Cathepsin S is a novel target for age-related dry eye.

Cathepsin S (Ctss) is a protease that is proinflammatory on epithelial cells. The purpose of this study was to investigate the role of Ctss in age-related dry eye disease. Ctss-/- mice [in a C57BL/6 (B6) background] of different ages were compared to B6 mice. Ctss activity in tears and lacrimal gland (LG) lysates was measured. The corneal barrier function was investigated in naïve mice or after topical administration of Ctss eye drops 5X/day for two days. Eyes were collected, and conjunctival goblet cell density was measured in PAS-stained sections. Immunoreactivity of the tight junction proteins, ZO-1 and occludin, was investigated in primary human cultured corneal epithelial cells (HCEC) without or with Ctss, with or without a Ctss inhibitor. A significant increase in Ctss activity was observed in the tears and LG lysates in aged B6 compared to young mice. This was accompanied by higher Ctss transcripts and protein expression in LG and spleen. Compared to B6, 12 and 24-month-old Ctss-/- mice did not display age-related corneal barrier disruption and goblet cell loss. Treatment of HCEC with Ctss for 48 h disrupted occludin and ZO-1 immunoreactivity compared to control cells. This was prevented by the Ctss inhibitor LY3000328 or Ctss-heat inactivation. Topical reconstitution of Ctss in Ctss-/- mice for two days disrupted corneal barrier function. Aging on the ocular surface is accompanied by increased expression and activity of the protease Ctss. Our results suggest that cathepsin S modulation might be a novel target for age-related dry eye disease.

Phenylephrine increases tear cathepsin S secretion in healthy murine lacrimal gland acinar cells through an alternative secretory pathway.

Little is known about the relationship between stimulation of lacrimal gland (LG) tear protein secretion by parasympathetic versus sympathetic nerves, particularly whether the spectrum of tear proteins evoked through each innervation pathway varies. We have previously shown that activity and abundance of cathepsin S (CTSS), a cysteine protease, is greatly increased in tears of Sjögren's syndrome (SS) patients and in tears from the male NOD mouse of autoimmune dacryoadenitis that recapitulates SS-associated dry eye disease. Beyond the increased synthesis of CTSS detected in the diseased NOD mouse LG, increased tear CTSS secretion in NOD mouse tears was recently linked to increased exocytosis from a novel endolysosomal secretory pathway. Here, we have compared secretion and trafficking of CTSS in healthy mouse LG acinar cells stimulated with either the parasympathetic acetylcholine receptor agonist, carbachol (CCh), or the sympathetic α1-adrenergic agonist, phenylephrine (PE). In situ secretion studies show that PE significantly increases CTSS activity and protein in tears relative to CCh stimulation by 1.2-fold (***, p = 0.0009) and ∼5-fold (*, p-0.0319), respectively. A similar significant increase in CTSS activity with PE relative to CCh is observed when cultured LGAC are stimulated in vitro. CCh stimulation significantly elevates intracellular [Ca2+], an effect associated with increases in the size of Rab3D-enriched vesicles consistent with compound fusion, and subsequently decreases in their intensity of labeling consistent with their exocytosis. PE stimulation induces a lower [Ca2+] response and has minimal effects on Rab3D-enriched SV diameter or the intensity of Rab3D-enriched SV labeling. LG deficient in Rab3D exhibit a higher sensitivity to PE stimulation, and secrete more CTSS activity. Significant increases in the colocalization of endolysosomal vesicle markers (Lamp1, Lamp2, Rab7) with the subapical actin suggestive of fusion of endolysosomal vesicles at the apical membrane occur both with CCh and PE stimulation, but PE demonstrates increased colocalization. In conclusion, the α1-adrenergic agonist, PE, increases CTSS secretion into tears through a pathway independent of the exocytosis of Rab3D-enriched mature SV, possibly representing an alternative endolysosomal secretory pathway.

Intralacrimal Sustained Delivery of Rapamycin Shows Therapeutic Effects without Systemic Toxicity in a Mouse Model of Autoimmune Dacryoadenitis Characteristic of Sjögren's Syndrome.

Sjögren's syndrome (SS) is an autoimmune disease associated with severe exocrinopathy, which is characterized by profound lymphocytic infiltration (dacryoadenitis) and loss of function of the tear-producing lacrimal glands (LGs). Systemic administration of Rapamycin (Rapa) significantly reduces LG inflammation in the male Nonobese Diabetic (NOD) model of SS-associated autoimmune dacryoadenitis. However, the systemic toxicity of this potent immunosuppressant limits its application. As an alternative, this paper reports an intra-LG delivery method using a depot formulation comprised of a thermoresponsive elastin-like polypeptide (ELP) and FKBP, the cognate receptor for Rapa (5FV). Depot formation was confirmed in excised whole LG using cleared tissue and observation by both laser-scanning confocal and lightsheet microscopy. The LG depot was evaluated for safety, efficacy, and intra-LG pharmacokinetics in the NOD mouse disease model. Intra-LG injection with the depot formulation (5FV) retained Rapa in the LG for a mean residence time (MRT) of 75.6 h compared to Rapa delivery complexed with a soluble carrier control (5FA), which had a MRT of 11.7 h in the LG. Compared to systemic delivery of Rapa every other day for 2 weeks (seven doses), a single intra-LG depot of Rapa representing 16-fold less total drug was sufficient to inhibit LG inflammation and improve tear production. This treatment modality further reduced markers of hyperglycemia and hyperlipidemia while showing no evidence of necrosis or fibrosis in the LG. This approach represents a potential new therapy for SS-related autoimmune dacryoadenitis, which may be adapted for local delivery at other sites of inflammation; furthermore, these findings reveal the utility of optical imaging for monitoring the disposition of locally administered therapeutics.

Rab27a Contributes to Cathepsin S Secretion in Lacrimal Gland Acinar Cells.

Altered lacrimal gland (LG) secretion is a feature of autoimmune dacryoadenitis in Sjögren's syndrome (SS). Cathepsin S (CTSS) is increased in tears of SS patients, which may contribute to disease. Rab3D and Rab27a/b isoforms are effectors of exocytosis in LG, but Rab27a is poorly studied. To investigate whether Rab27a mediates CTSS secretion, we utilized quantitative confocal fluorescence microscopy of LG from SS-model male NOD and control male BALB/c mice, showing that Rab27a-enriched vesicles containing CTSS were increased in NOD mouse LG. Live-cell imaging of cultured lacrimal gland acinar cells (LGAC) transduced with adenovirus encoding wild-type (WT) mCFP-Rab27a revealed carbachol-stimulated fusion and depletion of mCFP-Rab27a-enriched vesicles. LGAC transduced with dominant-negative (DN) mCFP-Rab27a exhibited significantly reduced carbachol-stimulated CTSS secretion by 0.5-fold and ß-hexosaminidase by 0.3-fold, relative to stimulated LGAC transduced with WT mCFP-Rab27a. Colocalization of Rab27a and endolysosomal markers (Rab7, Lamp2) with the apical membrane was increased in both stimulated BALB/c and NOD mouse LG, but the extent of colocalization was much greater in NOD mouse LG. Following stimulation, Rab27a colocalization with endolysosomal membranes was decreased. In conclusion, Rab27a participates in CTSS secretion in LGAC though the major regulated pathway, and through a novel endolysosomal pathway that is increased in SS.

Tear Proteases and Protease Inhibitors: Potential Biomarkers and Disease Drivers in Ocular Surface Disease.

Tears are highly concentrated in proteins relative to other biofluids, and a notable fraction of tear proteins are proteases and protease inhibitors. These components are present in a delicate equilibrium that maintains ocular surface homeostasis in response to physiological and temporal cues. Dysregulation of the activity of protease and protease inhibitors in tears occurs in ocular surface diseases including dry eye and infection, and ocular surface conditions including wound healing after refractive surgery and contact lens (CL) wear. Measurement of these changes can provide general information regarding ocular surface health and, increasingly, has the potential to give specific clues regarding disease diagnosis and guidance for treatment. Here, we review three major categories of tear proteases (matrix metalloproteinases, cathepsins, and plasminogen activators [PAs]) and their endogenous inhibitors (tissue inhibitors of metalloproteinases, cystatins, and PA inhibitors), and the changes in these factors associated with dry eye, infection and allergy, refractive surgery, and CLs. We highlight suggestions for development of these and other protease/protease inhibitor biomarkers in this promising field.

Biosynthesized Multivalent Lacritin Peptides Stimulate Exosome Production in Human Corneal Epithelium.

Lacripep is a therapeutic peptide derived from the human tear protein, Lacritin. Lacripep interacts with syndecan-1 and induces mitogenesis upon the removal of heparan sulfates (HS) that are attached at the extracellular domain of syndecan-1. The presence of HS is a prerequisite for the syndecan-1 clustering that stimulates exosome biogenesis and release. Therefore, syndecan-1-mediated mitogenesis versus HS-mediated exosome biogenesis are assumed to be mutually exclusive. This study introduces a biosynthesized fusion between Lacripep and an elastin-like polypeptide named LP-A96, and evaluates its activity on cell motility enhancement versus exosome biogenesis. LP-A96 activates both downstream pathways in a dose-dependent manner. HCE-T cells at high confluence treated with 1 µM LP-A96 enhanced cell motility equipotent to Lacripep. However, cells at low density treated with 1 µM LP-A96 generated a 210-fold higher number of exosomes compared to those treated at low density with Lacripep. As monovalent Lacripep is capable of enhancing cell motility but not exosome biogenesis, activation of exosome biogenesis by LP-A96 not only suggests its utility as a novel molecular tool to study the Lacritin biology in the corneal epithelium but also implies activity as a potential therapeutic peptide that can further improve ocular surface health through the induction of exosomes.

A Multivalent ICAM-1 Binding Nanoparticle which Inhibits ICAM-1 and LFA-1 Interaction Represents a New Tool for the Investigation of Autoimmune-Mediated Dry Eye.

The autoimmune disorder, Sjögren's syndrome (SS), is characterized by lymphocytic infiltration and loss of function of exocrine glands such as the lacrimal gland (LG) and salivary gland. SS-associated changes in the LG are associated with the development of autoimmune-mediated dry eye disease. We have previously reported the accumulation of intercellular adhesion molecule 1 (ICAM-1) in the LG of Non-Obese Diabetic (NOD) mice, a murine model of autoimmune-mediated dry eye in SS, in both LG acinar cells and infiltrating lymphocytes. ICAM-1 initiates T-cell activation and can trigger T-cell migration through binding to lymphocyte function-associated 1 antigen (LFA). To modulate this interaction, this study introduces a new tool, a multivalent biopolymeric nanoparticle assembled from a diblock elastin-like polypeptide (ELP) using the S48I48 (SI) ELP scaffold fused with a mouse ICAM-1 targeting peptide to form IBP-SI. IBP-SI forms a multivalent, monodisperse nanoparticle with a radius of 21.9 nm. Unlike the parent SI, IBP-SI binds mouse ICAM-1 and is internalized by endocytosis into transfected HeLa cells before it accumulates in lysosomes. In vitro assays measuring lymphocyte adhesion to Tumor Necrosis Factor TNF-α-treated bEnd.3 cells, which express high levels of ICAM-1, show that adhesion is inhibited by IBP-SI but not by SI, with IC50 values of 62.7 µM and 81.2 µM, respectively, in two different assay formats. IBP-SI, but not SI, also blocked T-cell proliferation in a mixed lymphocyte reaction by 74% relative to proliferation in an untreated mixed cell reaction. These data suggest that a biopolymeric nanoparticle with affinity for ICAM-1 can disrupt ICAM-1 and LFA interactions in vitro and may have further utility as an in vivo tool or potential therapeutic.

Molecular Targeting of Immunosuppressants Using a Bifunctional Elastin-Like Polypeptide.

Elastin-Like Polypeptides (ELP) are environmentally responsive protein polymers which are easy to engineer and biocompatible, making them ideal candidates as drug carriers. Our team has recently utilized ELPs fused to FKBP12 to carry Rapamycin (Rapa), a potent immunosuppressant. Through high affinity binding to Rapa, FKBP carriers can yield beneficial therapeutic effects and reduce the off-site toxicity of Rapa. Since ICAM-1 is significantly elevated at sites of inflammation in diverse diseases, we hypothesized that a molecularly targeted ELP carrier capable of binding ICAM-1 might have advantageous properties. Here we report on the design, characterization, pharmacokinetics, and biodistribution of a new ICAM-1-targeted ELP Rapa carrier (IBPAF) and its preliminary characterization in a murine model exhibiting elevated ICAM-1. Lacrimal glands (LG) of male NOD mice, a disease model recapitulating the autoimmune dacryoadenitis seen in Sjögren's Syndrome patients, were analyzed to confirm that ICAM-1 was significantly elevated in the LG relative to control male BALB/c mice (3.5-fold, p < 0.05, n = 6). In vitro studies showed that IBPAF had significantly higher binding to TNF-α-stimulated bEnd.3 cells which overexpress surface ICAM-1, relative to nontargeted control ELP (AF)(4.0-fold, p < 0.05). A pharmacokinetics study in male NOD mice showed no significant differences between AF and IBPAF for plasma half-life, clearance, and volume of distribution. However, both constructs maintained a higher level of Rapa in systemic circulation compared to free Rapa. Interestingly, in the male NOD mouse, the accumulation of IBPAF was significantly higher in homogenized LG extracts compared to AF at 2 h (8.6 ± 6.6% versus 1.3 ± 1.3%, respectively, n = 5, p < 0.05). This accumulation was transient with no differences detected at 8 or 24 h. This study describes the first ICAM-1 targeted protein-polymer carrier for Rapa that specifically binds to ICAM-1 in vitro and accumulates in ICAM-1 overexpressing tissue in vivo, which may be useful for molecular targeting in diverse inflammatory diseases where ICAM-1 is elevated.

Berunda Polypeptides: Biheaded Rapamycin Carriers for Subcutaneous Treatment of Autoimmune Dry Eye Disease.

The USFDA-approved immunosuppressive drug rapamycin (Rapa), despite its potency, is limited by poor bioavailability and a narrow therapeutic index. In this study, we sought to improve bioavailability of Rapa with subcutaneous (SC) administration and to test its therapeutic feasibility and practicality in a murine model of Sjögren's syndrome (SS), a systemic autoimmune disease with no approved therapies. To improve its therapeutic index, we formulated Rapa with a carrier termed FAF, a fusion of the human cytosolic FK506-binding protein 12 (FKBP12) and an elastin-like polypeptide (ELP). The resulting 97 kDa FAF (i) has minimal burst release, (ii) is "humanized", (iii) is biodegradable, (iv) solubilizes two Rapa per FAF, and (v) avoids organic solvents or amphiphilic carriers. Demonstrating high stability, FAF remained soluble and monodisperse with a hydrodynamic radius of 8 nm at physiological temperature. A complete pharmacokinetic (PK) analysis of FAF revealed that the bioavailability of SC FAF was 60%, with significantly higher blood concentration during the elimination phase compared to IV FAF. The plasma concentration of Rapa delivered by FAF was 8-fold higher with a significantly increased plasma-to-whole blood ratio relative to free Rapa, 24 h after injection. To evaluate therapeutic effects, FAF-Rapa was administered SC every other day for 2 weeks to male non-obese diabetic (NOD) mice, which develop an SS-like autoimmune-mediated lacrimal gland (LG) inflammation and other characteristic features of SS. Both FAF-Rapa and free Rapa exhibited immunomodulatory effects by significantly suppressing lymphocytic infiltration, gene expression of IFN-γ, MHC II, type I collagen and IL-12a, and cathepsin S (CTSS) activity in LG compared to controls. Serum chemistry and histopathological analyses in major organs revealed no apparent toxicity of FAF-Rapa. Given its improved PK and equipotent therapeutic efficacy compared to free Rapa, FAF-Rapa is of further interest for systemic treatments for autoimmune diseases like SS.

Longitudinal analysis of tear cathepsin S activity levels in male non-obese diabetic mice suggests its potential as an early stage biomarker of Sjögren's Syndrome.

CONTEXT: Cathepsin S (CTSS) activity is elevated in Sjögren's Syndrome (SS) patient tears. OBJECTIVE: To evaluate longitudinal expression of tear and tissue CTSS activity relative to other disease indicators in Non-Obese Diabetic (NOD) mice. METHODS: CTSS activity was measured in tears and lacrimal glands (LG) from male 1-6 month (M) NOD and 1 and 6 M BALB/c mice. Lymphocytic infiltration was quantified by histopathology, while disease-related proteins (Rab3D, CTSS, collagen 1) were quantified using q-PCR and immunofluorescence. RESULTS: In NOD LG, lymphocytic infiltration was noted by 2 M and established by 3 M (p < 0.01). IFN-É£, TNF-α, and MHC II expression were increased by 2 M (p < 0.01). Tear CTSS activity was significantly elevated at 2 M (p < 0.001) to a maximum of 10.1-fold by 6 M (p < 0.001). CTSS activity in LG lysates was significantly elevated by 2 M (p < 0.001) to a maximum of 14-fold by 3 M (p < 0.001). CTSS and Rab3D immunofluorescence were significantly increased and decreased maximally in LG acini by 3 M and 2 M, respectively. Comparable changes were not detected between 1 and 6 M BALB/c mouse LG, although Collagen 1 was decreased by 6 M in LG of both strains. CONCLUSION: Tear CTSS activity is elevated with other early disease indicators, suggesting potential as an early stage biomarker for SS.

NOD and NOR mice exhibit comparable development of lacrimal gland secretory dysfunction but NOD mice have more severe autoimmune dacryoadenitis.

The male Non-Obese Diabetic (NOD) mouse is an established model of autoimmune dacryoadenitis characteristic of Sjögren's Syndrome (SS), but development of diabetes may complicate studies. The Non-Obese Diabetes Resistant (NOR) mouse is a MHC-II matched diabetes-resistant alternative, but development of autoimmune dacryoadenitis is not well-characterized. We compare features of SS in male NOD and NOR mice at 12 and 20 weeks. Stimulated tear secretion was decreased in 12 week NOD relative to BALB/c mice (p < 0.05), while by 20 weeks both NOD and NOR showed decreased stimulated tear secretion relative to BALB/c mice (p < 0.001). Tear CTSS activity was elevated in NOD and NOR relative to BALB/c mice (p < 0.05) at 12 and 20 weeks. While NOD and NOR lacrimal glands (LG) showed increased LG lymphocytic infiltration at 12 and 20 weeks relative to BALB/c mouse LG (p < 0.05), the percentage in NOD was higher relative to NOR at each age (p < 0.05). Gene expression of CTSS, MHC II and IFN-γ in LG were significantly increased in NOD but not NOR relative to BALB/c at 12 and 20 weeks. Redistribution of the secretory effector, Rab3D in acinar cells was observed at both time points in NOD and NOR, but thinning of myoepithelial cells at 12 weeks in NOD and NOR mice was restored by 20 weeks in NOR mice. NOD and NOR mice share features of SS-like autoimmune dacryoadenitis, suggesting common disease etiology. Other findings suggest more pronounced lymphocytic infiltration in NOD mouse LG including increased pro-inflammatory factors that may be unique to this model.

Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease-Activated Receptor-2, in Human Corneal Epithelial Cells.

Cathepsin S (CTSS) activity is increased in tears of Sjögren's syndrome (SS) patients. This elevated CTSS may contribute to ocular surface inflammation. Human corneal epithelial cells (HCE-T cells) were treated with recombinant human CTSS at activity comparable to that in SS patient tears for 2, 4, 8, and 24 h. Acute CTSS significantly increased HCE-T cell gene and protein expression of interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) from 2 to 4 h, while matrix metalloproteinase 9 (MMP-9), CTSS, and protease-activated receptor-2 (PAR-2) were increased by chronic CTSS (24 h). To investigate whether the increased pro-inflammatory cytokines and proteases were induced by CTSS activation of PAR-2, HCE-T cells were transfected with PAR-2 siRNA, reducing cellular PAR-2 by 45%. Cells with reduced PAR-2 expression showed significantly reduced release of IL-6, TNF-α, IL-1ß, and MMP-9 into culture medium in response to acute CTSS, while IL-6, TNF-α, and MMP-9 were reduced in culture medium, and IL-6 and MMP-9 in cell lysates, after chronic CTSS. Moreover, cells with reduced PAR-2 expression showed reduced ability of chronic CTSS to induce gene expression of pro-inflammatory cytokines and proteases. CTSS activation of PAR-2 may represent a potential therapeutic target for amelioration of ocular surface inflammation in SS patients.

Imbalanced Rab3D versus Rab27 increases cathepsin S secretion from lacrimal acini in a mouse model of Sjögren's Syndrome.

The mechanism responsible for the altered spectrum of tear proteins secreted by lacrimal gland acinar cells (LGAC) in patients with Sjögren's Syndrome (SS) remains unknown. We have previously identified increased cathepsin S (CTSS) activity as a unique characteristic of tears of patients with SS. Here, we investigated the role of Rab3D, Rab27a, and Rab27b proteins in the enhanced release of CTSS from LGAC. Similar to patients with SS and to the male nonobese diabetic (NOD) mouse model of SS, CTSS activity was elevated in tears of mice lacking Rab3D. Findings of lower gene expression and altered localization of Rab3D in NOD LGAC reinforce a role for Rab3D in suppressing excess CTSS release under physiological conditions. However, CTSS activity was significantly reduced in tears of mice lacking Rab27 isoforms. The reliance of CTSS secretion on Rab27 activity was supported by in vitro findings that newly synthesized CTSS was detected in and secreted from Rab27-enriched secretory vesicles and that expression of dominant negative Rab27b reduced carbachol-stimulated secretion of CTSS in cultured LGAC. High-resolution 3D-structured illumination microscopy revealed microdomains of Rab3D and Rab27 isoforms on the same secretory vesicles but present in different proportions on different vesicles, suggesting that changes in their relative association with secretory vesicles may tailor the vesicle contents. We propose that a loss of Rab3D from secretory vesicles, leading to disproportionate Rab27-to-Rab3D activity, may contribute to the enhanced release of CTSS in tears of patients with SS.

Polymeric immunoglobulin receptor traffics through two distinct apically targeted pathways in primary lacrimal gland acinar cells.

The polymeric immunoglobulin receptor (pIgR) mediates transcytosis of dimeric immunoglobulin A (dIgA) and its release into mucosal secretions. The present study reveals the complexity of the trafficking of pIgR to the apical plasma membrane in epithelial cells with exocrine secretory functions; in rabbit lacrimal gland acinar cells (LGACs), trafficking of pIgR involves both the transcytotic pathway and one arm of the regulated secretory pathway. By specifically tracking pIgR endocytosed from the basolateral membrane, we show here that the Rab11a-regulated transcytotic pathway mediates the basal-to-apical transport of pIgR, and that pIgR sorted into the transcytotic pathway does not access the regulated secretory pathway. However, previous work in LGACs expanded in the present study has shown that some pIgR is localized to Rab3D-enriched mature secretory vesicles (SVs). Myosin Vb and myosin Vc motors modulate release of proteins from the Rab11a-regulated transcytotic pathway and the Rab3D-enriched secretory pathway in LGACs, respectively. Confocal fluorescence microscopy and biochemical assays showed that inhibition of myosin Vb and myosin Vc activity by overexpression of their dominant-negative mutants each significantly but differentially impaired aspects of apically targeted pIgR trafficking and secretory component release, suggesting that these motors function to regulate pIgR trafficking in both the transcytotic and exocytotic pathways. Intriguingly, a second mature SV population enriched in Rab27b was devoid of pIgR cargo, suggesting the specialization of Rab3D-enriched mature SVs to carry a particular subset of cargo proteins from the trans-Golgi network to the apical plasma membrane.

Flipping the Switch on Clathrin-Mediated Endocytosis using Thermally Responsive Protein Microdomains.

A ubiquitous approach to study protein function is to knock down activity (gene deletions, siRNA, small molecule inhibitors, etc) and study the cellular effects. Using a new methodology, this manuscript describes how to rapidly and specifically switch off cellular pathways using thermally responsive protein polymers. A small increase in temperature stimulates cytosolic elastin-like polypeptides (ELPs) to assemble microdomains. We hypothesize that ELPs fused to a key effector in a target macromolecular complex will sequester the complex within these microdomains, which will bring the pathway to a halt. To test this hypothesis, we fused ELPs to clathrin-light chain (CLC), a protein associated with clathrin-mediated endocytosis. Prior to thermal stimulation, the ELP fusion is soluble and clathrin-mediated endocytosis remains 'on.' Increasing the temperature induces the assembly of ELP fusion proteins into organelle-sized microdomains that switches clathrin-mediated endocytosis 'off.' These microdomains can be thermally activated and inactivated within minutes, are reversible, do not require exogenous chemical stimulation, and are specific for components trafficked within the clathrin-mediated endocytosis pathway. This temperature-triggered cell switch system represents a new platform for the temporal manipulation of trafficking mechanisms in normal and disease cell models and has applications for manipulating other intracellular pathways.

The Aging Lacrimal Gland of Female C57BL/6J Mice Exhibits Multinucleate Macrophage Infiltration Associated With Lipid Dysregulation.

Purpose: Loss of function of the lacrimal gland (LG), which produces the aqueous tear film, is implicated in age-related dry eye. To better understand this deterioration, we evaluated changes in lipid metabolism and inflammation in LGs from an aging model. Methods: LG sections from female C57BL/6J mice of different ages (young, 2-3 months; intermediate, 10-14 months; old,  ≥24 months) were stained with Oil Red-O or Toluidine blue to detect lipids. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blotting of LG lysates determined differences in the expression of genes and proteins related to lipid metabolism. A photobleaching protocol to quench age-related autofluorescence was used in LG sections to evaluate changes in immunofluorescence associated with NPC1, NPC2, CTSL, and macrophages (F4/80, CD11b) with age using confocal fluorescence microscopy. Results: Old LGs showed increased lipids prominent in basal aggregates in acinar cells and in extra-acinar sites. LG gene expression of Npc1, Npc2, Lipa, and Mcoln2, encoding proteins involved in lipid metabolism, was increased with age. NPC1 was also significantly increased in old LGs by western blotting. In photobleached LG sections, confocal fluorescence microscopy imaging of NPC1, NPC2, and CTSL immunofluorescence showed age-associated enrichment in macrophages labeled to detect F4/80. Although mononuclear macrophages were detectable in LG at all ages, this novel multinucleate macrophage population containing NPC1, NPC2, and CTSL and enriched in F4/80 and some CD11b was increased with age at extra-acinar sites. Conclusions: Lipid-metabolizing proteins enriched in F4/80-positive multinucleated macrophages are increased in old LGs adjacent to sites of lipid deposition in acini.

Nanoparticle translocation across mouse alveolar epithelial cell monolayers: species-specific mechanisms.

Studies of polystyrene nanoparticle (PNP) trafficking across mouse alveolar epithelial cell monolayers (MAECM) show apical-to-basolateral flux of 20 and 120nm amidine-modified PNP is ~65 times faster than that of 20 and 100nm carboxylate-modified PNP, respectively. Calcium chelation with EGTA has little effect on amidine-modified PNP flux, but increases carboxylate-modified PNP flux ~50-fold. PNP flux is unaffected by methyl-ß-cyclodextrin, while ~70% decrease in amidine- (but not carboxylate-) modified PNP flux occurs across chlorpromazine- or dynasore-treated MAECM. Confocal microscopy reveals intracellular amidine- and carboxylate-modified PNP and association of amidine- (but not carboxylate-) modified PNP with clathrin heavy chain. These data indicate (1) amidine-modified PNP translocate across MAECM primarily via clathrin-mediated endocytosis and (2) physicochemical properties (e.g., surface charge) determine PNP interactions with mouse alveolar epithelium. Uptake/trafficking of nanoparticles into/across epithelial barriers is dependent on both nanoparticle physicochemical properties and (based on comparison with our prior results) specific epithelial cell type. FROM THE CLINICAL EDITOR: In this study of polystyrene nanoparticle trafficking across mouse alveolar epithelial cell monolayers, the authors determined that uptake/trafficking of nanoparticles into/across epithelial barriers is dependent on both nanoparticle physicochemical properties and the specific type of epithelial cells.

The miRNA Landscape of Lacrimal Glands in a Murine Model of Autoimmune Dacryoadenitis.

Purpose: To analyze the changes in the lacrimal gland (LG) miRNAome from male nonobese diabetic (NOD) mice with autoimmune dacryoadenitis compared with LG from healthy male BALB/c and dacryoadenitis-free female NOD mice. Methods: LG from these mice were collected for small RNA sequencing to identify dysregulated miRNAs; hits were validated by RT-qPCR in male NOD and BALB/c LG. Dysregulation of validated species within immune cell-enriched cell fractions and epithelial-enriched cell fractions from LG was probed by RT-qPCR. Ingenuity pathway analysis identified putative miRNA targets, which were examined in publicly available mRNA-seq datasets. Western blotting and confocal imaging of immunofluorescence enabled validation of some molecular changes at the protein level. Results: Male NOD LG exhibited 15 and 13 significantly up- and downregulated miRNAs, respectively. Dysregulated expression of 14 of these miRNAs (9 upregulated, 5 downregulated) was validated in male NOD versus BALB/c LG by RT-qPCR. Seven of the upregulated miRNAs were increased owing to their abundance in immune cell-enriched cell fractions, whereas four downregulated miRNAs were largely expressed in epithelial-enriched cell fractions. Ingenuity pathway analysis predicted the upregulation of IL-6 and IL-6-like pathways as an outcome of miRNA dysregulation. Increased expression of several genes in these pathways was confirmed by mRNA-seq analysis, whereas immunoblotting and immunofluorescence confirmed Ingenuity pathway analysis-predicted changes for IL-6Rα and gp130/IL-6st. Conclusions: Male NOD mouse LG exhibit multiple dysregulated miRNAs owing to the presence of infiltrating immune cells, and decreased acinar cell content. The observed dysregulation may increase IL-6Rα and gp130/IL-6st on acini and IL-6Rα on specific lymphocytes, enhancing IL-6 and IL-6-like cytokine signaling.

Tear miRNAs Identified in a Murine Model of Sjögren's Syndrome as Potential Diagnostic Biomarkers and Indicators of Disease Mechanism.

Objective: The tear miRNAome of the male NOD mouse, a model of ocular symptoms of Sjögren's syndrome (SS), was analyzed to identify unique miRNAs. Methods: Male NOD mice, aged 12-14 weeks, were used to identify tear miRNAs associated with development of autoimmune dacryoadenitis. Age- and sex-matched male BALB/c mice served as healthy controls while age-matched female NOD mice that do not develop the autoimmune dacryoadenitis characteristic of SS were used as additional controls. Total RNA was isolated from stimulated tears pooled from 5 mice per sample and tear miRNAs were sequenced and analyzed. Putative miRNA hits were validated in additional mouse cohorts as well as in tears of SS patients versus patients with another form of dry eye disease, meibomian gland disease (MGD) using qRT-PCR. The pathways influenced by the validated hits were identified using Ingenuity Pathway Analysis. Results: In comparison to tears from both healthy (male BALB/c) and additional control (female NOD) mice, initial analy1sis identified 7 upregulated and 7 downregulated miRNAs in male NOD mouse tears. Of these, 8 were validated by RT-qPCR in tears from additional mouse cohorts. miRNAs previously implicated in SS pathology included mmu-miR-146a/b-5p, which were significantly downregulated, as well as mmu-miR-150-5p and mmu-miR-181a-5p, which were upregulated in male NOD mouse tears. All other validated hits including the upregulated miR-181b-5p and mmu-miR-203-3p, as well as the downregulated mmu-miR-322-5p and mmu-miR-503-5p, represent novel putative indicators of autoimmune dacryoadenitis in SS. When compared to tears from patients with MGD, miRNAs hsa-miR-203a-3p, hsa-miR-181a-5p and hsa-miR-181b-5p were also significantly increased in tears of SS patients. Conclusions: A panel of differentially expressed miRNAs were identified in tears of male NOD mice, with some preliminary validation in SS patients, including some never previously linked to SS. These may have potential utility as indicators of ocular symptoms of SS; evaluation of the pathways influenced by these dysregulated miRNAs may also provide further insights into SS pathogenesis.