Abstract
Corneal limbal stem cell deficiency (LSCD) may be treated using ex vivo limbal epithelial stem cells (LESCs) derived from cadaveric donor tissue. However, continuing challenges exist around tissue availability, inflammation, and transplant rejection. Lipopolysaccharide (LPS) or recombinant human IL-1β stimulated primary human keratocyte and LESC models were used to investigate the anti-inflammatory properties of a short chain, IL-1 receptor antagonist peptide for use in LESC sheet growth to control inflammation. The peptide was characterized using mass spectroscopy and high performance liquid chromatography. Peptide cytotoxicity, patterns of cell cytokine expression in response to LPS or IL-1β stimulation, and peptide suppression of this response were investigated by MTS/LDH assays, ELISA, and q-PCR. Cell differences in LPS stimulated toll-like receptor 4 expression were investigated using immunocytochemistry. A significant reduction in rIL-1β stimulated inflammatory cytokine production occurred following LESC and keratocyte incubation with anti-inflammatory peptide and in LPS stimulated IL-6 and IL-8 production following keratocyte incubation with peptide (1 mg/mL) (P < 0.05). LESCs produced no cytokine response to LPS stimulation and showed no TLR4 expression. The peptide supported LESC growth when adhered to a silicone hydrogel contact lens indicating potential use in improved LESC grafting through suppression of inflammation.
Highlights
4.9 million people worldwide are bilaterally blind due to corneal opacity and corneal blindness is the fifth most common cause of blindness globally [1]
Limbal epithelial stem cells (LESCs) may be destroyed by injury, infection or disease [4,5,6,7] resulting in a condition known as limbal epithelial stem cell deficiency (LSCD)
Using a cell model of corneal inflammation, we show a reduction in proinflammatory cytokines IL-6 and IL-8 with the peptide when cells are stimulated with LPS or IL-1β
Summary
4.9 million people worldwide are bilaterally blind due to corneal opacity and corneal blindness is the fifth most common cause of blindness globally [1]. Limbal epithelial stem cells (LESCs) are located in the basal region of the limbus, where the corneal epithelium meets the sclera, and are important in maintaining the structural integrity and transparency of the cornea [2]. LESCs are responsible for maintenance of corneal epithelial integrity through provision of a continuously renewed corneal epithelium, by producing a steady supply of daughter transient amplifying cells which differentiate into basal, wing, and squamous epithelial cells [3]. Cells move in an inwardly spiraling pattern from the basal to apical layers of the epithelium where the squamous cells at the surface of the cornea are continuously lost into the tear film.
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