Protein Transduction of Hoxa3 Transcription Factor to Regulate Diabetic Macrophage Differentiation

Abstract

Protein Transduction of Hoxa3 Transcription Factor to Regulate Diabetic Macrophage Differentiation Diabetes is a metabolic condition that has a significant economic and social burden for people across the world. Among the complications associated with the increasing rate of diabetes is delayed wound healing that often leads to non-traumatic limb amputations. Macrophages play a vital role in wound healing because of their functional mechanistic changes at the wound site from the time of injury. Delay in wound healing may occur as chronic systemic inflammation promotes defects in macrophages via chromatin modifications. These defects pre-program macrophages to a pro-inflammatory phenotype, while the local wound environment inhibits a pro-healing phenotype. Previous work from our lab found that transient transfection of a Hoxa3 gene expression vector to treat murine diabetic wounds helps to maintain a balance between inflammatory and pro-healing cells in the wound, hence decreasing inflammation and promoting the phenotypic switch to wound healing. This thesis focuses on bringing additional understanding of the link between diabetes and the hyper activation of macrophages towards M1/pro-inflammatory phenotype. In vitro culture was used to characterize intrinsic changes to human and murine diabetic macrophages and to assess the impact of Hoxa3 treatment on diabetic macrophages as well as how Hoxa3 transcription factor function in macrophages may contribute to restoring wound healing in vivo. Previous studies from this lab and others showed that in vitro cultured diabetic macrophages show persistent classical activation and may contribute to inflammation in the diabetic wound. Therefore, this research aimed at understanding different epigenetic changes, particularly to histone modification enzymes, that might contribute towards dysregulated macrophages and delayed wound healing. Here data is presented from murine macrophages showing HDAC and HAT enzymes are elevated in levels and activity at the mRNA level and protein level, respectively. The data also uncovers alterations in NF-kB p65 activity in diabetic macrophages, with diminished early response and heightened late response. Utilisation of purified Hoxa3 protein to treat diabetic macrophages shows enhanced macrophage maturation and diminished classical activation responses. This investigation uncovers potential underlying mechanisms of Hoxa3 to rescue diabetic macrophage defects using RNA sequencing, qRT-PCR, and protein analysis. Our data demonstrate that Hoxa3 regulates the activity of NF-kB p65 and works as an inhibitor for upregulated HAT and HDAC enzymes, which may be associated with a decrease in the production of inflammatory cytokines. In vivo examination investigating the effect of Hoxa3 protein transduction in diabetic wounds shows that Hoxa3 accelerates wound healing. Altogether, the unique role of Hoxa3 in rescuing phenotypic defects of diabetic macrophages thereby provides a better understanding of the significance and mechanisms employed by Hoxa3 in regulating macrophage dysregulation in diabetes.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Karel Dorey (Supervisor) & Kimberly Mace (Supervisor)