Toll-Like Receptor Signalling In Diabetic Wound Healing

UoM administered thesis: Phd

  • Authors:
  • Robert Harrison

Abstract

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.

Details

Original languageEnglish
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Award date1 Aug 2019