Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages

Abstract

Eosinophils in visceral adipose tissue (VAT) have been implicated in metabolic homeostasis and the maintenance of alternatively activated macrophages (AAMs). The absence of eosinophils can lead to adiposity and systemic insulin resistance in experimental animals, but what maintains eosinophils in adipose tissue is unknown. We show that interleukin-5 (IL-5) deficiency profoundly impairs VAT eosinophil accumulation and results in increased adiposity and insulin resistance when animals are placed on a high-fat diet. Innate lymphoid type 2 cells (ILC2s) are resident in VAT and are the major source of IL-5 and IL-13, which promote the accumulation of eosinophils and AAM. Deletion of ILC2s causes significant reductions in VAT eosinophils and AAMs, and also impairs the expansion of VAT eosinophils after infection with Nippostrongylus brasiliensis, an intestinal parasite associated with increased adipose ILC2 cytokine production and enhanced insulin sensitivity. Further, IL-33, a cytokine previously shown to promote cytokine production by ILC2s, leads to rapid ILC2-dependent increases in VAT eosinophils and AAMs. Thus, ILC2s are resident in VAT and promote eosinophils and AAM implicated in metabolic homeostasis, and this axis is enhanced during Th2-associated immune stimulation.

Figure 1.

Deficiency of IL-5 or eosinophils promotes obesity and insulin resistance and decreases oxidative respiration and heat production in mice on HFD. (a–c) Mice of the indicated genotype were fed HFD or ND for 18–20 wk, and then total weight (a), percent adiposity by EchoMRI (b), and terminal perigonadal VAT weight (c) were determined. Results are representative of three independent experiments and include four to six animals per cohort. Fasting blood glucose (d), glucose tolerance testing (e) and insulin tolerance testing (f) were performed in mice on ND or HFD for 18–20 wk. Results are representative of three experiments. IL-5^+/−, Red5 C57BL/6 R^/+ heterozygotes; IL-5^−/−, Red5R/R homozygous IL-5 knockouts. (g and h) CLAMS analysis was performed using individually housed groups of six C57BL/6 or C57BL/6 dblGata1 eosinophil-deficient mice after maintenance on HFD for 12 wk. Variations in oxygen consumption (g) and energy expenditure over time (h) were pooled among animals in each group and statistical analysis was performed using pairwise comparisons. Error bars are the mean ± SEM. P-values are shown.

Figure 2.

ILC2s are resident within VAT and are the primary cells expressing IL-5 and IL-13. (a and b) Representative ILC2s FACS plots (a and b) and frequency (c) of ILC2s from the VAT SVF of Rag2-deficient, WT, IL7Ra-deficient, and Rag2× γ_c–deficient C57BL/6 mice. Cells were pregated on lin⁻ lymphoid cells (CD11b⁻, F4/80⁻, SiglecF⁻, SSC-lo, FSC-lo, CD45⁺; a) or lin⁻ CD3e⁻ CD4⁻ (b). (d) Representative flow cytometry plots showing frequencies of IL-13+ and IL-5+ cells among various cell populations in VAT. (e) Expression of the indicated surface markers on VAT IL-5⁺ lin⁻ cells (ILC2, red line) compared with VAT CD3ε⁺ T cells (blue line) and isotype controls (gray; a–e) Data are representative of two or more experiments. (f and g) IL-5 and IL-13 expression on the following VAT populations: CD4⁺ T cells (CD4), iNKT (aGC-loaded tetramer), CD8⁺ T cells (CD8), NK cells (NK1.1), CD3ε⁺ double-negative T cells (CD3ε), B cells (CD19), macrophages (CD11b), eosinophils (SiglecF), and lin⁻ cells (SSC). Cells were pregated as shown in Fig. S2. Data are representative of two or more experiments.

Figure 3.

VAT ILC2s spontaneously produce IL-5 and IL-13 in vivo and ex vivo, and respond robustly to IL-33. Reporter cytokine expression by VAT ILC2s (lin⁻ IL7Rα⁺ T1/ST2⁺) from 4get (IL-4 competence), Red5 (IL-5), and YetCre13 x ROSA-YFP (IL-13 reporter) mice (a), with percentages of VAT ILC2s positive for each cytokine marker (b) are shown. (c) Representative image shows spontaneous IL-13 reporter⁺ cells (YetCre13 Y/⁺ x ROSA-ZsGreen) in freshly isolated, whole mounted VAT. (d) VAT total ILC2s (lin⁻ thy1.2⁺ CD25⁺) were sorted and cultured in vitro for 72 h with the indicated combinations of IL-2, IL-7, IL-33, and PMA/ionomycin, and supernatant cytokine levels were determined (picogram per milliliter). (e) VAT IL-5⁺ ILC2s (lin⁻ thy1.2⁺ Red5⁺), IL-5⁺ (Red5⁺) CD4⁺ T cells, and IL-5–negative (Red5⁻) CD4⁺ T cells were cultured with IL-7 (first bar) or PMA/Ionomycin (second bar; d and e) Results are representative of two or more experiments. (a) Numbers in brackets or over lines indicate percentage of cells within the gate. Nd, not detected.

Figure 4.

VAT eosinophils and AAMs are dependent on ILC2s. (a) C57BL/6 male mice were injected i.p. for the indicated number of days shown with 250 µg Edu per mouse. FACS analysis was performed after pre-gating on eosinophils (Fig. S1). Data are from one experiment with three animals per group, and are representative of two independent experiments. (b) Frequency of eosinophils among total viable VAT, lung, or spleen cells from WT, Rag2-deficient, and Rag2× γ_c–deficient C57BL/6 mice. Data are representative of three experiments. (c) WT C57BL/6 mice were fed a ND or HFD for 3–4 mo, and VAT SVF was examined for immune cell composition. Pooled data from three independent experiments are shown. (d) Correlation between VAT ILC2s or VAT CD4⁺ T cells and VAT eosinophils. Mouse strains shown include Rag x γc (Rag2 deficient x γc deficient), WT B6 (WT C57BL/6), WT BALB (WT BALB/c), Rag1^−/− (Rag1 deficient), WT B6 HFD (WT C57BL/6 fed HFD for 3–4 mo), IL-13 deleter (YetCre13 Y/Y x ROSA-DTA BALB/c), and IL-5 deleter (Red5 R/R x ROSA-DTA C57BL/6). Strains were fed ND unless indicated. Each data point represents pooled data from at least five mice over multiple experiments. Pearson correlation coefficient is shown with significance. CD4⁺ T cell data are not shown for strains on the Rag-deficient background. (e–i) ILC2s, CD4⁺ T cells, CD8⁺ T cells, macrophages, and eosinophils were enumerated from the VAT (or indicated compartment) from the indicated strains and tissues on a BALB/c background (e–g) or C57BL/6 background (h and i). Data were pooled from two or more experiments. (j) VAT IL-5⁺ (Red5⁺) ILC2s or IL-5⁺ (Red5⁺) CD4⁺ T cells from the strains indicated. (k and l) Arginase-1⁺ (YFP⁺) AAMs were enumerated from WT YARG or γ_c-deficient YARG C57BL/6 basal VAT (k) or WT YARG or YetCre13 x ROSA-DTA YARG (IL-13 deleter) BALB/c (l) homeostatic VAT. Results contain pooled data from two or more experiments with 2–4 mice per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

IL-33 promotes ILC2 activation with IL-5 and IL-13 production and rapid VAT eosinophil accumulation. (a) IL-33 (500 ng, gray circles) or PBS control (black circles) was administered i.p., and then, 12 h later, frequency of eosinophils was determined from VAT SVF, spleen, and bone marrow. Data are representative of three or more experiments. (b) Representative histograms of WT (red line) VAT ILC2s (lin⁻ IL7Rα⁺ CD25⁺), eosinophils (Eos), macrophages (Mac), and CD4⁺ T cells (gating in Fig. S1), assessed for expression of T1/ST2 (IL-33R) and compared with T1/ST2-deficient (black lines) control animals (c and d) Representative FACS plots 24 h after IL-33 or PBS administration, pregated on CD4⁺ T cells (c) or lin⁻, non–B cells, and non–T cells (d) in IL-13 lineage-tracking mice (YetCre13 Y/⁺ x Rosa-YFP) or IL-5 reporter mice (Red5 R/⁺ heterozygotes). Histograms in d are pregated on total lin⁻ IL7Rα⁺ CD25⁺ VAT ILC2s. (e–h) IL-33 (500 ng, gray circles) or PBS (black circles) was administered daily for three consecutive days (e and f) or every other day for three doses (g and h), after which spleen (e and g) or VAT (f and h) eosinophils (Eos), CD4⁺ T cells (CD4), macrophages (Macs), ILC2s (lin⁻ IL7Rα⁺ CD25⁺), and total cells were enumerated. Results are representative of two or more independent experiments. Numbers indicate percentages of cells within gates. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

IL-33 induces ILC2-dependent VAT accumulation of eosinophils and Arginase-1⁺ AAMs. (a–c) VAT eosinophils or VAT YARG⁺ (YFP⁺) AAM (e-g) determined as a percentage of CD45⁺ cells (a and b), total viable cells per g (c), or as a percentage of total macrophages (d–g) 24 h after administration of 500 ng IL-33 or PBS. (d) Representative FACS plots of YARG⁺ AAM from the strains indicated, pregated on total macrophages (Fig. S1). IL-13 deleter mice, YetCre13 Y/Y x ROSA-DTA D/⁺ BALB/c; IL-5 deleter mice, Red5 R/⁺ x ROSA-DTA D/⁺ C57BL/6; mice with YARG reporter as noted (e–g). (a–c and e–g) Data were pooled from two or more experiments. Numbers indicate percentages of cells in gate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7.

N. brasiliensis infection promotes ILC2-dependent accumulation of VAT eosinophils. (a–e) Mice were infected with N. brasiliensis and VAT was harvested ∼2 wk post-infection (a–e) and analyzed by flow cytometry. (a) VAT IL-13 lineage-tracked ILC2s or IL-13⁺ CD4 T cells (Yetcre13 Y/⁺ x ROSA-YFP) were enumerated. (b) IL-5⁺ (Red5⁺) ILC2s were pregated and the median fluorescence intensity of Red5 (tdTomato) was determined. (c) VAT eosinophil frequency in IL-5^+/− (Red5 R/⁺ heterozygotes) or IL-5 deleter (Red5 R/R x ROSA-DTA D/D) animals, (d) WT BALB/c or IL-13 deleter mice, or (e) WT C57BL/6 or Rag1-deficient C57BL/6 mice. Data were pooled from two to three experiments (a and c–e) or are representative of three experiments (b). *, P < 0.05; **, P < 0.01; ***, P < 0.001.