Research ArticleSTROMAL CELLS

Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose tissue immune and metabolic tenors

See allHide authors and affiliations

Science Immunology  03 May 2019:
Vol. 4, Issue 35, eaaw3658
DOI: 10.1126/sciimmunol.aaw3658
  • Fig. 1 PDGFRα+Sca-1+mSCs are the main source of IL-33 in eVAT.

    (A to D) Cytofluorimetric identification of IL-33–producing cells within eVAT of 8- to 10-week-old B6 mice using a polyclonal anti–IL-33 Ab. (A) Gating strategy to delineate cell fractions. SSC, side scatter. (B) Representative dot plots of control-Ab (top) or anti–IL-33 staining (bottom) of the cell fractions. (C) As per (B) except that whole-body IL-33–deficient (Il33−/−) mice were assessed. (D) Frequencies of IL-33+ cells in each cell fraction (left) and its contribution to total IL-33+ cells within the tissue (right). n ≥ 7 from at least three experiments. (E to G) Confocal microscopic images of IL-33+ VmSCs. (E) The eVAT depot is bordered by a ring of high PDPN positivity, presumably the mesothelium. (F) IL-33+ cells bordered in close proximity to CD31+ endothelial cells. (G) PDPN positivity outlines a large blood vessel surrounded by β3-tubulin+ nerves. Color code for Ab staining as indicated on top of each picture. PDPN, podoplanin; DNs, PDGFRα-Sca-1 cells; VmSCs, PDGFRα+Sca-1+ VAT mesenchymal stromal cells.

  • Fig. 2 PDGFRα+Sca-1+mSCs are biologically relevant IL-33 producers.

    (A) Quantitative PCR on whole eVAT of Il33 transcripts in Pdgfra-Cre.Il33f/f and corresponding wild-type littermates aged 12 to 16 weeks. (B and C) Fractions of Tregs in Pdgfra-Cre.Il33f/f (B) and Il1rl1−/− mice (C) compared with their matching wild-type controls in eVAT (top) and iSAT (bottom). n ≥ 3 from at least two independent experiments. Mean ± SEM. *P < 0.05, ****P < 0.0001, according to the one-tailed (A) and two-tailed (B and C) unpaired Student’s t test. AU, arbitrary units.

  • Fig. 3 mSCs exhibit extensive transcriptional heterogeneity.

    (A) Two-dimensional t-distributed stochastic neighbor embedding (tSNE) plot of scRNA data from VAT (orange), muscle (green), and lymph node (blue) PDGFRα+Sca-1+ mSCs. VmSC subtypes 1 to 5 were delineated by k-means clustering; their fractional contributions to total VmSCs are indicated. (B) Heatmap showing genes differentially expressed between the VmSC subtypes [false discovery rate (FDR) < 5%]. k-means clustered. (C) VmSC dynamics (velocity field). The projected next cell state for each single cell is represented by an arrow and projected on the tSNE plot. (D) Same tSNE plot as in (A) overlain with heatmaps of the density of cells expressing various transcript markers. (E) Heatmap of transcript expression within the combined single-cell data of each VmSC subtype. (F) Representative dot plots delineating the VmSC subtypes (left) and indicating their IL-33 expression levels (right) in PpargTdtIl33Egfp double-reporter mice. TdT, TdTomato; eGFP, enhanced green fluorescent protein. (G) Comparison of VmSC subtype frequencies obtained by scRNA-seq versus flow cytometry. (H) Matrix of Spearman correlation coefficients in head-to-head comparisons of ≥2-fold differentially expressed genes from the scRNA-seq and matching population (pop)-level RNA-seq datasets. LN, lymph node.

  • Fig. 4 mSC subtypes exhibit different functional properties.

    (A) Gene expression of pan-fibroblast genes across VmSC populations. (B) Three-dimensional PCA plot depicting the VmSC subsets. (C) GSEA-derived hallmarks for individual VmSC subtypes compared with the rest. The size of the circles denotes the number of genes significantly enriched in each individual pathway; rainbow color code indicates FDR values, and NES (normalized enrichment score) is depicted on the x axis of the plot. IFN-α, interferon-α. (D) Expression heatmaps for transcripts considered cardinal for the indicated cell types. All should be overexpressed, except Cd34 and Ncam1, which were underexpressed in pericytes. Precise expression values appear in table S3. (E) Representative images of three-dimensional in vitro cultured VmSCs in the absence (−) or in the presence (+) of an adipogenic medium (Adipo med) (top). Quantification of the spheroid diameter (bottom). All data from 8- to 10-week-old males of B6 genotype unless indicated otherwise. *P < 0.05. GSEA pathways were filtered based on a P nominal value <0.05. Other abbreviations as per Fig. 1.

  • Fig. 5 Age strongly influences immunocyte-promoting VmSCs and thereby Treg accumulation.

    (A) Representative plots (top) and frequencies and numbers (bottom) of total VmSCs from mice of different ages. (B) Frequencies (top) and numbers (bottom) of total IL-33+ VmSCs. (C and D) Cytofluorometric dot plots of VmSC subtypes (C) and corresponding fractions (top) and numbers (bottom) (D) from lean B6 males of the indicated ages. Pooled data from three independent experiments. Numbers of cells were normalized per tissue weight. One-way ANOVA analysis was performed to compare three or more groups. For all relevant plots, mean ± SEM. *P < 0.05, **P < 0.01. SVF, stromal vascular fraction. Other abbreviations as per Fig. 1.

  • Fig. 6 Gender determines drastically distinct VmSC subtype distribution and VAT Treg abundance.

    (A) Frequencies (top) and numbers (bottom) of total IL-33+ VmSCs. (B) Cytofluorimetric dot plots (left) and corresponding quantification of subtypes thereof (right) in gVAT and iSAT of lean male (M) and female (F) mice aged 18 to 20 weeks old. Pooled data from at least two independent experiments. (C) Three-dimensional PCA plot of the transcriptomes (population-level RNA-seq) from coincidently prepared male and female VmSC subtypes from B6 mice 8 to 10 weeks old. (D) Esr1 (top) and Ar (bottom) transcripts levels for the individual VmSC subtypes from male (white bars) and female (black bars) mice aged 8 to 10 weeks old. Each dot represents an individual biological replicate from two or more pooled mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Correlation curves for numbers of IL-33+ VmSCs versus ST2+ Treg numbers in gVAT using metadata from all male mice of various ages (6, 16, 18 to 20, and 32 weeks) and female mice 18 to 20 weeks old, i.e., all mice of normal physiologic state. In all cases, numbers of cells were normalized to tissue weight. r and P from Pearson’s correlation coefficient. All other statistics and abbreviations as per Figs. 1 and 5.

  • Fig. 7 An IL-33–focused VAT Treg:VmSC regulatory loop is uncovered in obese mice.

    (A and B) Fractions (top) and numbers (bottom) of total IL-33+ VmSCs (A) and VmSC subtypes (B) subsequent to 4 or 16 weeks of high-fat feeding of 14-week-old B6 males. Data from at least two independent experiments. (C and D). Effects of IL-33 administration to 8- to 12-week-old B6 males. Frequency (top) and numbers (bottom) of total IL-33+ VmSCs (C) or of individual VmSC subtypes (D). Data shown correspond to at least two pooled independent experiments. Numbers of cells were tissue weight–normalized in all cases. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. LFD, low-fat diet. All other statistics and abbreviations as per Figs. 1 and 5.

  • Fig. 8 Specific ST2 expression on VAT Tregs is involved in their interaction with VmSC subtypes.

    (A and B) IL-33 was administered to wild-type B6 males aged 8 to 12 weeks. Frequencies (top) and numbers (bottom) of total (left) and ST2+ (right) Tregs (A) or ILC2s (B). (C) Confocal images (group of left panels) showing Tregs and IL-33–expressing cells within eVAT at low magnification (left), corresponding delineation of adipocyte edges based on autofluorescence of DAPI channel (middle), and high magnification of the squared indicated area (right). Distance quantification between Foxp3+ Tregs and the nearest IL-33–expressing cell (group of right panels) from whole eVAT tissue sections taken from male VAT Treg TCR-transgenic mice at 7 (top) and 17 (bottom) weeks of age. Arrows in (C) depict Treg:IL-33+ cell proximity. Color code for Ab staining as indicated within the picture. (D to F) IL-33 was injected into mice lacking ST2 expression specifically by Tregs versus wild-type littermate controls. Frequencies (top) and numbers (bottom) corresponding to total and KLRG1+ Tregs (D), ILC2s (E), and IL-33+ VmSCs (F). All numbers were calculated relative to total tissue weight. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) Graphic scheme of the proposed VmSC:Treg negative regulatory loop model. All other abbreviations and statistics as per Figs. 1, 5, and 7.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/35/eaaw3658/DC1

    Materials and Methods

    Fig. S1. Controls for cytofluorimetric assessment of IL-33 expression.

    Fig. S2. IL-33 expression primarily by mSCs is a common feature across male fat depots.

    Fig. S3. Pdgfra-driven Il33 ablation affects the amount of mature VAT Tregs without affecting body, fat depot weights, or VmSC quantities in young lean male mice.

    Fig. S4. Intracellular anti–IL-33 staining is an alternative to IL-33 reporter fluorescence for VmSC subtype quantification.

    Fig. S5. Distinct gene expression profiles of VmSC subsets reveal functional heterogeneity.

    Fig. S6. Aging affects VAT Tregs, weights, and VmSCs.

    Fig. S7. Gender-specific differences in Tregs.

    Fig. S8. VmSCs constitute the main source of IL-33 in different adipose depot in females.

    Fig. S9. Diet-induced obesity provokes VAT Treg loss and VmSC remodeling.

    Fig. S10. Specific IL-33 sensing by VAT Tregs alters VmSC dynamics.

    Table S1. Specific and shared gene clusters reveal VmSC subsets’ heterogeneity.

    Table S2. Overrepresented pathways of VmSC subsets from 8-week-old male mice.

    Table S3. Cardinal gene expression profiles in VmSC subsets.

    Table S4. Differential expression of extracellular matrix and soluble protein on VmSC subtypes.

    Table S5. GSEA of VmSC subsets from female vis-à-vis male mice aged 8 to 10 weeks.

    Table S6. Raw data.

    References (3746)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. Controls for cytofluorimetric assessment of IL-33 expression.
    • Fig. S2. IL-33 expression primarily by mSCs is a common feature across male fat depots.
    • Fig. S3. Pdgfra-driven Il33 ablation affects the amount of mature VAT Tregs without affecting body, fat depot weights, or VmSC quantities in young lean male mice.
    • Fig. S4. Intracellular anti–IL-33 staining is an alternative to IL-33 reporter fluorescence for VmSC subtype quantification.
    • Fig. S5. Distinct gene expression profiles of VmSC subsets reveal functional heterogeneity.
    • Fig. S6. Aging affects VAT Tregs, weights, and VmSCs.
    • Fig. S7. Gender-specific differences in Tregs.
    • Fig. S8. VmSCs constitute the main source of IL-33 in different adipose depot in females.
    • Fig. S9. Diet-induced obesity provokes VAT Treg loss and VmSC remodeling.
    • Fig. S10. Specific IL-33 sensing by VAT Tregs alters VmSC dynamics.
    • Legends for tables S1 to S6
    • References (3746)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Specific and shared gene clusters reveal VmSC subsets’ heterogeneity.
    • Table S2 (Microsoft Excel format). Overrepresented pathways of VmSC subsets from 8-week-old male mice.
    • Table S3 (Microsoft Excel format). Cardinal gene expression profiles in VmSC subsets.
    • Table S4 (Microsoft Excel format). Differential expression of extracellular matrix and soluble protein on VmSC subtypes.
    • Table S5 (Microsoft Excel format). GSEA of VmSC subsets from female vis-à-vis male mice aged 8 to 10 weeks.
    • Table S6 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

Navigate This Article