Wood, William

Henderson, Neil

Todd, Henry

2023-08-15T16:10:38Z

2023-08-15T16:10:38Z

2023-08-15

Inflammation is a complex, dynamic process, which despite extensive research, is still far from fully understood. The use of model organisms provides unique insights into the mechanisms underlying inflammatory cell recruitment. Previous work using the fruit fly Drosophila melanogaster has demonstrated that following laser-induced epithelial wounding, hydrogen peroxide is rapidly produced, and it is this early damage signal that leads to an inflammatory response, via direct activation of the redox sensitive tyrosine kinase Src42a within responding macrophages. Src42a phosphorylates Draper, the key damage receptor in fly macrophages, which in turn recruits a downstream kinase, Shark, resulting in macrophage recruitment to wounds. More recently, the phosphatase Pez has been demonstrated to interact with both Src42a and Draper and is required for efficient inflammatory macrophage recruitment to sites of damage in vivo. Importantly, macrophages are insensitive to tissue damage until they become ‘primed’, by engulfment of an apoptotic corpse, leading to an increase in Draper expression. Given macrophage insensitivity to tissue damage prior to priming, we utilised the tractable genetics of Drosophila and undertook RNA sequencing of both primed and un-primed cells in vivo, identifying many differentially expressed genes. Selected candidates were used in macrophage specific, RNAi knock down wounding studies, revealing novel players in the regulation of macrophage recruitment to wounds. The mechanism underlying Pez signalling within the macrophage is poorly understood; we utilised live imaging in Drosophila embryos to investigate Pez intracellular dynamics during inflammatory cell migration. Microtubule mediated transport and clathrin mediated endocytosis have been excluded, however, we show that dynamic Pez puncta correlate with retrograde actin flow providing a potential mechanism for the regulation of Pez trafficking in macrophages in vivo. Evolutionary conservation of Pez and Draper signalling in modulating wound induced, inflammatory cell migration has previously been demonstrated using zebrafish larvae in a tail fin transection model. Here, we show that the Pez homologue in mouse, PTPN21, and the murine Draper homologues, MEGF11 and PEAR1, play a role in blood monocyte and tissue resident macrophage population dynamics through the use of congenic CD45 bone marrow chimeric mice. Specifically, PEAR1 knock out leads to a decrease in circulating classical monocytes, whilst PTPN21, MEGF11 and PEAR1 knock out leads to a decrease in alveolar macrophage, but not peritoneal macrophage, numbers. The scope for inflammation research is almost unlimited and in order to further dissect the cellular processes that underlie the inflammatory response in vivo, model organisms must be utilised. Here, we have demonstrated that different model organisms can be exploited in a synergistic manner, facilitating experiments and answering questions that would be impossible to undertake using a single system.

https://hdl.handle.net/1842/40869

http://dx.doi.org/10.7488/era/3622

en

The University of Edinburgh

Campbell, Jennie S, Andrew J Davidson, Henry Todd, Frederico SLM Rodrigues, Abigail M Elliot, Jason J Early, David A Lyons, Yi Feng, and Will Wood. 2021. “PTPN21/Pez Is a Novel and Evolutionarily Conserved Key Regulator of Inflammation In Vivo.” Current Biology 31: 875-883.e5. https://doi.org/10.1016/j.cub.2020.11.014.

inflammatory cell migration

evolutionary conservation

Inflammation

Drosophila melanogaster

hydrogen peroxide

redox sensitive tyrosine kinase Src42a

Src42a phosphorylates Draper

phosphatase Pez

inflammatory macrophage recruitment

Pez signalling

Drosophila embryos

Microtubule mediated transport

clathrin mediated endocytosis

Investigating the evolutionary conservation of inflammatory cell migration in vivo

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy