Abstract
Dry eye disease (DED) is a multifactorial disease of the ocular surface, characterized by loss of tear film homeostasis and ocular symptoms, in which neurosensory abnormalities have recently been shown to play an etiological role. Although the role of inflammation has been widely studied in DED, the kinetics of immune cells of the ocular surface in this complex disease are hereto unclear. Herein, we utilized intravital multiphoton imaging on transgenic mice to investigate the 3D morphology and kinetics of conventional dendritic cells (cDCs) and the role of ocular surface sensory nerves in regulating them in both the naïve state and experimental DED. Mice with DED had significantly lower tear secretion (p < 0.01), greater corneal fluorescein staining (p < 0.001), and higher cDC density in the ocular surface (p < 0.05), compared to naïve mice. cDCs in DED mice showed morphological alterations in the limbus, exhibiting smaller surface area (p < 0.001) and volume (p < 0.001) compared to naïve mice. Furthermore, corneal cDCs showed greater sphericity in DED mice compared to naïve mice (p < 0.01). In addition, limbal cDCs displayed significantly increased migratory kinetics in DED, including mean track speed, 3D instantaneous velocity, track length, and displacement, compared to naïve mice (all p < 0.05). In mice with DED, cDCs showed a higher meandering index in the limbus compared to central cornea (p < 0.05). In DED, cDCs were less frequently found in contact with nerves in the limbus, peripheral, and central cornea (p < 0.05). cDCs in contact with nerves demonstrated a larger surface area (p < 0.001) and volume (p < 0.001), however, they exhibited less sphericity (p < 0.05) as compared to cDCs not in contact with nerves in naïve mice. Importantly, cDCs in contact with nerves during DED had a decreased track length, displacement, mean track speed, and 3D instantaneous velocity compared to those not in contact with nerves (all p < 0.05). Taken together, we present in vivo evidence of altered cDC kinetics and 3D morphology in DED. Furthermore, apparent neuronal contact significantly alters cDC kinetics and morphological characteristics, suggesting that ocular surface nerves may play a direct role in mediating immune responses in DED.
Highlights
Dry eye disease (DED) is one of the most common causes of clinical ophthalmic visits, since it is accompanied by ocular discomfort and results in diminished quality of life [1]
Using fluorescent minus one controls, we observed that yellow fluorescent protein (YFP)+ cells in the bone marrow of CD11cYFP × Thy1YFP mice were conventional dendritic cells (cDCs), as they expressed the pan-leukocyte marker, CD45, myeloid markers, CD11b and CX3CR1, dendritic cell marker, CD11c; but were majorly negative for plasmacytoid dendritic cell marker, PDCA-1, as well as macrophage marker, CD68 (Supplementary Figure S1D)
Through the use of intravital multiphoton microscopy (IV-MPM) on double-transgenic mice, we presented detailed alterations in the 3D morphology and kinetics of ocular surface cDCs following desiccating stress-induced DED and cDC-nerves interplay in this process
Summary
Dry eye disease (DED) is one of the most common causes of clinical ophthalmic visits, since it is accompanied by ocular discomfort and results in diminished quality of life [1]. DED is “a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film, and accompanied by ocular symptoms, in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles” [1]. In addition to its dense innervation, the cornea is an immune privileged tissue and hosts antigen-presenting cells (APCs), including epithelial and stromal conventional dendritic cells (cDCs) and stromal macrophages during steady state [7,8,9,10,11,12]
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