Association of IL-8 and IL-18 Expression With Histone-Associated NETosis in Aqueous-Deficient and Evaporative Dry Eye...

Dry eye disease (DED) is a chronic inflammatory disorder of the ocular surface; however, upstream immune mechanisms contributing to DED are complex and unclear. This study aimed to identify differentially expressed tear proteins in patients with DED and elucidate the underlying disease mechanism.

Tear fluid from healthy controls (HC; n = 26), aqueous-deficient DED (ADDE; n = 29), and evaporative DED (EDE; n = 26) patients were collected using Schirmer strips. Proteomic profiling was performed using nano-ESI LC-MS/MS. Differential proteins were identified using LIMMA and MBQN with Gene Ontology pathway enrichment. Key candidates were validated by ELISA (IL-8, IL-18, histones), Western blotting for citrullinated H3, PicoGreen for extracellular DNA, and in vitro assays in human corneal epithelial cells.

A total of 2596 tear proteins were identified across groups, of which 101 were significantly altered (P < 0.001) in both ADDE and EDE groups. Principal component analysis and heatmap analyses revealed differential protein abundance patterns between HC and disease groups, with increased inflammatory and oxidative stress proteins (MMP9, MAPK3, S100A8/A9, and SOD1) and decreased lactoferrin, lysozyme, lipocalin-1, and mucins in ADDE and EDE. Pathway enrichment indicated activation of neutrophil-mediated inflammation and extracellular chromatin release. Validation confirmed significant upregulation of NETosis-associated markers, including IL-8 (P < 0.01), IL-18 (P < 0.001), and histone proteins (H1, H2A/B, H3, H4) (P < 0.001) in disease groups, which was further confirmed by in vitro DED model.

Our study shows that neutrophil-mediated inflammation and NETosis in DED may be associated with elevated IL-8, IL-18, and histone proteins in ADDE and EDE, which could be potential biomarkers or therapeutic targets.