Meibomian Gland Dysfunction in Type 2 Diabetic Patients

Purpose. To investigate meibomian gland and tear film function in patients with type 2 diabetes. Methods. This prospective study compared changes in meibomian gland and tear film function in type 2 diabetic patients with nondiabetic patients. Meibomian gland function was evaluated by measuring lipid layer thickness (LLT), grading of meibomian gland loss, lid margin abnormalities, and expression of meibum. Tear film function was assessed by measuring tear breakup time (TBUT), the Schirmer I test, noninvasive breakup time (NIBUT), tear meniscus height (TMH), and corneal fluorescein staining. Results. Meibography scores were significantly higher in the diabetic group compared with the nondiabetic group (p=0.004). The number of expressible glands was significantly lower in the diabetic group in temporal, central, and nasal third of the lower eyelid (nasal: p=0.002; central: p=0.040; and temporal: p=0.039). The lid margin abnormality score was significantly higher in the diabetic group than in the nondiabetic group (p=0.04). There was no statistically significant difference in the tear film function parameters between the two groups. Conclusions. Meibomian gland dysfunction (MGD) in type 2 diabetic patients is more severe compared with nondiabetic patients. Overall, most of the diabetic patients manifest as having asymptomatic MGD

IL-13 Stimulates Proliferation and Expression of Mucin and Immunomodulatory Genes in Cultured Conjunctival Goblet Cells

Citation: Tukler Henriksson J, Coursey TG, Corry DB, De Paiva CS, Pflugfelder SC. IL-13 stimulates proliferation and expression of mucin and immunomodulatory genes in cultured conjunctival goblet cells. Invest Ophthalmol Vis Sci. 2015;56:4186-4197. DOI:10.1167/iovs.14-15496 PURPOSE. To investigate the effects of IL-13 on goblet cell proliferation, differentiation, and expression of mucin and immunomodulatory genes. METHODS. Explants were excised from the conjunctiva of young C57BL/6 mice. Cultures received 200 lL per week of either Keratinocyte media (KSFM) or KSFM supplemented with 10 ng/mL IL-13 and were incubated for 3 (D3), 7 (D7), or 14 (D14) days. Subsequently, cell proliferation was assessed or cultures were immunostained, collected for dot blot, or for reverse transcription (RT) and quantitative real-time PCR (qPCR) or for RT-PCR gene array. RESULTS. The cultured conjunctival epithelium expressed goblet cell associated keratin 7 and mucins MUC5AC and MUC2 and when stimulated with IL-13 showed increased proliferation at D3 and D7 (P < 0.05) compared with control. MUC5AC expression was increased in the IL-13-treated group at D3 and D14 (P < 0.05). IL-13-treated cultures showed increased chemokine ligand 26 (CCL26), chloride channel calcium activated channel 3 (CLCA3), fas ligand (FasL), and Relm-b at D7. All conjunctival cultures expressed MUC2, and its expression was decreased at D3 (P < 0.05) and increased at D14 (P < 0.05) with IL-13 treatment. CONCLUSIONS. This study demonstrated that conjunctival goblet cells are IL-13 responsive cells that produce factors known to maintain epithelial barrier, stimulate mucin production, and modulate immune response in nonocular mucosa when treated with IL-13. The functional significance of IL-13-stimulated factors remains to be determined. Keywords: conjunctiva, goblet cells, interleukin-13, cell culture T he conjunctiva covers two-thirds of the ocular surface and functions as a support tissue for cornea. 2,3 Conjunctival goblet cells are surrounded by lymphocytes and dendritic cells and their density has been found to change in certain ocular surface immune/inflammatory conditions. 4 Goblet cell density has been reported to decrease in aqueous tear deficiency, a condition where T helper 1 (Th1) and Th17 cells infiltrate the conjunctiva, and increase in atopic keratoconjunctivitis and vernal keratoconjunctivitis, predominantly Th2-mediated diseases. 5-8 The mucus stimulating activity of the Th2 cytokine IL-13 has been reported to have a defensive role in the intestines by eliminating helminthic parasites and in the airways by protecting from particles or allergens. 9,10 However, excessive IL-13 expression is associated with goblet cell hyperplasia and mucous hypersecretion, both in the gut and in the airways where it can result in airway obstruction. The purpose of the present study was to investigate whether the Th2 cytokine IL-13 can modulate proliferation, differentiation, and expression of mucin and immunomodulatory gene

Chemokine receptors CCR6 and CXCR3 are necessary for CD4(+) T cell mediated ocular surface disease in experimental dry eye disease.

CD4(+) T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4(+) T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4(+) T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4(+) T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease

A Novel Innate Response of Human Corneal Epithelium to Heat-killed Candida albicans by Producing Peptidoglycan Recognition Proteins.

Fungal infections of the cornea can be sight-threatening and have a worse prognosis than other types of microbial corneal infections. Peptidoglycan recognition proteins (PGLYRP), which are expressed on the ocular surface, play an important role in the immune response against bacterial corneal infections by activating toll-like receptors (TLRs) or increasing phagocytosis. However, the role of PGLYRPs in innate immune response to fungal pathogens has not been investigated. In this study, we observed a significant induction of three PGLYRPs 2-4 in primary human corneal epithelial cells (HCECs) exposed to live or heat-killed Candida albicans (HKCA). The C-type lectin receptor dectin-1 plays a critical role in controlling Candida albicans infections by promoting phagocytic activity and cytokine production in macrophages and dendritic cells. Here, we demonstrate that dectin-1 is expressed by normal human corneal tissue and primary HCECs. HKCA exposure increased expression of dectin-1 on HCECs at mRNA and protein levels. Interestingly, dectin-1 neutralizing antibody, IκB-α inhibitor BAY11-7082, and NF-κB activation inhibitor quinazoline blocked NF-κB p65 nuclear translocation, as well as the induction of the PGLYRPs by HKCA in HCECs. Furthermore, rhPGLYRP-2 was found to suppress colony-forming units of Candida albicans in vitro. In conclusion, these findings demonstrate that dectin-1 is expressed by human corneal epithelial cells, and dectin-1/NF-κB signaling pathway plays an important role in regulating Candida albicans/HKCA-induced PGLYRP secretion by HCECs

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

CCR6KO and CXCR3KO mice are resistant to dry eye disease.

<p><b>A.</b> Corneal permeability - Mean±SD of Oregon green dextran (OGD) permeability into the corneal epithelium of wild-type (WT), CCR6KO and CXCR3KO mice without (NS) or with 5 days of desiccating stress (DS5). OGD permeability was measured in five to seven mice per strain/time point/experiment. The graph represents the combined results of two independent experiments with a total of 10-14 mice per experimental group. <b>B.</b> Representative pictures of OGD staining of each strain. <b>C.</b> CD4⁺ T cell density – Mean±SD of the number of CD4⁺ cells per 100 µm of conjunctival epithelium of WT, CCR6KO, and CXCR3KO mice without (NS) or with 5 (DS5) or 10 (DS10) days of desiccating stress. <b>D.</b> Representative pictures of CD4⁺ T cell infiltration in each strain. * - indicates goblet cells; ↓ - indicates CD4⁺ T cells; CJ – conjunctiva; K – cornea. <b>E.</b> Goblet cell density - Mean±SD of the number of goblet cells per mm of conjunctival epithelium of WT, CCR6KO and CXCR3KO mice without (NS) or with 5 (DS5) or 10 (DS10) days of desiccating stress. Three sections from three animals were examined (N = 9) for each time point/mouse strain for both CD4⁺ T cell and goblet cell density. <b>F.</b> Representative pictures of PAS staining of each strain.</p

Dectin-1 and NF-κB signaling pathways involve PGLYRPs induction in HCECs exposed to HKCA.

<p>A. The HCECs were exposed to 10⁶ cells/ml HKCA with prior incubation in the absence or presence of isotype IgG (10μg/ml), dectin-1 Ab (10μg/ml), BAY11-7082 (10μM) or NF-κB activation inhibitor quinazoline (NF-κB-I, 10μM) for 1 h. Cultures treated by HKCA for 4 h were subjected to RT-qPCR to measure mRNA. B. Total protein of HCECs treated for 48 hours was extracted with RIPA buffer for western blot to examine PGLYRP-2 production. C. Protein levels of PGLYRP-2 were evaluated by western blot using β-actin as control with quantitative ratio of PGLYRP-2/β-actin. Results shown are the mean ± SD of four independent experiments; *** p<0.001, as compared with normal control; ^^ p<0.005, ^^^ p<0.001, as compared with HCECs exposed to HKCA. Magnification: 400Х (bar = 25μm).</p

Inflammatory cytokines are not produced in response to DS in the cornea and conjunctiva of CCR6KO and CXCR3KO CD4⁺ T cell recipient mice.

<p>Wild-type (WT), CCR6KO, or CXCR3KO CD4⁺ T cells from NS or DS5 mice were adoptively transferred to T cell deficient RAG1KO mice. mRNA analysis for inflammatory cytokines and matrix metalloproteinases (MMP) was performed on the corneal (A) and conjunctival (B) tissues of CD4⁺ T cell RAG1KO recipient mice. Mean±SD of transcript levels of corneal and conjunctival tissues. Results represent the mean±SD expression of 2-3 animals per strain/time point per experiment in two independent experiments for a total of 5-6 mice. *, P<0.05, **, P<0.001, ***, P<0.0001.</p

NF-κB p65 activation was induced by HKCA and inhibited by dectin-1 neutralizing antibody and NF-κB activation inhibitor quinazoline (NF-κB-I) in HCECs.

<p>A. HCECs were exposed to HKCA (10⁶ cells/ml) with prior incubation in the absence or presence of isotype IgG (10μg/ml), dectin-1 neutralizing Ab (10μg/ml), BAY11-7082 (10μM) or NF-κB activation inhibitor quinazoline (NF-κB-I, 10μM) for 1 h. HCECs were treated with 10⁶ cells/ml HKCA for 48 hours in 8-chamber slides and examined by immunofluorescent staining for PGLYRPs 2–4. B. HCECs were treated for 4 hours in 8-chamber slides and were fixed in acetone for immunofluroscent staining total p65 (nuclear translocation) (green). C. The percentages of positive cells of PGLYRPs 2–4 staining in HCECs in A was quantified. D. The percentages of NF-ĸB p65 nuclear staining positive cells in B was quantified. Images are representatives from three independent experiments. Results shown are the mean ± SD of four independent experiments; *** p<0.001, as compared with normal control; ^^ p<0.005, ^^^ p<0.001, as compared with HCECs exposed to HKCA. Magnification: 400Х (bar = 25μm).</p

Dectin-1 expression in corneal epithelial tissue and primary cultured HCECs.

<p>Representative images showed immunofluorescent staining of dectin-1 on human corneal (A) and limbal tissue (B), as well as in HCECs (C). D. Dose-dependent stimulation of dectin-1 mRNA in HCECs by HKCA for 4 hours. E. Total protein of HCECs treated for 48 hours was extracted with RIPA buffer for western blot with dectin-1 or β-actin antibody. F. Quantitative ratio of the dectin-1/β-actin protein, evaluated by western blotting, in HCECs with or without exposure to 10⁶ cell/ml of HKCA. Propidium iodide (PI) was used as nuclear counterstaining (red color). Magnification: 400Х (bar = 25μm). Data are presented as mean ± SD, n = 5; * p< 0.05, ** p< 0.01, vs. controls.</p