Tear fluid proteome reveals inflammation and immune response proteins as potential predictive biomarkers of the effects of desiccating stress and dry eye treatments

Abstract

Purpose : To determine if tear protein biomarkers would predict the effects of topical steroid treatment and desiccating stress (DS) in dry eye (DE) patients.

Methods : In a randomized double-masked controlled clinical trial (Pinto-Fraga, Ophthalmology 2015), patients (n=28) were treated topically with either 0.1% fluorometholone (FML) or polyvinyl alcohol (PA). Tear samples were collected at baseline (V1) and after 21-day treatment period (before (V2) and after (V3) a 2-hour exposure to DS in a controlled environment laboratory) with 1μl capillary tubes. Proteins were analyzed for library generation and relative quantification of expression levels in 2.6 μg of tear proteins was carried out by NanoLC-MSTripleTOF using SWATH acquisition. Ocular surface integrity (corneal and conjunctival staining) was selected as the key DE-related sign along with proteomic data. Statistical analysis was performed with R software.

Results : A protein identification library consisting of >870 proteins (FDR 1 %) was established. In total 770 proteins were identified and relatively quantified. Protein baseline values and treatment, FML or PA, were used to predict the DE-related sign changes between initial and visits before and after a 2-hour controlled adverse environment exposure. We identified 32 potentially predictive proteins for conjunctival staining change (V1-V2, treatment effect) and 73 for V2-V3-change (DS effect). Two tear proteins, lysozyme C LYZ (V1-V2: R²=0.44, V2-V3: R²=0.56) and prolactin-inducible protein PIP (V1-V2: R²=0.441, V2-V3: R²=0.506), were found in common between the two results (both proteins had p-value<0.001 for the regression models). When the baseline protein expression value was higher, conjunctival staining of FML-treated patients was predicted to decrease more than in equivalent PA-treated patients. We also identified 22 potentially predictive proteins for corneal staining change between baseline and V2 and 14 of these proteins were immunoglobulins connected to complement activation.

Conclusions : Using only a 1μl sample, we reliably identified 770 proteins from each analysed sample for DE patients. Potential biomarkers were identified for predicting treatment effects with desiccating stress in DE patients.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.