The Role of Burn Tissue and Denatured Small Leucine Rich Proteoglycans in the Activation of the Toll-like Receptor 4 Pathway
Ambika Agrawal, Jie Ding, MD PhD, Babita Agrawal, PhD, Edward E. Tredget, MD, MSc, FRCSC.
University of Alberta, Edmonton, AB, Canada.
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. HTS formation is a dynamic, complex process that involves interactions between multiple factors such as fibroblasts, extracellular matrix molecules, inflammatory cells, cytokines, growth factors, and chemokines. Toll-like receptors (TLRs) are innate immune receptors that respond to microbes to initiate innate immunological defense, and detect and initiate tissue repair after injury. They are expressed in immune cells, gingival, synovial and dermal fibroblasts. 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) such as decorin, biglycan, fibromodulin, and lumican, are molecules involved in wound healing that modify the ECM by altering fibroblast proliferation, collagen organization, and growth factors. We hypothesized that the endogenous molecules released from damaged burn tissue could activate the TLR4 pathway in dermal cells, which may lead to a cascade of fibrogenic growth factors and collagen deposition following the activation of resident fibroblasts. Therefore, in this study, we determined the role of burn tissue and denatured SLRPs in the stimulation of the TLR4 pathway in vitro, to elucidate immunological mechanisms preceding HTS.
METHODS: Burn tissues, including eschar and exudate, were collected from patients (n=8) at the University of Alberta Hospital. A normal skin sample was collected from a patient who underwent abdominoplasty, as a control. Total cells were isolated from solid tissue by collagenase-digestion. HEK-Blue hTLR4 cells, human embryonic kidney cells that were co-transfected with human TLR4, MD-2 and CD14 co-receptor genes and are used to determine activation of the TLR4 pathway, were treated with the solid tissue, exudate, cells, denatured SLRPs decorin and biglycan, and bacterial lipopolysaccharide (LPS) as a positive control. Secreted embryonic alkaline phosphatase (SEAP) assay was used to measure NF-κB activation as an indicator of TLR4 activity.
RESULTS: 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. Normal skin tissue stimulated TLR4 pathway to some extent, possibly due to the presence of skin microbiota. Denatured decorin and biglycan caused lower TLR4 activity than controls, possibly due to endotoxin in the recombinant reagents.
CONCLUSION: The results indicate that there may be endogenous molecules released from the burned tissue and cells that are able to stimulate the TLR4 pathway. Stimulation of the TLR4 pathway would lead to production of Type 1 interferons and proinflammatory cytokines, such as IL-1β, IL-6, TNF-α, which may promote fibrosis and HTS formation. We will further explore the mechanism by culturing burn tissues and SLRPs with deep dermal fibroblasts. These studies will help in the development of future therapeutics, providing benefits to patients suffering with HTS and other fibroproliferative disorders.
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