Impaired Wound Healing and Inflammation: The Role of the Dermal Fibroblast. Phenotypic Changes in the Human Dermal Fibroblast with Inflammation; Potential Impact on Wound Healing

Abstract

Dermal fibroblasts positively contribute throughout the wounding response by secreting a profile of pro- and anti-inflammatory cytokines in the wound milieu. However, a chronically inflamed environment will, cause detrimental effects on the functional, secretory, and molecular properties of these cells. This study aims to understand how the effect of the pro-inflammatory cytokine TNF-α modulates both healthy and diabetic dermal fibroblast phenotype. To mimic a chronic inflammatory environment and assess whether fibroblasts respond similarly in different anatomical sites, donor-matched fibroblasts from face and scalp were pre-incubated for 3 days with different concentrations (2.5, 25 or 250 ng/ml) of TNF-α. All concentrations significantly impaired proliferation by day 14 in cells from both sites and stimulated (papillary) metabolic activity at day 14. However, this did not correlate with an increase in papillary cell senescence since this did not appear until passage 17, and then only at a supra pathophysiological concentration. Migration of dermal fibroblasts, assessed by the scratch assay. TNF-α significantly inhibited the cells migration, particularly in diabetic fibroblasts, suggesting they are more sensitive to TNF-α. Since TNF-α may stimulate the secretion of soluble paracrine factors by dermal fibroblasts, conditioned medium was collected to assess its effect on other dermal fibroblasts, however, this had no significant effect on migration. However, using gelatin zymography, it was found that TNF-α did stimulate the secretion of soluble paracrine factors that induce MMP activity in non-diabetic fibroblasts, mirroring previous observations that a pro-inflammatory environment can increase proteolytic activity, and indicating that diabetic fibroblasts were again more sensitive than healthy. No difference was observed with MMP-9 activity and nor did the results with dermal fibroblasts reach statistical significance, perhaps because of a relatively low n-number. The ability of TNF-α to modulate the expression of genes associated with the ECM (MMP-1, -2, -9, TIMP-1, and -2) and senescence (Sirt1 and 6) was investigated. There was no change in Sirt1 and Sirt6 expression and no evidence of paracrine effects (conditioned medium) on any of the genes. TNF-α significantly induced mRNA expression of MMP-1 in healthy non-scratched and scratched diabetic fibroblasts, and TIMP-1 in healthy non-scratched cells. There was also considerable donor variability that prevented statistical significance being achieved under the other conditions. The secretion of various cytokines associated with inflammation was compared in healthy and diabetic fibroblasts in the presence and absence of TNF-α. Seven cytokines were secreted, by healthy and diabetic male and female fibroblasts, although diabetic female fibroblasts did not secrete two of them. TNF-α stimulated secretion of cytokines in healthy and diabetic, male and female cells but the profiles of those released were different between the different groups. There was no TNF-α induced paracrine effect on cytokine secretion by healthy dermal fibroblasts. In conclusion, changes in the microenvironment and the influx of pro inflammatory cytokines may significantly alter the dermal fibroblast phenotype. Understanding these functional and molecular changes in response to inflammatory cytokines will give a better understanding of the differences between fibroblast activity in normal physiological wound healing and chronic or diabetic non-healing wounds.