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A protective Langerhans cell–keratinocyte axis that is dysfunctional in photosensitivity

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Science Translational Medicine  15 Aug 2018:
Vol. 10, Issue 454, eaap9527
DOI: 10.1126/scitranslmed.aap9527
  • Fig. 1 LCs limit UVR-induced keratinocyte apoptosis and skin injury.

    (A to H) WT and Langerin-DTA mice were exposed to UVR and examined. (A) Experimental scheme for (B) and (C); ears were harvested 24 hours after UVR. (B) Activated (Activ.) caspase-3+ keratinocytes per high-powered field (HPF). Left: Representative images of Langerin (red) and activated caspase-3 (green) stain. Right: Quantification (n = 3 to 9 mice). Scale bars, 50 μm. (C) Absolute (left) and normalized (right) monocyte numbers assessed by flow cytometry (n = 3 to 7 mice ). (D) Experimental scheme for (E) and (F); ears were harvested 5 days (d5) after UVR. (E) Epidermal thickness (n = 3 to 7 mice). (F) Epidermal permeability as assessed by toluidine blue penetrance. Left: Representative images. Right: Quantification (n = 3 to 5 mice). (G) Experimental scheme for (H); mice were exposed to UVR for 3 days and examined 24 hours later. (H) Left: Representative images of back skin. Right: Lesional area quantification (n = 3 to 5 mice). Bars represent means (B, C, F, and H) or medians (E). Error bars depict SDs (B, C, F, and H) or interquartile ranges (E). *P < 0.05, **P < 0.01, ***P < 0.001 using two-tailed unpaired Student’s t test (B, C, F, and H) or nonparametric nondirectional Mann-Whitney U test (E) after one-way analysis of variance (ANOVA). Data are from nine (B), five (C), four (E), two (F), and three (H) independent experiments.

  • Fig. 2 LCs limit UVR-induced keratinocyte apoptosis directly.

    (A) Whole-mount stain of homeostatic mouse epidermis for CD3 (red), Langerin (green), and 4′,6-diamidino-2-phenylindole (DAPI; blue). (B and C) Rag1−/−, Rag1−/− Langerin-DTA, WT, and Langerin-DTA mice were exposed to UVR, and ears were harvested 24 hours later (n = 3 to 8 mice). (B) Activated caspase-3+ keratinocytes. n.s., not significant. (C) Absolute (left) and normalized (right) monocyte numbers. (D and E) Effect of LCs on keratinocyte survival in vitro. Murine keratinocyte cultures without and with LCs were exposed to UVR and examined 24 hours later (n = 3 mice). (D) Representative images of cultures stained for Langerin (green), activated caspase-3 (red), and DAPI (blue). (E) Activated caspase-3+ keratinocytes. (E) Data are from five (B and C) and three (A, D, and E) independent experiments. Scale bars, 50 μm. (B, C, and E) Bars represent means. Error bars depict SDs. *P < 0.05, **P < 0.01, ***P < 0.001 using two-tailed unpaired Student’s t test after one-way ANOVA.

  • Fig. 3 LCs are required for UVR-induced epidermal EGFR activation and protect keratinocytes via EGFR stimulation.

    (A and B) Epidermal EGFR phosphorylation at homeostasis (A) and 1 hour after UVR (B) (n = 4 to 5 mice). Left: Representative Western blot for phosphoEGFR (pEGFR), total EGFR (tEGFR), and hsp90 (loading control). Right: pEGFR/tEGFR relative density ratio. Uncropped Western blots are shown in fig. S13. (C and D) Mouse ears were treated with vehicle or HB-EGF before UVR and examined 24 hours after UVR (n = 3 to 4 mice). (C) Activated caspase-3+ keratinocytes. (D) Absolute (left) and normalized (right) monocyte numbers. (E) Effect of human LCs on UVR-induced keratinocyte apoptosis. Primary human keratinocytes without or with indicated cells or recombinant HB-EGF were exposed to UVR and examined 24 hours later (n = 3 human donors). (F and G) Effect of keratinocyte EGFR knockdown and inhibition on LC-mediated protection. Primary murine keratinocytes were treated with EGFR-targeted or control siRNAs (F) or PD168393 (G) before LC coculture and UVR exposure (n = 3 mice). Bars represent means. Error bars depict SDs. Data are from two (A, B, F, and G), four (C and D), and three (E) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 using two-tailed unpaired Student’s t test. Student’s t test was performed after one-way ANOVA for (B) to (G).

  • Fig. 4 LCs express EGFR ligands, and LC-derived ADAM17 mediates UVR-induced epidermal EGFR phosphorylation.

    (A and B) Murine (A) and human (B) LC EGFR ligand expression (n = 3 to 4 mice or human donors). Murine LCs were sorted from control or UVR-exposed mice. Expression of each ligand was normalized to control murine Epgn or human Epgn expression. n.d., not detectable. (C and D) WT and LC-Ad17 mice were treated with UVR and analyzed at indicated time points. (C) LC numbers (n = 3 to 5 mice). (D) Epidermal EGFR phosphorylation. Left: Representative Western blot. Right: pEGFR/tEGFR ratio. Dashed lines are the values for the UVR-exposed WT (blue) and Langerin-DTA (red) mice shown in the blot. Uncropped Western blots are shown in fig. S13. Data are from three (A and B), four (C), and two (D) independent experiments. Bars represent means. Error bars depict SDs. *P < 0.05, **P < 0.01 using two-tailed unpaired Student’s t test. Student’s t test was performed after one-way ANOVA for (C) and (D).

  • Fig. 5 LC-derived ADAM17 limits UVR-induced keratinocyte apoptosis and skin injury.

    (A to D) WT and LC-Ad17 mice were treated with UVR and analyzed at indicated time points. (A) Activated caspase-3+ keratinocytes (n = 3 to 5 mice). (B) Absolute (left) and normalized (right) monocyte numbers (n = 4 to 7 mice). (C) Epidermal thickness (n = 3 to 4 mice). (D) Epidermal permeability (n = 3 to 5 mice). Left: Representative images. Right: Quantification. (E and F) Vehicle or HB-EGF was applied on the ears before UVR exposure (n = 3 to 4 mice). (E) Activated caspase-3+ keratinocytes. (F) Absolute (left) and normalized (right) monocyte numbers. (G and H) Effect of LC Adam17 deletion or ADAM17 blockade on keratinocyte survival in vitro. Murine keratinocytes with LCs from indicated mice (G) and human keratinocytes with control immunoglobulin G (IgG) or anti-ADAM17–treated LCs (H) were exposed to UVR and examined at 24 hours (n = 3 mice, n = 4 human donors). Data are from three (E to G), four (A), two (H), five (B), and one (C and D) independent experiments. Bars represent means (A, B, and D to H) or medians (C). Error bars depict SDs (A, B, and D to H) or interquartile ranges (C). *P < 0.05, **P < 0.01, ***P < 0.001 using two-tailed unpaired Student’s t test (A, B, and D to H) or nonparametric nondirectional Mann-Whitney U test after one-way ANOVA (C).

  • Fig. 6 UVR directly activates LC ADAM17 and EGFR ligand release.

    (A and B) Effect of UVR on ADAM17 activity in sorted murine (A) and human (B) LCs as measured by change in TNFR1 mean fluorescence intensity (MFI) 45 min after the indicated treatments. PMA is a positive control (n = 5 to 6 mice, n = 4 human donors). (C and D) Conditioned supernatants from murine (C) or human LCs (D) were added to A431 EGFR indicator cells, and phosphoEGFR was measured 10 min later by flow cytometry. Murine LC supernatants were from (A); human LC supernatants were from cells treated similarly to (B), except that the antibody was washed out before UVR (see the Supplementary Materials). Left: Representative histogram. Right: Quantification relative to cells treated with control WT LC supernatants (C) or control IgG-treated LC supernatants (D). Results are from six (A), two (B and D), and three (C) independent experiments. Bars represent means. Error bars depict SDs. *P < 0.05, **P < 0.01, ***P < 0.001 using two-tailed unpaired Student’s t test after one-way ANOVA.

  • Fig. 7 Photosensitive SLE mouse models and human SLE skin show a dysfunctional LC-keratinocyte axis.

    WT and MRL-Faslpr (n = 2 to 4 mice) (A to C) or B6.Sle1Yaa mice (n = 3 to 5 mice) (E to G) were treated and examined as indicated. (A and E) Activated caspase-3+ keratinocytes. (B and F) Epidermal EGFR phosphorylation 1 hour after UVR. Left: Representative Western blot. Right: pEGFR/tEGFR ratio. Uncropped Western blots are shown in fig. S13. (C and G) LC Adam17 expression. (D) Effect of MRL-Faslpr LCs on keratinocyte apoptosis. Balb/c or MRL-Faslpr keratinocytes were exposed to UVR without or with indicated LCs (n = 3 mice). (H and I) LC numbers and epidermal EGFR phosphorylation in human SLE skin (n = 3 healthy controls, n = 10 to 13 SLE patients). Scale bars, 50 μm. (H) Left: Representative images of anti-Langerin (purple) and DAPI (blue) staining. Right: LC numbers per millimeter of tissue. (I) Left: Representative images of anti-pEGFR (red), anti-tEGFR (green), and DAPI (blue) staining. Middle: Relative pEGFR/tEGFR fluorescence (fluor.) intensity normalized to healthy control skin. Right: Magnified inset from pEGFR and DAPI stain. Data are from three (A, B, D, E, and G to I) and two (C and F) independent experiments. Bars represent means. Error bars depict SDs. *P < 0.05, **P < 0.01, ***P < 0.001 using two-tailed unpaired Student’s t test. Student's t test was performed after one-way ANOVA for (A) to (D) and (F).

  • Fig. 8 Topical EGFR ligand reduces photosensitivity.

    (A) Experimental scheme for (B) to (E) (n = 4 mice). MRL-Faslpr mouse ears and back skin were topically treated with HB-EGF for 2 days before and on the first day of UVR exposure and examined 24 hours after the final exposure. (B) Representative images of ears. The MRL-MpJ ear represents a non-SLE control. (C) Representative images of back skin; boxes outline lesional areas. Magnified images of back skin are shown in fig. S11. (D) Ear histopathology score. (E) Absolute monocyte numbers. (B to E) Data are from three independent experiments. Bars represent means. Error bars depict SDs. *P < 0.05, ***P < 0.001 using two-tailed unpaired Student’s t test after one-way ANOVA.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/454/eaap9527/DC1

    Materials and Methods

    Fig. S1. Additional features of LC-mediated protection from UVR-induced keratinocyte apoptosis and skin injury.

    Fig. S2. The role of accumulated monocytes and monocyte-derived DCs in UVR-induced skin injury.

    Fig. S3. Additional features of LC-mediated protection of keratinocytes in vitro.

    Fig. S4. Mice treated with EGFR inhibitor resemble Langerin-DTA mice and timing of epidermal EGFR activation after UVR exposure.

    Fig. S5. Effect of human LCs on human keratinocytes without UVR and further characterization of in vitro LC-keratinocyte EGFR signaling.

    Fig. S6. Characterization of mouse and human ADAM17 expression, LC-Ad17 mice, and Langerin-Cre mice.

    Fig. S7. Effects of inducible ADAM17 deletion in LCs.

    Fig. S8. Validation of EGFR ligand release assay and characterization of keratinocyte EGFR ligand release.

    Fig. S9. Photosensitive MRL-Faslpr mice have more skin plasma cells and reduced LC EGFR ligand expression, LC ADAM17 activity, and LC EGFR ligand release.

    Fig. S10. B6.Sle1yaa mice exhibit photosensitivity and characterization of EGFR ligand expression by their LCs.

    Fig. S11. EGFR ligand application reduces the severity of UVR-induced skin lesions and lymph node B cell responses in SLE model mice.

    Fig. S12. Model of protective LC-keratinocyte axis and dysfunction of this axis in lupus photosensitivity.

    Fig. S13. Uncropped Western blot images.

    Table S1. List of primary antibodies.

    Table S2. Secondary antibodies and other staining reagents.

    Table S3. Primary data.

    Reference (53)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Additional features of LC-mediated protection from UVR-induced keratinocyte apoptosis and skin injury.
    • Fig. S2. The role of accumulated monocytes and monocyte-derived DCs in UVR-induced skin injury.
    • Fig. S3. Additional features of LC-mediated protection of keratinocytes in vitro.
    • Fig. S4. Mice treated with EGFR inhibitor resemble Langerin-DTA mice and timing of epidermal EGFR activation after UVR exposure.
    • Fig. S5. Effect of human LCs on human keratinocytes without UVR and further characterization of in vitro LC-keratinocyte EGFR signaling.
    • Fig. S6. Characterization of mouse and human ADAM17 expression, LC-Ad17 mice, and Langerin-Cre mice.
    • Fig. S7. Effects of inducible ADAM17 deletion in LCs.
    • Fig. S8. Validation of EGFR ligand release assay and characterization of keratinocyte EGFR ligand release.
    • Fig. S9. Photosensitive MRL-Faslpr mice have more skin plasma cells and reduced LC EGFR ligand expression, LC ADAM17 activity, and LC EGFR ligand release.
    • Fig. S10. B6.Sle1yaa mice exhibit photosensitivity and characterization of EGFR ligand expression by their LCs.
    • Fig. S11. EGFR ligand application reduces the severity of UVR-induced skin lesions and lymph node B cell responses in SLE model mice.
    • Fig. S12. Model of protective LC-keratinocyte axis and dysfunction of this axis in lupus photosensitivity.
    • Fig. S13. Uncropped Western blot images.
    • Table S1. List of primary antibodies.
    • Table S2. Secondary antibodies and other staining reagents.
    • Reference (53)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Primary data.

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