The NF-κB pathway is a master regulator of inflammation and controls expression of many pro- and anti-inflammatory genes, including many known to act as chemokines. However, it is unclear whether NF-κB activity regulates migration of immune cells in response to inflammatory cues in vivo. Moreover, NF-κB signalling is important for the resolution of inflammation and the subsequent regeneration. The role of the NF-κB signalling in macrophage migration during inflammation and in regeneration was investigated. Caudal fin transections of zebrafish larvae were used to model injury and to test the importance of NF-κB activity for controlling macrophage migration. Timelapse imaging of a transcriptional reporter for NF-κΒ activity showed that the pathway is active in macrophages responding to injury. Gene expression analysis revealed a general activation of NF-κB target genes in the wounded tissue including il-1β, tnfα, ikbaa and ikbab. To assess the requirement for NF-κB activity during macrophage responses to injury small molecule inhibitors were used and macrophages observed by time-lapsed microscopy. NF-κB pathway inhibition in the context of injury lead to down-regulation of direct pathway targets and to an unexpected increase in the directionality and speed of macrophages. Additionally, changes to biphasic cytokine expression were observed, including an early up regulation of anti-inflammatory cytokines. Altered macrophage behaviour was also observed in the tnfα receptor 1 mutant (tnfr1) suggesting that TNFα and NF-κB interact to regulate macrophage migration. A dominant active NF-κB inhibitor IκBα (IKBASR) acts to inhibit NF-κB activity and when expressed in macrophages revealed a cell autonomous requirement for NF-κB in controlling macrophage migration to injured tissue. The role of TNF and NF-κB signalling in caudal fin regeneration were also evaluated. Elevated blastema proliferation was observed in response to inhibition of NF-κB at early stages of regeneration. This was correlated with an overall delay in regeneration of the fin in tnfr1 mutant larvae and larvae with IKBASR globally expressed suggesting that effective fin regeneration is dependent on NF-κB activity. Moreover, a cell autonomous requirement for the NF-κB signalling in macrophages for regeneration was found. In order to measure NF-κB activity in real time Förster Resonance Energy Transfer (FRET) reporters for RelA and IκBα were generated. Fluorescent Lifetime Imaging Microscopy (FLIM) was used to measure the association of the reporters, and to confirm the activity of the pathway in cultured human cells and in zebrafish larvae. Overall my results suggest that the NF-κB/TNFα signalling acts to fine-tune the migratory response of macrophages to injury, and subsequently regeneration. This active involvement of the NF-κB pathway in the macrophage migratory behaviour is very important in understanding dynamics of the inflammatory response and how this might be perturbed in diseases that involve acute or chronic inflammation.
Directional macrophage migration in response to injury is regulated by NF-κB signalling
Arkoudi, K. (Author). 1 Feb 2020
Student thesis: Doctoral Thesis › Doctor of Philosophy