Wound healing is a complex process defined by three distinct yet overlapping stages: inflammation, proliferation and remodelling. Macrophages play a key role in all three stages, as they can switch between pro-inflammatory 'M1-like' states and anti-inflammatory 'M2-like' states. In approximately 4-10% of patients with diabetes, aberrant inflammation leads to the formation of nonhealing diabetic foot ulcers (DFUs). One of the reasons for DFU formation is a failure to switch between these M1 and M2 states. In this thesis, I aim to elucidate whether dysregulation within the toll-like receptor (TLR) signalling pathway plays a role in the formation of an excessive pro-inflammatory phenotype. An in vitro functional screen of TLRs in which macrophages were stimulated with the specific TLR agonists showed that diabetic macrophages express significantly higher levels of pro-inflammatory cytokines. However, the fold increase of cytokine production is much greater in non-diabetic macrophages. The expression levels of TLRs were not different between non-diabetic and diabetic macrophages. Next, non-diabetic and diabetic mice were given a bone marrow (BM) transplant of cells with a knockout of the TLR adaptor protein Myd88. Interestingly, although ndb mice with Myd88-/- BM healed wounds significantly slower than their counterparts with wild-type BM, there was no difference between the rate of wound healing in diabetic mice with wild-type BM and those with Myd88-/- BM, implying a significant role for Myd88- independent inflammatory signalling. Finally, ChIP-seq was used to examine the differences in NF-kB binding. It was discovered that NF-kB binds to DNA overwhelmingly in distal intergenic regions in diabetic macrophages compared with non-diabetic macrophages, suggesting dysregulation of chromatin conformation in diabetic macrophages. Together these results demonstrate that even though diabetic macrophages are inherently more pro-inflammatory, they fail to actively mount an inflammatory response in the same way as non-diabetic macrophages. This primarily seems to be because of great changes in the regulation of inflammatory transcription factor binding rather than aberrant TLR activity.
Date of Award | 1 Aug 2019 |
---|
Original language | English |
---|
Awarding Institution | - The University of Manchester
|
---|
Supervisor | Nicoletta Bobola (Supervisor) & Kimberly Mace (Supervisor) |
---|
- Macrophages
- Inflammation
- Wound healing
- Diabetes
- Toll-like receptor
Toll-Like Receptor Signalling In Diabetic Wound Healing
Harrison, R. (Author). 1 Aug 2019
Student thesis: Phd