Epidermal growth factor effect on lipopolysaccharide-induced inflammation in fibroblasts derived from diabetic foot ulcer

Abstract

Background Diabetic foot ulcers (DFU) are characterised by high levels of inflammatory mediators, resulting from sustained hyperglycaemic insult and the local microbial biofilm. The intralesional administration of epidermal growth factor (EGF) has emerged as an effective treatment that stimulates granulation and closure of DFU, reducing the risk of amputation. Within the wound, fibroblasts play key roles during the healing process, promoting granulation and contraction. The aim of the present study was to examine the anti-inflammatory effect of EGF in DFU-derived fibroblasts, challenged with lipopolysaccharide (LPS), under hyperglycaemic conditions, recreating in vitro what happens in a clinical scenario. Methods Healthy skin (HS) and DFU granulation tissue biopsies were used to isolate primary fibroblasts. The effect of LPS on cell proliferation was analysed. Transcriptional expression of toll-like receptor (TLR) pathway mediators (TLR4, TLR2, CD14, MYD88 and NFKB) and pro-inflammatory cytokines (TNF, IL-6 and IL-1B) were measured by semi-quantitative polymerase chain reaction (qPCR), in cells treated with appropriate concentrations of LPS, EGF and their combination. IL-6 protein concentration was quantified by ELISA. Results LPS stimulated proliferation of HS-derived fibroblasts, while inhibiting the proliferation of cells derived from DFU at the highest assayed concentration of 1 µg/mL. Regarding the TLR signalling pathway, LPS increased messenger RNA levels of mediators and pro-inflammatory genes, while EGF, alone or in the presence of LPS, downregulated them, except for IL-1B. Conclusion The results suggest that EGF might elicit an anti-inflammatory response in LPS-challenged fibroblasts, even in a hyperglycaemic milieu. Collectively, our findings contribute to explain newly observed effects of EGF in the clinical arena. Lay Summary In this research article, we analyse the putative anti-inflammatory effect of epidermal growth factor (EGF) on fibroblast isolated from diabetic foot ulcer (DFU) granulation tissue. To induce the inflammatory response, the cells were treated with lipopolysaccharide (LPS), simulating the gram-negative bacterial infection that takes place in the wounds of diabetic patients. We studied the expression of genes involved in bacterial recognition receptors signalling pathway and those that code for different pro-inflammatory cytokines. We obtained primary fibroblasts from biopsies of a neuropathic diabetic ulcer and from healthy skin, the former was used as the control. Cells were isolated and grown in high glucose Dulbecco’s Modified Eagle Medium (DMEM) culture medium, to simulate the hyperglycaemic insult. The effect of increasing concentrations of LPS on cell proliferation was analysed. Relative transcriptional expression of genes in the study was quantified by quantitative polymerase chain reaction (qPCR) in cells treated with LPS, EGF or a combination. Untreated cells served to normalise the expression. In the present study, we demonstrated that EGF modulated the primary immune response by reducing the activation of pathogen-recognition receptors and common genes involved in these signalling pathways, even in hyperglycaemic conditions. This effect translated in a decreased expression of pro-inflammatory cytokines. These results contribute to explain our previous observations about the reduction of circulating levels of inflammatory cytokines after local administration of human recombinant EGF in DFU. Further molecular studies should be carried out to fully understand the biological mechanisms elicited by EGF in this clinical scenario.

Sequential treatment for diabetic foot ulcers in dialysis patients: A case report

“…In the case of deep wounds, the tissue-engineered skin can be overlaid repeatedly to thicken the new dermis, which was the method used in this case, in which the tissue-engineered skin was repeatedly grafted to the heel, and the wound healed before skin grafting. There are many clinical reports on the treatment of DFUs using tissue-engineered skin[ 14 , 15 ], but the application to weight-bearing areas and dialysis patients with ischemic DFUs has not previously been reported (Table 1 ).…”

Secretion of IL-6 by fibroblasts exposed to Australian honeys involves lipopolysaccharide and is independent of floral source

“…As TLR4 is a sensor for LPS, we determined whether grey ironbark honey might activate TLR4 signalling. TLR4 is expressed in fibroblasts and macrophages 16 , and its activation by LPS stimulates nuclear factor-kappa B (NF-κB), which regulates inflammatory responses including induction of the gene encoding for IL-6 17 . To test this hypothesis, we used RAW 264.7 murine macrophages which express TLR4 and a knock-in reporter gene that encodes for SEAP.…”

Fibroblasts in Diabetic Foot Ulcers

“…Increased levels of TNF-α in DWs have been reported [54] as well as increased levels of other proinflammatory cytokines like (IL)-1β, IL-6, IL-8, in more recent studies [3,54,77,95]. The elevated amount of ROS, AGEs products from necrotic debris as well as presence of lipopolysaccharides (LPS) from biofilm can trigger and maintain abnormally increased levels of local and circulating inflammatory mediators [13,[96][97][98]. The secretion of these pro-inflammatory mediators in the DW is thought to be a consequence of the continuous infiltration of monocytes and neutrophils in the wound bed [1,11,13,99] or to be generated by a negative feedback loop from an initial alteration of the innate immune system [100].…”