed to use a zebrafish model to identify chromatin remodelling enzymes responsible for chromatin conformational changes regulating neutrophil function. Zebrafish provide an excellent model for neutrophil function as they can be easily chemically and genetically modified, have numerous transgenic reporters for immune function, and transparent larvae can be easily imaged. Here, the TLR8 agonist R848 – previously shown in vitro to exert its function on human neutrophils by chromatin remodelling at the il6 locus – was first shown to manipulate neutrophil function by promoting inflammation resolution in a tailfin injury model. R848 also promoted expression of the pro- inflammatory transcription factor NF-κB and cytokine TNFα. This provided evidence that zebrafish neutrophils could be manipulated by similar pathways to human neutrophils. CRISPR/Cas9 was then used to knockdown chromatin remodelling enzymes kdm6b and ezh2. In the tailfin injury model, knockdown of kdm6b resulted in a delay to inflammation resolution, while knockdown of ezh2 increased recruitment of neutrophils and delayed inflammation resolution. RNA sequencing was then performed following kdm6b knockdown. In the injury model, kdm6b knockdown showed a reduction in pro-inflammatory pathways and specifically reduction in expression of il1b and tnf. These data presented in this thesis suggest that Kdm6b plays a role in neutrophil response to injury and provides a target for exploration in neutrophil samples from aged individuals and chronic inflammatory disease patients for potential drivers of neutrophil dysfunction.