Novel signalling pathways resolving the inflammatory response during peripheral nerve regeneration

Abstract

Introduction. The peripheral nervous system has the remarkable capacity to regenerate and restore function following damage or disease. Instrumental in this regenerative process are macrophages, Schwann cells and the production of inflammatory cytokines. The production of inflammatory cytokines from macrophages and Schwann cells needs to be tightly regulated to ensure efficient regeneration and to prevent chronic inflammation. Currently there is limited knowledge regarding which signalling pathways act to control both the inflammatory response and macrophage efflux that allows tissue homeostasis to resume within the peripheral nerves. This thesis aims to investigate the expression pattern and distinct function of VIP and PACAP and their receptors VPAC1, VPAC2 and PAC1 in the peripheral nerve post injury and how signalling through these receptors can act to promote resolution of the inflammatory response. Once macrophages have executed their function, they are required to leave the peripheral nerve to prevent chronic inflammation. There is only one macrophage efflux mechanism currently described in the peripheral nerves, where the NOGO receptors interacts with myelin to promote macrophage clearance. EphA5 has been shown to be upregulated in various RNA sequencing data sets and can induce contact dependent migratory behaviour within cells. EphA5-EphrinA signalling as a potential mechanism to induce macrophage migration and efflux out of the peripheral nerve was investigated. Methods. This project uses a combination of in vitro, ex vivo and in vivo models, including primary macrophage and Schwann cell cultures and mouse sciatic nerve cut and crush injury models to establish the function of VIP and PACAP and EphA5-EphrinA signalling at various time points throughout peripheral nerve regeneration. Results. My results show VPAC1, VPAC2, and PAC1 to be up-regulated in the mouse distal nerve following peripheral nerve injury and are highly expressed in Schwann cells and macrophages within the distal sciatic nerve. VIP and PACAP can act on cultured rat Schwann cells, to promote myelin gene expression but can also inhibit the release of pro-inflammatory cytokines by Schwann cells. In addition to this, we show VIP and PACAP can act through macrophages to inhibit pro-inflammatory cytokine expression. EphA5 and EphrinA ligands expression is also upregulated after injury. EphA5 is expressed from macrophages and Schwann cells in the distal nerve stump and EphA5 global knock out (KO) leads to increased macrophage numbers in the distal nerve stump up to 60 days post injury and a decrease in axon regeneration into the nerve bridge. Additionally, migration assays further showed Schwann cell and macrophage migration to be decreased upon inhibition of EphA5 in vitro. Finally, following a crush injury, EphA5 KO mice have a slower recovery of sensory and motor function and decreased g-ratios at 28DPI suggesting a role for EphA5-dependent signalling in functional recovery. Conclusions. This data provides evidence that VIP and PACAP have important functions in the distal nerve stump following injury to promote remyelination and regulate the inflammatory response. Consequently, VIP and PACAP receptors represent important potential targets to promote peripheral nerve repair following injury. In addition, my results also shows a novel function for the upregulated expression of EphA5 and EphrinA in injured peripheral nerves. EphA5 KO mice revealed that EphA5 signalling is important for both macrophage and Schwann cell migratory function following injury. EphA signalling through EphA5 expressed on macrophages is revealed to be an important signalling pathway for macrophage efflux out of the peripheral nerve following peripheral nerve injury, whereas EphA5 signaliing through Schwann cells could act to promote Schwann cell elongation and migration into the nerve bridge.