Research ArticleTHYMUS

Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration

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Science Immunology  12 Jan 2018:
Vol. 3, Issue 19, eaal2736
DOI: 10.1126/sciimmunol.aal2736
  • Fig. 1 BMP signaling pathways are up-regulated in the thymus after thymic damage.

    (A and B) Thymuses were pooled from 6-week-old C57BL/6 mice were pooled, and microarray analysis was performed on CD45 cells enriched from either untreated mice (d0) or 4 and 7 days (d4 and d7, respectively) after TBI (550 cGy; n = 3 per time point with each n pooled from three to five mice). (A) Volcano plot outlining genes that changed >1.5-fold, P < 0.05 with some key thymus-related genes highlighted. (B) GSEA was performed on the transcriptome derived from CD45 cells after TBI (Fig. 1A) with BMP target genes (GO: 0030510). NES, normalized enrichment score. (C and D) Thymuses were harvested at days 0, 2, 4, 7, 10, 14, and 21 after TBI (n = 5 to 14 per time point), and BMP4 levels were measured by ELISA. (C) Absolute amount of BMP4 in the thymus. (D) Amount of BMP4 normalized to the weight of the thymus (ng BMP4/μg thymus). Data combined from two to three independent experiments. *P < 0.05; **P < 0.01, ***P < 0.001. ns, not significant.

  • Fig. 2 BMP4 targets TECs and induces the expression of Foxn1 and its downstream targets after damage.

    (A) TEC subsets were FACS-purified from untreated 6-week-old C57BL/6 mice, and the expression of the BMPR subunits Bmpr1a, Bmpr1b, and Bmpr2 was measured by qPCR (n = 3 per subset). ND, not detectable. (B) cTECs or mTECs were FACS-purified from the thymus at days 0, 4, and 7 after TBI, and the expression of Foxn1 was assessed by qPCR (n = 4 to 6 per time point from two to three independent experiments). (C to E) Thymuses were pooled from 6-week-old C57BL/6 mice, and microarray analysis was performed on CD45 cells enriched from either untreated mice or 4 and 7 days after TBI (550 cGy; n = 3 per time point with each n pooled from three to five mice). (C) Heat map of high-confidence FOXN1 target genes in cTECs (21) showing clustering between samples and between days 0, 4, and 7. (D) Dot plots showing normalized expression values comparing days 0, 4, and 7 after TBI. Green dots represent significantly different (P < 0.05) individual genes, and dotted lines (and shaded section) represent genes with a fold change of <1.5 (±). The proportion of significantly changed genes is denoted within each plot. (E) GSEA was performed comparing gene changes with a FOXN1 target gene signature comprising the list of high-confidence FOXN1 gene targets (table S1). Bar graphs represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 BMP4 production by ECs represents a nonredundant pathway of regeneration after thymic damage.

    (A) Six-week-old C57BL/6 mice were administered with the BMPR1 inhibitor dorsomorphin dihydrochloride [12.5 mg/kg, intraperitoneally (ip)] at day −1 before TBI and twice daily after TBI. The thymus was harvested, and total cellularity was assessed at day 7 after TBI (n = 10 mice per treatment). (B) Tamoxifen was administered to iGremlin::K5-CreER+ and iGremlin::K5-CreER mice on days −1, 0, and +1 surrounding TBI (550 cGy). Thymus was harvested, and total cellularity was assessed on day 9. (C) Cell subsets (n = 3 per population) comprising about 99.5% of the known cellular subsets in the thymus were FACS-purified and assessed for their expression of Bmp4 at steady state by qPCR: double negative (DN; CD4CD8), double positive (DP; CD4+CD8+), CD4 single positive (SP4; CD4+CD8CD3+), SP8 (CD4CD8+CD3+), DCs (CD11c+MHCII+), cTEC (CD45EpCAM+MHCII+Ly51+UEA1lo), mTEC (CD45EpCAM+MHCII+Ly51loUEA1+), fibroblasts (CD45EpCAMPDGFRa+), and ECs (CD45EpCAMVE-cadherin+). (D) ECs and fibroblasts were FACS-sorted at days 0 and 4 after TBI, and the expression of Bmp4 was assessed by qPCR (n = 6 per population per time point). (E) Tamoxifen was administered to BMP4fl/fl::Cdh5-CreERT2+ (BMP4ΔEC; n = 18) and BMP4fl/fl::Cdh5-CreERT2 (BMP4fl/fl; n = 17) mice on days −2, −1, 0, 1, and 2 surrounding TBI (550 cGy). The thymus was harvested, and total cellularity was measured on day 7. Bar graphs represent means ± SEM of at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 ECs form a damage-resistant regenerative niche in the thymus.

    (A) Cell subsets in the thymus were assessed at day 7 after TBI, and the depletion was calculated compared with an untreated age-matched control cohort (n = 10 to 25 per subset). Subsets analyzed include DN1 (CD44+CD25), early T-lineage progenitor (ETP; CD44+CD25c-kit+), DN2 (CD44+CD25), DN3 (CD44+CD25), DN4 (CD44+CD25), DP, SP4, SP8, CD8+ or CD8 DCs, MHCIIhi or MHCIIlo cTEChi/lo (CD45EpCAM+MHCII+Ly51+UEA1lo), MHCIIhi or MHCIIlo mTEChi/lo (CD45EpCAM+MHCII+Ly51loUEA1+), fibroblast, ECs, and ILCs (CD45+CD3CD8IL7Rα+CD4+RORγt+CCR6+). (B and C) Six-week-old female C57BL/6 mice were treated with PBS (n = 10), Dex (50 mg/kg, ip, on day 0; n = 10), Cyclo (100 mg/kg per day, ip, on days −1 and 0; n = 10), or TBI (550 cGy on day 0; n = 10). On day 4, mice were perfused with 25 μg of anti–VE-cadherin antibody (BV13) conjugated to Alexa Fluor 647 and sacrificed, and total thymic cellularity and EC number were assessed. (B) Total thymic cellularity and absolute number of ECs. (C) Concatenated flow cytometry plots detailing the proportion of VE-cadherin+CD45 cells in the thymus. SL-TBI, sublethal TBI. (D and E) C57BL/6 mice were given TBI (n = 10 to 15 per group) and assessed on days 4, 7, and 14. On the day of harvest, mice were perfused with 25 μg of anti–VE-cadherin antibody (BV13) conjugated to Alexa Fluor 647. (D) Total cellularity (open circles) and absolute number of ECs (closed circles) in the thymus were calculated using flow cytometry. (E) Proportion of VE-cadherin+ ECs as a function of CD45 stromal cells. Flow cytometry plots represent concatenated data from one experiment. (F) Proportion of ECs as a function of total thymic cellularity (n = 14 to 20 from four independent experiments). (G to M) Three-dimensional reconstruction of thymus vasculature at days 0, 4, 7, and 14 after TBI using LSFM (n = 3 per time point). (G) Visualization of VE-cadherin staining in the thymus. (H) Calculation of the volume of whole thymus and vasculature. (I) Total number of vessel segments in the thymus vasculature after damage where segments were defined as the length of the vessel between two branching points. (J) Vessel segments were binned according to their length. Total vasculature length was calculated. (K) Vascular segments were color-coded on the basis of branch level. (L) Number of segments per branch level and the total number of vessel branches. (M) Vascular density was calculated as a ratio of vascular network volume and as a function of total thymus volume [from (H)]. Bar graphs represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 TECs can be cultivated ex vivo and mediate enhanced thymic regeneration upon exogenous administration after damage.

    ECs were FACS-sorted from the thymus (n = 25), heart (n = 10), or kidney (n = 10) on the basis of the expression of VE-cadherin and transduced with the viral gene E4ORF1. These cells are referred to as exECs. To model immune injury, we exposed 6- to 8-week-old C57BL/6 mice to TBI, and 1 × 106 exECs were administered intravenously at day 3 after TBI (n = 25 in control group). (A) Experiment schematic. (B) Total thymic cellularity at day 9 after TBI. (C and D) Concatenated flow cytometry plots detailing the proportion of TECs (C) and thymocyte subsets (D). (E) Absolute number of cTECs and mTECs. (F) TEC proliferation was measured on day 3 by Ki-67 staining. (G) C9 or TE-71 cells were stimulated with BMP4 (100 ng/ml) for 24 hours after which proliferation was assessed (n = 6 to 7 independent experiments). (H) Absolute number of ECs. (I) EC proliferation on day 3 after exEC administration. (J) exECs were generated and labeled with CFSE, and 10 × 106 cells were transferred on day 3 after TBI. Four hours after transfer, CFSE expression was assessed by VE-cadherin+ cells in the thymus. Displayed are the total EC number and proportion of CFSE+ ECs (n = 13 to 15 from three independent experiments). (K) Total thymic cellularity and absolute number of cTECs and mTECs 28 days after TBI and administration of 1 × 106 thymus-derived exECs on day 3 (n = 10 per group). Bar graphs represent means ± SEM of at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 6 exEC-produced BMP4 mediates thymic regeneration via activation of Foxn1 in TECs.

    (A and B) Six- to 8-week-old C57BL/6 mice were given TBI, and 1 × 106 exECs were administered intravenously at day 3 after TBI. Thymus was harvested 4 days later, cTECs and mTECs were FACS-purified, and the expression of Foxn1 (A) and its downstream target genes Dll4, Kitl, and Cxcl12 in cTECs (B) was measured by qPCR (n = 8 to 9 per group). (C and D) The CM from in vitro cultures of exECs derived from the thymus was incubated with C9 or TE-71 cells for 20 min after which phosphorylation of Smad1/8 was measured by flow cytometry (n = 3 per group). MFI, mean fluorescence intensity. (E and F) The CM from in vitro cultures of exECs derived from the thymus was incubated with the C9 cTEC cell line for 24 hours (n = 7 to 10). Recombinant BMP4 (30 ng/ml) and/or Noggin (100 ng/ml) were added to the marked wells as controls. (E) Foxn1 measured by qPCR. (F) Expression of Dll4 and Kitl measured by qPCR. (G) Bmp4 expression in the heart, kidney, and thymus exECs. (H) BMP4 protein was measured by ELISA in exEC CM derived from the thymus, heart, or kidney (n = 4 per group). (I) The CM from exECs generated from the heart, kidney, or thymus was incubated with C9 cells for 24 hours after which the expression of Foxn1 was measured by qPCR. (J to L) exECs were generated from thymus-derived ECs and transduced to express either an shBMP4 or shScram control. (J and K) The CM derived from the thymus shBMP4 or shScram exEC cultures was incubated with C9 cells for 24 hours when the expression of Foxn1 (J) and/or the Foxn1 downstream genes Dll4 and Kitl (K) was assessed by qPCR. (L) Transduced exECs were transplanted into mice previously given TBI on day 3. Total thymus cellularity at day 9 after shBmp4 or shScram was administered 3 days after TBI (n = 10 per group). Bar graphs represent means ± SEM of at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/19/eaal2736/DC1

    Fig. S1. Thymic response to damage.

    Fig. S2. Distribution of BMPR subunits on thymic cell populations.

    Fig. S3. Inhibition of BMP signaling abrogates endogenous thymic regeneration.

    Fig. S4. Deletion of BMP4 in ECs abrogates endogenous thymic regeneration.

    Fig. S5. Damage response in the thymus to corticosteroids, chemotherapy, and TBI.

    Fig. S6. exECs can be propagated ex vivo and maintain an EC phenotype.

    Fig. S7. Validating methods of inducing Foxn1 and silencing Bmp4.

    Table S1. High-confidence FOXN1 target gene changes after TBI.

    Movie S1. Thymic vascular architecture and branching by LSFM (day 0).

    Movie S2. Thymic vascular architecture and branching by LSFM (day 4).

    Movie S3. Thymic vascular architecture and branching by LSFM (day 7).

    Movie S4. Thymic vascular architecture and branching by LSFM (day 14).

  • Supplementary Materials

    Supplementary Material for:

    Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration

    Tobias Wertheimer, Enrico Velardi, Jennifer Tsai, Kirsten Cooper, Shiyun Xiao, Christopher C. Kloss, Katja J. Ottmüller, Zeinab Mokhtari, Christian Brede, Paul deRoos, Sin?ad Kinsella, Brisa Palikuqi, Michael Ginsberg, Lauren F. Young, Fabiana Kreines, Sophia R. Lieberman, Amina Lazrak, Peipei Guo, Florent Malard, Odette M. Smith, Yusuke Shono, Robert R. Jenq, Alan M. Hanash, Daniel J. Nolan, Jason M. Butler, Andreas Beilhack, Nancy R. Manley, Shahin Rafii, Jarrod A. Dudakov,* Marcel R. M. van den Brink*

    *Corresponding author. Email: jdudakov{at}fredhutch.org; vandenbm{at}mskcc.org

    Published 12 January 2018, Sci. Immunol. 2, eaal2736 (2017)
    DOI: 10.1126/sciimmunol.aal2736

    This PDF file includes:

    • Fig. S1. Thymic response to damage.
    • Fig. S2. Distribution of BMPR subunits on thymic cell populations.
    • Fig. S3. Inhibition of BMP signaling abrogates endogenous thymic regeneration.
    • Fig. S4. Deletion of BMP4 in ECs abrogates endogenous thymic regeneration.
    • Fig. S5. Damage response in the thymus to corticosteroids, chemotherapy, and TBI.
    • Fig. S6. exECs can be propagated ex vivo and maintain an EC phenotype.
    • Fig. S7. Validating methods of inducing Foxn1 and silencing Bmp4.
    • Table S1. High-confidence FOXN1 target gene changes after TBI.

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1. (.mp4 format). Thymic vascular architecture and branching by LSFM (day 0).
    • Movie S2. (.mp4 format). Thymic vascular architecture and branching by LSFM (day 4).
    • Movie S3. (.mp4 format). Thymic vascular architecture and branching by LSFM (day 7).
    • Movie S4. (.mp4 format). Thymic vascular architecture and branching by LSFM (day 14).

    Files in this Data Supplement: