Research ArticleEYE DISEASE

Neutrophils cause obstruction of eyelid sebaceous glands in inflammatory eye disease in mice

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Science Translational Medicine  25 Jul 2018:
Vol. 10, Issue 451, eaas9164
DOI: 10.1126/scitranslmed.aas9164
  • Fig. 1 MG orifice obstruction in AED model is a key disease manifestation.

    (A) Clinical disease was scored at 20 min (immediate hypersensitivity) and 6 hours after allergen challenge (late phase/chronic response) in AED and the mild allergy model (n = 20 mice per group). (B) Representative flow cytometry histograms showing total leukocyte (CD45+) numbers in conjunctiva. Quantification is shown on the right. (C) Representative flow cytometry scatterplots showing eosinophil (CD45+Siglec F+) numbers in conjunctiva. Quantification is shown on the right (n = 5 per group). (D) Representative images of orifice plugging from mild allergy and AED mice. Incidence of eyes with MG plugs is graphed (n = 5 per group). Data are expressed as means ± SEM from at least three independent experiments. Differences in clinical scores were determined by two-way analysis of variance (ANOVA) with Tukey’s post hoc test. One-way ANOVA and unpaired Student’s two-tailed t tests were performed to compare the cell numbers and MG plug percentages, respectively. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 AED model exhibits meibographical and histological evidence of underlying MGD.

    (A) Representative image of binarized keratograph of eyelids (superior and inferior) with corresponding area calculation (in square millimeters). On the right, scatterplots of full, superior, and inferior lid area measurements of MGs are shown (n = 10 eyelids per group). Data are expressed as means ± SEM. Unpaired Student’s two-tailed t tests were used. **P < 0.01, ***P < 0.001. (B) Representative serial sections of eyelids from naive and AED at anatomically matched locations (orifice, terminal duct, and central duct). Black double-headed arrows indicate central duct size. Images are representative of n = 3 plugged MGs in each mouse, with at least three mice per group.

  • Fig. 3 TH17 responses generated in AED mice play a central role in mediating MG obstruction.

    (A) Representative flow cytometry scatterplots (left) and quantification (right) of cervical LN TH17 cells (naive, n = 3; mild, n = 12; AED, n = 11 mice). (B and C) Representative flow cytometry data of LN TH17 cells in Il17a−/− mice with AED in (B) (WT, n = 5; Il17a−/−, n = 4 mice), and corresponding eyelid data shown as number of plugs per eye in (C) (WT, n = 28; Il17a−/−, n = 12 eyelids). (D and E) Flow cytometry data of LN TH17 cells (untreated, n = 22; isotype, n = 6; anti–IL-23, n = 16 mice) in (D) and corresponding eyelid data (untreated, n = 22; isotype, n = 6; anti–IL-23, n = 16 eyelids) in (E) after anti–IL-23 blockade or isotype control (100 μg) given during sensitization period only. (F and G) Flow cytometry data of LN TH17 cells (mild only, n = 4; +TH2, n = 5; +TH17, n = 4 mice) from mild allergy mice in (F) and corresponding eyelid data (mild only, n = 8; +TH2, n = 8; +TH17, n = 10 eyelids) in (G) after adoptive transfer of in vitro generated TH2 or TH17 cells. Data are expressed as means ± SEM from at least two independent experiments. One-way ANOVA was performed for comparisons among three groups, and unpaired Student’s two-tailed t tests were used for comparisons between two groups. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 TH17 cells are associated with PMN recruitment to the conjunctiva in AED, and these PMNs cause MG obstruction.

    (A) Representative flow cytometry scatterplots of PMN in conjunctiva and quantifications of PMN counts on the right (untreated, n = 5; anti–IL-23, n = 6 mice), after anti–IL-23 blockade or isotype control given during sensitization period only. (B) Quantifications of conjunctival PMN counts after adoptive transfer of in vitro generated TH2 or TH17 cells to mild allergy model (mild only, n = 4; +TH2, n = 7; +TH17, n = 6 mice). (C and D) Flow cytometry data of PMNs in conjunctiva of AED mice (untreated, n = 3; isotype, n = 4; anti-Ly6G, n = 6 mice) and corresponding eyelid data (untreated, n = 18; isotype, n = 15; anti-Ly6G, n = 28 eyelids) after anti-Ly6G blockade or isotype control. Data are expressed as means ± SEM from at least two independent experiments. One-way ANOVA was performed for comparisons among three groups, and unpaired Student’s two-tailed t tests were used for comparisons between two groups. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 Increased PMN quantity in patient tear fluids strongly correlates with clinical severity of MGD.

    (A) Flow cytometry analysis of tear collections and gating strategy for leukocytes (CD45+), myeloid cells (CD45+CD11b+), and PMNs (CD45+CD11b+CD14CD15+CD16+SSchi). (B) Bar graphs show increased total leukocytes, myeloid cells, and PMNs in tear fluids of MGD subjects, as compared to controls (no MGD). (C) Stratification data of MGD subjects into those with MGD only or MGD with underlying immune disease (MGD only or MGD + ImD, respectively). Red dashed box indicates PMN+ cases. See table S1. (D) Pie charts show the breakdown of PMN+ cases in both MGD groups (MGD only, n = 38; MGD + ImD, n = 12). (E and F) Correlation analyses between tear PMN numbers and MGD severity in PMN+ subjects. Severity is based on masked scoring on a 0 to 4 scale for expressed meibum quality (0 = healthy, clear, and normal meibum; 4 = obstructed, opaque, and thick meibum). Data were collected from n = 50 MGD patients and n = 14 controls. Data of dot plots are expressed as means ± SEM. Mann-Whitney U tests were used for two-group comparisons, and Kruskal-Wallis test with Conover-Inman tests were used for three-group comparisons. *P < 0.05, ***P < 0.001. Spearman’s rank correlation was used.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/451/eaas9164/DC1

    Materials and Methods

    Fig. S1. Ocular manifestations are observed in AED mice.

    Fig. S2. Meibography and histology of MGs in normal and AED mice.

    Fig. S3. Quantifications of TH2 and TH1 cells in LN and TH17 cells in conjunctiva.

    Fig. S4. Conjunctival PMN infiltration is reduced by blockade of IL-17a antibody.

    Fig. S5. Tear cytology reveals leukocyte populations in MGD patients with underlying AKC or BKC.

    Fig. S6. Flow cytometry application to analyze MGD patient tears.

    Table S1. Patient demographics.

    Table S2. Raw data (Excel file).

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Ocular manifestations are observed in AED mice.
    • Fig. S2. Meibography and histology of MGs in normal and AED mice.
    • Fig. S3. Quantifications of TH2 and TH1 cells in LN and TH17 cells in conjunctiva.
    • Fig. S4. Conjunctival PMN infiltration is reduced by blockade of IL-17a antibody.
    • Fig. S5. Tear cytology reveals leukocyte populations in MGD patients with underlying AKC or BKC.
    • Fig. S6. Flow cytometry application to analyze MGD patient tears.
    • Table S1. Patient demographics.
    • Legend for table S2

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    Other Supplementary Material for this manuscript includes the following:

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