Human Amnion/Chorion Grafts Modulate the Inflammatory Response of Fibroblasts Stimulated with Pro-Inflammatory Cytokines TNF-α and IL-1β

Chronic wounds frequently remain in an inflammatory state, which is associated with high levels of pro-inflammatory cytokines, including tissue necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β).  Inflammation can be downregulated through suppression of pro-inflammatory cytokines by applying placental membranes^1,2, with a further decrease in inflammation from driving macrophages towards a M2 phenotype.³ This study was designed to investigate the ability of dehydrated amnion/chorion membrane (dACM) to reduce inflammation caused by TNF-α and IL-1β in human dermal fibroblasts. Downregulation of inflammation was determined by evaluating levels of pro-inflammatory cytokine RANTES and chemoattractant cytokine MCP-1, which are both upregulated by fibroblasts in response to TNF-α and IL-1β. While upregulation of RANTES is pro-inflammatory, the effect of MCP-1 on inflammation is more complex and dependent on downstream macrophage phenotype.

Fibroblasts were stimulated with either 1ng/mL or 0.1ng/mL TNF-α or IL-1β with or without media conditioned with releasate from dACM. Following culture, cell supernatant was used to determine RANTES and MCP-1 production from fibroblasts via ELISAs, and production was normalized to the overall cell number.

 Production of RANTES was slightly increased with the addition of IL-1β and 0.1ng/mL TNF-α, with the largest increase with 1ng/mL TNF-α (5780±3312-fold increase). The addition of dACM conditioned media to 1ng/mL TNF-α, 0.1ng/mL IL-1β and 1ng/mL IL-1β groups led to statistically significant reductions in RANTES production (88.8%±9.7%, 49.6%±26.8%, and 54.5%±17.6% compared to controls without CM; n=9; p<0.05).  MCP-1 production from fibroblasts was significantly increased when CM was added in both control and 0.1ng/mL TNF-α groups, however, no other groups were shown to be significantly different from controls.

These results show that dACM alters the inflammatory signaling pathways for fibroblasts stimulated with TNF-α or IL-1β.  Additionally, dACM is not only involved in the downregulation of these pathways, but may play a more complex role in inflammation modulation.