The Role of Burn Tissue and Denatured Small Leucine Rich Proteoglycans in the Activation of the Toll
University of Alberta
INTRODUCTION & PURPOSE: Hypertrophic scar (HTS), a common and significant consequence of burn injury to deep dermis with prolonged inflammation, causes reduced range of motion, intense pruritis, heat intolerance, and cosmetic problems. It does not respond well to current treatment options. Activation of toll-like receptor 4 (TLR4), a proinflammatory pathway, has been suggested to be associated with HTS by responding to extracellular matrix (ECM) and endogenous cellular ligands to promote inflammation. Small leucine-rich proteoglycans (SLRPs) are molecules involved in wound healing that modify the ECM by altering fibroblast proliferation, collagen organization, and growth factors. We hypothesized that endogenous molecules released from damaged burn tissue could activate the TLR4 pathway in dermal cells. In this study, we determined the role of burn tissue and denatured SLRPs in the stimulation of the TLR4 pathway in vitro. METHODS: Skin tissues from burn patients (n = 10) and healthy donors (n = 2) were collected at the University of Alberta Hospital. HEK-Blue hTLR4 cells, human embryonic kidney cells expressing the TLR4 pathway, were treated with the tissue including eschar, exudate and cells and denatured SLRPs decorin and biglycan. Secreted embryonic alkaline phosphatase (SEAP) assay was used to measure NF-kB activation as an indicator of TLR4 activity. Endotoxin quantification was done on burn and control skin samples. RESULTS: We found that HEK-Blue hTLR4 cells treated with solid tissue, exudate, and burn tissue-isolated cells showed higher TLR4 activity compared to untreated cells. Burn site microbiology and days post-burn injury are clinical patient factors that influenced TLR4 activity. Interestingly, denatured SLPRs, decorin and biglycan also led to the stimulation of TLR4 pathway. Further, normal skin tissue stimulated TLR4 pathway to some extent, possibly due to the presence of skin microbiota. Endotoxin levels varied across burn samples and were undetectable in control skin. CONCLUSIONS: The results indicate that there may be non-LPS endogenous molecules released from the burned tissue and cells that can stimulate the TLR4 pathway. This would lead to production of Type 1 interferons and proinflammatory cytokines which may promote fibrosis and HTS formation. These studies will help in the development of future therapeutics, providing benefits to patients suffering with HTS and other fibroproliferative disorders.