Toll-like receptor activation modulates antimicrobial peptide expression by ocular surface cells

Area of Science:

• Immunology
• Ophthalmology
• Microbiology

Background:

• The ocular surface possesses innate immunity mechanisms to combat microbial invasion.
• Toll-like receptors (TLRs) are key components of this innate immune system, recognizing conserved microbial patterns.
• Antimicrobial peptides (AMPs), such as human beta defensins (hBDs) and cathelicidin (hCAP-18/LL-37), are vital effectors against ocular pathogens like Pseudomonas aeruginosa (PA).

Purpose of the Study:

• To investigate the expression of TLR1-10 mRNA and protein on various ocular surface cells.
• To determine if TLR agonists modulate the expression and secretion of AMPs (hBDs and hCAP-18/LL-37).
• To assess if TLR agonists or AMPs influence TLR mRNA expression and to evaluate the antimicrobial activity of induced AMPs against PA.

Main Methods:

• RT-PCR was used to examine TLR mRNA expression in corneal epithelial, stromal, and conjunctival epithelial cells.
• Flow cytometry and immunostaining confirmed protein expression for selected TLRs.
• Ocular surface cells were stimulated with TLR agonists, and AMP expression (mRNA and protein) was quantified.
• Antimicrobial activity of cell culture media against PA was tested after agonist stimulation.
• TLR mRNA expression was analyzed in cells treated with AMPs or TLR agonists.

Main Results:

• Ocular surface cells expressed a range of TLRs, with notable cell-specific differences (e.g., TLR4 absence in HCEC, TLR8 absence in all tested cells).
• TLR agonists upregulated hBD-2 and, in some cases, hCAP-18/LL-37 expression and secretion.
• Combined TLR3, 5, and 6/2 agonist stimulation in primary HCEC significantly enhanced LL-37 secretion, leading to potent antimicrobial activity against PA.
• AMPs (LL-37, hBD-2) downregulated certain TLR mRNA expressions (TLR5, 7, 9) in a cell-dependent manner.

Conclusions:

• The ocular surface expresses functional TLRs capable of initiating innate immune responses.
• TLR activation effectively stimulates the production and secretion of protective AMPs, particularly LL-37.
• LL-37 plays a critical role in defending the ocular surface against PA infection, highlighting a key TLR-AMP axis in ocular immunity.