Fig. 1

Dry eye disease (DED) dysregulated neutrophils and Th17/Treg imbalance in the corneal limbus of a murine model. (A) Schematic diagram showing dry eye disease (DED) mouse model establishment. (Created with bioRender.com). (B) Representative fluorescein-stained images of corneas from healthy control mice (Control) and mice after 15 days of desiccating stress (DED). Scale bars, 1 mm. (C) Clinical fluorescence scores for corneal epithelial defects (n = 5). (D) Representative immunohistochemistry images of the corneal limbus. CD31 (magenta) indicates blood vessels, and Ly6G (yellow) indicates neutrophils. Scale bars, 10 μm. (E) Quantification of Ly6G⁺ neutrophils in flat-mounted corneas, focusing on the limbus region (n = 5). The total number of corneal neutrophils was counted in nine 40 × fields of view that comprised the diameter of the cornea. (F) The proportions of Th17 (CD4⁺CD196⁺) and Treg (CD4⁺CD25⁺) cells in eye-draining lymph nodes were analyzed by flow cytometry. t-SNE plots were generated from 2 × 10⁴ cells from the Control and DED groups (n = 3), with data downsampling and concatenation for inter-subject comparison. (G) Frequencies of Th17 cells and Treg cells and the Th17/Treg ratio in the eye-draining lymph nodes (n = 5). Data are presented as mean ± SEM for each group (n = 5). Statistical significance is denoted as ^***P < 0.001, ^****P < 0.0001 for DED versus Control groups and ^{^}P < 0.05, ^{^^}P < 0.01 for Aged versus Young groups; P values were calculated using Student’s t-test.