TOLL-LIKE RECEPTOR EXPRESSION AND FUNCTION

Purpose: Ocular surface inflammation presents from various etiologies (e.g. dry eye and Pseudomonas aeruginosa (PA) infection), yet little is known about the role of toll-like receptors (TLR) which, upon activation, stimulate inflammation. The objective of this research was to determine: (1) baseline expression of TLRs in ocular surface cells, if TLR activation or products thereof (antimicrobial peptides, AMPs) can modulate TLR expression, and if TLR agonists can stimulate the expression of AMPs human β–defensin (hBD-2) or LL-37 which can kill PA; (2) if ocular surface cell TLR expression is modulated in response to dry eye associated conditions, desiccation and in dry eye patients; (3) TLR expression in mice with experimental dry eye (EDE), AMP expression in EDE mice and dry eye subjects and if topical TLR agonist application modulates the inflammatory response in mice with EDE in vivo.

Methods: (1) TLR expression was examined by RT-PCR, flow cytometry or immunostaining. Cells were exposed to AMPs or TLR agonists to determine TLR mRNA expression or the antimicrobial activity of the culture media against PA. (2) TLR mRNA or protein expression was examined by real-time RT- PCR or western blotting in cells cultured with hyperosmolar stress (HOS), or dry eye associated cytokines (IL-1α/β, TNFα, TGFβ), in a human corneal organ culture model following desiccation and in conjunctival impression cytology (CIC) samples from human dry eye patients. (3) TLR and/or AMP expression was determined at the ocular surface and/or lacrimal gland in mice with EDE using quantitative RT-PCR and immunostaining. AMP mRNA expression was examined in human dry eye CIC samples. A TLR agonist cocktail was applied to the ocular surface of mice with EDE. Central corneal thickness (CCT), recruitment of inflammatory cells into to the corneal stroma and corneal fluorescein staining were determined by in vivo imaging and corneal epithelial AMP expression was determined by quantitative RT-PCR and immunostaining 24 hours later.

Results: (1) Ocular surface cells express most TLRs with the exception of TLR8. Some AMPs but not TLR agonists modulated TLR mRNA expression. TLR agonists increased the secretion of hBD-2 and LL-37 into the growth media, which was able to significantly kill PA. (2) HOS significantly increased TLR4 mRNA but decreased TLR4 protein. Dry eye associated cytokines had no effect on TLR expression. TLR4 and TLR5 were upregulated in response to desiccation. TLR9 expression was downregulated in response to HOS, desiccation and in dry eye subjects. (3) EDE modulated TLR mRNA and protein expression by the ocular surface and lacrimal gland, compromised corneal epithelial integrity and decreased the expression of mRNA and protein of CRAMP, the LL-37 mouse homolog. hBD-2 was significantly upregulated in dry eye subjects. TLR agonist treatment downregulated mBD-4 protein in the cornea, decreased CCT, but did not increase inflammatory cell recruitment into the stroma.

Conclusions: (1) TLRs are expressed on the ocular surface and their activation triggers the production of LL-37 and hBD-2, with LL-37 being particularly important in killing PA. (2) Dry eye and associated conditions modulated TLR expression which may alter the ocular surface immune/inflammatory response. (3) EDE significantly disrupts the mouse corneal epithelium, increases TLR expression and decreases the expression of CRAMP. Mice with EDE were resistant to TLR induced corneal inflammation