Research ArticleSTROMAL CELLS

Stromal cells maintain immune cell homeostasis in adipose tissue via production of interleukin-33

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Science Immunology  03 May 2019:
Vol. 4, Issue 35, eaax0416
DOI: 10.1126/sciimmunol.aax0416
  • Fig. 1 IL-33 regulates ILC2 activity and eosinophil numbers.

    (A) Body weight of WT and Il33−/− mice on conventional diet at 16 weeks of age (n = 4). (B) Mass of indicated adipose depots in WT and Il33−/− mice at 16 weeks of age (n = 4). (C) Proportion of combined WAT mass of ewat, mwat, and iwat depots as percent of body weight in WT and Il33−/− mice at 16 weeks of age (n = 4). (D) ILC2 numbers and proportions in visceral adipose compartments of WT and Il33−/− mice (n ≥ 3). Data were pooled from two individual experiments. (E and F) Representative dot plots (E) and quantification (F) showing intracellular IL-5 and IL-13 staining in ILC2s from visceral adipose depots of WT and Il33−/− mice (n = 3). (G and H) Representative dot plots (G) and quantification (H) of macrophages and eosinophils in visceral adipose depots of WT and Il33−/− mice (n = 3). Means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant, Student’s t test.

  • Fig. 2 Visceral WAT ASPCs produce IL-33.

    (A and B) Expression of Il33 in (A) isolated adipocytes (Adip.) and SVF, and in (B) indicated sort-purified cells of murine ewat, as determined by qRT-PCR analysis (n ≥ 5). (C) Representative histogram overlay and dot plots of cells in ewat SVF fraction of WT mice, stained with anti–IL-33 (α–IL-33) and IgG control (ctrl) antibodies. Data depicted are gated on live cells. Numbers in gates indicate proportion (%) of parent gate. (D) Histogram demonstrating mean normalized counts from RNA-seq of genes expressed in sort-purified ASPCs from ewat. Markers of ASPCs are indicated in black, highly expressed cytokine genes are indicated in blue, and Il33 is shown in red. Genes with mean normalized counts <10 are not shown (n = 3). (E) Expression of Il33 in adipocytes and sort-purified cells from ewat as determined by qRT-PCR analysis (n ≥ 5). (F) Representative dot plots showing eGFP-expressing cells in ewat of the IL-33–eGFP mouse. Numbers in gates indicate proportion (%) of parent gate. Data shown are gated on live cells. (G) Representative dot plot and histogram overlay of live eGFP+ lin (CD31+ CD45+ Ter119+) cells in the ewat of the IL-33–eGFP mouse. (H) Expression of indicated genes in isolated adipocytes and sort-purified cells from the ewat as determined by qRT-PCR analysis (n ≥ 5). Mesoth, mesothelial cell. (I) Representative dot plots of MSLN staining in WT mouse SVF from ewat and subcutaneous WAT (iwat) first gated on live lin cells. Numbers in gates indicate proportion (%) of the parent gate. (J) Quantification of eGFP+ cell populations in indicated WAT compartments. Data were compiled from three independent experiments (n ≥ 13). Means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test.

  • Fig. 3 Localization of IL-33–expressing cells in visceral WAT.

    (A) Surface view of the ewat from the IL-33–eGFP reporter mouse (right) and a WT ctrl animal (left). (B) Immunostaining of IL-33 (green) in paraffin-embedded WT mouse ewat. Note the IL-33 localization to DAPI-stained nuclei (blue) scattered between perilipin+ adipocytes (purple), as well as IL-33 in the PDPN+ (red) membrane lining the adipose compartment. (C) Immunostaining of paraffin-embedded WT mouse ewat. PDGFRα-expressing ASPCs (green) embedded between perilipin+ adipocytes (purple). (D) Immunostained transverse sections (side view) of whole-mount WT mouse ewat demonstrating PDGFRα-stained (purple) ASPCs that express IL-33 (green) in the nucleus (blue). The PDPN-stained (red) cell layer expressing nuclear IL-33 (green) envelops the adipose compartment. Arrowheads indicate magnified areas in adjacent panels. (E) Side and surface view of the same sample of WT mouse whole-mount stained ewat. (F and G) Surface view of whole-mount stained ewat. ZO-1 (green) staining on PDPN+ (red) cobblestone-shaped cells in (F) and colocalization of IL-33 (green) and WT1 (purple) in the nucleus (blue) in (G). Scale bars, 100 μm, unless indicated otherwise. All immunostaining experiments are representative of two mice, and the staining protocol has been repeated a minimum of three times.

  • Fig. 4 ASPC-derived IL-33 controls ILC2 activity.

    (A) Representative histogram and dot plots showing ST2+ cells in the lymphocyte population first gated on live CD45+ cells. (B) Representative histogram of ST2 expression on ILC2 (defined as shown in fig. S9A) from the SI LP and ewat. (B and C) Representative histogram (B) and bar graph (C) showing mean fluorescence intensity (MFI) of ST2 on ILC2s from ewat and SI LP (n = 6). (D) Schematic indicating experimental design for (E) and (F). (E) Sort-purified ILC2s from WT or Il1rl1−/− mouse visceral WAT (pooled ewat and mwat) ILC2s were cultured in ASPC or ctrl, and IL-5 secretion was analyzed by ELISA after 48 hours (n = 3). (F) Sort-purified ILC2s from visceral WAT or SI LP were cultured in ASPC or ctrl, and IL-5 and IL-13 secretion was analyzed by multiplex bead-based assay after 48 hours (n = 3). (G) Schematic indicating experimental design. Sort-purified ASPCs from WT or Il33−/− mice were injected into the peritoneum of Il33−/− mice and immunological analysis (H to J) performed after 6 weeks in visceral adipose compartments. (H) ILC2 numbers, (I) proportions of IL-5+ and IL-13+ ILC2s, and (J) eosinophil quantification (n = 4). Means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant, Student’s t test.

  • Fig. 5 IL-33 controls adipose tissue expansion and immunological homeostasis in short-term HFD feeding.

    (A and B) WT mice were fed with HFD or CD up to 7 days (d). (A) Representative dot plot of Ki67 staining in ASPCs [defined as live lin (CD31 CD45 Ter119) PDGFRα+Sca-1+ cells] on day 7. (B) Proportion of proliferating (Ki67+) cells among total ASPCs from indicated WAT depots (n = 3). (C) Expression of Il33 and Mcp1 in sort-purified ASPCs isolated from ewat of WT mice on CD or HFD for 3 days as determined by qRT-PCR (n = 3). (D to I) WT or Il33−/− mice were fed with CD or HFD and euthanized for analysis after 3 days. (D) Body weight change as a proportion (%) of initial weight (day 0). Data were compiled from two individual experiments (n ≥ 7). (E) Proportion of ewat mass as proportion (%) of body weight. Data were compiled from two individual experiments (n ≥ 7). (F) Proportion of Ki67+ and adipocyte progenitor cells (CD24+) among total ewat ASPCs. (G) MFI of forward scatter (FSC) and side scatter (SSC) of ewat ASPCs. (H and I) Quantification of macrophages (H) and DCs (I) in ewat (n ≥ 4). Means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test.

  • Fig. 6 Mesothelial IL-33 acts as an alarmin to activate the immune system upon peritoneal infection.

    (A) WT mice were injected intraperitoneally with N. brasiliensis (N.b.). L3 larvae, and ewat was extracted after 1 hour. Representative surface view of immunostained whole-mount ewat depicting IL-33 (green) and PDPN+ mesothelium (red). Note the IL-33 relocalization from the nucleus to the cytoplasm in the N. brasiliensis–treated condition. Boxed area indicates magnified area. Arrowheads point to diffuse IL-33 staining in the cytoplasm. (B and C) ELISA quantification of IL-33 and IL-5 in peritoneal lavage fluid (Perit. lavage) and serum of WT and Il33−/− mice 4 hours after N. brasiliensis infection (n ≥ 3). (D and E) Representative dot plots (D) and quantification (E) showing intracellular IL-5 and IL-13 staining in ILC2s from ewat of WT and Il33−/− mice treated with N. brasiliensis (n ≥ 2). (F) MFI of IL-13 and IL-5 in ILC2s from (D) and (E). (G) Quantification of eosinophils in peritoneal lavage fluid of WT and Il33−/− mice treated with N. brasiliensis at 4 hours (n ≥ 5). Means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant, Student’s t test.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/35/eaax0416/DC1

    Fig. S1. ST2 regulates ILC2 activity and eosinophil numbers.

    Fig. S2. IL-33 treatment reverses immunodeficiency in Il33−/− mice.

    Fig. S3. ASPCs and mesothelial cells express nuclear IL-33.

    Fig. S4. ASPCs highly express Il33 among various cytokine and chemokine genes.

    Fig. S5. IL-33–eGFP+ cells are detectable in all adipose compartments of the body.

    Fig. S6. Localization of IL-33–expressing cells in body WAT.

    Fig. S7. ASPCs express IL-33 in humans.

    Fig. S8. IL-33 controls adipose tissue expansion and immunological homeostasis in short-term HFD feeding.

    Fig. S9. Gating strategy for flow cytometric analysis.

    Table S1. Raw data.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. ST2 regulates ILC2 activity and eosinophil numbers.
    • Fig. S2. IL-33 treatment reverses immunodeficiency in Il33−/− mice.
    • Fig. S3. ASPCs and mesothelial cells express nuclear IL-33.
    • Fig. S4. ASPCs highly express Il33 among various cytokine and chemokine genes.
    • Fig. S5. IL-33–eGFP+ cells are detectable in all adipose compartments of the body.
    • Fig. S6. Localization of IL-33–expressing cells in body WAT.
    • Fig. S7. ASPCs express IL-33 in humans.
    • Fig. S8. IL-33 controls adipose tissue expansion and immunological homeostasis in short-term HFD feeding.
    • Fig. S9. Gating strategy for flow cytometric analysis.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

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