Abstract
The lacrimal gland (LG) is the main source of the tear film aqueous layer and its dysfunction results in dry eye disease (DED), a chronic immune-mediated disorder of the ocular surface. The desiccating stress (DS) murine model that mimics human DED, results in LG dysfunction, immune cell infiltration, and consequently insufficient tear production. To date, the immune cell kinetics in DED are poorly understood. The purpose of this study was to develop a murine model of intravital multi-photon microscopy (IV-MPM) for the LG, and to investigate the migratory kinetics and 3D morphological properties of conventional dendritic cells (cDCs), the professional antigen presenting cells of the ocular surface, in DED. Mice were placed in a controlled environmental chamber with low humidity and increased airflow rate for 2 and 4 weeks to induce DED, while control naïve transgenic mice were housed under standard conditions. DED mice had significantly decreased tear secretion and increased fluorescein staining (p < 0.01) compared to naïve controls. Histological analysis of the LG exhibited infiltrating mononuclear and polymorphonuclear cells (p < 0.05), as well as increased LG swelling (p < 0.001) in DED mice compared to controls. Immunofluorescence staining revealed increased density of cDCs in DED mice (p < 0.001). IV-MPM of the LG demonstrated increased density of cDCs in the LGs of DED mice, compared with controls (p < 0.001). cDCs were more spherical in DED at both time points compared to controls (p < 0.001); however, differences in surface area were found at 2 weeks in DED compared with naïve controls (p < 0.001). Similarly, 3D cell volume was significantly lower at 2 weeks in DED vs. the naïve controls (p < 0.001). 3D instantaneous velocity and mean track speed were significantly higher in DED compared to naïve mice (p < 0.001). Finally, the meandering index, an index for directionality, was significant increased at 4 weeks after DED compared with controls and 2 weeks of DED (p < 0.001). Our IV-MPM study sheds light into the 3D morphological alterations and cDC kinetics in the LG during DED. While in naïve 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.
Highlights
Dry eye disease (DED) is a significant public health concern affecting ∼16 million adults in the United States alone [1, 2]
Considering the key role of the sensory nerves as the afferent pathway of the lacrimal functional unit (LFU) in regulating tear production, we aimed to investigate if corneal nerve damage may affect the density and function of immune cells in the LG, in particular Conventional dendritic cells (cDCs), which serve as the professional antigen presenting cells (APCs) of the ocular surface (Figure 2)
We demonstrate that the cDC density in the LG is higher during DED, and that cDCs become smaller, more spherical, but more motile in Desiccating stress (DS)-induced DED compared to naïve mice
Summary
Dry eye disease (DED) is a significant public health concern affecting ∼16 million adults in the United States alone [1, 2]. The LFU is defined as the conjunction of lacrimal glands (main and accessory), the ocular surface and the communicating innervation [6] This innervation is responsible for the maintenance of the tear film through baseline and reflex tearing. The sensory nerves within the cornea represent the afferent arm, detecting changes in the tear film osmolarity or mechanical stimuli, necessitating lacrimation. These signals are relayed to the LG through the parasympathetic nerves via the efferent arm, resulting in lacrimation. We hypothesize that compromised corneal sensory nerves on the ocular surface (afferent pathways of the LFU) may, in addition to decreased tear secretion, directly lead to LG inflammation
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