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The lacrimal gland (LG) secretes aqueous tears. Previous studies have provided insights into the cell lineage relationships during tissue morphogenesis. However, little is known about the cell types composing the adult LG and their progenitors. Using scRNAseq, we established the first comprehensive cell atlas of the adult mouse LG to investigate the cell hierarchy, its secretory repertoire, and the sex differences. Our analysis uncovered the complexity of the stromal landscape. Epithelium subclustering revealed myoepithelial cells, acinar subsets, and two novel acinar subpopulations: Tfrc hi and Car6 hi cells. The ductal compartment contained Wfdc2+ multilayered ducts and an Ltf+ cluster formed by luminal and intercalated duct cells. Kit+ progenitors were identified as: Krt14+ basal ductal cells, Aldh1a1+ cells of Ltf+ ducts, and Sox10 + cells of the Car6 hi acinar and Ltf+ epithelial clusters. Lineage tracing experiments revealed that the Sox10 + adult populations contribute to the myoepithelial, acinar, and ductal lineages. Using scRNAseq data, we found that the postnatally developing LG epithelium harbored key features of putative adult progenitors. Finally, we showed that acinar cells produce most of the sex-biased lipocalins and secretoglobins detected in mouse tears. Our study provides a wealth of new data on LG maintenance and identifies the cellular origin of sex-biased tear components.

Lacrimal gland inflammation triggers dry eye disease through impaired tear secretion by the epithelium. As aberrant inflammasome activation occurs in autoimmune disorders including Sjögren's syndrome, we analyzed the inflammasome pathway during acute and chronic inflammation and investigated its potential regulators. Bacterial infection was mimicked by the intraglandular injection of lipopolysaccharide (LPS) and nigericin, known to activate the NLRP3 inflammasome. Acute injury of the lacrimal gland was induced by interleukin (IL)-1α injection. Chronic inflammation was studied using two Sjögren's syndrome models: diseased NOD.H2 b compared to healthy BALBc mice and Thrombospondin-1-null (TSP-1 -/- ) compared to TSP-1 WT C57BL/6J mice. Inflammasome activation was investigated by immunostaining using the R26 ASC-citrine reporter mouse, by Western blotting, and by RNAseq. LPS/Nigericin, IL-1α and chronic inflammation induced inflammasomes in lacrimal gland epithelial cells. Acute and chronic inflammation of the lacrimal gland upregulated multiple inflammasome sensors, caspases 1/4, and interleukins Il1b and Il18 . We also found increased IL-1β maturation in Sjögren's syndrome models compared with healthy control lacrimal glands. Using RNA-seq data of regenerating lacrimal glands, we found that lipogenic genes were upregulated during the resolution of inflammation following acute injury. In chronically inflamed NOD.H2 b lacrimal glands, an altered lipid metabolism was associated with disease progression: genes for cholesterol metabolism were upregulated, while genes involved in mitochondrial metabolism and fatty acid synthesis were downregulated, including peroxisome proliferator-activated receptor alpha (PPARα)/sterol regulatory element-binding 1 (SREBP-1)-dependent signaling. We conclude that epithelial cells can promote immune responses by forming inflammasomes, and that sustained inflammasome activation, together with an altered lipid metabolism, are key players of Sjögren's syndrome-like pathogenesis in the NOD.H2 b mouse lacrimal gland by promoting epithelial dysfunction and inflammation.

Purpose: Fibroblast growth factor 10 (FGF10) is involved in eye, meibomian, and lacrimal gland (LG) development, but its function in adult eye structures remains unknown. This study aimed to characterize the role of FGF10 in homeostasis and regeneration of adult LG and corneal epithelium proliferation., Methods: Quantitative reverse transcription PCR was used for analysis of FGF10 expression in both early postnatal and adult mouse LG, and RNA sequencing was used to analyze gene expression during LG inflammation. FGF10 was injected into the LG of two mouse models of Sjögren's syndrome and healthy controls. Flow cytometry, BrdU cell proliferation assay, immunostaining, and hematoxylin and eosin staining were used to evaluate the effects of FGF10 injection on inflammation and cell proliferation in vivo. Mouse and human epithelial cell cultures were treated with FGF10 in vitro, and cell viability was assessed using WST-8 and adenosine triphosphate (ATP) quantification assays., Results: The level of Fgf10 mRNA expression was lower in adult LG compared to early postnatal LG and was downregulated in chronic inflammation. FGF10 injection into diseased LGs significantly increased cell proliferation and decreased the number of B cells. Mouse and human corneal epithelial cell cultures treated with FGF10 showed significantly higher cell viability and greater cell proliferation., Conclusions: FGF10 appears to promote regeneration in damaged adult LGs. These findings have therapeutic potential for developing new treatments for dry eye disease targeting the ability of the cornea and LG to regenerate.

The lacrimal gland (LG) is an exocrine gland that produces the watery part of the tear film that lubricates the ocular surface. Chronic inflammation, such as Sjögren's syndrome (SS), is one of the leading causes of aqueous-deficiency dry eye (ADDE) disease worldwide. In this study we analyzed the chronic inflammation in the LGs of the NOD.B10Sn-H2b/J ( NOD.H-2b ) mice, a mouse model of SS, utilizing bulk RNAseq and Visium spatial gene expression. With Seurat we performed unsupervised clustering and analyzed the spatial cell distribution and gene expression changes in all cell clusters within the LG sections. Moreover, for the first time, we analyzed and validated specific pathways defined by bulk RNAseq using Visium technology to determine activation of these pathways within the LG sections. This analysis suggests that altered metabolism and the hallmarks of inflammatory responses from both epithelial and immune cells drive inflammation. The most significant pathway enriched in upregulated DEGs was the "TYROBP Causal Network", that has not been described previously in SS. We also noted a significant decrease in lipid metabolism in the LG of the NOD.H-2b mice. Our data suggests that modulation of these pathways can provide a therapeutic strategy to treat ADDE., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mauduit, Delcroix, Umazume, de Paiva, Dartt and Makarenkova.)

The purpose of the present studies was to investigate the impact of chronic inflammation of the lacrimal gland, as occurs in Sjögren's syndrome, on the morphology and function of myoepithelial cells (MECs). In spite of the importance of MECs for lacrimal gland function, the effect of inflammation on MECs has not been well defined. We studied changes in MEC structure and function in two animal models of aqueous deficient dry eye, NOD and MRL/lpr mice. We found a statistically significant reduction in the size of MECs in diseased compared to control lacrimal glands. We also found that oxytocin receptor was highly expressed in MECs of mouse and human lacrimal glands and that its expression was strongly reduced in diseased glands. Furthermore, we found a significant decrease in the amount of two MEC contractile proteins, α-smooth muscle actin (SMA) and calponin. Finally, oxytocin-mediated contraction was impaired in lacrimal gland acini from diseased glands. We conclude that chronic inflammation of the lacrimal gland leads to a substantial thinning of MECs, down-regulation of contractile proteins and oxytocin receptor expression, and therefore impaired acini contraction. This is the first study highlighting the role of oxytocin mediated MEC contraction on lacrimal gland function.

The purpose of this study is to determine neural, vascular, protein secretion, and cellular signaling changes with disease progression in lacrimal glands of the thrombospondin-1 -/- (TSP-1 -/- ) mouse model of dry eye compared to C57BL/6 wild-type (WT) mice. Neural innervation was reduced in TSP-1 -/- lacrimal glands compared to WT controls, whereas the number of blood vessels was increased. Intracellular Ca 2+ stores and the amount of lysosomes, mitochondria, and secretory granules, but not the endoplasmic reticulum, were reduced in TSP-1 -/- compared to WT acini at 12 weeks of age. Ex vivo high KCl-evoked secretion was decreased in TSP-1 -/- compared to WT lacrimal gland tissue pieces. The α 1D -adrenergic agonist-stimulated response was increased in TSP-1 -/- at 4 and 24 weeks but decreased at 12 weeks, and the ATP and MeSATP-stimulated peak [Ca 2+ ] i responses were decreased at 24 weeks. These changes were observed prior to the appearance of mononuclear infiltrates. We conclude that in the lacrimal gland the absence of TSP-1: injures peripheral nerves; blocks efferent nerve activation; decreases protein secretion; and alters intracellular Ca 2+ stores. Through these effects the absence of TSP-1 leads to disruption of ocular surface homeostasis and development of dry eye.

Purpose: Sjögren's syndrome is a systemic chronic autoimmune inflammatory disease that primarily targets the salivary and lacrimal glands (LGs). Currently there is no cure; therefore, cell-based regenerative therapy may be a viable option. LG inflammation is facilitated by extracellular ATP and mediated by the Pannexin-1 (Panx1) membrane channel glycoprotein. We propose that suppression of inflammation through manipulation of Panx1 activity can stimulate epithelial cell progenitor (EPCP) engraftment., Methods: The expression of pannexins in the mouse and human LG was assayed by qRT-PCR and immunostaining. Acute LG inflammation was induced by interleukin-1α (IL1α) injection. Prior to EPCP transplantation, IL1α-injured or chronically inflamed LGs of thrombospondin-1-null mice (TSP-1-/-) were treated with the Panx1-specific blocking peptide (10panx) or the self-deliverable RNAi (sdRNAi). The efficacy of cell engraftment and the area of inflammation were analyzed by microscopy., Results: Panx1 and Panx2 were detected in the mouse and human LGs. Panx1 and proinflammatory factors were upregulated during acute inflammation at days 1 to 3 after the IL1α injection. The analysis of EPCP engraftment demonstrated a significant and reproducible positive correlation between the 10panx peptide or Panx1 sdRNAi treatment and the number of engrafted cells. Similarly, treatment of the LG of the TSP-1-/- mouse (mouse model of chronic LG inflammation) by either Panx1 or Caspase-4 (also known as Casp11) sdRNAi showed a significant decrease in expression of proinflammatory markers and the lymphocyte infiltration., Conclusions: Our results suggest that blocking Panx1 and/or Casp4 activities is a beneficial strategy to enhance donor cell engraftment and LG regeneration through the reduction of inflammation.

In humans, the lacrimal gland (LG) is the primary contributor to the aqueous layer of the tear film. Production of tears in insufficient quantity or of inadequate quality may lead to aqueous-deficiency dry eye (ADDE). Currently there is no cure for ADDE. The development of strategies to reliably isolate LG stem/progenitor cells from the LG tissue brings great promise for the design of cell replacement therapies for patients with ADDE. We analyzed the therapeutic potential of epithelial progenitor cells (EPCPs) isolated from adult wild-type mouse LGs by transplanting them into the LGs of TSP -1 -/- mice, which represent a novel mouse model for ADDE. TSP-1 -/- mice are normal at birth but progressively develop a chronic form of ocular surface disease, characterized by deterioration, inflammation, and secretory dysfunction of the lacrimal gland. Our study shows that, among c-kit-positive epithelial cell adhesion molecule (EpCAM + ) populations sorted from mouse LGs, the c-kit + dim/EpCAM + /Sca1 - /CD34 - /CD45 - cells have the hallmarks of an epithelial cell progenitor population. Isolated EPCPs express pluripotency factors and markers of the epithelial cell lineage Runx1 and EpCAM, and they form acini and ducts when grown in reaggregated three-dimensional cultures. Moreover, when transplanted into injured or "diseased" LGs, they engraft into acinar and ductal compartments. EPCP-injected TSP-1 -/- LGs showed reduction of cell infiltration, differentiation of the donor EPCPs within secretory acini, and substantial improvement in LG structural integrity and function. This study provides the first evidence for the effective use of adult EPCP cell transplantation to rescue LG dysfunction in a model system. Stem Cells Translational Medicine 2017;6:88-98., (© 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Purpose: The adult lacrimal gland (LG) is highly regenerative and is able to repair itself even after substantial damage; however, this ability to regenerate is lost with the development of dry eye conditions in chronically inflamed LGs.This study compares changes in the cell adhesion and cell matrix molecules and stem cell transcription factors in the LGs of healthy mice and of two mouse models of Sjögren's syndrome: nonobese diabetic (NOD) and MRL-lpr/lpr (MRL/lpr) mice during the early stage of inflammation., Methods: The LGs from 12- to 13-week-old female MRL/lpr and male NOD mice along with their respective control strains were harvested and divided into three pieces and processed for quantitative (q) RT-PCR and qRT-PCR Arrays, histology, immunohistochemistry, and Western blotting., Results: The extracellular matrix (ECM) and adhesion molecules RT2-PCR array combined with protein expression data revealed changes in the expression of integrins, matrix metalloproteinases, and other molecules, which are associated largely with invasion, attachment, and expansion of the lymphocytic cells, whereas changes in the stem cell transcription factors revealed substantial decrease in expression of transcription factors associated with epithelial stem/progenitor cell lineage., Conclusions: We concluded that the expression of several important ECM components is significantly deregulated in the LG of two murine models of Sjögren's syndrome, suggesting an alteration of the epithelial stem/progenitor cell niche. This may result in profound effects on localization, activation, proliferation, and differentiation of the LG stem/progenitor cells and, therefore, LG regeneration.

Purpose: Chronic inflammation of the lacrimal gland results in changes in the composition of the extracellular matrix (ECM), which is believed to compromise tissue repair. We hypothesized that increased production/activity of matrix metalloproteinases (MMPs), especially MMP-2 and -9, in inflamed lacrimal glands modifies the ECM environment, therefore disrupting tissue repair., Methods: The lacrimal glands from female MRL/lpr and male NOD mice along with their respective control strains were harvested and divided into three pieces and processed for histology, immunohistochemistry, zymography, Western blotting, and RNA analyses. In another study, MRL/lpr mice were treated for 5 weeks with a selective MMP2/9 inhibitor peptide or a control peptide. At the end of treatment, the lacrimal glands were excised and the tissue was processed as described above., Results: There was a 2.5- and 2.7-fold increase in MMP2 gene expression levels in MRL/lpr and NOD mice, respectively. Matrix metalloproteinase 2 and 9 enzymatic activities and protein expression levels were significantly upregulated in the lacrimal glands of MRL/lpr and NOD mice compared to controls. Treatment with the MMP2/9 inhibitor resulted in decreased activity of MMP-2 and -9 both in vitro and in vivo. Importantly, MMP2/9 inhibitor treatment of MRL/lpr mice improved aqueous tear production and resulted in reduced number and size of lymphocytic foci in diseased lacrimal glands., Conclusions: We conclude that MMP2/9 expression and activity are elevated in lacrimal glands of two murine models of Sjögren's syndrome, suggesting that manipulation of MMP2/9 activity might be a potential therapeutic target in chronically inflamed lacrimal glands.

Purpose: Lacrimal gland (LG) morphogenesis and repair are regulated by a complex interplay of intrinsic factors (e.g., transcription factors) and extrinsic signals (e.g., soluble growth/signaling factors). Many of these interconnections remain poorly characterized. Runt-related (Runx) factors belong to a small family of heterodimeric transcription factors known to regulate lineage-specific proliferation and differentiation of stem cells. The purpose of this study was to define the expression pattern and the role of Runx proteins in LG development and regeneration., Methods: Expression of epithelial-restricted transcription factors in murine LG was examined using immunostaining, qRT-PCR, and RT(2)Profiler PCR microarrays. The role of Runx transcription factors in LG morphogenesis was studied using siRNA and ex vivo LG cultures. Expression of Runx transcription factors during LG regeneration was assessed using in vivo model of LG regeneration., Results: We found that Runx factors are expressed in the epithelial compartment of the LG; in particular, Runx1 was restricted to the epithelium with highest level of expression in ductal and centroacinar cells. Downregulation of Runx1 to 3 expression using Runx-specific siRNAs abolished LG growth and branching and our data suggest that Runx1, 2, and 3 are partially redundant in LG development. In siRNA-treated LG, reduction of branching correlated with reduction of epithelial proliferation, as well as expression of cyclin D1 and the putative epithelial progenitor cell marker cytokeratin-5. Runx1, Runx3, and cytokeratin-5 expression increased significantly in regenerating LG and there was modest increase in Runx2 expression during LG differentiation., Conclusions: Runx1 and 2 are new markers of the LG epithelial lineage and Runx factors are important for normal LG morphogenesis and regeneration.

The lacrimal gland (LG) develops through branching morphogenesis and produces secretions, including tears, that lubricate and protect the ocular surface. Despite the prevalence of LG disorders such as dry eye, relatively little is known about the regulation of LG development. In this study, we show that the homeobox transcription factor Barx2 is highly expressed in conjunctival epithelium, eyelids and ocular [lacrimal, harderian (HG), and meibomian (MG)] glands and is necessary for normal ocular gland and eyelid development. Barx2(-/-) mice show defective LG morphogenesis, absence of the HG, and defects in MG and eyelid fusion. Ex vivo antisense assays confirm the requirement for Barx2 in LG bud elongation and branching. Gene expression profiles reveal decreased expression of several adhesion and matrix remodeling molecules in Barx2(-/-) LGs. In culture, Barx2 regulates expression of matrix metalloproteinases (MMPs) and epithelial cell migration through the extracellular matrix. Fibroblast growth factors are crucial regulators of LG development and we show that Barx2 is required for Fgf10-induced LG bud elongation and that both Barx2 and Fgf10 cooperate in the regulation of MMPs. Together, these data suggest a mechanism for the effects of loss of Barx2 on ocular gland development. Intriguingly, salivary glands that also express a high level of Barx2 develop normally in Barx2(-/-) mice and do not show altered levels of MMPs. Thus, the function of Barx2 is specific to ocular gland development. Based on our data, we propose a functional network involving Barx2, Fgf10 and MMPs that plays an essential role in regulating branching morphogenesis of the ocular glands.

The lacrimal gland provides an excellent model with which to study the epithelial-mesenchymal interactions that are crucial to the process of branching morphogenesis. In the current study, we show that bone morphogenetic protein 7 (Bmp7) is expressed with a complex pattern in the developing gland and has an important role in regulating branching. In loss-of-function analyses, we find that Bmp7-null mice have distinctive reductions in lacrimal gland branch number, and that inhibition of Bmp activity in gland explant cultures has a very similar consequence. Consistent with this, exposure of whole-gland explants to recombinant Bmp7 results in increased branch number. In determining which cells of the gland respond directly to Bmp7, we have tested isolated mesenchyme and epithelium. We find that, as expected, Bmp4 can suppress bud extension in isolated epithelium stimulated by Fgf10, but interestingly, Bmp7 has no discernible effect. Bmp7 does, however, stimulate a distinct response in mesenchymal cells. This manifests as a promotion of cell division and formation of aggregates, and upregulation of cadherin adhesion molecules, the junctional protein connexin 43 and of alpha-smooth muscle actin. These data suggest that in this branching system, mesenchyme is the primary target of Bmp7 and that formation of mesenchymal condensations characteristic of signaling centers may be enhanced by Bmp7. Based on the activity of Bmp7 in promoting branching, we also propose a model suggesting that a discrete region of Bmp7-expressing head mesenchyme may be crucial in determining the location of the exorbital lobe of the gland.

Fibroblast growth factor (FGF) signaling plays an important role in controlling cell proliferation, survival, and cell movements during branching morphogenesis of many organs. In mammals branching morphogenesis is primarily regulated by members of the FGF7-subfamily (FGF7 and FGF10), which are expressed in the mesenchyme, and signal to the epithelial cells through the "b" isoform of fibroblast growth factor receptor-2 (FGFR2). Our previous work demonstrated that FGF7 and FGF10 form different gradients in the extracellular matrix (ECM) and induce distinct cellular responses and gene expression profiles in the lacrimal and submandibular glands. The last finding was the most surprising since both FGF7 and FGF10 bind signal most strongly through the same fibroblast growth factor receptor-2b isoform (FGFR2b). Here we revisit this question to gain an explanation of how the different FGFs regulate gene expression. For this purpose, we employed our ex vivo epithelial explant migration assay in which isolated epithelial explants are grown near the FGF loaded beads. We demonstrate that the graded distribution of FGF induces activation of ERK1/2 MAP kinases that define the position of the boundary between proliferating "bud" and differentiating "stalk" cells of growing lacrimal gland epithelium. Moreover, we showed that gene expression profiles of the epithelial explants exposed to distinct FGFs strictly depend on the ratio between "bud" and "stalk" area. Our data also suggests that differentiation of "stalk" and "bud" regions within the epithelial explants is necessary for directional and persistent epithelial migration. Gaining a better understanding of FGF functions is important for development of new approaches to enhance tissue regeneration. [ABSTRACT FROM AUTHOR]

Sjögren's syndrome (SS) is a systemic autoimmune disorder affecting approximately 3% of the population in the United States. This disease has a female predilection and affects exocrine glands, including lacrimal and salivary glands. Dry eyes and dry mouths are the most common symptoms due to the loss of salivary and lacrimal gland function. Symptoms become more severe in secondary SS, where SS is present along with other autoimmune diseases like systemic lupus erythematosus, systemic sclerosis, or rheumatoid arthritis. It is known that aberrant activation of immune cells plays an important role in disease progression, however, the mechanism for these pathological changes in the immune system remains largely unknown. This review highlights the role of different immune cells in disease development, therapeutic treatments, and future strategies that are available to target various immune cells to cure the disease. [ABSTRACT FROM AUTHOR]