Imbalanced IL-37/TNF-α/CTSS signaling disrupts corneal epithelial barrier in a dry eye model in vitro.

PURPOSE: To explore novel role and molecular mechanism of a natural anti-inflammatory cytokine interleukin (IL) 37 in preventing corneal epithelial barrier disruption from hyperosmolar stress as can occur in dry eye disease. METHODS: Primary human corneal epithelial cells (HCECs) were cultured from fresh donor limbal explants. An in vitro dry eye model with hyperosmolar stress was established by switching HCECs from isosmolar (312mOsM) to hyperosmolar medium (350-500 mOsM), and some cells were treated with rhIL-37 or rhTNF-α, for different periods (2-48 h). The expression of cytokines and cathepsin S, and barrier protein integrity were evaluated by RT-qPCR, ELISA, and immunofluorescent staining with confocal microscopy. RESULTS: The integrity of epithelial barrier was significantly disrupted in HCECs exposed to hyperosmolar medium, as shown by immunofluorescent images of tight junction (TJ, ZO-1, occludin and claudin-1) and adheren junction (E-cadherin) proteins. TNF-α accentuated hyperosmolar-induced disruption of TJ barrier functional integrity whereas exposure to IL-37 blunted or even reversed these changes. Cathepsin S, encoded by CTSS gene, was found to directly disrupt epithelial barrier integrity. Interestingly, CTSS expression was significantly induced by TNF-α and hyperosmolarity, while exogenous rhIL-37 inhibited TNF-α and CTSS expression at mRNA and protein levels following hyperosmolar stress. Furthermore, rhIL-37 restored barrier protein integrity, observed in 2D and 3D confocal immunofluorescent images, in HCECs under hyperosmolar stress. CONCLUSION: Our findings demonstrate a novel signaling pathway by which anti-inflammatory cytokine IL-37 prevents corneal epithelial barrier disruption under hyperosmotic stress via suppressing TNF-α and CTSS expression. This study provides new insight into mechanisms protecting corneal barrier in dry eye disease.

IL-36α/IL-36RA/IL-38 signaling mediates inflammation and barrier disruption in human corneal epithelial cells under hyperosmotic stress.

PURPOSE: To explore the distinct expression and diverse roles of IL-36 cytokines in dry eye disease using an in vitro hyperosmolarity model of human corneal epithelial cells (HCECs). METHODS: Primary HCECs were cultured from fresh donor limbal explants. Hyperosmolarity model was established by switching HCECs from isosmotic (312 mOsM) to hyperosmotic medium (350-500 mOsM) alone or with addition of rhIL-36RA or rhIL-38 for 2-48 h. Some cultures were treated with IL-36α (1-10 ng/ml) with or without rhIL-36RA or rhIL-38. Gene expression was detected by RT-qPCR; and protein production and barrier disruption were evaluated by ELISA and/or immunofluorescent staining. RESULTS: IL-36 cytokines were differential expressed in primary HCECs. Among 3 pro-inflammatory agonists, IL-36α, but not IL-36ß and IL-36γ, was distinctly induced at osmolarity-dependent manner while two antagonist IL-36RA and IL-38 were significantly suppressed in HCECs exposed to hyperosmotic stress. IL-36α increased to 4.4-fold in mRNA and 6.9-fold at protein levels (116.0 ± 36.33 pg/ml vs 16.79 ± 6.51 pg/ml in controls) by 450 mOsM, but dramatically inhibited by addition of rhIL-36RA or rhIL-38. Exogenous rhIL-36α stimulated expression of TNF-α and IL-1ß at mRNA and protein levels and disrupted tight junction proteins ZO-1 and occludin. However, rhIL-36RA or rhIL-38 suppressed TNF-α and IL-1ß production and protected HCECs from barrier disruption in response to IL-36α or hyperosmolarity. CONCLUSIONS: Our findings demonstrate that the stimulated pro-inflammatory IL-36α with the suppressed antagonists IL-36RA and IL-38 is a novel mechanism by which hyperosmolarity induces inflammation in dry eye. IL-36RA and IL-38 may have a therapeutic potential in dry eye.

Dry eye disease flares: A rapid evidence assessment.

PURPOSE: Characteristics of periodic flares of dry eye disease (DED) are not well understood. We conducted a rapid evidence assessment to identify evidence for and characteristics of DED flares. METHODS: Literature searches were performed in Embase® via Ovid®, MEDLINE®, and PubMed®. Clinical trials and observational studies published 2009-2019 were included if they investigated patients aged ≥18 years with clinically diagnosed DED who experienced a flare, defined as a temporary or transient episode of increased ocular discomfort, typically lasting days to a few weeks. Triggers of flares, patient-reported outcomes (symptoms), clinician-measured outcomes (signs), and changes in tear molecules were captured. RESULTS: Twenty-one publications that included 22 studies met inclusion criteria. Five observational studies described evidence of DED flares in daily life, 5 studies reported changes following cataract/refractive surgery in patients with preoperative DED, and 12 studies employed controlled environment (CE) models. Real-world triggers of DED flares included air conditioning, wind, reading, low humidity, watching television, and pollution. CE chambers (dry, moving air) and surgery also triggered DED flares. Exacerbations of symptoms and signs of DED, assessed through varied measures, were reported during flares. Across studies, matrix metalloproteinase-9 and interleukin-6 increased and epidermal growth factor decreased during DED flares. CONCLUSIONS: Evidence from 22 studies identified triggers and characteristics of DED flares. Further research is needed to assist clinicians in early diagnosis and treatment of patients experiencing flares.

Conjunctivochalasis and Tear Osmolarity Are Associated With Reduced Conjunctival Epithelial Thickness in Dry Eye.

PURPOSE: To investigate the hypothesis that ocular surface epithelial thickness is correlated with tear osmolarity, conjunctivochalasis (CCh), and dry eye clinical tests. DESIGN: A case-control study. METHODS: A total of 50 patients with different types of dry eye disease (DED) and 15 age- and sex-matched control subjects were enrolled in this study. We performed a detailed diagnostic assessment of tear film and ocular surface parameters, including Ocular Surface Disease Index questionnaire, visual analog scale questionnaire, topographic surface regularity and surface asymmetry indices, tear film break-up time, corneal and conjunctival dye staining, Schirmer I test, and tear osmolarity. The corneal and bulbar conjunctival epithelial thickness (CET) were obtained using the latest version of spectral domain optical coherence tomography (SDOCT). RESULTS: Patients with aqueous-deficient DED (ADDED) had lower bulbar CET values, particularly in the temporal region, than those of normal subjects and patients with evaporative-type DED (EDED); however, the difference did not quite reach a statistically significant level. Patients with DED and CCh had lower bulbar CET values in temporal (38.52 ± 9.58 µm) and inferior regions (50.79 ± 9.10 µm) compared to those with DED without CCh (47.39 ± 11.71 µm, 60.38 ± 14.36 µm, respectively, P < .02). In the DED group, tear osmolarity was found to be negatively correlated with CET values in temporal bulbar region (P = .006 and r = -0.403) and central corneal epithelial thickness values (P = .029 and r = -0.325). CONCLUSIONS: CCh and tear osmolarity are associated with reduced conjunctival epithelial thickness in DED.

Rapamycin Eyedrops Increased CD4+Foxp3+ Cells and Prevented Goblet Cell Loss in the Aged Ocular Surface.

Dry eye disease (DED), one of the most prevalent conditions among the elderly, is a chronic inflammatory disorder that disrupts tear film stability and causes ocular surface damage. Aged C57BL/6J mice spontaneously develop DED. Rapamycin is a potent immunosuppressant that prolongs the lifespan of several species. Here, we compared the effects of daily instillation of eyedrops containing rapamycin or empty micelles for three months on the aged mice. Tear cytokine/chemokine profile showed a pronounced increase in vascular endothelial cell growth factor-A (VEGF-A) and a trend towards decreased concentration of Interferon gamma (IFN)-γ in rapamycin-treated groups. A significant decrease in inflammatory markers in the lacrimal gland was also evident (IFN-γ, IL-12, CIITA and Ctss); this was accompanied by slightly diminished Unc-51 Like Autophagy Activating Kinase 1 (ULK1) transcripts. In the lacrimal gland and draining lymph nodes, we also observed a significant increase in the CD45+CD4+Foxp3+ cells in the rapamycin-treated mice. More importantly, rapamycin eyedrops increased conjunctival goblet cell density and area compared to the empty micelles. Taken together, evidence from these studies indicates that topical rapamycin has therapeutic efficacy for age-associated ocular surface inflammation and goblet cell loss and opens the venue for new investigations on its role in the aging process of the eye.

Autophagy Activation Protects Ocular Surface from Inflammation in a Dry Eye Model In Vitro.

Inflammation is the main pathophysiology of dry eye, characterized by tear film instability and hyperosmolarity. The aim of this study was to investigate the association of inflammation and cellular autophagy using an in vitro dry eye model with primary cultured human corneal epithelial cells (HCECs). Primary HCECs cultured with fresh limbal explants from donors were switched to a hyperosmotic medium (450 mOsM) by adding sodium chloride into the culture medium. We observed the stimulated inflammatory mediators, TNF-α, IL-1ß, IL-6 and IL-8, as well as the increased expression of autophagy related genes, Ulk1, Beclin1, Atg5 and LC3B, as evaluated by RT-qPCR and ELISA. The immunofluorescent staining of LC3B and Western blotting revealed the activated autophagosome formation and autophagic flux, as evidenced by the increased LC3B autophagic cells with activated Beclin1, Atg5, Atg7 and LC3B proteins, and the decreased levels of P62 protein in HCECs. Interestingly, the autophagy activation was later at 24 h than inflammation induced at 4 h in HCECs exposed to 450 mOsM. Furthermore, application of rapamycin enhanced autophagy activation also reduced the inflammatory mediators and restored cell viability in HCECs exposed to the hyperosmotic medium. Our findings for the first time demonstrate that the autophagy activation is a late phase response to hyperosmotic stress, and is enhanced by rapamycin, which protects HCECs by suppressing inflammation and promoting cells survival, suggesting a new therapeutic potential to treat dry eye diseases.

Trehalose Induces Autophagy Against Inflammation by Activating TFEB Signaling Pathway in Human Corneal Epithelial Cells Exposed to Hyperosmotic Stress.

Purpose: Autophagy plays an important role in balancing the inflammatory response to restore homeostasis. The aim of this study was to explore the mechanism by which trehalose suppresses inflammatory cytokines via autophagy activation in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress. Methods: An in vitro dry eye model was used in which HCECs were cultured in hyperosmolar medium with the addition of sodium chloride (NaCl). Trehalose was applied in different concentrations. The levels of TNF-α, IL-1ß, IL-6, and IL-8 were detected using RT-qPCR and ELISA. Cell viability assays, immunofluorescent staining of LC3B, and western blots of Beclin1, Atg5, Atg7, LC3B, and P62 were conducted. The key factors in upstream signaling pathways of autophagy activation were measured: P-Akt, Akt, and transcription factor EB (TFEB). Results: Trehalose reduced the proinflammatory mediators TNF-α, IL-1ß, IL-6, and IL-8 in primary HCECs at 450 mOsM. This effect was osmolarity dependent, and a level of 1.0% trehalose showed the most suppression. Trehalose promoted autophagosome formation and autophagic flux, as evidenced by increased production of Beclin1, Atg5, and Atg7, as well as higher LC3B I protein turnover to LC3B II, with decreased protein levels of P62/SQSTM1. The addition of 3-methyladenine blocked autophagy activation and increased the release of proinflammatory cytokines. Trehalose further activated TFEB, with translocation from cytoplasm to the nucleus, but diminished Akt activity. Conclusions: Our findings demonstrate that trehalose, functioning as an autophagy enhancer, suppresses the inflammatory response by promoting autophagic flux via TFEB activation in primary HCECs exposed to hyperosmotic stress, a process that is beneficial to dry eye.

Short ragweed pollen promotes M2 macrophage polarization via TSLP/TSLPR/OX40L signaling in allergic inflammation.

This study was to explore the role and mechanism of macrophages in pollen-triggered allergic inflammation. A murine model of short ragweed (SRW) pollen-induced experimental allergic conjunctivitis (EAC), and bone marrow (BM)-macrophages cultures were used. Typical allergic manifestations and TSLP-stimulated Th2 hyperresponse were observed in ocular surface of EAC model in wild-type (WT) mice induced by SRW. The M2 phenotype markers, Arg1, Ym1 and FIZZ1, were highly expressed by conjunctiva and draining cervical lymph nodes (CLNs) of WT-EAC mice when compared with controls, as evaluated by RT-qPCR and Immunofluorescent double staining with macrophage marker F4/80. The stimulated expression of TSLPR and OX40L by macrophage was detected in conjunctiva and CLNs by RT-qPCR, double staining, and flow cytometry. M2 macrophages were found to produce TARC and MDC. In contrast, EAC model with TSLPR-/- mice did not show allergic signs and any increase of Th2 cytokines (IL-4, IL-5 and IL-13) and M2 markers. In vitro cultures confirmed that SRW extract stimulates expression of TSLPR, OX40L, TARC, MDC, and three M2 markers by BM-macrophages from WT mice, but not from TSLPR-/- mice. These findings demonstrate that SRW pollen primes macrophage polarization toward to M2 phenotype via TSLP/TSLPR/OX40L signaling to amplify allergic inflammation.

IL-27 signaling deficiency develops Th17-enhanced Th2-dominant inflammation in murine allergic conjunctivitis model.

BACKGROUND: While most studies focus on pro-allergic cytokines, the protective role of immunosuppressive cytokines in allergic inflammation is not well elucidated. This study was to explore a novel anti-inflammatory role and cellular/molecular mechanism of IL-27 in allergic inflammation. METHODS: A murine model of experimental allergic conjunctivitis (EAC) was induced in BALB/c, C57BL/6 or IL-27Rα-deficient (WSX-1-/- ) mice by short ragweed pollen, with untreated or PBS-treated mice as controls. The serum, eyeballs, conjunctiva, cervical lymph nodes (CLNs) were used for study. Gene expression was determined by RT-qPCR, and protein production and activation were evaluated by immunostaining, ELISA and Western blotting. RESULTS: Typical allergic manifestations and stimulated thymic stromal lymphopoietin (TSLP) signaling and Th2 responses were observed in ocular surface of EAC models in BALB/c and C57BL/6 mice. The decrease of IL-27 at mRNA (IL-27/EBI3) and protein levels were detected in serum, conjunctiva and CLN, as evaluated by RT-qPCR, immunofluorescent staining, ELISA and Western blotting. EAC induced in WSX-1-/- mice showed aggravated allergic signs with higher TSLP-driven Th2-dominant inflammation, accompanied by stimulated Th17 responses, including IL-17A, IL-17F, and transcription factor RORγt. In contrast, Th1 cytokine IFNγ and Treg marker IL-10, with their respective transcription factors T-bet and foxp3, were largely suppressed. Interestingly, imbalanced activation between reduced phosphor (P)-STAT1 and stimulated P-STAT6 were revealed in EAC, especially WSX-1-/- -EAC mice. CONCLUSION: These findings demonstrated a natural protective mechanism by IL-27, of which signaling deficiency develops a Th17-type hyperresponse that further aggravates Th2-dominant allergic inflammation.

Reduced Corneal Innervation in the CD25 Null Model of Sjögren Syndrome.

Decreased corneal innervation is frequent in patients with Sjögren Syndrome (SS). To investigate the density and morphology of the intraepithelial corneal nerves (ICNs), corneal sensitivity, epithelial cell proliferation, and changes in mRNA expression of genes that are involved in autophagy and axon targeting and extension were assessed using the IL-2 receptor alpha chain (CD25 null) model of SS. ICN density and thickness in male and female wt and CD25 null corneas were assessed at 4, 6, 8, and 10/11 wk of age. Cell proliferation was assessed using ki67. Mechanical corneal sensitivity was measured. Quantitative PCR was performed to quantify expression of beclin 1, LC3, Lamp-1, Lamp-2, CXCL-1, BDNF, NTN1, DCC, Unc5b1, Efna4, Efna5, Rgma, and p21 in corneal epithelial mRNA. A significant reduction in corneal axon density and mechanical sensitivity were observed, which negatively correlate with epithelial cell proliferation. CD25 null mice have increased expression of genes regulating autophagy (beclin-1, LC3, LAMP-1, LAMP-2, CXCL1, and BDNF) and no change was observed in genes that were related to axonal targeting and extension. Decreased anatomic corneal innervation in the CD25 null SS model is accompanied by reduced corneal sensitivity, increased corneal epithelial cell proliferation, and increased expression of genes regulating phagocytosis and autophagy.

Goblet cell-produced retinoic acid suppresses CD86 expression and IL-12 production in bone marrow-derived cells.

Conjunctival goblet cell loss in ocular surface diseases is accompanied by increased number of interleukin-12 (IL-12)-producing antigen-presenting cells (APCs) and increased interferon-γ (IFN-γ) expression. This study tested the hypothesis that mouse conjunctival goblet cells produce biologically active retinoic acid (RA) that suppresses CD86 expression and IL-12 production by myeloid cells. We found that conditioned media from cultured conjunctival goblet cells (CjCM) suppressed stimulated CD86 expression, NF-κB p65 activation and IL-12 and IFN-γ production in unstimulated and lipopolysaccharide-stimulated cultured bone marrow-derived cells (BMDCs) containing a mixed population of APCs. Goblet cell-conditioned, ovalbumin-loaded APCs suppressed IFN-γ production and increased IL-13 production in co-cultured OTII cells. The goblet cell suppressive activity is due in part to their ability to synthesize RA from retinol. Conjunctival goblet cells had greater expression of aldehyde dehydrogenases Aldh1a1 and a3 and ALDEFLUOR activity than cornea epithelium lacking goblet cells. The conditioning activity was lost in goblet cells treated with an ALDH inhibitor, and a retinoid receptor alpha antagonist blocked the suppressive effects of CjCM on IL-12 production. Similar to RA, CjCM increased expression of suppressor of cytokine signaling 3 (SOCS3) in BMDCs. SOCS3 silencing reversed the IL-12-suppressive effects of CjCM. Our findings indicate that conjunctival goblet cells are capable of synthesizing RA from retinol secreted by the lacrimal gland into tears that can condition APCs. Evidence suggests goblet cell RA may function in maintaining conjunctival immune tolerance and loss of conjunctival goblet cells may contribute to increased Th1 priming in dry eye.

Near-infrared laser thermal conjunctivoplasty.

Conjunctivochalasis is a common cause of tear dysfunction due to the conjunctiva becoming loose and wrinkly with age. The current solutions to this disease include either surgical excision in the operating room, or thermoreduction of the loose tissue with hot wire in the clinic. We developed a near-infrared laser thermal conjunctivoplasty system. The system utilizes a 1460-nm programmable laser diode system as the light source. At this wavelength, a water absorption peak exists and the blood absorption is minimal, so the heating of redundant conjunctiva is even and there is no bleeding. A miniaturized handheld probe delivers the laser light and reshapes the laser into a 10 × 1 mm2 line on the working plane. A foot pedal is used to deliver a preset number of calibrated laser pulses. A fold of loose conjunctiva is grasped by a pair of forceps. The NIR laser light is delivered through an optical fiber and a laser line is aimed exactly on the conjunctival fold by a cylindrical lens. Ex vivo experiments using porcine eye was performed to investigate the induced shrinkage of conjunctiva and decide the optimal laser parameters. It was found that up to 45% of conjunctiva shrinkage could be achieved.

Assessment of the Prosthetic Replacement of Ocular Surface Ecosystem (PROSE) scleral lens on visual acuity for corneal irregularity and ocular surface disease.

PURPOSE: To describe changes in visual acuity in patients fit with the Prosthetic Replacement of Ocular Surface Ecosystem (PROSE) with irregular corneas compared to those with ocular surface disease, as well as describe patient demographics and fitting indications for PROSE at an academic medical center. METHODS: A retrospective chart review from 2010 to 2016 on a total of 825 eyes from 493 patients fitted with a PROSE lens, and grouped by etiology, either irregular cornea/dystrophy or ocular surface disease and associated sub-etiologies. We compared best corrected visual acuity (BCVA) before and after PROSE fitting. RESULTS: The irregular cornea group (n = 262 eyes) included corneal scar (n = 57), dystrophy (n = 17), post-operative corneal irregularity (n = 70), and primary ectasia (n = 118). The OSD group included 563 eyes. For all etiologies combined, mean BCVA prior to PROSE fitting compared to after improved by 0.28 logMAR (p < .001). In the irregular cornea group, the mean BCVA improved by 0.46 logMAR (p < .001), while the improvement in BCVA for the OSD group was 0.20 logMAR (p < .001). All etiologies in the irregular cornea group demonstrated improvement in BCVA (p < .05) with the exception of post-PRK (n = 2). BCVA improvement was found for all sub-etiologies in the OSD subgroup (p < .05). CONCLUSIONS: PROSE treatment had a positive impact on visual acuity for a wide range of corneal irregularities and ocular surface disease, suggesting that PROSE lenses may offer improvements in visual acuity without risks inherent to surgery.

Goblet cell loss abrogates ocular surface immune tolerance.

Intestinal epithelial cells condition tolerogenic properties in DCs. Aqueous-deficient dry eye is associated with goblet cell (GC) loss and increased IFN-γ expression in the conjunctiva. We hypothesized that loss of GCs reduces tolerance-inducing properties of antigen presenting cells (APCs) in the conjunctiva and draining nodes. Mice lacking the SAM pointed domain containing ETS transcription factor (Spdef) that is required for GC differentiation had an increased frequency of macrophages in the conjunctiva and CD11b+CD11c+ DCs in the conjunctiva and draining nodes, and these cells had greater IL-12 expression than WT mice. Conditioned media from cultured WT conjunctival GCs suppressed LPS-induced IL-12 production by conjunctival APCs. OVA antigen-specific OTII CD4+ T cells primed by Spdef-KO draining lymph node APCs showed greater proliferation, lower frequency of Foxp3+, increased frequency of IFN-γ+ and IL-17+ cells, and greater IFN-γ production than those primed by WT APCs. The immune tolerance to OVA antigen topically applied to the conjunctiva measured by cutaneous delayed type hypersensitivity (DTH) reaction, OVA-specific T cell proliferation, Foxp3 induction, and IFN-γ production observed in WT mice was lost in the Spdef-KO mice. We concluded that conjunctival GCs condition tolerogenic properties in APCs that suppress IL-12 production and Th1 polarization.

Study design and baseline findings from the progression of ocular findings (PROOF) natural history study of dry eye.

BACKGROUND: The aim of this research is to initiate a 5-year natural history study of dry eye disease (DED) using objectively assessed and patient-reported outcomes, to explore the hypothesis that DED is a progressive condition that has substantive and measurable impacts not only on the ocular surface, but on quality of life and visual functioning. Our objective for this report is to examine the baseline data. METHODS: A multicenter, prospective, controlled, observational study of Level 2 (mild-to-moderate) DED patients based on International Task Force Delphi Panel severity grading, and controls, documented baseline measures (including tear film biomarkers and quality of life). Tear cytokine concentrations were also measured in the tear film. Patients were using artificial tears as needed. RESULTS: Two hundred seventeen DED patients and 67 gender- and age-matched controls were enrolled. A majority were females and Caucasian and groups did not differ significantly in terms of gender, race, or age. Differences between DED and matched controls, at baseline, included mean scores for Ocular Surface Disease Index (31.7 vs 4.1, P < 0.0001), Schirmer test (5.7 vs 15.3 mm, P < 0.0001), corneal staining (1.4 vs 0.2, P < 0.0001), conjunctival staining (1.4 vs 0.3, P < 0.0001), and tear break-up time (5.7 vs 8.5 s, P < 0.0001). Tear cytokines levels were determined and included interferon-γ, interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor-α, epidermal growth factor, IL-13, IL-17, IL-1α, and inducible protein-10. The mean levels of IL-8 and IL-6 were slightly higher in the DED group at baseline. Blurred vision was reported as moderate/severe/very severe at baseline in 57.6% of DED patients vs.10.5% of normal controls (P < 0.0001). DED patients reported greater reductions in work and non-work productivity, as well as greater need for visits to ophthalmologists during the prior year. CONCLUSIONS: In this report of the baseline findings of a 5-year natural history study of DED, a striking disease burden is observed with regard to blurred vision, productivity, and visits to eye care practitioners in mild to moderate DED patients compared to normal subjects of similar ages and genders. TRIAL REGISTRATION: ClinicalTrials.gov NCT00833235 on January 30, 2009.

The Pathophysiology of Dry Eye Disease: What We Know and Future Directions for Research.

Clinical and laboratory studies performed over the past few decades have discovered that dry eye is a chronic inflammatory disease that can be initiated by numerous extrinsic or intrinsic factors that promote an unstable and hyperosmolar tear film. These changes in tear composition, in some cases combined with systemic factors, lead to an inflammatory cycle that causes ocular surface epithelial disease and neural stimulation. Acute desiccation activates stress signaling pathways in the ocular surface epithelium and resident immune cells. This triggers production of innate inflammatory mediators that stimulate the production of matrix metalloprotease, inflammatory cell recruitment, and dendritic cell maturation. These mediators, combined with exposure of autoantigens, can lead to an adaptive T cell-mediated response. Cornea barrier disruption develops by protease-mediated lysis of epithelial tight junctions, leading to accelerated cell death; desquamation; an irregular, poorly lubricated cornea surface; and exposure and sensitization of epithelial nociceptors. Conjunctival goblet cell dysfunction and death are promoted by the T helper 1 cytokine interferon gamma. These epithelial changes further destabilize the tear film, amplify inflammation, and create a vicious cycle. Cyclosporine and lifitegrast, the 2 US Food and Drug Administration-approved therapies, inhibit T-cell activation and cytokine production. Although these therapies represent a major advance in dry eye therapy, they are not effective in improving discomfort and corneal epithelial disease in all patients. Preclinical studies have identified other potential therapeutic targets, biomarkers, and strategies to bolster endogenous immunoregulatory pathways. These discoveries will, it is hoped, lead to further advances in diagnostic classification and treatment.

Goblet Cells Contribute to Ocular Surface Immune Tolerance-Implications for Dry Eye Disease.

Conjunctival goblet cell (GC) loss in dry eye is associated with ocular surface inflammation. This study investigated if conjunctival GCs contribute to ocular surface immune tolerance. Antigens applied to the ocular surface, imaged by confocal microscopy, passed into the conjunctival stroma through goblet cell associated passages (GAPs) in wild type C57BL/6 (WT), while ovalbumin (OVA) was retained in the epithelium of SAM pointed domain containing ETS transcription factor (Spdef) knockout mice (Spdef-/-) that lack GCs and are a novel model of dry eye. Stimulated GC degranulation increased antigen binding to GC mucins. Induction of tolerance to topically applied OVA measured by cutaneous delayed type hypersensitivity (DTH) was observed in WT, but not Spdef-/-. OTII CD4⁺ T cells primed by dendritic cells (DCs) from the conjunctival draining lymph nodes of Spdef-/- had greater IFN-γ production and lower Foxp3 positivity than those primed by WT DCs. These findings indicate that conjunctival GCs contribute to ocular surface immune tolerance by modulating antigen distribution and antigen specific immune response. GC loss may contribute to the abrogation of ocular surface immune tolerance that is observed in dry eye.

Evolving risk factors and antibiotic sensitivity patterns for microbial keratitis at a large county hospital.

Purpose: To identify the risk factors, causative organisms, antimicrobial susceptibility and outcomes of microbial keratitis in a large county hospital in Houston, Texas.Design: Case series. METHODS: Setting: A large county hospital in Houston, Texas. STUDY POPULATION: Patients with known diagnosis of microbial keratitis from January 2011 to May 2015. OBSERVATION PROCEDURE: Retrospective chart review. MAIN OUTCOMES: Epidemiology, risk factors, outcomes and antibiotic susceptibility of microbial keratitis. RESULTS: The most commonly identified risk factors were contact lens use (34.4%), ocular trauma (26.3%), diabetes mellitus (16.7%), ocular surgery (13.5%), ocular surface diseases (11.5%), previous keratitis (10.4%), glaucoma (6.3%), cocaine use (5.2%) and HIV-positive status (4.2%). Eyes with positive cultures (61.5%) were associated with worse visual outcomes (p=0.019) and a higher number of follow-up visits (p=0.007) than eyes with negative cultures (38.5%). Corneal perforation was the most common complication (11.5%). Gram-negative organisms (21.9%) were all susceptible to ceftazidime, tobramycin and fluoroquinolones. Gram-positive organisms (33.3%) had worse outcomes than Gram-negative organisms (21.9%) and exhibited a wide spectrum of antibiotic resistance, but all were susceptible to vancomycin. Twenty-seven per cent of the coagulase-negative Staphylococci were resistant to fluoroquinolones. CONCLUSION: This study identified a recent shift in risk factors and antibiotic resistance patterns in microbial keratitis at a County Hospital in Houston, Texas. In our patient population, fluoroquinolone monotherapy is not recommended for severe corneal ulcers. On the basis of these results, vancomycin and tobramycin should be used for empirical therapy until microbial identity and sensitivity results are available.

Long-term outcomes of ciliary sulcus versus capsular bag fixation of intraocular lenses in children: An ultrasound biomicroscopy study.

PURPOSE: To evaluate the long-term outcomes of ciliary sulcus versus capsular bag fixation of intraocular lenses (IOLs) in children after pediatric cataract surgery. METHODS: IOL was implanted in the ciliary sulcus in 21 eyes of 14 children, and in the capsular bag in 19 eyes of 12 children for the treatment of pediatric cataract in an institutional setting. Ultrasound biomicroscopy (UBM) was performed. Main outcome measures included IOL decentration, IOL tilt, anterior chamber depth (ACD), angle-opening distance at 500 µm (AOD500), trabecular-iris angle (TIA), best-corrected visual acuity (BCVA), intraocular pressure (IOP), and incidence of postoperative complications. RESULTS: The mean follow-up period was 6.81 ± 1.82 years. Comparing to the capsular bag fixation group, the ciliary sulcus fixation group had higher vertical IOL decentration, horizontal IOL tilt, and vertical IOL tilt (p = 0.02, 0.01,0.01, respectively), higher incidence of iris-IOL contact and peripheral anterior synechia (p = 0.001, 0.03, respectively), smaller ACD, AOD500, and TIA (p = 0.02, 0.03, 0.04, respectively), higher mean IOP (17.10 ±6.06 mmHg vs.14.15± 4.74 mmHg, p = 0.01), and higher incidence of secondary glaucoma (28.57% vs. 10.53%, p = 0.007).There was no significant difference between the two groups with regard to the BCVA, refractive errors, incidence of myopic shift, nystagmus, strabismus, and visual axis opacity. CONCLUSIONS: Ciliary sulcus fixation of IOLs in pediatric eyes may increase IOL malposition and crowding of the anterior segment, and may associate with a higher risk of secondary glaucoma compared to capsular bag fixation of IOLs.

Pollen/TLR4 Innate Immunity Signaling Initiates IL-33/ST2/Th2 Pathways in Allergic Inflammation.

Innate immunity has been extended to respond environmental pathogen other than microbial components. Here we explore a novel pollen/TLR4 innate immunity in allergic inflammation. In experimental allergic conjunctivitis induced by short ragweed (SRW) pollen, typical allergic signs, stimulated IL-33/ST2 signaling and overproduced Th2 cytokine were observed in ocular surface, cervical lymph nodes and isolated CD4+ T cells of BALB/c mice. These clinical, cellular and molecular changes were significantly reduced/eliminated in TLR4 deficient (Tlr4-d) or MyD88 knockout (MyD88-/-) mice. Aqueous SRW extract (SRWe) directly stimulated IL-33 mRNA and protein expression by corneal epithelium and conjunctiva in wild type, but not in Tlr4-d or MyD88-/- mice with topical challenge. Furthermore, SRWe-stimulated IL-33 production was blocked by TLR4 antibody and NF-kB inhibitor in mouse and human corneal epithelial cells. These findings for the first time uncovered a novel mechanism by which SRW pollen initiates TLR4-dependent IL-33/ST2 signaling that triggers Th2-dominant allergic inflammation.