Research ArticleWound Healing

The Fas/Fap-1/Cav-1 complex regulates IL-1RA secretion in mesenchymal stem cells to accelerate wound healing

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Science Translational Medicine  14 Mar 2018:
Vol. 10, Issue 432, eaai8524
DOI: 10.1126/scitranslmed.aai8524
  • Fig. 1 Murine gingival MSCs and skin MSCs produce and secrete IL-1RA–EV.

    (A) Total protein contained within small extracellular vesicles (sEVs) isolated from the culture supernatant of 1 × 106 murine human bone marrow mesenchymal stem cells (BMMSCs), gingiva-derived MSCs (GMSCs), and skin MSCs (SMSCs) (n = 3). (B) Western blotting and semi-quantification analysis of CD63, CD9, and CD81 expression from sEVs isolated from GMSCs and SMSCs. (C) Differential centrifugation and sucrose cushion procedure for the isolation of EVs from MSC culture supernatants (SN). (D) Interleukin-1 receptor antagonist (IL-1RA), CD63, CD9, and CD81 expression in lysates from fractions corresponding to (C). (E) Super-resolution stimulated emission depletion staining and quantification for IL-1RA–enhanced green fluorescent protein (EGFP) (green), CD63 (red), and CD81 (red) in GMSCs transfected with plasmids containing IL-1RA–EGFP fusion protein. The lower right box is a higher magnification of the boxed region in the merged image; colocalization of IL-1RA with CD63 or CD81 is shown in yellow (n = 5). Scale bar, 10 μm. (F) Total internal reflection fluorescence (TIRF) microscopy images from GMSCs cotransfected with plasmids expressing IL-1RA–EGFP (green) and CD63-mCherry (red). The top right panel is a higher magnification of the boxed region in the left image; colocalization of IL-1RA–EGFP and CD63-mCherry is shown in yellow. The bottom panels (1 to 4) show sequential images from live-cell imaging. Arrows indicate two individual IL-1RA–positive vesicle fusion events. Scale bar, 10 μm. (G) Enzyme-linked immunosorbent assay (ELISA) of IL-1RA from the culture supernatant of GMSCs and SMSCs (n = 3). (H) Western blotting and semi-quantification analysis of IL-1RA expressed by GMSCs and SMSCs. (I) Immunocytofluorescence staining of IL-1RA (green) and the MSC marker CD105 (red) in GMSCs and SMSCs. Scale bar, 20 μm. (J and K) Real-time polymerase chain reaction analysis of soluble IL-1RA (sIL-1RA) mRNA (J) and intracellular IL-1RA (icIL-1RA) mRNA (K) in GMSCs and SMSCs. All results are representative of data generated in at least three independent experiments (J and K) (n = 6). **P < 0.01, ***P < 0.001. Error bars are means ± SD. Data were analyzed using one-way analysis of variance (ANOVA) with Bonferroni correction (A), or independent unpaired two-tailed Student’s t tests (B, G, H, J, and K).

  • Fig. 2 Fas controls IL-1RA–sEV secretion in murine MSCs.

    (A) Western blotting and semi-quantification of Fas expression in GMSCs and SMSCs (n = 3). (B) Secreted sEV-associated protein quantification from Fas-deficient MRL/lpr and wild-type (WT) control GMSCs (n = 5). (C) Western blotting and semi-quantification analysis of CD63, CD9, CD81, and IL-1RA from sEV from Fas-deficient MRL/lpr and WT control GMSCs. sEV-associated proteins from culture supernatants of equal numbers of cells in control and MRL/lpr GMSC groups were loaded (n = 3). (D) ELISA analysis of secreted IL-1RA from the culture supernatant from WT control and Fas-deficient MRL/lpr GMSCs (n = 3). (E) Western blotting and semi-quantification analysis of cytoplasmic IL-1RA from WT control and Fas-deficient MRL/lpr GMSCs (n = 3). (F) Immunocytofluorescent double staining of IL-1RA (green) and Fas (red) in WT control and Fas-deficient MRL/lpr GMSCs. Dashed lines indicate the cell edge. Scale bar, 20 μm. (G) ELISA analysis of IL-1RA secretion in the culture supernatant of MRL/lpr and Fas-overexpressing MRL/lpr GMSCs (n = 5). (H) Western blotting and semi-quantification analysis of cytoplasmic IL-1RA from MRL/lpr and Fas-overexpressing MRL/lpr GMSCs (n = 3). (I) ELISA analysis of IL-1RA secretion in the culture supernatant from WT control GMSCs treated with and without Fas small interfering (siRNA) (n = 5). (J) Western blotting and semi-quantification of cytoplasmic IL-1RA and Fas from WT control GMSCs treated with or without Fas siRNA (n = 3). All results are representative of data generated from at least three independent experiments. **P < 0.01, ***P < 0.001. Error bars are means ± SD. All data were analyzed using independent unpaired two-tailed Student’s t tests.

  • Fig. 3 Fas binds with Fap-1 and Cav-1 to regulate IL-1RA–sEV release in murine GMSCs.

    (A) Fas co-IP of WT control and MRL/lpr GMSC lysate. (B) Immunocytofluorescence double staining for Fas, Fap-1, and Cav-1 in WT GMSCs. (C) Secreted sEV-associated protein quantification from WT control, Fap-1, and Cav-1 knockout GMSCs (n = 3). (D) Western blotting and semi-quantification of CD63, CD9, CD81, and IL-1RA in sEVs from WT control, Fap-1, and Cav-1 knockout GMSCs. Culture supernatants from equal numbers of cells from control and knockout GMSCs were loaded for Western blotting analysis (n = 3). (E) Western blotting and semi-quantification of Fas, Fap-1, Cav-1, and IL-1RA expression in WT control, MRL/lpr, Fas-deficient, Fap-1 knockout, and Cav-1 knockout GMSCs (n = 3). (F) ELISA analysis of secretion of IL-1RA in the culture supernatant from WT control, Fap-1–deficient, and Cav-1–deficient GMSCs (n = 5). (G) Immunoprecipitation (IP) and semi-quantification analysis of Fas from WT control, Fap-1–deficient, and Cav-1 knockout GMSC lysates (n = 3). (H) IP and semi-quantification analysis of Fap-1 from WT control, MRL/lpr, Cav-1 knockout, and Fap-1 knockout GMSC lysates (n = 3). (I) IP and semi-quantification analysis of Cav-1 from WT control, MRL/lpr, Fap-1 knockout, and Cav-1 knockout GMSCs (n = 3). (J) Immunocytofluorescence staining of Cav-1 in WT, Fas-deficient MRL/lpr, and Fap-1 knockout GMSCs, and double-staining of Fap-1 and Fas in WT and Cav-1 knockout GMSCs. For A-P, Fas-deficient GMSCs from MRL/lpr mice, GMSCs with Fap-1 knocked out using a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) plasmid, and Cav-1 knockout GMSCs from Cav-1−/− mice were used, and WT GMSCs served as a control. For IP, whole-cell lysates from indicated GMSCs were immunoprecipitated with corresponding antibodies, and the immunocomplexes were subjected to Western blotting with antibodies against Fas, Fap-1, and Cav-1. All results are representative of data generated from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars are means ± SD. Scale bars, 20 μm. All data were analyzed using independent unpaired two-tailed Student’s t tests. DAPI, 4′,6-diamidino-2-phenylindole; IgG, immunoglobulin G.

  • Fig. 4 TNF-α up-regulates Fas/Fap-1 expression to promote IL-1RA–sEV release in murine MSCs.

    (A) ELISA analysis of IL-1RA secretion into the culture supernatant from GMSCs treated with tumor necrosis factor–α (TNF-α) or interferon-γ (IFN-γ) (n = 3). (B) Secreted sEV-associated proteins from control or TNF-α (20 ng/ml)–treated GMSCs (n = 6). (C) Western blotting and semi-quantification of CD63, CD9, CD81, and IL-1RA expression in WT control GMSCs with or without TNF-α (20 ng/ml) treatment. sEV-associated proteins from culture supernatants of equal numbers of cells were loaded for Western blotting analysis (n = 3). (D) Western blotting and semi-quantification analysis of Fas, Fap-1, Cav-1, and IL-1RA expression in WT GMSCs (left) and MRL/lpr GMSCs (right) treated with or without TNF-α (n = 3). (E) ELISA analysis of secretion of IL-1RA in the culture supernatant in control or MRL/lpr GMSCs treated with and without TNF-α (20 ng/ml) (n = 3). (F) Western blotting and semi-quantification of Fas, Cav-1, and IL-1RA in Fap-1 knockout GMSCs with and without TNF-α (20 ng/ml) treatment (n = 3). (G) Western blotting and semi-quantification of Fas, Fap-1, and IL-1RA in Cav-1 knockout GMSCs with and without TNF-α (20 ng/ml) treatment (n = 3). (H) ELISA analysis of IL-1RA in the culture supernatant of WT control, Fap-1, and Cav-1 knockout GMSCs treated with and without TNF-α (20 ng/ml) (n = 3). (I) Immunocytofluorescence staining of GMSCs at various time points after TNF-α (20 ng/ml) treatment. Scale bar, 20 μm. (J) Western blotting and semi-quantification analysis of Fas, Fap-1, and Cav-1 in membrane and cytoplasmic fractions of GMSCs treated with and without TNF-α (20 ng/ml) (n = 3). (K) TIRF microscopy images of IL-1RA–EGFP (green) and CD63-mCherry (red) cotransfected into WT GMSCs treated with TNF-α (20 ng/ml) for 0.5 hours. The top left panel is a higher magnification of the boxed region in the left image to show colocalization (yellow); the bottom panels show sequential images (1 to 4). Arrows indicate two individual IL-1RA–positive vesicle fusion events. Scale bar, 10 μm. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars are means ± SD. Data were analyzed using independent unpaired two-tailed Student’s t tests (A to D, F, G, and J), or one-way ANOVA with Bonferroni correction (E and H).

  • Fig. 5 GMSCs produce IL-1RA, which contributes to gingival wound healing in mice.

    (A) Scheme illustrating the gingival wound procedure and treatment with IL-1RA neutralizing antibody. Full-thickness circular wounds were made in the palates of WT control mice and IL-1RA−/− mice and submucosally injected one time with placebo (0.9% saline) or IL-1RA neutralizing antibody (IL-1RA Ab,10 μg per mouse) 1 day after wound creation. (B) Representative macroscopic images and quantification of gingival wound area in WT control and IL-1RA−/− mice. All the gingival wound is outlined in a dashed line (n = 5). (C) Representative macroscopic images and quantification of gingival wound area in WT mice after treatment with or without IL-1RA Ab (n = 5). (D) Representative macroscopic images and quantification of gingival wound area in WT mice after treatment with or without IL-1RA drug (n = 5). (E) Scheme illustrating cutaneous wound procedure and treatment with IL-1RA drug anakinra. Full-thickness excision cutaneous wounds (1 cm × 1 cm) were created in the mid-backs of WT mice. One day after wound creation, the mice were subcutaneously injected with either placebo (0.9% saline) or the IL-1RA drug anakinra (500 μg per mouse). (F) Representative macroscopic images and quantification of closure of full-thickness cutaneous wounds in WT mice after treatment with or without anakinra (n = 5). (G) Scheme illustrating the gingival wound procedure and administration of sEVs. IL-1RA knockout mice were submucosally injected with placebo (0.9% saline) or WT GMSC–derived, IL-1RA knockout GMSC–derived, or TNF-α–activated GMSC-derived sEVs 1 day after wound creation. (H) Representative macroscopic images and quantification of gingival wound area in IL-1RA knockout mice after treatment with or without sEVs (n = 3 for day 7; n = 5 for day 5). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars are means ± SD. Data were analyzed using independent unpaired two-tailed Student’s t tests (B to F) or one-way ANOVA with Bonferroni correction (H).

  • Fig. 6 Fas-controlled IL-1RA secretion regulates wound healing in mice.

    (A) Scheme illustrating the gingival wound procedure in MRL/lpr mice and treatment with the IL-1RA drug anakinra. Full-thickness circular wounds were made in the palates of WT and MRL/lpr mice with a biopsy punch, and MRL/lpr mice were submucosally injected with either placebo (0.9% saline) or the IL-1RA drug anakinra (100 μg per mouse) 1 day after wound creation. (B) Representative macroscopic images and quantification of gingival wound area in WT and MRL/lpr mice after treatment with and without anakinra (n = 5). (C) Representative macroscopic images and quantification of dermal wound area in WT and MRL/lpr mice over time (n = 3). (D) Representative macroscopic images and quantification of dermal wound area in MRL/lpr mice treated with placebo (0.9% saline) or anakinra (500 μg per mice) injected 1 day after wound creation as in (A) (n = 3). **P < 0.01, ***P < 0.001. Error bars are means ± SD. Data were analyzed using one-way ANOVA with Bonferroni correction (B) or independent unpaired two-tailed Student’s t tests (C and D).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/432/eaai8524/DC1

    Materials and methods

    Fig. S1. GMSCs secrete higher amounts of sEVs and cytokines.

    Fig. S2. Fas controls IL-1RA–sEV secretion in murine SMSCs.

    Fig. S3. Fas/Fap-1 binds with Cav-1 to control SNAP25/VAMP5-mediated IL-1RA release in murine MSCs.

    Fig. S4. TNF-α promotes sEV and IL-1RA release in murine MSCs.

    Fig. S5. Histomorphology of IL-1RA in wound healing in mice.

    Fig. S6. sEVs containing IL-1RA ameliorate delayed wound healing in diabetic mice.

    Fig. S7. Histomorphology of Fas in wound healing in mice.

    Fig. S8. Schematic drawing of Fas/Fap-1/Cav-1–controlled IL-1RA–sEV secretion in MSCs.

    Fig. S9. Characterization of BMMSCs, GMSCs, and SMSCs.

    Table S1. Individual subject-level data.

    Movie S1. GMSCs secrete IL-1RA–positive exosome-like EVs.

    Movie S2. Exocytotic fusions of IL-1RA–positive vesicles in living GMSCs.

    Movie S3. TNF-α–activated GMSCs release IL-1RA–positive exosome-like EVs.

    Database S1. Western blotting films corresponding to Figs. 1 to 4.

  • Supplementary Material for:

    The Fas/Fap-1/Cav-1 complex regulates IL-1RA secretion in mesenchymal stem cells to accelerate wound healing

    Xiaoxing Kou, Xingtian Xu, Chider Chen, Maria Laura Sanmillan, Tao Cai, Yanheng Zhou, Claudio Giraudo, Anh Le, Songtao Shi*

    *Corresponding author. Email: songtaos{at}upenn.edu

    Published 14 March 2018, Sci. Transl. Med. 10, eaai8524 (2018)
    DOI: 10.1126/scitranslmed.aai8524

    This PDF file includes:

    • Materials and methods
    • Fig. S1. GMSCs secrete higher amounts of sEVs and cytokines.
    • Fig. S2. Fas controls IL-1RA–sEV secretion in murine SMSCs.
    • Fig. S3. Fas/Fap-1 binds with Cav-1 to control SNAP25/VAMP5-mediated IL-1RA release in murine MSCs.
    • Fig. S4. TNF-α promotes sEV and IL-1RA release in murine MSCs.
    • Fig. S5. Histomorphology of IL-1RA in wound healing in mice.
    • Fig. S6. sEVs containing IL-1RA ameliorate delayed wound healing in diabetic mice.
    • Fig. S7. Histomorphology of Fas in wound healing in mice.
    • Fig. S8. Schematic drawing of Fas/Fap-1/Cav-1–controlled IL-1RA–sEV secretion in MSCs.
    • Fig. S9. Characterization of BMMSCs, GMSCs, and SMSCs.
    • Legends for movies S1 to S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Individual subject-level data.
    • Movie S1 (.mov format). GMSCs secrete IL-1RA–positive exosome-like EVs.
    • Movie S2 (.mov format). Exocytotic fusions of IL-1RA–positive vesicles in living GMSCs.
    • Movie S3 (.mov format). TNF-α–activated GMSCs release IL-1RA–positive exosome-like EVs.
    • Database S1 (.pdf format). Western blotting films corresponding to Figs. 1 to 4.

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