Let-7 Regulation of Macrophage Differentiation and Inflammatory Response in Normal and Hyperglycaemic Conditions

Abstract

Macrophages orchestrate the transition between the key stages of wound healing through their ability to functionally differentiate or polarise towards different phenotypes, mainly pro- lnflammatory (M1) and pro-healing (M2) subtypes. Chronic wounds are characterised by an over- abundance of M1 macrophages that fail to polarise towards an M2 phenotype, particularly in chronic diabetic wounds. The let-7 family of microRNAs have been shown to modulate aspects of macrophage differentiation and behaviour in many biological processes. This project aims to understand the roles of specific let-7 family members in macrophage polarisation, and how differences in their expression and function in a hyperglycaemic or diabetic environment may be involved in the associated changes macrophage polarisation. In two macrophage models, bone marrow-derived macrophages (BMDM) and RAW.264.7 cells, let-7b is found to be the most significantly down-regulated let-7 in M1 macrophages cultured in high glucose media. Our functional studies on Let-7b revealed that its over expression is sufficient to drive macrophages towards an M2 phenotype as indicated by increased M2 and lowered M1 biomarker expression. Let-7b is shown to be involved in controlling aspects of TLR4/NF-kB signalling, the main pathway mediating M1 polarisation. Let-7b is shown to control TLR4, NF- kB (P65), and IL-6 at mRNA and protein levels. Let-7b was also found to be involved in TLR4 endocytosis, NF-kB phosphorylation and nuclear translocation, and direct targeting of Il-6 m- RNA. Moreover, let-7b is found to be involved in modulating a number of M1 behavioural characteristics, such as inhibiting autophagy and promoting phagocytosis. More specifically let-7b can mitigate the effects of hyperglycaemia on M1 inflammatory response and behaviour. This establishes let-7 as a potential therapeutic target for treating prolonged inflammatory conditions like chronic diabetic wounds.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Kimberly Mace (Supervisor) & Matthew Ronshaugen (Supervisor)