Dissecting the molecular mechanisms of neural–fibroblast interactions in wound healing

The skin and other epithelial surfaces such as the oral mucosa play a critical role as a barrier to pathogens and other environmental insults. These surfaces are vulnerable to injury, necessitating efficient wound healing responses, which can be affected by many factors including diseases such as diabetes, as well as cancer therapy and age. Defective wound healing is a major cause of ill health and poor quality of life, and poses a huge economic burden on the NHS. Peripheral neuropathy, specifically the loss of neurons from the connective tissue underlying epithelial barriers, is strongly implicated in defective wound healing. However, the mechanisms underlying this remain poorly understood.

We have evidence that fibroblasts are stimulated to differentiate into myofibroblasts – a process critical for effective wound healing – by signals generated by sensory neurons. We have also observed that isolated primary neurons are activated and stimulated to form neurites by myofibroblast-derived signals. In this project, we propose to comprehensively characterise the interactions between sensory neurons and fibroblasts, with the aim of elucidating fundamental mechanisms underlying the influence of neurons on wound healing and diseases such as cancer.

We will test the hypothesis that neural–fibroblast neuronal interactions are necessary for effective wound healing. The following aims will be addressed:

Aim 1. Generate trigeminal ganglion (TG) neuronal cultures and characterise the response of fibroblasts to TG-derived factors. This will be achieved using techniques including RNAseq, qPCR, western blot and immunocytochemistry for known myofibroblast markers (eg α smooth muscle actin), and bioinformatic analyses.

Aim 2. Determine which cells in the TG are involved in cross-talk with fibroblasts. The cellular composition of TG cultures will be assessed by immunocytochemistry and FACS using markers of relevant cell types (eg neuronal, glial, smooth muscle and endothelial cells) and responses to fibroblast-derived signals assessed by calcium flux imaging, capitalising on the expertise of the co-supervisor Anton Nikolaev, RNAseq (as described in Aim 1), and neurite outgrowth assessed by microscopy.

Aim 3. Assess the influence of neural–fibroblast interactions on wound healing in vivo. We have preliminary evidence of defective wound healing in vivo in an experimental model of denervation. Here, we will extend these findings to assess the abundance and phenotype of fibroblasts in innervated and denervated wounds. In addition, we will examine the effect of neuronal factors on wound healing using fibrin glue containing neuronal mediators and specific inhibitors previously optimised in vivo in Prof Boissonade’s laboratory.

Expected outcomes: This project will generate insight into the roles of neural–fibroblast interactions in the context of wound healing, and identify molecular pathways that may be amenable to therapeutic intervention. In addition, the potential of neuronal mediators as pro-regenerative agents will be determined, providing a platform for subsequent translational studies.