Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis

Abstract

Eosinophils are associated with helminth immunity and allergy, often in conjunction with alternatively activated macrophages (AAMs). Adipose tissue AAMs are necessary to maintain glucose homeostasis and are induced by the cytokine interleukin-4 (IL-4). Here, we show that eosinophils are the major IL-4-expressing cells in white adipose tissues of mice, and, in their absence, AAMs are greatly attenuated. Eosinophils migrate into adipose tissue by an integrin-dependent process and reconstitute AAMs through an IL-4- or IL-13-dependent process. Mice fed a high-fat diet develop increased body fat, impaired glucose tolerance, and insulin resistance in the absence of eosinophils, and helminth-induced adipose tissue eosinophilia enhances glucose tolerance. Our results suggest that eosinophils play an unexpected role in metabolic homeostasis through maintenance of adipose AAMs.

Fig. 1

IL-4-expressing cells in adipose tissue. (A) GFP-positive cells in perigonadal adipose from 4get mice on normal chow. Gating criteria delineated in Supplemental methods and fig. S1B. Data pooled from 3 independent experiments with 2 or more mice per group. (B) Hematoxylin and eosin stain of wildtype (WT) paraffin-embedded adipose (scale bar 20 μm), representative of 2 independent experiments. (C) Eosinophil numbers as ascertained by flow cytometry in perigonadal adipose from 8 wk-old male mice from ΔdblGATA, WT, and IL-5tg mice on normal chow diet. Data is representative of 3 or more independent experiments. (D) Representative immunofluorescent images of Siglec-F+ cells in perigonadal adipose from the strains indicated (scale bar 50 μm). Siglec-F, green; nuclei counterstain with DAPI, blue, representative of 2 experiments. (E–H) WT male C57BL/6 mice were fed high-fat diet for 10–14 wk and compared with normal chow WT C57BL/6 controls. Perigonadal adipose eosinophils were quantified by flow cytometry per g adipose tissue (E) or percent of total stromal vascular fraction (SVF) cells (F). Correlation is shown between mouse weight on high-fat chow and adipose eosinophil numbers (G, H), utilizing Pearson’s correlation coefficient. (E–H) Results are pooled data from two independent experiments with 20 total mice. *p<0.05, **p<0.01

Fig. 2

Eosinophil migration to adipose tissue is integrin-mediated. (A) Eosinophils in the left lobe of the lung, spleen and perigonadal adipose tissue 3 and 7 days after adoptive transfer into eosinophil-deficient ΔdblGATA mice on normal chow. Data shown is pooled from two independent experiments. (B) ΔdblGATA mice received antibodies to α4 and αL integrins (100 μg each) or control isotypes (IgG2a and IgG2b) 2 hrs prior to adoptive transfer of eosinophils. Tissues were harvested 16 hrs later. Data shown is representative of two independent experiments. *p<0.05, **p<0.01 as determined using Student’s t-test. n.s. = not significant.

Fig. 3

Adipose macrophage alternative activation is impaired in the absence of IL-4/IL-13 or eosinophils. (A) Flow cytometric analysis of adipose from indicated mice on normal chow diet. Gates show YFP-positive cells as a percent of total CD11b^highF4/80^high macrophages (A) and are quantitated (B) Results are pooled data from two or more independent experiments with 2–4 animals per experiment. *p<0.05, **p<0.01 as determined using ANOVA with Bonferonni’s post-test correction for multiple comparisons. (C) ΔdblGATA × YARG mice were sublethally irradiated and reconstituted with bone marrow cells from 4get × IL-5tg mice. After 4–6 weeks, perigonadal adipose tissues were analyzed for eosinophils (left gate; eosinophils were GFP-positive and side-scatter^high, not shown) and macrophages (CD11b^high F4/80^high, right gate). Macrophages were then analyzed for YFP. Eosinophil-reconstituted mice (red); non-reconstituted mice (blue); WT control (non-reporter) mice (gray). (D) Statistical correlation (Spearman’s rank correlation) between the total numbers of eosinophils reconstituting perigonadal adipose tissues in eosinophil-deficient mice and the total numbers of AAM expressing the marker arginase-1 allele. Results are pooled data from 5 independent experiments with 2–4 animals per experiment. (E) Mice reconstituted with IL-5tg bone marrow lacking IL-4 and IL-13 (IL-5tg × 4/13 DKO) display significantly fewer total YARG+ AAM (E) or total YARG+AAM per 1,000 tissue eosinophils (IL-5tg 2.5 ± 1.1; IL-5tg × 4/13 DKO 2.4×10⁻⁴ ± 6.2×10⁻⁵). Results are pooled data from two or more independent experiments with 2–5 animals per experiment. *p<0.05, **p<0.01 as determined using Student’s t-test.

Fig. 4

Metabolic analysis of eosinophil-deficient and hypereosinophilic mice. (A) Perigonadal fat tissues (testis attached) from IL-5 transgenic (IL-5tg) and wildtype (WT) littermate controls. (B) Fasting male 8 wk-old WT or IL-5tg littermates maintained on normal chow (NC) diet were challenged with intraperitoneal glucose and blood was sampled for glucose at times indicated. Data compiled from two independent experiments with 6–7 mice in each group. (C,D) DEXA analysis of total, lean and fat tissue composition (C) or percentage adiposity (D) in ΔdblGATA and wildtype (WT) mice on normal chow (NC) or high-fat (HF) diet for 15 wk. Data compiled from two experiments with 5–8 mice in each group. (E) Intraperitoneal glucose tolerance test in male ΔdblGATA and WT mice on HF diet for 15 wk. Data compiled from 3 independent experiments with 5–8 mice in each group. (F) Fasting blood glucose in male WT and ΔdblGATA mice maintained on HF diet for 20–22 wk. Data compiled from 2 independent experiments with 5 mice in each group. (G, H) Insulin signaling, as measured by the ratio of serine phosphorylated AKT to total AKT in adipose, muscle and liver of mice aged 24 wk on HF diet (n = 4–6 mice per genotype, 4 representative mouse adipose samples shown). (I, J) Twelve-wk old wild-type C57BL/6 mice on HF diet for 6 wk were infected with N. brasiliensis (Nippo) or unchallenged (Control) and monitored for insulin tolerance (I) and glucose tolerance (J) at the indicated times. Insulin tolerance results are normalized to baseline fasting glucose, which was statistically different between cohorts (WT control 207 mg/dL ± 6; Nippo 179 mg/dL ± 7; p < 0.05). (K) Adipose tissue collected at days 40–45 post N. brasiliensis infection or from uninfected control mice and analyzed by flow cytometry for eosinophils per g adipose (K) or percent eosinophils (WT Control 2.9% ± 0.41; Nippo 10.1% ± 0.36, p<0.01). Data (I, J, K) are representative of two independent experiments with 20–30 total mice per cohort. *p<0.05, **p<0.01 as determined using Student’s t-test (B, E, F, H–K) or ANOVA with Bonferroni’s post-test correction for multiple comparisons (C–D); error bars = SEM; n.s. = not significant.