Abstract
Purpose :
Chronic inflammation governed by the NF-κB signaling pathway is a hallmark of DED that contributes to the development of patient symptomology. Previous work has demonstrated that solubilized proteins derived from the Bombyx mori silkworm cocoon can be utilized to reduce DED related biomarkers in animal models. The data presented here support the hypothesis that the silk-derived biotherapeutic candidate, SDP-4, effectively inhibits NF-κB activation and downstream inflammatory signaling in vitro in both human corneal limbal-epithelial (HCLE) and embryonic kidney (HEK293) cell lines.
Methods :
SDP-4 was prepared by performing an alkaline extraction on Bombyx mori silkworm cocoons, which was followed by dissolution of extracted fibers in concentrated LiBr solution under heat and pressure. Final purification of the 20kDa SDP-4 molecule was achieved using membrane dialysis. NF-κB-mediated gene expression was stimulated in HCLEs using TNF-α (1ng/mL) in the presence/absence of SDP-4 (up to 1%) for up to 6-hours, and expression of inflammatory cytokines measured using quantitative PCR and ELISA. Coculture immune cell (HL-60) migration assays were performed to evaluate the functional impact of impaired HCLE signaling. To determine whether SDP-4 inhibits transcription factor activation directly, a HEK reporter cell line was used to quantitate NF-κB-driven chemiluminescence in the presence or absence of SDP-4.
Results :
SDP-4 significantly reduced (p<0.05, n=3) TNF-α induced expression of the inflammatory genes TNF-α, MMP-9, IL-1a/b and IL-8. Furthermore, there was a dose-dependent inhibition of IL-8 production by over 50% with increasing SDP-4 concentration. This reduction in cytokine release by HCLEs was functionally sufficient to significantly inhibit (p<0.05, n=3) activation/migration of immune-like HL-60 cells. Lastly, SDP-4 demonstrated a dose-dependent reduction in NF-κB-mediated chemiluminescence in stimulated reporter cells.
Conclusions :
These studies support that SDP-4 inhibits in vitro corneal inflammatory signaling mediated by NF-κB. Additionally, this reduced expression was effective to inhibit activation of immune-like cells, which may provide a novel opportunity to inhibit chronic inflammatory signaling in DED.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.