MyD88 Contributes to the Intrinsic Innate Defense of the Ocular Surface

Abstract

Purpose : MyD88 is a key signalling molecule necessary for Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) mediated immune defense. Previously, we demonstrated that TLR activation leads to enhanced cytokine, chemokine, and matrix metalloproteinase (MMP) expression in human corneal and conjunctival cells. Here, we examined the intrinsic role of TLR signalling in ocular surface tissues by determining baseline levels of inflammatory mediators, the response to mechanical stimuli, and corneal infection in MyD88-deficient mice (MyD88^-/-).

Methods : Corneal and conjunctival tissues were collected from 8-12 week old C57BL/6 wild-type (WT), MyD88^-/-, and IL-1R^-/- mice for MMP, cytokine, and chemokine RNA and protein quantitation by qRT-PCR and Luminex assay, respectively. Mouse corneal sensitivity was measured using a Cochet-Bonnet esthesiometer. For corneal infection, mice were inoculated with 10⁶ colony forming units (cfu) GFP-labeled Pseudomonas aeruginosa (PA). Clinical scores and viable corneal bacterial cfu were determined 24h post-infection.

Results : Compared to WT animals, MyD88^-/- mice had significantly lower MMP-9 levels in the cornea (23±5 vs. 36±2 pg/ml) and conjunctiva (531±92 vs.1345±87 pg/ml). Corneal IL-1α, TNFα (RNA and protein) and conjunctival IL-1α, IL-2, IL-6, and IL-9 levels were significantly reduced in MyD88^-/- mice. Additionally, CXCL1 and RANTES expression was lower in both MyD88^-/- tissues compared to WT and IL-1R^-/- (p<0.05) mice. Interestingly, MyD88^-/- mice had lower corneal sensitivities (1.01±0.31 gm/mm²) than both WT (0.59±0.16 gm/mm²) and IL-1R^-/- (0.52±0.08 gm/mm²). Following 24h PA infection, MyD88^-/- mice had better clinical scores (0.5±0.0) compared to IL-1R^-/- (1.5±0.6) and WT (2.3±0.3) animals, but had significantly more corneal cfu compared to WT and IL-1R^-/-.

Conclusions : MyD88 signaling is necessary for the intrinsic defense of the ocular surface through maintaining levels of cytokines and chemokines and, notably, regulating corneal sensitivity. This work furthers our understanding of the importance of TLR signalling in corneal defense, showing that a lack of MyD88 may compromise the baseline innate response to insult. A careful balance of TLR activation must be maintained to initiate an immune response to pathogens without generating sight threating inflammation.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.