Lacrimal Gland Epithelial Cells Research Articles

Modulation of Decellularized Lacrimal Gland Hydrogel Biodegradation by Genipin Crosslinking.

Hydrogels derived from decellularized tissues are promising biomaterials in tissue engineering, but their rapid biodegradation can hinder in vitro cultivation. This study aimed to retard biodegradation of a hydrogel derived from porcine decellularized lacrimal glands (dLG-HG) by crosslinking with genipin to increase the mechanical stability without affecting the function and viability of lacrimal gland (LG)-associated cells. The effect of different genipin concentrations on dLG-HG stiffness was measured rheologically. Cell-dependent biodegradation was quantified over 10days, and the impact on matrix metalloproteinase (MMP) activity was quantified by gelatin and collagen zymography. The viability of LG epithelial cells (EpCs), mesenchymal stem cells (MSCs), and endothelial cells (ECs) cultured on genipin-crosslinked dLG-HG was assessed after 10days, and EpC secretory activity was analyzed by β-hexosaminidase assay. The 0.5-mM genipin increased the stiffness of dLG-HG by about 46%, and concentrations > 0.25mM caused delayed cell-dependent biodegradation and reduced MMP activity. The viability of EpCs, MSCs, and ECs was not affected by genipin concentrations of up to 0.5mM after 10days. Moreover, up to 0.5-mM genipin did not negatively affect EpC secretory activity compared to control groups. A concentration of 0.5-mM genipin increased dLG-HG stiffness, and 0.25-mM genipin was sufficient to prevent MMP-dependent degradation. Importantly, concentrations of up to 0.5-mM genipin did not compromise the viability of LG-associated cells or the secretory activity of EpCs. Thus, crosslinking with genipin improves the properties of dLG-HG for use as a substrate in LG tissue engineering.

A New Immortalized Human Lacrimal Gland Cell Line.

The lacrimal gland is crucial for maintaining ocular health by producing the aqueous component of the tear film, which hydrates and nourishes the ocular surface. Decreased production of this component results in dry eye disease, a condition affecting over 250 million people worldwide. However, the scarcity of primary human material for studying its underlying mechanisms and the absence of a cell model for human lacrimal gland epithelial cells present significant challenges. Here, we describe the generation of immortalized human lacrimal gland cell lines through the introduction of an SV40 antigen. We successfully isolated and characterized three cell clones from a female lacrimal gland donor, confirming their epithelial identity through genomic and protein analyses, including PCR, RNAseq, immunofluorescence and cultivation in a 3D spheroid model. Our findings represent a significant advancement, providing improved accessibility to investigate the molecular pathogenesis mechanisms of dry eye disease and potential therapeutic interventions. We identified the expression of typical epithelial cell marker genes and demonstrated the cells' capability to form 2D cell sheets and 3D spheroids. This establishment of immortalized human lacrimal gland cells with epithelial characteristics holds promise for future comprehensive studies, contributing to a deeper understanding of dry eye disease and its cellular mechanisms.

Enhancement of lacrimal gland cell function by decellularized lacrimal gland derived hydrogel

Sustainable treatment of aqueous deficient dry eye (ADDE) represents an unmet medical need and therefore requires new curative and regenerative approaches based on appropriate in vitro models. Tissue specific hydrogels retain the individual biochemical composition of the extracellular matrix and thus promote the inherent cell´s physiological function. Hence, we created a decellularized lacrimal gland (LG) hydrogel (dLG-HG) meeting the requirements for a bioink as the basis of a LG model with potential for in vitro ADDE studies. Varying hydrolysis durations were compared to obtain dLG-HG with best possible physical and ultrastructural properties while preserving the original biochemical composition. A particular focus was placed on dLG-HG´s impact on viability and functionality of LG associated cell types with relevance for a future in vitro model in comparison to the unspecific single component hydrogel collagen type-I (Col) and the common cell culture substrate Matrigel. Proliferation of LG epithelial cells (EpC), LG mesenchymal stem cells, and endothelial cells cultured on dLG-HG was enhanced compared to culture on Matrigel. Most importantly with respect to a functional in vitro model, the secretion capacity of EpC cultured on dLG-HG was higher than that of EpC cultured on Col or Matrigel. In addition to these promising cell related properties, a rapid matrix metalloproteinase-dependent biodegradation was observed, which on the one hand suggests a lively cell–matrix interaction, but on the other hand limits the cultivation period. Concluding, dLG-HG possesses decisive properties for the tissue engineering of a LG in vitro model such as cytocompatibility and promotion of secretion, making it superior to unspecific cell culture substrates. However, deceleration of biodegradation should be addressed in future experiments.

Regulation of Axon Guidance by Slit2 and Netrin-1 Signaling in the Lacrimal Gland of Aqp5 Knockout Mice.

Dry eye disease (DED) is multifactorial and associated with nerve abnormalities. We explored an Aquaporin 5 (AQP5)-deficiency-induced JunB activation mechanism, which causes abnormal lacrimal gland (LG) nerve distribution through Slit2 upregulation and Netrin-1 repression. Aqp5 knockout (Aqp5-/-) and wild-type (Aqp5+/+) mice were studied. LGs were permeabilized and stained with neuronal class III β-tubulin, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). Whole-mount images were acquired through tissue clearing and 3D fluorescence imaging. Mouse primary trigeminal ganglion (TG) neurons were treated with LG extracts and Netrin-1/Slit2 neutralizing antibody. Transcription factor (TF) prediction and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) experiments verified the JunB binding and regulatory effect on Netrin-1 and Slit2. Three-dimensional tissue and section immunofluorescence showed reduced LG nerves in Aqp5-/- mice, with sympathetic and sensory nerves significantly decreased. Netrin-1 was reduced and Slit2 increased in Aqp5-/- mice LGs. Aqp5+/+ mice LG tissue extracts (TEs) promoted Aqp5-/- TG neurons axon growth, but Netrin-1 neutralizing antibody (NAb) could inhibit that promotion. Aqp5-/- mice LG TEs inhibited Aqp5+/+ TG axon growth, but Slit2 NAb alleviated that inhibition. Furthermore, JunB, a Netrin-1 and Slit2 TF, could bind them and regulate their expression. SR11302, meanwhile, reversed the Netrin-1 and Slit2 shifts caused by AQP5 deficiency. AQP5 deficiency causes LG nerve abnormalities. Persistent JunB activation, the common denominator for Netrin-1 suppression and Slit2 induction, was found in Aqp5-/- mice LG epithelial cells. This affected sensory and sympathetic nerve fibers' distribution in LGs. Our findings provide insights into preventing, reversing, and treating DED.

Lacrimal Gland Epithelial Cells Shape Immune Responses through the Modulation of Inflammasomes and Lipid Metabolism.

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.H2b compared to healthy BALBc mice and Thrombospondin-1-null (TSP-1-/-) compared to TSP-1WTC57BL/6J mice. Inflammasome activation was investigated by immunostaining using the R26ASC-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.H2b 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.H2b mouse lacrimal gland by promoting epithelial dysfunction and inflammation.

Lysine Demethylase 1B Promotes Tear Secretion Disorder in Sjogren’s Syndrome by Regulating the PAX6/CLU Axis

The impacts of lysine demethylase 1B (KDM1B) have been probed in multiple diseases, but the effects of KDM1B on SS remained obscure. The study aimed to unravel the efficiency of KDM1B on SS progression via the paired box 6 (PAX6)/clusterin (CLU) axis. NODB10. H2b mice were selected to establish the SS model. KDM1B, Pax6, and CLU expression in SS mice was assessed. Adeno-associated viruses carrying KDM1B, Pax6, and CLU were injected into the SS mice to detect tear secretion, epithelium corneal fluorescein staining scores, and levels of specific markers of lacrimal gland epithelial cells, neurotransmitter receptors that induce secretion from the lacrimal gland, and genes encoding normal tear components. The relation among KDM1B, Pax6, and CLU was examined. The rescue experiments were conducted for verifying the interaction among KDM1B, Pax6, and CLU. KDM1B expression was elevated, while Pax6 and CLU levels were decreased in the lacrimal gland tissues of SS mouse models. KDM1B decrement and Pax6 augmentation improved tear secretion, reduced corneal fluorescein staining score, decreased levels of specific markers of lacrimal gland epithelial cells, and increased levels of neurotransmitter receptors that induce secretion from the lacrimal gland and genes encoding normal tear components. KDM1D suppressed Pax6 expression by mediating H3K4me2 demethylation. Pax6 promoted the expression of CLU at the transcriptional level by binding to the CLU promoter. Silencing of Pax6 or CLU could reverse the effects of KDM1B reduction on improving the tear secretion disorder of SS mice. Silencing KDM1B mitigates the tear secretion disorder of SS mice via modulating the Pax6/CLU axis.

Growth factor dependence of the proliferation and survival of cultured lacrimal gland epithelial cells isolated from late-embryonic mice.

Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) regulate the growth and morphogenesis of various exocrine glands with branched morphologies. Their roles in lacrimal gland (LG) development remain unknown, but fibroblast growth factor (FGF) 10 is crucial for early LG organogenesis. To clarify the roles of EGF, HGF, and FGF10 in LG development, LG epithelial cells were isolated from late-embryonic and neonatal mice; cultured; and treated with EGF, HGF, or FGF10 and their respective receptor tyrosine kinase (RTK) inhibitors AG1478, PHA665752, or SU5402. EGF and HGF increased the number of viable cells by enhancing DNA synthesis, FGF10 and SU5402 showed no such effect, and RTK inhibitors exhibited the opposite effect. EGF and HGF receptors were immunostained in cultured late-embryonic LG epithelial cells and terminal LG acini from late embryos and adult mice. HGF was detected in neonatal LG epithelial cell culture supernatants by western blotting. In the absence of EGF and HGF RTK inhibitors, growth factor addition increased the number of viable cells and suppressed cell death. However, when one RTK was inhibited and a growth factor targeting an intact RTK was added, the number of dead cells increased as the number of viable cells increased. No cells survived when both RTKs were inhibited. In explant cultures of LGs from embryos, AG1478 or PHA665752 decreased the number of Ki67-positive proliferating epithelial cells in terminal acini. Thus, EGF and HGF may function in a cooperative autocrine manner, supporting cell proliferation and survival during LG development in late-embryonic and neonatal mice.

Expression Profiles of CircRNA and mRNA in Lacrimal Glands of AQP5-/- Mice With Primary Dry Eye.

Purpose: This work aimed to identify differentially expressed circular RNAs (circRNAs) and elucidate their potential function in aquaporin 5 (AQP5) knockout (AQP5–/–) mice with the primary dry eye phenotype.Methods: A slit lamp examination was performed on AQP5–/– mice to assess corneal epithelial defects using fluorescein sodium staining. Hematoxylin–eosin staining and transmission electron microscopy analysis were performed to identify structural changes in lacrimal gland epithelial cells due to AQP5 deficiency. The expression profiles of circRNA and messenger RNA (mRNA) were determined by a microarray analysis. The selected circRNA was verified by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to predict the biological functions and the potential pathways of parental genes involved in lacrimal gland epithelial cell changes. According to the bioinformatics analysis of identified circRNAs, we predicted a circRNA–miRNA–mRNA network of phagosomes.Results: The AQP5–/– mice spontaneously exhibit dry eye symptoms, wherein the AQP5 deficiency changes the structure of lacrimal gland epithelial cells. The analysis revealed that, compared to AQP5+/+ mice, 30 circRNAs in the lacrimal glands of AQP5–/– mice were differentially expressed (fold change ≥ 2.0, p < 0.05). Nine upregulated circRNAs were identified using qRT-PCR, and nine upregulated validated circRNAs, 40 altered microRNAs (miRNAs), and nine upregulated mRNAs were identified through a network analysis. The KEGG analysis showed that these nine target genes were expressed in phagosomes.Conclusion: The AQP5–/– mice have primary and stable dry eye phenotypes from birth. We identified differently expressed circRNAs in the lacrimal glands of AQP5–/– and AQP5+/+ mice, predicting a circRNA–miRNA–mRNA network of phagosomes. CircRNA likely plays an important role in lacrimal gland epithelial cell pathogenesis. Therefore, it is reasonable to use circRNA as a potential therapeutic agent for the treatment of dry eyes.

Developmental change in the gene expression of transient receptor potential melastatin channel 3 (TRPM3) in murine lacrimal gland

Transient receptor potential (TRP) channels are cation channels with ubiquitous expression. Various TRP channels are functionally active at the ocular surface and are involved in tear secretion and multiple inflammatory processes. So far, the impact of TRP channels regarding the development of the lacrimal gland (LG) is unclear. While investigating TRP channels in the LG, the TRPM3 channel presented itself as a promising candidate to play a role in the development and functioning of the LG. Therefore, Trpm3 expression was analyzed in different embryonic and postembryonic LGs. Thus, gene expression of TRPM channels including Trpm2, Trpm3, Trpm4 and Trpm6 was analyzed by quantitative RT-PCR in murine LGs at different developmental stages. Localization of TRPM3 in LGs was examined by immunohistochemistry. Primary LG epithelial cells (LGEC) and mesenchymal cells (MC) from newborn mice were cultured (either separately or collectively) for three days, and Trpm3 expression was analyzed in LGEC and MC. As a result, gene expression of Trpm2, Trpm4 and Trpm6 showed no significant difference in LGs in the different stages of development. However, Trpm3 gene expression was significantly higher in the embryonic stage than in the postnatal stage with the peak at E18. Postnatal, Trpm3 expression significantly decreased up to 28-fold until two years of age. Immunohistochemistry for TRPM3 revealed apical membranous expression in the excretory ducts, as well as in the acini of up to P7 old mice. Trpm3 expression in LGEC were significantly higher than that of MC. Our results indicate that Trpm3 expression in murine LG is age-dependent and peaks at age E18. Its expression is localized in the apical membrane of the glandular epithelium. However, its functional role still requires additional study in the LG.

Clinical histopathologic characteristics of lacrimal glands in lacrimal gland prolapse with blepharochalasis

Objective: To analyze the clinical histopathologic characteristics of lacrimal glands and possible mechanisms of lacrimal gland prolapse in blepharochalasis (BC). Methods: A case-controlled study of 23 consecutive patients with prolapse of lacrimal glands in BC was performed. All samples were obtained during surgery from the Department of Ophthalmology, Beijing Tongren Hospital, Capital Medical University between January 2009 and December 2016. The lacrimal tissue included prolapsed lacrimal glands (30 samples) and controls from the donors in the eye bank of Beijing Tongren Hospital, Capital Medical University (8 samples). Hematoxylin-eosin staining, special staining, immunohistochemistry and colloidal gold-labeled pre-embedded indirect immunogold electron microscopy (Gold-IIEM) were performed to analyze the histopathologic characteristics of the samples. The nonparametric Wilcoxon signed-ranks test was carried out for statistical analysis. Results: Among the 23 patients with lacrimal gland prolapse in BC, there were 3 males and 20 females. The mean age of morbidity was 11 years old (7-16 years). In the 8 normal control cases, 3 males and 5 females were included. The mean age was 15 years (10-20 years). In the 30 prolapsed lacrimal gland samples, hematoxylin-eosin staining showed enlargement of glandular lumina accompanied by inflammatory infiltrates of interstitial tissue in 2 samples. Marked loosening of collagen fibers of the obtainable lacrimal fascia was observed. The results on immunohistochemical staining demonstrated an increased level of immunocytes in the 30 prolapsed lacrimal gland samples, including IgA (+++, ++, +, -; 12, 11, 4, 3 vs. 0, 0, 1, 7; Z=-3.892), CD3(+)T cells (+++, ++, +, -; 2, 19, 7, 2 vs. 0, 0, 1, 7; Z=-4.168), matrix metalloproteinase (MMP)-3 (+++, ++, +, -; 0, 0, 11, 19 vs. 0, 0, 0, 8; Z=-2.005) and MMP-9 (+++, ++, +, -; 14, 14, 0, 2 vs. 0, 0, 0, 8; Z=-4.552) (all P<0.05). IgG, IgM, CD20 and C1-inhibitor were either absent or expressed at background level in the 30 prolapsed lacrimal gland samples (all P>0.05). Gold-IIEM showed zymogon granules in lacrimal glands were out of shape. MMP-3 and MMP-9 colloidal gold particles existed on the zymogon granules, and MMP-3 colloidal gold particles also existed on the surface of lacrimal gland epithelial cells. Conclusions: The histopathological changes in the lacrimal glands of lacrimal gland prolapse with BC include inflammatory infiltration, elastic fiber degeneration, marked loosening of the supporting fascia tissue, and an increased level of immunocytes, including IgA, CD3(+)T cells, MMP-3 and MMP-9. The results suggest that lacrimal gland prolapse with BC may result in the immuno-pathogenetic mechanisms with the involvement of cell-mediated immune responses. (Chin J Ophthalmol, 2020, 56: 205-210).

Three Dimensional Culture of Potential Epithelial Progenitor Cells in Human Lacrimal Gland.

PurposeWe investigate human lacrimal gland tissue to determine the presence of progenitor cells in this adult human tissue.MethodsSix human lacrimal gland tissues from donors were collected and stored immediately in the culture medium at 4°C until the next procedure. One part of the lacrimal gland tissue was prepared for immunofluorescence staining and the other part was prepared for primary cell culture. Immunofluorescence analysis was conducted to evaluate cultured lacrimal epithelial phenotype and progenitor cell markers for five passages. Real-time polymerase chain reaction (PCR) was performed to assess proliferation markers in the different passages. Three-dimensional culture and PCR were conducted to determine the differentiation potential of cultured human lacrimal gland cells.ResultsHuman lacrimal gland tissue expressed a number of epithelial progenitor cell markers. Precursor cell markers C-Kit, K15, Nestin, and P63 were observed in lacrimal gland tissues. Lacrimal gland epithelial cells were cultured successfully and passaged to P5. The cultured lacrimal gland epithelial cells were positive for pan-cytokeratin (PCK), AQP5, Rab3D, ABCB5, C-kit, K15, Ki67, and P63. Human lacrimal gland cells could form spheroids in vitro and then grow into mini-gland-like structures. PCR results showed proliferation and differentiation capability of those cultured cells.ConclusionsHuman lacrimal gland tissues contain precursor marker-positive cells and marker expression also was detected in ex vivo cultured cells, which showed differentiation capability.Translational RelevanceFuture studies of differentiation in human lacrimal gland tissue may aid in developing stem cell-based therapies for dry eye disease.

Analysis of lacrimal gland derived mesenchymal stem cell secretome and its impact on epithelial cell survival

In situ regeneration of lacrimal gland (LG) tissue would be a promising approach to curatively treat dry eye disease (DED). Mesenchymal stem cells (MSC) exhibit therapeutic effects in a variety of pathological conditions and our group recently reported that their number increases in regenerating mouse LG. Since the therapeutic effects are suggested to arise from secreted trophic factors, the application of MSC-secreted proteins seems to be a promising approach to induce/enhance LG regeneration. Therefore, this study aims to optimize the isolation of murine LG-MSC and analyze their secretome to investigate its potential for LG epithelial cell survival in vitro. For optimization, LG-MSC were isolated by an explant technique or cell sorting and their secretome was investigated under normal and inflammatory conditions. Results showed that the secretome of MSC had beneficial effects on the viability of ethanol-damaged LG epithelial cells. Additional, Lipocalin-2, prosaposin, ras GTPase-activating protein-binding protein 1 (Rac1) and signal transducer and activator of transcription 1 (STAT1), proteins that were up-regulated under inflammatory conditions, further improved the cell survival of ethanol-damaged LG epithelial cells. Interestingly, recovery of cell viability was highest, when the cells were incubated with STAT1. Summarizing, this study identified promising proteins for further studies on LG regeneration.

Isolation and Propagation of Lacrimal Gland Putative Epithelial Progenitor Cells.

We present a protocol for isolation of putative epithelial progenitor cells from mouse lacrimal gland (LG) by fluorescence-activated cell sorting (FACS). Isolated LG epithelial progenitor cells can be cultured as 3D reaggregates within extracellular matrix gel or plated as a monolayer. 3D cultures could be maintained for several days and then dissociated with trypsin and plated as monolayer cultures, processed for analysis (e.g., mRNA/protein expression) and/or used for transplantations. Our goal is to provide researchers with a method that can be used as is or modified if isolation of other LG epithelial cell types is required.

Development of lacrimal gland spheroids for lacrimal gland tissue regeneration.

Severe dry eye syndrome resulting from lacrimal gland (LG) dysfunction can cause blindness, yet treatments remain palliative. In vitro reconstruction of LG tissue could provide a curative treatment. We aimed to combine epithelial cells with endothelial cells and mesenchymal stem cells (MSCs) to form a 3D functional unit. Epithelial cells and MSCs were isolated from porcine LG; endothelial cells were isolated from human foreskin. MSCs were characterised (flow cytometry and differentiation potential assays). All 3 cell types were combined on Matrigel and spheroid formation observed. Spheroids were characterised [immunohistochemistry (IHC) and transmission electron microscopy] and function assessed (β-hexosaminidase assay). Spheroids were transferred to decellularised jejunum (SIS-Muc) in dynamic cultures for 1week before further characterisation. MSCs did not express CD31 but expressed CD44 and CD105 and differentiated towards osteogenic and adipogenic lineages. Spheroids formed on Matrigel within 18hr, contracting to ~10% of the well area (p<.005). IHC revealed presence of all 3 cells within spheroids. Transmission electron microscopy revealed cell-cell contacts and polarisation at the apical surface. In static cultures, function was increased in spheroids cf. monolayer controls (p<.05) but over 72hr, spheroid function (p<.05), viability (p<.05), and proliferation decreased, whilst apoptosis increased. On SIS-Muc under dynamic culture, however, spheroids continued to proliferate to repopulate SIS-Muc. IHC revealed LG epithelial cells coexpressing pan-cytokeratin and lysozyme, as well as endothelial cells and MSCs and cells remained capable of responding to carbachol (p<.05). These spheroids could form the basis of a regenerative medicine treatment approach for dry eye syndrome. In vivo studies are required to evaluate this further.

Manipulation of Panx1 Activity Increases the Engraftment of Transplanted Lacrimal Gland Epithelial Progenitor Cells.

PurposeSjögren'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.MethodsThe 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.ResultsPanx1 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.ConclusionsOur 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.

Evaluation of Decellularized Porcine Jejunum as a Matrix for Lacrimal Gland Reconstruction In Vitro for Treatment of Dry Eye Syndrome.

Dry eye syndrome (DES) can cause blindness in severe cases, but mainly palliative treatments exist. A tissue-engineered lacrimal gland (LG) could provide a curative treatment. We aimed to evaluate decellularized porcine jejunum (SIS-Muc) as a scaffold for porcine LG epithelial cells. To evaluate SIS-Muc as a potential scaffold, basement membrane proteins in SIS-Muc and native LG were compared (immunohistochemistry [IHC]). Porcine LG epithelial cells cultured on plastic were characterized (immunocytochemistry), and their culture supernatant was compared with porcine tears (proteomics). Epithelial cells were then seeded onto SIS-Muc in either a static (cell crown) or dynamic culture (within a perfusion chamber) and metabolic (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and secretory capacities (β-hexosaminidase assay), protein expression (IHC), and ultrastructure transmission electron microscopy (TEM) compared in each. Collagen IV and laminin were found in both native LG and SIS-Muc. When cultured on plastic, LG epithelial cells expressed pan-cytokeratin, Rab3D, HexA, and produced mucins, but lysozyme and lactoferrin expression was nearly absent. Some porcine tear proteins (lipocalin-2 and lactoferrin) were found in LG epithelial cell culture supernatants. When LG cells were cultured on SIS-Muc, metabolic and β-hexosaminidase activities were greater in dynamic cultures than static cultures (P < 0.05). In both static and dynamic cultures, cells expressed pan-cytokeratin, Rab3D, lysozyme, and lactoferrin and produced mucins, and TEM revealed cell polarization at the apical surface and cell-cell and cell-scaffold contacts. SIS-Muc is a suitable scaffold for LG cell expansion and may be useful toward reconstruction of LG tissue to provide a curative treatment for DES. Dynamic culture enhances cell metabolic and functional activities.

Strategies for Regenerating the Lacrimal Gland.

Aqueous deficient dry eye disease, a significant cause of morbidity worldwide, is due to dysfunction of the main and accessory lacrimal glands. Recent advances in efforts to regenerate lacrimal gland are reviewed. Several strategies are being explored: ex vivo culture models of human and non-human lacrimal gland epithelial and myoepithelial cells, isolation and characterization of adult precursor cells within lacrimal glands, directed differentiation of stem cells to lacrimal gland cells, and organogenesis and engraftment techniques. Conditions for primary cell culture and expansion are being established and will help in the characterization of lacrimal cells. Presumed adult precursor cells have been isolated, laying down foundations for regeneration. Stem cells have been induced to express features of lacrimal gland cells. Engraftment of ex vivo cultured lacrimal tissue is proof of concept that lacrimal gland regeneration and repopulation is possible.

A pilot molecular mechanism study on lacrimal gland as a potential human immunodeficiency virus reservoir

Background Acquired immune deficiency syndrome (AIDS) is an infectious disease caused by human immunodeficiency virus (HIV). Highly active antiretroviral therapy (HAART) is an effective treatment for AIDS, but it cannot completely eliminate the viral load in the body for the existence of HIV reservoir.Previous studies demonstrated that HIV could be detected in tears of virus load negative AIDS patients who received effective HAART, suggesting that lacrimal gland is another member of HIV reservoirs. Objective The aim of this study was to explore whether lacrimal gland has a molecular basis of HIV infection and the mechanism of lacrimal gland infection of HIV. Methods Fourteen specimens of lacrimal gland were collected during the surgery from 14 patients with lacrimal gland diseases in Peking Union Medical College Hospital from November 2013 to December 2015, including 13 non-HIV-infected patients and 1 HIV-infected patient.In 13 non-HIV infected patients, lacrimal glands prolapse was in 12 patients with the normal pathological tissue structure and dacryoadenitis was in 1 patient with the histopathological diagnosis of interstitial lymphoid tissue hyperplasia.The clinical manifestation of HIV-infected patient was dacryoadenitis with the histopathological diagnosis of interstitial lymphoid tissue hyperplasia.The paraffin sections of 12 non-HIV-infected specimens and 1 HIV-infected specimen were prepared, and the expressions of CD4, C-X-C chemokine receptor 4 (CXCR4)and C-C chemokine receptor type 5 (CCR5) in lacrimal gland specimens were detected by immunohistochemistry and verified in 1 specimen of non-HIV-infected specimen by immunofluorescence technology. Results Immunohistochemistry showed that CD4 was suspiciously positive expression in non-HIV-infected specimens with the strong background staining.CXCR4 was positively expressed in cytoplasm and nuclei of most lacrimal epithelial cells of lacrimal gland epithelial cells in each specimen, and CCR5 was focally expressed in few lacrimal gland epithelial cells in each specimen.In addition, CD4, CXCR4 and CCR5 were positively expressed in intercellular scattered lymphocytes on the specimens.Immunofluorescence assay showed that CD4, CXCR4 and CCR5 were expressed in the specimens with the red fluorescence, with the linear- and patchy-like distribution mainly in cellular membrane for CD4 or spot-like distribution for CXCR4 and CCR5 in the cytoplasm. Conclusions HIV receptor CD4 and accessory receptor CXCR4, CCR5 are positively expressed in the lacrimal gland epithelial cells, which is the molecular basis of HIV infection and become a potential HIV reservoir preventing HIV eradication. Key words: Acquired immunodeficiency syndrome/immunology; AIDS-associated ophthalmopathy/metabolism; HIV Infections/metabolism; Virus latency; Tears/virology; Receptors, CD4+ T-lymphocytes/virology; Receptors, CXCR4/metabolism; Receptors, CCR5/metabolism; Lacrimal gland epithelial cells

A Ligation of the Lacrimal Excretory Duct in Mouse Induces Lacrimal Gland Inflammation with Proliferative Cells.

The lacrimal gland secretes tear fluids to ocular surface, which plays an indispensable role in maintaining the health of the ocular epithelia and protecting the ocular surface from the external environment. The dysfunction of the lacrimal glands causes dry eye disease due to a reduction in tear volume. The dry eye disease is becoming a popular public disease, for the number of patients is increasing, who have subjective symptom and loss of vision, which affect the quality of life. Inflammatory change in the damaged lacrimal gland has been reported; however, a major challenge is to establish a simple animal model to observe the changes. Here, we demonstrated an injury model by ligating the main excretory duct of the lacrimal gland, which is a simple and stable way to clearly understand the mechanism of lacrimal gland inflammation. We observed the process of injury and proliferation of the lacrimal gland and detected a population of lacrimal gland epithelial cells with proliferation potential which were also nestin-positive cells following duct ligation. This study successfully established an injury model to observe the tissue injury process of the lacrimal gland, and this model will be useful for analysis of the inflammation and proliferation mechanism in the future.

Cryopreservation and hypothermic storage of lacrimal gland: towards enabling delivery of regenerative medicine therapies for treatment of dry eye syndrome.

Severe dry eye syndrome (DES) can cause painful loss of vision and may result from lacrimal gland dysfunction. Current treatments are palliative, so a causative therapy is desirable. The ability to (cryo)preserve lacrimal gland tissue or epithelial cells would simplify this. Here, lacrimal gland tissue was cryopreserved in 10% dimethylsulphoxide in liquid nitrogen, or stored at 4 °C in culture medium for up to 7 days, and compared with fresh tissue using immunohistochemistry. Cultures were initiated from fresh and stored tissue, and cells characterised in P1 for proliferation (WST-1), colony-forming efficiency (CFE) and secretory capacity (immunocytochemistry and β-hexosaminidase activity assay). Tissue stored for > 3 days at 4 °C displayed grossly altered tissue architecture when compared with fresh tissue, decreased acinus density and increased caspase-3 activity. Cryopreserved tissue showed less obvious signs of damage without caspase-3 activation. Storage at 4 °C and cryopreservation delayed epithelial outgrowth compared with that from fresh tissue initially (p < 0.05) but, by day 9, all explants showed comparable outgrowth (~90%), except tissue stored at 4 °C for 3 or 7 days (p < 0.05 compared with fresh tissue). Epithelial cell yields per explant were similar from fresh and stored tissue, apart from tissue stored at 4 °C for 7 days (p < 0.01). In P1, epithelial cells from fresh and stored tissue were largely equivalent in terms of: proliferation; CFE (~21%); Rab3D, HexA and lysozyme expression; mucin production; and β-hexosaminidase activity. These data demonstrate that cryo(preservation) of lacrimal gland tissue and cells is possible, which may enable use of autologous cells in regenerative medicine approaches to treating DES. Copyright © 2016 John Wiley & Sons, Ltd.