Thymosin ß4 Alleviates Autoimmune Dacryoadenitis via Suppressing Th17 Cell Response.

Purpose: We investigated the therapeutic effect of recombinant thymosin ß4 (rTß4) on rabbit autoimmune dacryoadenitis, an animal model of SS dry eye, and explore its mechanisms. Methods: Rabbits were treated topically with rTß4 or PBS solution after disease onset for 28 days, and clinical scores were determined by assessing tear secretion, break-up time, fluorescein, hematoxylin and eosin staining, and periodic acid-Schiff. The expression of inflammatory mediators in the lacrimal glands were measured by real-time PCR. The expression of T helper 17 (Th17) cell-related transcription factors and cytokines were detected by real-time PCR and Western blotting. The molecular mechanism underlying the effects of rTß4 on Th17 cell responses was investigated by Western blotting. Results: Topical administration of rTß4 after disease onset efficiently ameliorated the ocular surface inflammation and relieved the clinical symptoms. Further analysis revealed that rTß4 treatment significantly inhibited the expression of Th17-related genes (RORC, IL-17A, IL-17F, IL-1R1, IL-23R, and granulocyte-macrophage colony-stimulating factor) and IL-17 protein in lacrimal glands, and meanwhile decreased the inflammatory mediators expression. Mechanistically, we demonstrated that rTß4 repressed the phosphorylation of signal transducer and activator of transcription 3 (STAT3) both in vivo and in vitro. Activation of the STAT3 signal pathway by Colivelin partly reversed the suppressive effects of rTß4 on IL-17 expression in vitro. Conclusions: rTß4 could alleviate ongoing autoimmune dacryoadenitis in rabbits, probably by suppressing Th17 response via partly affecting the STAT3 pathway. These data may provide a new insight into the therapeutic effect and mechanism of rTß4 in dry eye associated with Sjögren's syndrome.

Marginal Zone B (MZB) Cells: Comparison of the Initial Identification of Immune Activity Leading to Dacryoadenitis and Sialadenitis in Experimental Sjögren's Syndrome.

Although multiple mouse strains have been advanced as models for Sjögren's syndrome (SS), which is a human systemic autoimmune disease characterized primarily as the loss of lacrimal and salivary gland functions, the C57BL/6.NOD-Aec1Aec2 recombinant inbred (RI) mouse derived from the NOD/ShiLtJ line is considered one of the more appropriate models exhibiting virtually all the characteristics of the human disease. This mouse model, as well as other mouse models of SS, have shown that B lymphocytes are essential for the onset and development of observed clinical manifestations. Recently, studies carried out in the C57BL/6.IL14α transgenic mouse have provided clear evidence that the marginal zone B (MZB) cell population is directly involved in the early pathological events initiating the development of the clinical SS disease, as well as late-stage lymphomagenesis resulting in B-cell lymphomas. Since MZB cells are difficult to study in vivo and in vitro, we carried out a series of ex vivo investigations that utilize temporal global RNA transcriptomic analyses to profile differentially expressed genes exhibiting temporal upregulation during the initial onset and subsequent development of pathophysiological events within the lacrimal and salivary gland tissues per se or associated with the leukocyte cell migrations into these glands. The initial transcriptomic analyses revealed that while the upregulated gene expression profiles obtained from lacrimal and salivary glands overlap, multiple genetic differences exist between the defined activated pathways. In the current study, we present a concept suggesting that the initial pathological events differ between the two glands, yet the subsequent upregulated TLR4/TLR3 signal transduction pathway that activates the type-1 interferon signature appears to be identical in the two glands and indicates an autoimmune response against dsRNA, possibly a virus. Here, we attempt to put these findings into perspective and determine how they can impact the design of future therapeutic protocols.

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.

Heterochronic Parabiosis Causes Dacryoadenitis in Young Lacrimal Glands.

Aging is associated with inflammation and oxidative stress in the lacrimal gland (LG). We investigated if heterochronic parabiosis of mice could modulate age-related LG alterations. In both males and females, there were significant increases in total immune infiltration in isochronic aged LGs compared to that in isochronic young LGs. Male heterochronic young LGs were significantly more infiltrated compared to male isochronic young LGs. While both females and males had significant increases in inflammatory and B-cell-related transcripts in isochronic and heterochronic aged LGs compared to levels isochronic and heterochronic young LGs, females had a greater fold expression of some of these transcripts than males. Through flow cytometry, specific subsets of B cells were increased in the male heterochronic aged LGs compared to those in male isochronic aged LGs. Our results indicate that serum soluble factors from young mice were not enough to reverse inflammation and infiltrating immune cells in aged tissues and that there were specific sex-related differences in parabiosis treatment. This suggests that age-related changes in the LG microenvironment/architecture participate in perpetuating inflammation, which is not reversible by exposure to youthful systemic factors. In contrast, male young heterochronic LGs were significantly worse than their isochronic counterparts, suggesting that aged soluble factors can enhance inflammation in the young host. Therapies that aim at improving cellular health may have a stronger impact on improving inflammation and cellular inflammation in LGs than parabiosis.

Toll-Like Receptor Signaling in the Pathogenesis of Chronic Dacryocystitis: Implication of c-FOS Transcription Factor and its Downstream Effector Chemokine Genes CCL2, CCL4, CXCL3, CXCR4 with a Shift of the M1/M2 Macrophage Phenotype.

INTRODUCTION: TLRs are fundamental elements in the orchestration of the innate immune system. These receptors seem to be responsible for the inflammation and fibrosis in chronic dacryocystitis. The aim of the present study was to investigate the role of the toll-Like receptors (TLR2 and TLR4) signaling pathway and its downstream effector chemokine genes in the pathogenesis of chronic dacryocystitis. METHODS: This study was conducted on 20 patients diagnosed with chronic dacryocystitis and underwent external dacryocystorhinostomy. Estimation of gene expression of TLR2, TLR4, CCL2, CCL4, CXCL3, CXCR4, and c-FOS genes in the lacrimal sac tissues was performed together with the assessment of the inflammatory markers TNFα, IL-1ß, IFN-γ, and IL-22. Histopathological examination of the lacrimal sac walls using hematoxylin and eosin (H&E) stain, in addition to immunohistochemical staining of the CD68 and CD163 macrophage markers, was also performed. RESULTS: Our results showed that TLR2, TLR4, and c-FOS gene expressions were significantly increased in the chronic dacryocystitis group with a subsequent increase in their downstream effector chemokine genes CCL2, CCL4, and CXCL3. This up-regulation of genes was accompanied by macrophage shift of polarization toward the M1 pro-inflammatory phenotype (increased CD68 and decreased CD163 expression), leading to increased levels of the pro-inflammatory cytokines (TNF- α, IL-1ß and IFN-γ) and decreased anti-inflammatory marker IL-22 with chronic dacryocystitis. CONCLUSION: It is essential to fine-tune TLR activation through emerging therapeutic approaches. Targeting TLR signaling at the level of receptors or downstream adaptor molecules represents a new challenge for treating chronic dacryocystitis.

Altered Prostaglandin E Receptor Subtype 3 Expression in Lacrimal Glands of Patients with Chronic Stevens-Johnson Syndrome.

PURPOSE: To compare the prostaglandin E2 receptor subtype 3 (EP3) distribution in the lacrimal glands of normals, non-specific dacryoadenitis, and chronic Stevens-Johnson syndrome (SJS) patients. METHODS: Biopsies from lacrimal glands of four chronic SJS patients with severe dry eye disease, four dacryoadenitis patients, and five fresh body donors were assessed for EP3 expression using immunohistochemistry. RESULTS: In normal main and accessory lacrimal glands, EP3 is expressed strongly in nuclei and cytoplasm of majority (>75%) of acini with no ductular expression. In dacryoadenitis, EP3 expression was similar to normal glands. However, lacrimal glands from SJS patients (5-20/HPF mononuclear cells) showed a weak and reduced (<10% acini) EP3 expression within acinar cells. The reduction in intensity was more in glands with higher mononuclear cell infiltration (>10/HPF). CONCLUSION: There is downregulation of EP3 expression in the lacrimal glands of SJS patients, whereas EP3 expression is preserved in non-specific lacrimal gland inflammations.

MSC-Derived Small Extracellular Vesicles Attenuate Autoimmune Dacryoadenitis by Promoting M2 Macrophage Polarization and Inducing Tregs via miR-100-5p.

Background: Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have been increasingly proved as promising immunomodulators against some autoimmune disorders. However, the possible effect and the underlying mechanism of MSC-sEVs in autoimmune dry eye have been rarely studied. Methods: Small extracellular vesicles from human umbilical cord mesenchymal stem cells (hUC-MSC-sEVs) were subconjunctivally injected to rabbit dry eye model, and their preventive or therapeutical effects were assessed by recording the clinical and histological scores. Quantitative real-time PCR (Q-PCR), western blot and flow cytometry were performed to evaluate the immunomodulatory effects of hUC-MSC-sEVs on macrophages and T regulatory cells (Tregs) both in vivo and in vitro, and the in vitro T cell proliferation was detected by Bromodeoxyuridine (BrdU) assay. In addition, high expression of miR-100-5p in hUC-MSC-sEVs was identified by Q-PCR, and the functional role of sEVs-miR-100-5p on macrophages was explored by a series of co-culture experiments using sEVs derived from hUC-MSCs transfected with miR-100-5p inhibitor. Results: We firstly demonstrated that hUC-MSC-sEVs had the preventive and therapeutical effects on rabbit autoimmune dacryoadenitis, an animal model of Sjögren's syndrome (SS) dry eye. Further investigation revealed that hUC-MSC-sEVs administration effectively elicited macrophages into an anti-inflammatory M2 phenotype and elevated the proportion of Tregs both in vivo and in vitro, which contributed to reduced inflammation and improved tissue damage. Importantly, hUC-MSC-sEVs-educated macrophages with M2-like phenotype exhibited strong capacity to inhibit CD4+ T cell proliferation and promote Treg generation in vitro. Mechanistically, miR-100-5p was highly enriched in hUC-MSC-sEVs, and knockdown of miR-100-5p in hUC-MSC-sEVs partially blunted the promotion of hUC-MSC-sEVs on M2 macrophage polarization and even attenuated the effect of hUC-MSC-sEVs-educated macrophages on T cell suppression and Treg expansion. Conclusion: Our data indicated that hUC-MSC-sEVs alleviated autoimmune dacryoadenitis by promoting M2 macrophage polarization and Treg generation possibly through shuttling miR-100-5p. This study sheds new light on the application of MSC-sEVs as a promising therapeutic method for SS dry eye.

PPAR-α Agonist Fenofibrate Ameliorates Sjögren Syndrome-Like Dacryoadenitis by Modulating Th1/Th17 and Treg Cell Responses in NOD Mice.

Purpose: To investigate the effects and mechanisms of fenofibrate, a synthetic ligand of peroxisome proliferator-activated receptor α (PPAR-α), on autoimmune dacryoadenitis in a mouse model of Sjögren syndrome (SS) dry eye. Methods: Male nonobese diabetic (NOD) mice were fed chow with or without 0.03% fenofibrate for 8 weeks, and clinical scores were determined by assessing tear secretion, fluorescein, and hematoxylin and eosin staining. Intracellular IFN-γ, IL-17, and Foxp3 in CD4+ T cells were measured by flow cytometry. The expressions of Th1, Th17, and Treg cell-related transcription factors and cytokines were detected by real-time PCR. The levels of PPAR-α and liver X receptor ß (LXR-ß) were detected with real-time PCR and Western blotting. Results: Fenofibrate efficiently diminished the lymphocytic inflammation in lacrimal glands (LGs), increased tear secretion, and decreased corneal fluorescein staining in NOD mice. Meanwhile, treatment of fenofibrate evidently reduced the proportion of Th1 and Th17 cells and increased the proportion of Treg cells in vivo and vitro, together with decreased expression of T-bet, IFN-γ, RORγt, and IL-17, as well as increased expression of Foxp3 and TGF-ß1 in LGs. Furthermore, fenofibrate significantly upregulated the expressions of PPAR-α and LXR-ß at the protein and mRNA levels. Conclusions: Fenofibrate potently attenuated LG inflammation in a model of autoimmune dry eye, and this effect might partially result from regulating Th1/Th17/Treg cell responses by activating PPAR-α/LXR-ß signaling. These data suggest that fenofibrate may be a novel class of therapeutic agent for SS-associated dacryoadenitis.

The FcεRI signaling pathway is involved in the pathogenesis of lacrimal gland benign lymphoepithelial lesions as shown by transcriptomic analysis.

This study aimed to analyze the role of the FcepsilonRI (FcεRI) signaling pathway in the pathogenesis of benign lymphoepithelial lesion of lacrimal gland (LGBLEL). Transcriptomic analysis was performed on LGBLEL and orbital cavernous hemangioma (CH) patients diagnosed via histopathology in Beijing Tongren Hospital, Capital Medical University, between July 2010 and October 2013. Four LGBLEL and three orbital CH patients, diagnosed between October 2018 and August 2019, were randomly selected as experimental and control groups, respectively. RT-PCR, immunohistochemical staining, and western blotting were used to verify genes and proteins related to the FcεRI signaling pathway. Transcriptomic analysis showed that the FcεRI signaling pathway was upregulated in the LGBLEL compared with the CH group. The mRNA expression levels of important genes including SYK, p38, JNK, PI3K, and ERK were significantly increased in the LGBLEL group (P = 0.0066, P = 0.0002, P = 0.0003, P < 0.0001, P < 0.0001, respectively). Immunohistochemical staining results showed that SYK, p38, and ERK were positively expressed in LGBLEL, while JNK and PI3K were not. The protein contents of P-SYK, P-p38, P-JNK, P-PI3K, and P-ERK were significantly higher in the LGBLEL than in the CH group (P = 0.0169, P = 0.0074, P = 0.0046, P = 0.0157, P = 0.0156, respectively). The FcεRI signaling pathway participates in the pathogenesis of LGBLEL.

Programmed Cell Death-1 Pathway Deficiency Enhances Autoimmunity Leading to Dacryoadenitis of Mice.

Programmed cell death protein (PD)-1 is a coinhibitory molecule that suppresses immune response and maintains immune homeostasis. Moreover, the PD-1 pathway blocks cancers from being attacked by immune cells. Anti-PD-1 antibody therapy such as nivolumab improves survival in cancer patients. However, the occurrence of autoimmune inflammatory disorders in various organs has been increasingly reported as an adverse effect of nivolumab. Of the disorders associated with nivolumab, Sicca syndrome occurs in 3% to 11% of cases and has unknown pathologic mechanisms. Whether the absence of the PD-1 pathway causes functional and morphologic disorders in lacrimal glands was determined by analyzing PD-1 gene-knockout (Pdcd1-/-) mice. Histopathologic analysis showed that Pdcd1-/- mice developed dacryoadenitis beginning at 3 to 4 months of age, and deteriorated with age. Flow-cytometric analysis confirmed that cells infiltrating the affected lacrimal glands consisted mainly of CD3+ T cells and only a small proportion of CD19+ B cells. Among infiltrating T cells, the CD4+ Th-cell subset consisted of Th1 cells producing interferon-γ in an early stage of dacryoadenitis in Pdcd1-/- mice. Experiments of lymphocyte transfer from Pdcd1-/- into irradiated wild-type mice confirmed that CD4+ T cells from Pdcd1-/- mice induced dacryoadenitis. These results indicate that PD-1 plays an important role in the prevention of autoimmune inflammatory disorders in lacrimal glands caused by activated CD4+ Th1 cells.

Lacrimal Fossa Bony Changes in Chronic Primary Acquired Nasolacrimal Duct Obstruction and Acute Dacryocystitis.

PURPOSE: This study aims to assess the bony lacrimal fossa changes in chronic cases of primary acquired nasolacrimal duct obstruction versus acute dacryocystitis. METHODS: A prospective study was performed on 25 bony lacrimal fossae of 25 eyes of 15 patients who underwent endoscopic dacryocystorhinostomy at a tertiary care Dacryology service over a period of 6 months. Ten patients with chronic PANDO (> 1 year) with bilateral involvement and five patients of unilateral acute dacryocystitis were recruited in the study. None of the patients had a history of trauma or previous surgeries or nasal disease in the past. The bone samples from the frontal process of the maxilla and the lacrimal bone were obtained during the osteotomy and subjected to routine histopathological examination. Special stains used were von Kossa, Masson trichrome, periodic acid Schiff, and Alcian blue. Immunohistochemistry was performed using CD68 antibodies. Patient demographics, clinical presentation, duration of the disease, and bony changes were analyzed in different patient subsets. RESULTS: The mean disease duration in the chronic PANDO subset was 3.1 years, whereas acute dacryocystitis was 6.8 days. There was no correlation between the bony changes and the laterality in the chronic subset. Periosteal thickness and fibrosis were universal in the chronic group but not in the acute dacryocystitis. There were also differences in the number of osteocytes per sq mm, osteoblast, osteoclast, bony remodeling, bony canals structure, and intrastromal fibrosis between the subsets. These changes within the chronic group increased with the duration of the disease. Interestingly, there was no evidence of any bony inflammation across the subsets in all the samples studied. CONCLUSION: Characteristic bony changes can be demonstrated in patients with chronic PANDO but not in acute dacryocystitis. The lack of bony inflammatory infiltrates may provide clues in understanding the peri-sac disease pathogenesis in acute dacryocystitis.

HIF1α-mediated TRAIL Expression Regulates Lacrimal Gland Inflammation in Dry Eye Disease.

PURPOSE: The purpose of this study was to investigate the expression of death ligands in the lacrimal glands (LGs), identify upstream factors that regulate their expression, and determine the functional roles of these factors in the pathogenesis of dry eye disease (DED). METHODS: For DED experiment, ex vivo coculture system with LG and in vivo murine model using a controlled environment chamber were utilized. C57BL/6 mice and hypoxia-inducible factor (HIF)-1α conditional knockout (CKO) mice were used. Immunohistochemical staining, polymerase chain reaction, and immunoblotting were performed to determine levels of death ligands including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in DED-induced LGs. Additionally, acinar cell and CD45+ cell apoptosis was determined with neutralizing TRAIL treatment. RESULTS: Desiccating stress significantly increased HIF-1α expression in LG-acinar cells. Furthermore, HIF-1α deficiency significantly enhanced the infiltration of CD45+ inflammatory cells in LG and induced LG-acinar cell death. Meanwhile, only TRAIL expression was increased in DED-LG, but abrogated in HIF-1α CKO. Interestingly, the main source of TRAIL was the CD45- LG-acinar cells, but not CD45+ immune cells after DED induction. Using ex vivo coculture system, we confirmed LG-induced apoptosis of immune cells via HIF-1α-mediated TRAIL secretion following DED. Consistent with ex vivo, the insufficiency of HIF-1α and TRAIL enhanced recruitment of inflammatory cells to the LG and subsequently exacerbated ocular surface damage in DED mice. CONCLUSIONS: Our findings offer novel insight into the regulatory function of acinar cell-derived TRAIL in limiting inflammatory damage and could be implicated in the development of potential therapeutic strategies for DED.

A 6-Week, Prospective, Randomized, Single-Masked Study of Lifitegrast Ophthalmic Solution 5% Versus Thermal Pulsation Procedure for Treatment of Inflammatory Meibomian Gland Dysfunction.

PURPOSE: Meibomian gland dysfunction (MGD) is present in most cases of dry eye disease. MGD involves both inflammatory and obstructive etiologies. We compared efficacy and safety of treatment to reduce inflammation (lifitegrast) versus obstruction [thermal pulsation procedure (TPP)] in patients with inflammatory MGD over 42 days. METHODS: This was a single-center, 6-week, prospective, randomized, single-masked study of adults with inflammatory MGD, defined as having all of the following: burning, stinging, dryness; thickened secretions or occlusion of glands; eyelid redness; and elevated matrix metalloproteinase-9. Patients received lifitegrast ophthalmic solution 5% twice daily for 42 days or one TPP treatment at day 0. Seven symptoms and 8 objective measures of dry eye disease were assessed. RESULTS: Overall, 40 of 50 randomized patients (80%) were women with mean (SD) age 65.8 (8.9) years. Lifitegrast-treated (n = 25) versus TPP-treated (n = 25) patients had greater improvement from baseline to day 42 in eye dryness [mean (SD) change from baseline: -1.05 (0.79), lifitegrast; -0.48 (0.96), TPP; P = 0.0340], corneal staining [-0.55 (0.80), lifitegrast; 0.12 (1.09), TPP; P = 0.0230], and eyelid redness [-0.77 (0.43), lifitegrast; -0.38 (0.58), TPP; P = 0.0115]; trend favored lifitegrast for best corrected visual acuity and gland patency. Unexpectedly, TPP treatment did not improve lipid layer thickness or gland patency compared with lifitegrast. No adverse events were reported. CONCLUSIONS: Although MGD is often considered a disease of gland obstruction, these findings demonstrate antiinflammatory treatment with lifitegrast significantly improved patient symptoms and signs compared with treatment for obstruction (TPP). Lifitegrast should be included in treatment for inflammatory MGD.

Inhibition of Cathepsin S Reduces Lacrimal Gland Inflammation and Increases Tear Flow in a Mouse Model of Sjögren's Syndrome.

Cathepsin S (CTSS) is highly increased in Sjögren's syndrome (SS) patients tears and in tears and lacrimal glands (LG) of male non-obese diabetic (NOD) mice, a murine model of SS. To explore CTSS's utility as a therapeutic target for mitigating ocular manifestations of SS in sites where CTSS is increased in disease, the tears and the LG (systemically), the peptide-based inhibitor, Z-FL-COCHO (Z-FL), was administered to 14-15 week male NOD mice. Systemic intraperitoneal (i.p.) injection for 2 weeks significantly reduced CTSS activity in tears, LG and spleen, significantly reduced total lymphocytic infiltration into LG, reduced CD3+ and CD68+ cell abundance within lymphocytic infiltrates, and significantly increased stimulated tear secretion. Topical administration of Z-FL to a different cohort of 14-15 week male NOD mice for 6 weeks significantly reduced only tear CTSS while not affecting LG and spleen CTSS and attenuated the disease-progression related reduction of basal tear secretion, while not significantly impacting lymphocytic infiltration of the LG. These findings suggest that CTSS inhibitors administered either topically or systemically can mitigate aspects of the ocular manifestations of SS.

Surfactant proteins: Role in lacrimal drainage disorders.

Surfactants are complex mixtures of phospholipids and proteins produced by type II alveolar cells of the lungs and play a crucial role in pulmonary physiology. Six types of surfactant proteins (SP) are known; SP-A, SP-B, SP-C, SP-D, SP-G and SP-H. The major role of SP is in reducing surface tension and various immunological functions. SP-A, SP-B, SP-C and SP-D have been demonstrated in the tear film and the epithelium of the lacrimal sac (LS) and nasolacrimal ducts (NLD). All surfactant proteins except SP-G were also isolated from the canalicular tissues. The authors hypothesize that surfactant proteins play a significant role in the pathogenesis of lacrimal drainage disorders; functional nasolacrimal duct obstruction (FNLDO) and infective dacryocystitis.

Type I Interferon Signaling Is Required for Dacryoadenitis in the Nonobese Diabetic Mouse Model of Sjögren Syndrome.

Nonobese diabetic (NOD) mice spontaneously develop lacrimal and salivary gland autoimmunity similar to human Sjögren syndrome. In both humans and NOD mice, the early immune response that drives T-cell infiltration into lacrimal and salivary glands is poorly understood. In NOD mice, lacrimal gland autoimmunity spontaneously occurs only in males with testosterone playing a role in promoting lacrimal gland inflammation, while female lacrimal glands are protected by regulatory T cells (Tregs). The mechanisms of this male-specific lacrimal gland autoimmunity are not known. Here, we studied the effects of Treg depletion in hormone-manipulated NOD mice and lacrimal gland gene expression to determine early signals required for lacrimal gland inflammation. While Treg-depletion was not sufficient to drive dacryoadenitis in castrated male NOD mice, chemokines (Cxcl9, Ccl19) and other potentially disease-relevant genes (Epsti1, Ubd) were upregulated in male lacrimal glands. Expression of Cxcl9 and Ccl19, in particular, remained significantly upregulated in the lacrimal glands of lymphocyte-deficient NOD-severe combined immunodeficiency (SCID) mice and their expression was modulated by type I interferon signaling. Notably, Ifnar1-deficient NOD mice did not develop dacryoadenitis. Together these data identify disease-relevant genes upregulated in the context of male-specific dacryoadenitis and demonstrate a requisite role for type I interferon signaling in lacrimal gland autoimmunity in NOD mice.

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.

The Imbalance of Lymphocyte Subsets and Cytokines: Potential Immunologic Insights Into the Pathogenesis of Chronic Dacryocystitis.

Purpose: To explore the quantitative distributions of different lymphocyte subsets in the lacrimal sac mucosa and identify changes of Th1- and Th2-associated cytokines in the tears of patients with chronic dacryocystitis. Methods: Lacrimal sac mucosal specimens from patients with chronic dacryocystitis were analyzed. Hematoxylin-eosin staining and Masson staining were performed for pathologic analysis, and immunohistochemical staining was performed for the detection of CD3+, CD4+, CD8+, CD20+, Th1, and Th2 lymphocytes. Quantitative real-time PCR was performed to analyze IFN-γ and IL-4 mRNA expression. In addition, tear samples from patients with chronic dacryocystitis and healthy volunteers were collected and analyzed with an antibody array system for Th1- and Th2-associated cytokines and chemokines. Results: Different distribution patterns of lymphocyte subsets were observed in the lacrimal sac walls. Both CD20+ B lymphocytes and CD3+ T lymphocytes accumulated in organized lymphoid follicles, and CD3+ T cells were also distributed in a diffuse manner. Among the two subsets of T cells, CD4+ T cells were more abundant than CD8+ T cells. Both the immunohistochemical staining and real-time PCR results revealed significantly higher expression levels of IFN-γ than those of IL-4. The levels of Th1- and Th2-related cytokines and chemokines measured were significantly higher in the tears of patients than in those of controls. Conclusions: The different distribution patterns of lymphocyte subsets provide insight into a potential immunologic mechanism for dacryocystitis. The cytokines secreted by Th1 or Th2 cells may play a major role in the pathogenesis of dacryocystitis and could be explored as therapeutic targets.

Immunostaining of Immunoglobulin G4 in the Lacrimal Sac.

PURPOSE: To immunohistochemically examine the lacrimal sac walls harvested during dacryocystorhinostomy using an immunostain for immunoglobulin G4 (IgG4). METHODS: Forty-four lacrimal sac walls were evaluated. We determined "intensively positive," "sparsely positive," or "negative" staining when the specimens showed IgG4-positive lymphoplasmacytic staining with or without >50 IgG4-positive cells/high-power field, or the absence of stained IgG4-positive cells. RESULTS: Intensively positive, sparsely positive, or negative staining was observed in 8 (18.2%), 14 (31.8%), and 22 specimens (50.0%), respectively. Stained cells infiltrated the subepithelial layer in all specimens with positive staining. Four of the 8 specimens demonstrated partial epithelial denudation with loss of goblet cells. CONCLUSIONS: IgG4-positive lymphoplasmacytic infiltration was observed in the subepithelial layer in specimens with intensively positive staining, of which some showed a partially denuded epithelium with loss of goblet cells. These may lead to narrowing of the lacrimal sac lumen and adhesions of the sac walls.