Intravital Multi-Photon Microscopy Reveals Alterations in Interactions of Conventional Dendritic Cells and Corneal Nerves in Dry Eye Disease

Abstract

Purpose : To study the impact of neuro-immune interactions in alterations in conventional dendritic cells (cDC) kinetics in dry eye disease (DED).

Methods : CD11c^YFP×Thy1^YFP double transgenic mice (in which both cDCs and nerves are YFP⁺), were generated. Six-8-week old female mice were placed in a custom-made controlled environment chamber (humidity: 15%, air circulation: 15L/min, and temperature: 21-23^○C) for 4 weeks to induce DED. Mice in standard housing served as controls. Tear volume was assessed via phenol red thread and corneal fluorescein staining (CFS) was performed and scored based on NEI scale. Intravital multi-photon microscopy was performed on both limbus and the cornea. Imaris was used to analyzed data. T test was used to assess statistical significance. p<0.05 was considered significant.

Results : Compared to controls, mice with DED exhibited lower tear volume (3.4±0.9 vs. 8.8±0.5 mm, p<0.01) and developed greater CFS (0.5±0.2 vs. 13.8±0.3, p<0.01). DED led to increased density of YFP⁺ cDCs compared to controls (1.8-fold in limbus and 1.9-fold in the peripheral cornea; both p<0.05). In DED, YFP⁺ cDCs showed smaller 3D surface area (1,127.2±55.0 vs. 1,761.9±104.114 μm2, p<0.01) and volume (1,880.8±184.3 vs. 3,371.5±186.3 μm3, p<0.01) in the limbus and higher sphericity in the peripheral and central cornea (both p<0.01) compared with controls. In DED, YFP⁺ cDCs in the limbus displaced longer (8.87±0.68 vs.5.23±0.56 μm, p<0.01) with higher mean track speed compared to controls (2.44±0.11 vs. 1.98±0.08 μm/min, p<0.05). While in controls, 72.7±6.1% of YFP⁺ cDCs were in contact with nerves, in DED, only 34.0±2.1% of them remained in contact with nerves (p<0.01). Only in controls, but not in DED, YFP⁺ cDCs in contact with nerves demonstrated a larger 3D surface area (2,359.0±177.75 vs. 1,481.7±90.0 μm2, p<0.01), volume (4,522.9±422.8 vs. 2,466.0±175.7 μm3, p<0.01), and greater sphericity (0.57±0.01 vs. 0.61±0.01, p<0.05) compared to cells without nerve contact. In DED, YFP⁺ cDCs without nerve contact displayed longer displacement (3.15±0.36 vs. 5.62±0.43 μm, p<0.01) with higher mean track speed (1.60±0.07 vs. 1.93±0.06 μm/min, p<0.01) compared to cells without nerve contact.

Conclusions : We present in vivo evidence that apparent neuronal contact significantly alters cDC kinetics and morphology in DED, indicating its role in mediating immune responses.

This is a 2020 ARVO Annual Meeting abstract.