Effect of Dry Eye Disease on the Kinetics of Lacrimal Gland Dendritic Cells as Visualized by Intravital Multi-Photon Microscopy

Abstract

Purpose : Ocular surface dryness induced by desiccating stress (DS) compromises afferent nerve pathways in the lacrimal function unit, which may lead to lacrimal gland (LG) inflammation and reduced tear secretion. We aimed to evaluate the impact of DS-induced DED on conventional dendritic cell (cDC) density, morphology and kinetics in the LG, using intravital multiphoton microscopy (IV-MPM).

Methods : DED was induced by housing 6-8 week old CD11c^YFP×Thy1^YFP double transgenic mice in a controlled environmental chamber for a period of 2 and 4 weeks (w). DED severity was assessed by corneal fluorescein staining score (CFS) using NEI scale, and tear secretion using phenol red thread test. CD11c^YFP cell phenotype and density was quantified by flow cytometry in LG single cell suspensions. A novel approach was developed to image CD11c^YFP cells in the LG by IV-MPM, by placing mice onto a custom-designed stage. The LG was exposed carefully and the area to be imaged was covered by a sealed chamber. Analysis of CD11c^YFP cell density, 3D morphology, and kinetics was performed using Imaris software. One-way ANOVA with Tukey’s post-hoc was used to assess statistical significance.

Results : DED mice had a 3-fold decreased tear secretion and 12-fold increased fluorescein staining score (p<0.01) compared to naïve mice. IV-MPM and flow cytometry of the LG demonstrated 2-fold increase in the density of cDCs in the LGs of DED mice, compared with controls (p<0.001). cDCs were more spherical in DED at 2w and 4w compared to naïve mice (p<0.001); surface area were found at 2w in DED compared with naïve mice (p<0.001). Mean track speed was 1.8-fold and 1.3-fold higher at 2w and 4w compared to naïve mice (p<0.001). Finally, the meandering index, an index for directionality (scale from 0 to 1), was increased at 4w (0.20±0.01), compared with naïve (0.14±0.01) and 2w (0.15±0.01) of DED (p<0.001).

Conclusions : Our IV-MPM study sheds light into the 3D morphological alterations and cDC kinetics in the LG during DED. While in normal LGs, cDCs exhibit a more dendritic morphology and are less motile, they became more spherical with enhanced motility during DED. This study shows that IV-MPM represents a robust tool to study immune cell trafficking and kinetics in the LG, which might elucidate cellular alterations in immunological diseases, such as DED.

This is a 2020 ARVO Annual Meeting abstract.