Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaEGFP (mCherry-EGFP-LC3B) was quenched by autolysosomalacidity , unlike pH insensitive mCherry. No lacritin stimulatedautophagy was evident in Htt25Q cells in keeping with the absence ofstress.Conclusions: Lacritin signaling rids cells of toxic aggregatedproteins that develop during stress by rapidly and transientlystimulating autophagy. Autolysosomal digestion products then feedinto and restore oxidative phosphorylation.Commercial Relationships: Keith B. Zimmerman, None; MiltonF. Tyler, None; Ningning Wang, None; Ronald Raab, None;Robert L. McKown, EyeRx Research, Inc. (I); Gordon W. Laurie,UVa Patent Foundation (F)Support: NH Grant EY018222Program Number: 909 Poster Board Number: B0214Presentation Time: 1:00 PM - 2:45 PMLacritin as the Primary Epithelial Survival Activity in BasalHuman Tears Reveals Novel Features About AutophagicSurvival SignalingNingning Wang 1 , Ronald Raab 2 , Robert L. McKown 2 , Cindy M.Hutnik 3 , Gordon W. Laurie 1 . 1 Cell Biology, University of Virginia,Charlottesville, VA; 2 2Integrated Science and Technology, jamesmadison university, harrisonburg, VA; 3 Depts. of Ophthalmology &Pathology, Ivey Eye Institute, London, ON, Canada.Purpose: As the fluid thought to support the survival of the avascularcorneal epithelium, tears are rich in proteins with growth and survivalcapabilities - including lacritin, a tear prosecretory mitogen thatsupports the survival of stressed corneal epithelial cells in culture,and promotes basal tearing in rabbits and monkeys. Here we askwhether tears are indeed prosurvival, if so does tear lacritincontribute, and what is the FOXO3 survival mechanism?Methods: Human corneal epithelial (HCE; Riken) cells weresensitized with INFG, and then treated with TNF in the in thepresence of normal or dry eye tears, normal tears mock or lacritinimmunodepleted, or with dry eye tears supplemented withrecombinant lacritin or negative control C-25. FOXO3 translocationserved as a readout for cell stress (nuclear) or survival (cytoplasmic).FOXO3 immunoprecipitates were used to screen for FOXO3 bindingpartners.Results: Normal and mock depleted tears, but not lacritin depletedtears promoted the survival of INFG/TNF stressed HCE cells. Tearsfrom dry eye patients supplemented lacritin promoted cell survival,but not dry eye tears alone nor C-25 supplemented dry eye tears. Wediscovered that autophagy mediator ATG101 binds FOXO3 instressed cells treated with lacritin, and that shRNA depletion ofATG101 or FOXO3 abrogates lacritin stimulated autophagy andsurvival.Conclusions: Lacritin appears to be the primary survival factor inbasal human tears. Surprisingly it is not compensated by other tearfactors when depleted. Lack of dry eye tear survival activity can berescued with recombinant lacritin, suggesting efficacy in treatinghuman dry eye. Lacritin rapidly stimulates FOXO3/ATG101 ligation.ATG101 is known to bind and stabilize the ATG13/ULK1 complexresponsible for initiating autophagy - thus explaining how lacritin sorapidly stimulates autophagy.Commercial Relationships: Ningning Wang, None; Ronald Raab,None; Robert L. McKown, EyeRx Research, Inc. (I); Cindy M.Hutnik, None; Gordon W. Laurie, UVa Patent Foundation (F)Support: EY018222, EY013143 (to GWL).Neutralization of Ocular Surface TNF-α Reduces Ocular Surfaceand Lacrimal Gland Inflammation Induced by In Vivo Dry EyeYongwoo Ji, Yu Jeong Byun, Jin Sun Kim, Eunae Jeong, Hyung KeunLee. Institute of Vision Research, Department of Ophthalmology,Yonsei University College of Medicine, Seoul, Republic of Korea.Purpose: The purposes of this study are to investigate theeffectiveness of tumor necrosis factor (TNF)-α blocker for treatmentof dry eye (DE)-induced inflammation and determine a moreeffective method to suppress lacrimal gland inflammation. Efficacyof topical vs. systemic administration of TNF-α blockers wasdetermined using a murine DE model.Methods: The TNF-α blocker HL036 was developed by modificationof the TNF receptor I. Protein purity, binding affinity, and clearanceof TNF-α was compared with etanercept. Using DE inducedC57BL/6 mice, corneal erosion, tear secretion, and goblet cell countswere measured after subcutaneous or topical treatment withetanercept or HL036. Inflammatory cytokines in cornea and lacrimalglands were determined by quantitative reverse transcriptionpolymerase chain reaction and enzyme-linked immune absorbentassay.Results: HL036 showed TNF-α binding affinity comparable toetanercept, as measured by surface plasmon resonance. HL036concentration was significantly higher in cornea and anterior segmentthan etanercept, and effectively eliminated TNF-α on ocular surfaces.Etanercept was preferentially concentrated in the posterior segment.Corneal erosion, goblet cell counts, and tear secretion were improvedonly with topically applied etanercept and HL036. Ocular surfaceinterferon-γ (IFN-γ), interleukin-6 (IL-6), IL-21, and IL-22 weresignificantly decreased by topical HL036. DE induced lacrimal glandIFN-γ and IL-6 expression was effectively suppressed by topicaletanercept, glucocorticoid, and HL036.Conclusions: Topical TNF-α blockers effectively suppressedlacrimal gland and corneal inflammation by suppressing IFN-γ, IL-21, and IL-6. Differences in TNF-α affinity, clearance, and localconcentration of blockers may account for the anti-inflammatoryeffects in different ocular regions.Program Number: 910 Poster Board Number: B0215Presentation Time: 1:00 PM - 2:45 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.