Interferon-γ–Induced Unfolded Protein Response in Conjunctival Goblet Cells as a Cause of Mucin Deficiency in Sjögren Syndrome
2016; Elsevier BV; Volume: 186; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2016.02.004
ISSN1525-2191
AutoresTerry G. Coursey, Johanna Tukler Henriksson, Flavia L. Barbosa, Cintia S. de Paiva, Stephen C. Pflugfelder,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoGoblet cells (GCs) are specialized secretory cells that produce mucins and a variety of other proteins. Significant conjunctival GC loss occurs in both experimental dry eye models and patients with keratoconjunctivitis sicca due to the induction of interferon (IFN)-γ. With the use of a primary murine culture model, we found that GCs are highly sensitive to IFN-γ with significantly reduced proliferation and altered structure with low concentrations. GC cultures treated with IFN-γ have increased gene expression of Muc2 and Muc5AC but do not express these mucin glycoproteins. We hypothesized that IFN-γ induces endoplasmic reticulum stress and the unfolded protein response (UPR) in GCs. Cultures treated with IFN-γ increased expression of UPR-associated genes and proteins. Increased GRP78 and sXBP1 expression was found in experimental dry eye and Sjögren syndrome models and was GC specific. Increased GRP78 was also found in the conjunctiva of patients with Sjögren syndrome at the gene and protein levels. Treatment with dexamethasone inhibited expression of UPR-associated genes and increased mucin production. These results indicate that induction of UPR by IFN-γ is an important cause of GC-associated mucin deficiency observed in aqueous-deficient dry eye. Therapies to block the effects of IFN-γ on the metabolically active endoplasmic reticulum in these cells might enhance synthesis and secretion of the protective GC mucins on the ocular surface. Goblet cells (GCs) are specialized secretory cells that produce mucins and a variety of other proteins. Significant conjunctival GC loss occurs in both experimental dry eye models and patients with keratoconjunctivitis sicca due to the induction of interferon (IFN)-γ. With the use of a primary murine culture model, we found that GCs are highly sensitive to IFN-γ with significantly reduced proliferation and altered structure with low concentrations. GC cultures treated with IFN-γ have increased gene expression of Muc2 and Muc5AC but do not express these mucin glycoproteins. We hypothesized that IFN-γ induces endoplasmic reticulum stress and the unfolded protein response (UPR) in GCs. Cultures treated with IFN-γ increased expression of UPR-associated genes and proteins. Increased GRP78 and sXBP1 expression was found in experimental dry eye and Sjögren syndrome models and was GC specific. Increased GRP78 was also found in the conjunctiva of patients with Sjögren syndrome at the gene and protein levels. Treatment with dexamethasone inhibited expression of UPR-associated genes and increased mucin production. These results indicate that induction of UPR by IFN-γ is an important cause of GC-associated mucin deficiency observed in aqueous-deficient dry eye. Therapies to block the effects of IFN-γ on the metabolically active endoplasmic reticulum in these cells might enhance synthesis and secretion of the protective GC mucins on the ocular surface. It is now recognized that T helper (Th)1 and Th17 cells contribute to the ocular surface epithelial disease that develops in dry eye.1De Paiva C.S. Villarreal A.L. Corrales R.M. Rahman H.T. Chang V.Y. Farley W.J. Stern M.E. Niederkorn J.Y. Li D.Q. Pflugfelder S.C. Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-{gamma}.Invest Ophthalmol Vis Sci. 2007; 48: 2553-2560Crossref PubMed Scopus (238) Google Scholar, 2De Paiva C.S. Chotikavanich S. Pangelinan S.B. Pitcher III, J.I. Fang B. Zheng X. Ma P. Farley W.J. Siemasko K.S. Niederkorn J.Y. Stern M.E. Li D.Q. Pflugfelder S.C. IL-17 disrupts corneal barrier following desiccating stress.Mucosal Immunol. 2009; 2: 243-253Crossref PubMed Scopus (301) Google Scholar Th17 cells produce IL-17 that stimulates the expression of matrix metalloprotease-3 and -9 that breakdown corneal epithelial tight junctions that lead to altered barrier function.2De Paiva C.S. Chotikavanich S. Pangelinan S.B. Pitcher III, J.I. Fang B. Zheng X. Ma P. Farley W.J. Siemasko K.S. Niederkorn J.Y. Stern M.E. Li D.Q. Pflugfelder S.C. IL-17 disrupts corneal barrier following desiccating stress.Mucosal Immunol. 2009; 2: 243-253Crossref PubMed Scopus (301) Google Scholar Th1 cells predominantly produce interferon (IFN)-γ that causes apoptosis and cornification of corneal and conjunctival epithelial cells.1De Paiva C.S. Villarreal A.L. Corrales R.M. Rahman H.T. Chang V.Y. Farley W.J. Stern M.E. Niederkorn J.Y. Li D.Q. Pflugfelder S.C. Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-{gamma}.Invest Ophthalmol Vis Sci. 2007; 48: 2553-2560Crossref PubMed Scopus (238) Google Scholar, 3Corrales R.M. de Paiva C.S. Li D.Q. Farley W.J. Henriksson J.T. Bergmanson J.P. Pflugfelder S.C. Entrapment of conjunctival goblet cells by desiccation-induced cornification.Invest Ophthalmol Vis Sci. 2011; 52: 3492-3499Crossref PubMed Scopus (47) Google Scholar Within the stratified columnar conjunctival epithelia are goblet cells (GCs), which are specialized epithelial cells that secrete mucins and other proteins, that are integral components of the tear film. IFN-γ induces the progressive loss of conjunctival GCs in dry eye animal models and increased IFN-γ expression was detected in patients with aqueous tear deficiency.1De Paiva C.S. Villarreal A.L. Corrales R.M. Rahman H.T. Chang V.Y. Farley W.J. Stern M.E. Niederkorn J.Y. Li D.Q. Pflugfelder S.C. Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-{gamma}.Invest Ophthalmol Vis Sci. 2007; 48: 2553-2560Crossref PubMed Scopus (238) Google Scholar, 4Pflugfelder S.C. De Paiva C.S. Moore Q.L. Volpe E.A. Li D.Q. Gumus K. Zaheer M.L. Corrales R.M. Aqueous tear deficiency increases conjunctival interferon-gamma (IFN-gamma) expression and goblet cell loss.Invest Ophthalmol Vis Sci. 2015; 56: 7545-7550Crossref Scopus (84) Google Scholar One of the aims of this study was to determine the molecular mechanism for the loss of GC secretory function in response to IFN-γ. GCs, like other secretory cells, are reliant on the endoplasmic reticulum (ER) for robust protein production and secretion. This makes secretory cells particularly vulnerable to ER stress, which results in the accumulation of unfolded and misfolded proteins in the ER lumen. GCs predominantly produce high-molecular weight mucin glycoproteins that are difficult to fold. Accumulation of misfolded proteins results in the induction of the unfolded protein response (UPR) in an attempt to rescue the cell from impending apoptosis. Under homeostatic conditions the ER chaperone 78-kDa glucose-regulated protein [GRP78; also called binding immunoglobulin protein (BiP) or heat shock 70-kDa protein 5] holds the ER stress indicator proteins, inositol-requiring kinase 1α (IRE1α), pancreatic ER eIF2α kinase, and activating transcription factor 6α (ATF6α) in an inactive state.5McGuckin M.A. Eri R.D. Das I. Lourie R. Florin T.H. ER stress and the unfolded protein response in intestinal inflammation.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G820-G832Crossref PubMed Scopus (139) Google Scholar When the induction of UPR fails to compensate for accumulation of unfolded proteins, cell death is induced, typically apoptosis. The mechanisms that trigger cell death that are a result of ER stress vary and may involve caspase-dependent apoptosis or caspase-independent necrosis.5McGuckin M.A. Eri R.D. Das I. Lourie R. Florin T.H. ER stress and the unfolded protein response in intestinal inflammation.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G820-G832Crossref PubMed Scopus (139) Google Scholar Many other downstream players are involved in this highly developed and complicated mechanism to prevent and deal with ER stress. For example, chaperones bound to misfolded proteins in the ER initiate a cascade of events, including activation of stress kinases (p38 and Jun kinase), ubiquitination enzymes, and caspases. One of these players is X-box protein-1 (XBP1). During UPR, XBP1 is spliced into a 26-nucleotide isoform, spliced XBP1 (sXBP1), by the action of inositol-requiring kinase 1α. sXBP1 is a strong transcription factor and a regulator of protein folding in the UPR.6Lee A.H. Iwakoshi N.N. Glimcher L.H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response.Mol Cell Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1617) Google Scholar Detection of sXBP1 is a valuable marker for increased expression of proteins involved in UPR.7Samali A. Fitzgerald U. Deegan S. Gupta S. Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.Int J Cell Biol. 2010; 2010: 830307PubMed Google Scholar Here, we hypothesize that IFN-γ produced by lymphocytes in the pathogenesis of dry eye disease (DED) induces ER stress in conjunctival GCs, leading to the UPR in both mouse dry eye models and patients with Sjögren syndrome (SS)-associated dry eye. All experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The following stains of mice were used in this study: C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME; stock 000664), IFN-γ knock out (KO; The Jackson Laboratory; B6.129S7-Ifngtm1Ts/J; stock 002287), IFN-γ receptor KO (IFN-γRKO; The Jackson Laboratory; B6.129S7-Ifngr1tm1Agt/J; stock 003288), and CD25KO (The Jackson Laboratory; B6.129S4-Il2ratm1Dw/J; stock 002952). Breeder pairs were established for colonies in our facility. Briefly, explants were excised from the forniceal conjunctiva of 6- to 8-week-old female C57BL/6 or KO mice and incubated for 15 to 20 minutes at 37°C in keratinocyte serum-free media (Thermo Fisher, Waltham, MA; catalog 10724-011) supplemented with 3% fetal bovine serum defined, 1.25 μg/mL amphotericin B (Thermo Fisher; catalog 15290-018), 0.5 μL/mL gentamicin (Thermo Fisher; catalog 15750-060), and 5 μg/mL dispase II (Roche, Basil, Switzerland; 04942078001), as previously described.8Tukler Henriksson J. Coursey T.G. Corry D.B. De Paiva C.S. Pflugfelder S.C. IL-13 stimulates proliferation and expression of mucin and immunomodulatory genes in cultured conjunctival goblet cells.Invest Ophthalmol Vis Sci. 2015; 56: 4186-4197Crossref Scopus (71) Google Scholar Conjunctival explants were plated 1 explant per well in 48-well plates and 200 μL of keratinocyte serum-free media supplemented with 80 ng/mL mouse epidermal growth factor (BD Biosciences, Franklin Lakes, NJ; catalog 354001). Four explants (upper and lower conjunctival fornix from each eye) were obtained per animal. Cultures were treated with media that contained various concentrations of recombinant murine IFN-γ (PeproTech, Rocky Hill, NJ; catalog 315-05). Cultures were incubated for 7 or 14 days. As a positive control for UPR, cultures were treated with 0.5 μmol/L thapsigargin (Sigma-Aldrich, St. Louis, MO; T9033) for 24 hours, as previously described.7Samali A. Fitzgerald U. Deegan S. Gupta S. Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.Int J Cell Biol. 2010; 2010: 830307PubMed Google Scholar Cultures were treated with 40 μmol/L dexamethasone (Dexa; Sigma-Aldrich; D4902)9Das I. Png C.W. Oancea I. Hasnain S.Z. Lourie R. Proctor M. Eri R.D. Sheng Y. Crane D.I. Florin T.H. McGuckin M.A. Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins.J Exp Med. 2013; 210: 1201-1216Crossref PubMed Scopus (72) Google Scholar in addition to IFN-γ for 7 days. Corneal epithelial cultures were established by excision of the central cornea and incubation in 5 μg/mL of dispase II (Roche; 04942078001) for 20 minutes at 37°C. Explants were placed epithelial side down in a 24-well plate. Tissues were cultured in CnT Corneal Epithelium Medium (CnT-20; Zen-Bio Research, Triangle Park, NC) for 7 days before harvesting for RNA isolation. After 7 or 14 days media and explants were removed, and fresh media (200 μL) was added to each well. Cell proliferation reagent WST-1 (Roche; 5015944001) was added at a final concentration of 1:10, and plates were incubated at 37°C for 24 hours. Absorbance was measured at wavelengths 440 and 690 nm with a plate reader (infinite M200; Techan, Durham, NC), as previously described.8Tukler Henriksson J. Coursey T.G. Corry D.B. De Paiva C.S. Pflugfelder S.C. IL-13 stimulates proliferation and expression of mucin and immunomodulatory genes in cultured conjunctival goblet cells.Invest Ophthalmol Vis Sci. 2015; 56: 4186-4197Crossref Scopus (71) Google Scholar Caspase-3 activity was measured with a Caspase-3/CPP32 Fluorometric Assay Kit (BioVision, Milpitas, CA; catalog K105). Cell lysate (50 μg) in 50 μL of cell lysis buffer (supplied) per well was added to a 96-well plate before addition of 50 μL of 2× reaction buffer (containing 10 mmol/L DTT and 100 μmol/L YVAD-AFC) to each sample well. Samples were incubated at 37°C for 2 hours and measured at wavelengths 400 and 505 nm with a plate reader (Techan; infinite M200). Fold increase in caspase-3 activity was determined by comparing the results of IFN-γ–treated samples with the level of the untreated samples. For mouse conjunctival cultures, cell culture media were aspirated, and conjunctival explants were removed. Cultures were fixed in the plate with cold methanol or 4% paraformaldehyde. Cultures were washed in phosphate-buffered saline (PBS) and treated with 0.1% Triton-X for 10 minutes. After blocking with 20% goat serum, anti-Muc2 (dilution 1:50; Santa Cruz Biotechnology, Dallas, TX; SC-15334;), anti-Muc5AC (Abmart, Berkeley Heights, NJ; 14906-1-4), anti-GRP78 (dilution 1:50; Santa Cruz Biotechnology; SC-1050) were applied overnight. Cultures were washed with PBS, and fluorescent secondary antibodies were applied to antibody-treated wells. The cultures were then counterstained with Hoechst (Sigma-Aldrich; 33342 DNA dye; B2261) or propidium iodide (Sigma-Aldrich; P4864), 1 drop of gel mount was applied, and, finally, the cultures were coverslipped with no. 1.5 (8-mm diameter) round cover glass (Electron Microscopy Sciences, Hatfield, PA; catalog 72296-08). For human samples, human conjunctival samples were obtained by impression cytology of bulbar conjunctiva with the use of Biopore membranes, as previously described.10Moore Q.L. De Paiva C.S. Pflugfelder S.C. Effects of dry eye therapies on environmentally induced ocular surface disease.Am J Ophthalmol. 2015; 160: 135-142Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The samples were fixed in cold methanol for 10 minutes, washed in PBS, and blocked with 1% bovine serum albumin to reduce nonspecific labeling for 1 hour. GRP78 primary antibody (Santa Cruz Biotechnology; sc-1050) diluted 1:50 in 1% bovine serum albumin was applied for 1 hour at room temperature. Tissues were washed with 1% PBS and then incubated with donkey anti-goat IgG (H + L; Molecular Probes, Thermo Fisher; A11055) diluted 1:250 in PBS for 60 minutes at room temperature in a dark room. Samples were counterstained with Hoechst dye (dilution 1:500; 33342 DNA dye;) for 10 minutes. After washing with PBS, coverslip was applied with Gel/Mount (Sigma-Aldrich; G0918). Digital confocal images were captured with a laser scanning confocal microscope (Nikon A1 RMP; Nikon, Neville, NY) with wavelength 400 to 750 nm. The images were processed with NIS Elements 4.20 version (Nikon). A bicinchoninic acid assay (Thermo Fisher; catalog 23225) was initially performed to determine total protein concentration per sample. On the basis of the results, the volume that was equal to 1.5 μg of protein per sample was used for the dot blot assay. Samples were collected in RIPA buffer (Sigma-Aldrich; R0278), and additional RIPA buffer was added to bring the total volume of each sample to 50 μL before proceeding to dot blot. Polyvinylidene difluoride membranes were prewet with PBS + 0.05% Tween 20 (TBST) and applied to the dot blot apparatus. Samples were loaded and left to incubate on the apparatus for 1 hour and subsequently were washed for 30 minutes in Carbo-Free Blocking Solution (catalog no. SP-5040). The membranes were then incubated overnight with 10 μg/mL anti-Muc5AC (Santa Cruz Biotechnology; SC-20118) or anti-Muc2 (Santa Cruz Biotechnology; SC-15334) at 4°C. The specificity of these antibodies for the respective GC mucins in the conjunctiva was previously confirmed.8Tukler Henriksson J. Coursey T.G. Corry D.B. De Paiva C.S. Pflugfelder S.C. IL-13 stimulates proliferation and expression of mucin and immunomodulatory genes in cultured conjunctival goblet cells.Invest Ophthalmol Vis Sci. 2015; 56: 4186-4197Crossref Scopus (71) Google Scholar Blots were washed with TBST and incubated for 1 hour with horseradish peroxidase–goat–anti-rabbit (Invitrogen, Carlsbad, CA; 656120). Blots were washed with TBST. Horseradish peroxidase-conjugated proteins were detected by electrochemiluminescence reagent. The mean intensity of the blots from the different samples was measured with NIS Elements (Nikon). A bicinchoninic acid assay was performed to measure total protein concentration of each sample. Samples (25 μL, equal to a protein concentration 90 to 100 μg) were diluted with one part 2× sample buffer (2× Laemmli Sample Buffer; Bio-Rad Laboratories, Hercules, CA; 161-0737), boiled for 5 minutes, and loaded onto the polyacrylamide gel. The gel (mini-PROTEAN TGX stain-free Precast Gel 4% to 15%; Bio-Rad Laboratories; catalog 456-8024) was run at constant current at 150 V for 45 to 60 minutes at room temperature before the proteins were transferred onto a polyvinylidene difluoride membrane (Immobilion Transfer membranes; EMD Millipore, Darmstadt, Germany; catalog IVPH07850) at 100 V for 60 minutes. Membranes were incubated in TBST with 5% fat-free milk for 60 minutes, followed by incubation in anti-GRP78 antibody (Santa Cruz Biotechnology; SC-1050; 1:200) overnight at 4°C. Membranes were washed in TBST, incubated in secondary horseradish peroxidase–rabbit–anti-goat (dilution 1:5000; Thermo Fisher; catalog 61-1620) for 1 hour, washed 3× with TBST, and developed with Clarity Western electrochemiluminescence blotting substrate (Bio-Rad Laboratories; 1705060). Stain-free gels were analyzed on a ChemiDoc Touch Imaging System (Bio-Rad Laboratories). Bands were normalized to total protein per lane as previously described.11Rivero-Gutierrez B. Anzola A. Martinez-Augustin O. de Medina F.S. Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting.Anal Biochem. 2014; 467: 1-3Crossref PubMed Scopus (131) Google Scholar For real-time quantitative PCR (qPCR), total RNA from approximately 10 cultures per sample was extracted with an RNeasy Micro Kit (Qiagen, Valencia, CA), cDNA was synthesized by Ready-To-Go You-Prime First-Strand Beads according to the manufacturer's instructions (GE Healthcare, Chalfont St. Giles, UK; 27-9264-01). RT-PCR was then performed on a Step One Plus system (Thermo Fisher/Applied Biosystems). The RT-PCR was run three independent times with at least five samples per group and time point. Real-time PCR was performed with specific TaqMan probes (Applied Biosystems) for mucin 2 (Muc2; Muc2, Mm00458299_m1), mucin 5AC (Muc5AC; Muc5AC, Mm01276718_m1), GRP78 (BiP) (mouse: Hspa5; Mm00517690_g1 and human: HSPA5; Hs00607129_gH), C/EBP homologous protein (CHOP; Ddit3; Ddit3, Mm01135937_g1), ATF4 (Atf4; Mm00515325_g1), and ATF6 (Atf6; Mm01295319_m1). Mouse sXbp1 TaqMan primers were custom primers. Sequences were: forward, 5′-GAGTCCGCAGCAGTGC-3′; reverse, 5′-CAAAAGGATATCAGACTCAGAATCTGAA-3′; and probe, 5′-CCATGGACTCTGACACTGTTGCCT-3′. Sequences for forward and reverse primers were previously published.12Heazlewood C.K. Cook M.C. Eri R. Price G.R. Tauro S.B. Taupin D. Thornton D.J. Png C.W. Crockford T.L. Cornall R.J. Adams R. Kato M. Nelms K.A. Hong N.A. Florin T.H. Goodnow C.C. McGuckin M.A. Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis.PLoS Med. 2008; 5: e54Crossref PubMed Scopus (548) Google Scholar Human sXbp1 TaqMan primers were also custom primers. Sequences are forward, 5′-TGCTGAGTCCGCAGCAGGTA-3′; reverse, 5′-GCTGGCAGGCTCTGGGGAAG-3′; and probe, 5′-TCCTGTTGGGCATTCTGGACAACT-3′. The results were analyzed by the comparative threshold cycle method and normalized by HPRT-1 (human: HPRT1; Hs02800695_m1; mouse: Hprt; Mm00446968_m1) as the housekeeping control gene. C57BL/6J mice were exposed to desiccating stress (DS) by subcutaneous injection of scopolamine hydrobromide (0.5 mg/0.2 mL; Sigma-Aldrich) four times a day (8 AM, Noon, 2 PM, and 5 PM), alternating flanks for consecutive 5 days of DS (DS5) as previously described.13Niederkorn J.Y. Stern M.E. Pflugfelder S.C. De Paiva C.S. Corrales R.M. Gao J. Siemasko K. Desiccating stress induces T cell-mediated Sjogren's Syndrome-like lacrimal keratoconjunctivitis.J Immunol. 2006; 176: 3950-3957Crossref PubMed Scopus (282) Google Scholar Mice were placed in a cage with a perforated plastic screen on one side to allow airflow from a fan placed approximately 15 cm in front of it for 16 h/d. Room humidity was maintained at 20% to 30%. Control mice were maintained in a nonstressed (NS) environment at 50% to 75% relative humidity without exposure to a forced air draft. In vivo neutralization of IFN-γR was performed with topical application of rat anti-mouse IFN-γR IgG1 (1 mg/mL, purified from hybridoma GR-20; catalog no. CRL-2024; ATCC, Rockville, MD). Anti–IFN-γR antibody (10 μL) or isotype control (rat IgG, 1 mg/mL; Vector Laboratories, Burlingame, CA) was applied to the ocular surface four times daily from −3 to 5 days of DS. Samples were collected from the conjunctival epithelial layer of animals that were exposed to DS5 and NS controls. This was accomplished by cutting the medial and lateral eyelids after euthanasia to expose the conjunctival epithelium. A Supor 450 membrane cut to fit a mouse conjunctiva (Pall, Port Washington, NY) was placed on the conjunctival epithelium with gentle pressure for 4 to 5 seconds. The membranes were then placed in RNA lysis buffer for RNA isolation. Four mice were used per sample; three samples were used per experimental group. Impression cytology samples were taken from exposed temporal conjunctiva of SS patients and control subjects with the use of EMD Millipore Biopore membranes for immunofluorescence staining, as previously described.10Moore Q.L. De Paiva C.S. Pflugfelder S.C. Effects of dry eye therapies on environmentally induced ocular surface disease.Am J Ophthalmol. 2015; 160: 135-142Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Supor 450 membranes from the exposed nasal conjunctiva were used for extracting RNA for measuring gene expression. Samples were obtained from eight female patients (ages 59 to 67 years) diagnosed with SS. Healthy controls were obtained from four female subjects (ages 28 to 38 years) that had no conjunctival staining or corneal fluorescein staining. Prism 6.0 software (GraphPad Software Inc., La Jolla, CA) was used for statistical analysis. An unpaired t-test was used to compare control with treated cultures at day 7 and day 14. The statistical significance was set to P ≤ 0.05, and data are presented as means ± SEM. Previous studies in our laboratory reported that significant GC loss occurs in experimental dry eye models when IFN-γ expression increases.1De Paiva C.S. Villarreal A.L. Corrales R.M. Rahman H.T. Chang V.Y. Farley W.J. Stern M.E. Niederkorn J.Y. Li D.Q. Pflugfelder S.C. Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-{gamma}.Invest Ophthalmol Vis Sci. 2007; 48: 2553-2560Crossref PubMed Scopus (238) Google Scholar, 14Zhang X. Chen W. De Paiva C.S. Corrales R.M. Volpe E.A. McClellan A.J. Farley W.J. Li D.Q. Pflugfelder S.C. Interferon-{gamma} exacerbates dry eye-induced apoptosis in conjunctiva via dual apoptotic pathways.Invest Ophthalmol Vis Sci. 2011; 52: 6279-6285Crossref PubMed Scopus (96) Google Scholar, 15Zhang X. De Paiva C.S. Su Z. Volpe E.A. Li D.Q. Pflugfelder S.C. Topical interferon-gamma neutralization prevents conjunctival goblet cell loss in experimental murine dry eye.Exp Eye Res. 2014; 118: 117-124Crossref PubMed Scopus (61) Google Scholar To examine the effect of IFN-γ specifically on GCs, we established primary cultures from mouse conjunctiva that become GCs, as previously described.8Tukler Henriksson J. Coursey T.G. Corry D.B. De Paiva C.S. Pflugfelder S.C. IL-13 stimulates proliferation and expression of mucin and immunomodulatory genes in cultured conjunctival goblet cells.Invest Ophthalmol Vis Sci. 2015; 56: 4186-4197Crossref Scopus (71) Google Scholar To investigate the effect of IFN-γ on these cells, GC cultures were treated with 5 and 10 ng/mL IFN-γ. At these concentrations, the cultured cells did not grow (data not shown). To determine the appropriate dose of IFN-γ that did not inhibit growth, cultures were treated with 1.0, 0.5, or 0.1 ng/mL IFN-γ, and cell proliferation was measured by WST-1 cell proliferation assay after 7 and 14 days. All three concentrations caused a significant reduction in proliferation (Figure 1A). To determine whether the decreased cell number was due to the apoptosis in IFN-γ–treated cultures, a caspase-3 activity assay was performed. Cultures treated with 0.5 and 1.0 ng/mL IFN-γ had a significant increase in caspase-3 activity. Caspase-3 activity was significantly decreased by blockage of the IFN-γR with a neutralizing antibody (50 μg/mL) (Figure 1B). Likewise, we demonstrated that blockage of the IFN-γR restored cell proliferation to untreated culture levels (Figure 1C). To confirm the effect of IFN-γ, GC cultures were initiated with IFN-γKO and IFN-γRKO mice. By showing the high sensitivity of GCs to IFN-γ, cultures from IFN-γKO and IFN-γRKO mice had a significant increase in cell proliferation compared with those from wild-type mice (Figure 1, D and E). This suggests that the low basal level of IFN-γ found in a normal wild-type mouse conjunctiva inhibits GC growth. Previous studies have shown loss of mucin-filled GCs by histochemistry or immunohistochemistry in experimental dry eye and human aqueous tear deficiency.1De Paiva C.S. Villarreal A.L. Corrales R.M. Rahman H.T. Chang V.Y. Farley W.J. Stern M.E. Niederkorn J.Y. Li D.Q. Pflugfelder S.C. Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-{gamma}.Invest Ophthalmol Vis Sci. 2007; 48: 2553-2560Crossref PubMed Scopus (238) Google Scholar, 2De Paiva C.S. Chotikavanich S. Pangelinan S.B. Pitcher III, J.I. Fang B. Zheng X. Ma P. Farley W.J. Siemasko K.S. Niederkorn J.Y. Stern M.E. Li D.Q. Pflugfelder S.C. IL-17 disrupts corneal barrier following desiccating stress.Mucosal Immunol. 2009; 2: 243-253Crossref PubMed Scopus (301) Google Scholar, 4Pflugfelder S.C. De Paiva C.S. Moore Q.L. Volpe E.A. Li D.Q. Gumus K. Zaheer M.L. Corrales R.M. Aqueous tear deficiency increases conjunctival interferon-gamma (IFN-gamma) expression and goblet cell loss.Invest Ophthalmol Vis Sci. 2015; 56: 7545-7550Crossref Scopus (84) Google Scholar To assess the effects of IFN-γ on gene expression of Muc2 and Muc5AC in GC cultures qPCR was performed for Muc2 and Muc5AC in cultures treated with 0.5 ng/mL IFN-γ. Paradoxically, Muc5AC gene expression increased at day 7, and both Muc2 and Muc5AC transcripts increased at day 14 (Figure 2A). Interestingly, loss of Muc2 and Muc5AC expression at the protein level was seen by immunostaining at concentrations of 0.5 and 1.0 ng/mL IFN-γ (Figure 2B). GCs treated with IFN-γ >0.1 ng/mL were also noted to have a dramatic change in cell structure. These cells assumed a spindle-like appearance with condensed nuclei. To confirm the loss mucins at the protein level, dot blots for Muc2 and Muc5AC were performed. A significant decrease of Muc2 (Figure 2C) and Muc5AC (Figure 2D) was observed in cultures treated with 0.5 ng/mL IFN-γ. Confirming the specificity of the Muc2 and Muc5AC antibodies, protein concentration (from untreated GC cultures) was positively correlated to mean intensity (lower panels). The primary function of GCs is production and secretion of mucins and other proteins. We hypothesized that the high secretory activity of GCs would make them particularly susceptible to UPR and would explain the dichotomy of increased expression of mucin genes in the face of decreased production of mucin glycoproteins. To test this hypothesis, we assessed the expression of UPR-related genes. Day 7 cultures treated with 0.5 ng/mL IFN-γ had a significant increase in Grp78 (Hsp5a/BiP), sXBP1, CHOP, and ATF4 (Figure 3A). GRP78 (BiP) is a key chaperone in the UPR and is commonly used as an ER stress marker.16Kozutsumi Y. Segal M. Normington K. Gething M.J. Sambrook J. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins.Nature. 1988; 332: 462-464Crossref PubMed Scopus (983) Google Scholar As a positive control, cultures were treated with 0.5 μmol/L ER stress inducer thapsigargin for 24 hours before harvesting. Expression of GRP78, sXBP1, CHOP, ATF4, and ATF6 increased in IFN-γ–treated cultures at day 14 (Figure 3B). To confirm the results by protein expression, immunostaining for GRP78 and mucins was performed. Cultures treated with 0.5 ng/mL IFN-γ had dramatically increased GRP78 and loss or Muc2 or Muc5AC immunoreactivity compared with untreated and 0.1 ng/mL IFN-γ–treated cultures (Figure 3C). To confirm increased expression of GRP78 in IFN-γ–treated cultures, a Western blot analysis for GRP78 was performed. Cultures treated with IFN-γ for 7 days had a single band that corresponded to the appropriate size, whereas a faint barely detectable band was observed in the untreated sample. Lysates from Jurkat cells were used as a posi
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