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Lacrimal Gland Myoepithelial Cells Are Altered in a Mouse Model of Dry Eye Disease.

ABSTRACT: The purpose of this study was to determine the pathogenic changes that occur in myoepithelial cells (MECs) from lacrimal glands of a mouse model of Sjögren syndrome. MECs were cultured from lacrimal glands of C57BL/6J [wild type (WT)] and thrombospondin 1 null (TSP1^-/-, alias Thbs1^-/-) mice and from mice expressing α-smooth muscle actin-green fluorescent protein that labels MECs. MECs were stimulated with cholinergic and α₁-adrenergic agonists, vasoactive intestinal peptide (VIP), and the purinergic agonists ATP and UTP. Then intracellular [Ca²⁺] was measured using fura-2, and contraction was observed using live cell imaging. Expression of purinergic receptors was determined by Western blot analysis, and mRNA expression was analyzed by microarray. The increase in intracellular [Ca²⁺]_I with VIP and UTP was significantly smaller in MECs from TSP1^-/- compared with WT mice. Cholinergic agonists, ATP, and UTP stimulated contraction in MECs, although contraction of MECs from TSP1^-/- mice was reduced compared with WT mice. The amount of purinergic receptors P2Y1, P2Y11, and P2Y13 was significantly decreased in MECs from TSP1^-/- compared with WT mice, whereas several extracellular matrix and inflammation genes were up-regulated in MECs from TSP1^-/- mice. We conclude that lacrimal gland MEC function is altered by inflammation because the functions regulated by cholinergic agonists, VIP, and purinergic receptors are decreased in TSP1^-/- compared with WT mice.

SUBMITTER: Garcia-Posadas L

PROVIDER: S-EPMC7520664 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In the lacrimal gland, myoepithelial cells (MEC) express muscle contractile proteins such as alpha smooth muscle actin (SMA) and calponin and therefore can contract to help expel lacrimal fluid. In a previous study, we demonstrated that lacrimal gland MEC express the oxytocin receptor (OXTR) and they contract under oxytocin (OXT) stimulation. Using NOD and MRL/lpr mice (animal models of Sjogren's syndrome), we reported a decrease in SMA and calponin protein levels plus a decline in acini contraction after stimulation with OXT. It is known that proinflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α) or interferon gamma (IFN-γ), can affect OXTR expression and signaling capacity and inhibit MEC contraction. The aim of the current study was to investigate if proinflammatory cytokines are implicated in the loss of MEC contractile ability. Thus, lacrimal gland MEC from a SMA-GFP transgenic mouse were treated with IL-1β (10 ng/ml) for a total of 7 days. At days 0, 2, 4 and 7, GFP intensity, cell size/area, contractile proteins amounts and MEC contraction were assessed. At day 0, control and treated cells showed no differences in GFP intensity and cell size. GFP intensity started to decrease in treated MEC at day 2 (20%; p=0.02), continuing after day 4 (25%; p=0.007) and 7 (30%; p=0.0001). Mean cell area was also reduced at day 2 (34%; p=0.0005), and after 4 (51%; p<0.0001) and 7 days (30%; p=0.0015). The contraction assay at day 2 showed a 70% decrease of contraction in treated MEC (p<0.0001), 73% (p<0.0001) at day 4 and 82% (p=0.0015) at day 7 when compared to control. Levels of contractile proteins were measured on day 7 showing a decrease in SMA and calponin amount in treated MEC compared with the control group (around 30%; p=0.0016 and p=0.0206; respectively). Similar results were observed when TNF-α and IFN-γ were added along with IL-1β. Taken together the present data and those from our previous studies with Sjogren's syndrome mouse models, they strongly suggest that proinflammatory cytokines affect lacrimal gland MEC contractile ability that may account for the reduced tear secretion associated with Sjogren's syndrome dry eye disease.

Project description: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.

Project description:Dry eye disease can be a consequence of lacrimal gland insufficiency in Sjögren's Syndrome or increased tear film evaporation despite normal lacrimal gland function. To determine if there is a correlation between severity effects in these models and underlying pathophysiological responses, we compared the time dependent changes in each of these parameters that occur during a 6 week period. Dry eye was induced in 6-week-old female C57BL/6 mice by exposing them to an Intelligently Controlled Environmental System (ICES). Sixty mice were housed in ICES for 1, 2, 4 and 6 weeks respectively. Twelve were raised in normal environment and received subcutaneous injections of scopolamine hydrobromide (SCOP) 3 times daily for 5 days. Another sixty mice were housed in a normal environment and received no treatment. Corneal fluorescein staining along with corneal MMP-9 and caspase-3 level measurements were performed in parallel with the TUNEL assay. Interleukin-17(IL-17), IL-23, IL-6, IL-1, TNF-α, IFN-γ and TGF-β2 levels were estimated by real-time PCR measurements of conjunctival and lacrimal gland samples (LGs). Immunohistochemistry of excised LGs along with flow cytometry in cervical lymph nodes evaluated immune cell infiltration. Light and transmission electron microscopy studies evaluated LGs cytoarchitectural changes. ICES induced corneal epithelial destruction and apoptosis peaked at 2 weeks and kept stable in the following 4 weeks. In the ICES group, lacrimal gland proinflammatory cytokine level increases were much lower than those in the SCOP group. In accord with the lower proinflammatory cytokine levels, in the ICES group, lacrimal gland cytosolic vesicular density and size exceeded that in the SCOP group. ICES and SCOP induced murine dry eye effects became progressively more severe over a two week period. Subsequently, the disease process stabilized for the next four weeks. ICES induced local effects in the ocular surface, but failed to elicit lacrimal gland inflammation and cytoarchitectural changes, which accounts for less dry eye severity in the ICES model than that in the SCOP model.

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Lacrimal Gland Myoepithelial Cells Are Altered in a Mouse Model of Dry Eye Disease.

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