Diverse immune cells participate in the regulation of visceral adipose tissue (VAT) and metabolic homeostasis. With obesity, pro-inflammatory macrophages, neutrophils, CD8+ T cells, CD4+ Th1 cells, and mast cells accumulate in VAT and contribute to local and systemic inflammation, ultimately promoting insulin resistance and the development of metabolic syndrome and type 2 diabetes; in contrast, normal lean VAT contains eosinophils, alternatively activated macrophages (AAM), invariant natural killer T cells (iNKTs), and regulatory T (T reg) cells that can promote insulin sensitivity and metabolic homeostasis (Chawla et al., 2011; Schipper et al., 2012; Wu et al., 2011). How lean, healthy VAT recruits and sustains these distinct immune cell types remains largely unknown. We previously reported that eosinophils reside in VAT and that eosinophil deficiency impairs Arginase-1+ AAM accumulation. VAT eosinophils are abundant in IL-5 transgenic mice and promote AAM accumulation and insulin sensitivity (Wu et al., 2011; Chawla et al., 2011). Prolonged VAT eosinophilia after helminth infection is also correlated with improved metabolic parameters in animals challenged with high-fat diet (HFD; Wu et al., 2011). Eosinophil production, bone marrow release and tissue recruitment and retention depend on several cytokines, chemokines, and integrins. IL-5 is integral at multiple levels, promoting eosinophil bone marrow production, release, and tissue recruitment, and is required for optimal systemic and local eosinophilia in diverse models of allergic inflammatory responses (Mould et al., 1997; Kopf et al., 1996; Foster et al., 1996). In contrast, IL-5 deficiency in unperturbed animals leads to a modest reduction in bone marrow, blood, and gastrointestinal tract eosinophil levels, indicating eosinophil production and recruitment to certain tissues can occur without IL-5 (Mishra et al., 1999; Kopf et al., 1996). Eotaxins (CCL11 and CCL24) are chemokines that recruit eosinophils, are central to eosinophil maintenance within the gastrointestinal tract, and can be up-regulated by IL-13 during allergic inflammation (Mishra et al., 1999; Rothenberg and Hogan, 2006; Voehringer et al., 2007). Eosinophils also use endothelial cell integrins, which can be increased by IL-4 and IL-13, to traffic into tissues (Blanchard and Rothenberg, 2009). The relative dependence of VAT eosinophils on these factors, including IL-4, IL-5, and IL-13, remains unknown. Innate lymphoid type 2 cells (ILC2s) are recently characterized innate cells widely distributed in mammalian tissues (Spits and Di Santo, 2011). Also, designated innate helper type 2 cells (Price et al., 2010), nuocytes (Neill et al., 2010), or natural helper cells (Moro et al., 2010), ILC2s share features with other populations of innate lymphocytes, including NK cells (ILC1) and ILC3, comprising the RORγt-dependent ILC: lymphoid tissue-inducer cells (LTic), innate IL-22 producing cells (also referred to as NK22, ILC22, NCR22, and NKR+ LTic) and innate IL-17-producing cells (Spits and Di Santo, 2011). ILCs all share a dependence on the transcription factor Id2 and the common-γ chain (γc) cytokine receptor (Spits and Di Santo, 2011). In response to the epithelial cytokines IL-25 and IL-33, ILC2s expand and produce large amounts of type 2 cytokines, particularly IL-13 and IL-5 (Hurst et al., 2002; Price et al., 2010; Moro et al., 2010; Neill et al., 2010), which can promote AAMs and eosinophils, respectively (Blanchard and Rothenberg, 2009; Martinez et al., 2009). Although ILC2s are functionally similar to CD4+ T helper type 2 (Th2) cells (Price et al., 2010), ILC2s are widely distributed within tissues independent of antigenic stimulation and appear poised to respond to epithelial signals. One of the earliest descriptions of ILC2s identified them within lymphoid structures in mouse and human mesenteric adipose tissues (Moro et al., 2010). With this in mind, we sought to quantify ILC2s in metabolically active perigonadal VAT and determine whether these cells and the cytokines they produce, including IL-5 and IL-13, were responsible for the localization of eosinophils and AAMs to this tissue under basal conditions and after their activation by cytokines or in response to intestinal helminth infection. RESULTS Eosinophils and IL-5 promote insulin sensitivity and lean physiology We previously reported metabolic consequences of eosinophil deficiency using dblGata1 mice (Wu et al., 2011). Because IL-5 can promote local and systemic eosinophilia, we compared metabolic parameters in eosinophil-deficient and IL-5–deficient C57BL/6 mice during HFD challenge. We used Red5 mice, which contain a tandem-dimer red fluorescent protein (tdTomato) linked by an internal ribosomal entry site (IRES) to a Cre element, replacing the first exon of the il5 gene (unpublished data), thus marking cells producing IL-5; Red5 homozygous mice are IL-5–deficient and the Cre element facilitates deletional studies based on IL-5 expression. To control for potential genetic or microbiome contributions to these phenotypes, we compared IL-5–deficient Red5 homozygote and IL-5-sufficient Red5 heterozygote mice. Eosinophil-deficient and IL-5-deficient animals fed HFD for 18–20 wk gained more weight (Fig. 1 a), with increased total body adiposity (Fig. 1 b) and perigonadal VAT weight (Fig. 1 c), as compared with IL-5–sufficient mice. Fasting glucose levels were elevated in both strains of mice (Fig. 1 d), and both had impaired glucose (Fig. 1 e) and insulin tolerance (Fig. 1 f and unpublished data). These findings support and extend our previous results (Wu et al., 2011) to implicate IL-5 in metabolic homeostasis. 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. To further understand the mechanisms by which eosinophils and IL-5 influence metabolism, we placed eosinophil-deficient and -sufficient animals on HFD for 12 wk in metabolic cages. Although food and water intake and physical activity were not altered (unpublished data), total oxygen consumption (VO2) and energy utilization (heat) were decreased in eosinophil-deficient mice (Fig. 1, g and h); similar results occurred in IL-5–deficient animals (unpublished data). Thus, eosinophils and IL-5 do not alter caloric intake or caloric expenditures by enhancing physical activity. Instead, they may act in metabolically relevant tissue to promote increased oxidative metabolism and limit inflammation. Consistent with these findings, activation of iNKT IL-4 production (Lynch et al., 2012; Ji et al., 2012a) or exogenous IL-4 administration (Ricardo-Gonzalez et al., 2010) each promoted loss of adiposity and insulin sensitivity. ILC2s are the major source of IL-5 and IL-13 in VAT ILC2s have been implicated in promoting eosinophil influx into tissues such as the lung and intestines during allergic inflammation (Neill et al., 2010; Price et al., 2010; Liang et al., 2012). We used flow cytometry to analyze perigonadal VAT to ascertain a potential role for ILC2s in controlling eosinophils in this tissue. Perigonadal adipose tissue was isolated and digested to yield the stromal vascular fraction (SVF) enriched for hematopoietic cells, endothelial cells, and other stromal components, but devoid of adipocytes. After using lineage markers to exclude B cells, T cells, and NK cells, we could readily identify a discrete population of lymphoid cells in the SVF-expressing receptors for IL-2 (CD25), IL-7, and IL-33 (Fig. 2, a and b), as well as intracellular Gata3 (Fig. 2 b). These markers were previously demonstrated for ILC2s (Moro et al., 2010; Neill et al., 2010; Price et al., 2010). Similar to other ILC2s, VAT ILC2s were present in Rag-deficient mice but absent in Rag x γc-deficient and IL-7Rα–deficient mice (Fig. 2, a–c), strains previously shown to lack ILC2s. VAT ILC2s were present in male and female mice and in C57BL/6 and BALB/c mice in both WT and Rag-deficient (T/B cell–deficient) backgrounds, although consistently more abundant in C57BL/6 mice (see also Fig. 4 d, bottom, and not depicted). Thus, the SVF of perigonadal adipose tissue contains innate lymphoid cells with the phenotype of previously described ILC2s (Moro et al., 2010; Neill et al., 2010; Price et al., 2010). 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× γc–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. To assess the contribution of VAT ILC2s to the total IL-5 and IL-13 cytokine production in VAT, we used reporter mice with knock-in fluorescent alleles at various gene loci, thus allowing interrogation of the cytokine expression of these cells without the need for restimulation ex vivo. Both adipose SVF cells from Red5 mice, which mark IL-5–expressing cells with tdTomato expression, and YetCre13 x ROSA-YFP mice, which functionally mark cells that have ever expressed IL-13 by establishing constitutive YFP expression from the ROSA26 locus (Price et al., 2010), each contained cells marked by in situ IL-5 and IL-13 expression (Fig. 2 d). IL-5–expressing cells were negative for the myeloid marker CD11b, and included a small subset of CD4+ CD3e+ IL33R+ (T1/ST2+) Th2 cells (5–15%) and a large population of lineage-negative cells (85–95%). These VAT lineage-negative cells expressed CD25 (IL2Rα), IL33R (T1/ST2), CD122 (IL2Rβ), Thy1.2 (CD90.2), c-Kit, Sca-1, and KRLG1, and were uniformly negative for T cell markers, including CD4, CD8, CD3ε, TCR-β, and TCR-γδ (Fig. 2 e), consistent with previously described ILC2s (Moro et al., 2010; Neill et al., 2010; Price et al., 2010). VAT B cells, CD8+ T cells, CD3ε+ CD4− CD8− “double-negative” T cells, macrophages, eosinophils, and α-galactosylceramide (αGC)-reactive invariant NKT cells (iNKT) did not show IL-5 fluorescence (Fig. 2 f and gating in Fig. S2), consistent with previous studies about lung IL-5+ cells (Ikutani et al., 2012). Similar results were found for VAT IL-13–expressing cells, although small percentages of eosinophils (0.2–0.4%) and iNKT cells (3–5%) expressed IL-13 using lineage-tracked expression (Fig. 2 g). After prolonged IL-33 administration or helminth infection, ILC2s remain the predominant IL-5– and IL-13–expressing cells, with no significant increased expression by macrophages, eosinophils, or other lymphocytes (Figs. S1 and S2 and unpublished data). Together, these results establish that ILC2s are the predominant IL-5– and IL-13–expressing cells in VAT and that rare Th2 cells account for most of the remaining cytokine-expressing cells. As assessed using these reporter alleles, significant proportions of VAT ILC2s spontaneously produced IL-5 and IL-13 (Fig. 3, a and b), and this was particularly striking for IL-5. We could identify no phenotypic differences between cytokine-positive and -negative ILC2s, suggesting a uniform population with variable cytokine expression. IL-13 cytokine-marked cells, the great majority of which are ILC2s (Fig. 2 d), were readily detected in close apposition to the adipose vasculature and dispersed within VAT (Fig. 3c). Unlike ILC2s reported in mesenteric lymph nodes and mesenteric lymphoid clusters (Moro et al., 2010), we were unable to identify discrete lymphoid structures within perigonadal adipose tissue (unpublished data). In contrast to VAT ILC2s, bone marrow ILC2s (lineage− IL7Rα+ T1/ST2+; Brickshawana et al., 2011), which were also described as ILC2 precursors (Hoyler et al., 2012), did not express basal IL-13 as assessed with IL-13 lineage tracking (2.0 ± 0.3%, n = 8), although marrow ILC2s were predominantly IL-4 competent, as assessed using cells from 4get mice (85.5 ± 7.4%, n = 3). Although a subset of VAT ILC2s were competent to make IL-4 (4get+; Fig. 3, a and b), they were unmarked by reporter expression in KN2 mice (unpublished data), whose cells contain an IL-4 replacement allele and reveal cells actively producing IL-4 in situ (Mohrs et al., 2001; Wu et al., 2011), as previously described (Price et al., 2010; Wu et al., 2011). 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. To confirm the fidelity of the cytokine reporters and confirm additional cytokines secreted by these cells, VAT ILC2s (lineage-negative Thy1.2+ CD25+) were purified by flow cytometry and placed in vitro for 72 h with various cytokines. Low amounts of IL-5, IL-6, IL-13, and GM-CSF spontaneously accumulated in the VAT ILC2 culture supernatants (Fig. 3, d and e, and unpublished data). After addition of IL-33, greater amounts of IL-5, IL-6, IL-9, IL-13, and GM-CSF accumulated (Fig. 3 d), and these cytokines increased further with the addition of IL-2 or IL-7, similar to results reported by ILC2s from other tissues (Moro et al., 2010; Halim et al., 2012). Together, these data suggest that VAT ILC2s spontaneously produce IL-5 and IL-13, and can respond to IL-33 with high levels of cytokine production, as shown for other ILC2s. Although rare in VAT, IL-5+ (Red5+) CD4+ T cells revealed a similar capacity to produce IL-2, IL-5, IL-6, IL-13, and GM-CSF after in vitro culture with PMA/ionomycin (Fig. 3 e). These data indicate IL-5+ ILC2s are numerically predominant within VAT, but otherwise have a similar cytokine capacity to IL-5+ Th2 cells. ILC2s are required to sustain adipose eosinophils and AAMs Eosinophils home to and are sustained in VAT, where they promote AAM maintenance and systemic insulin sensitivity (Wu et al., 2011). As assessed after mitotic labeling during bone marrow differentiation, eosinophils had significantly lower turnover in VAT as compared with spleen and lung, consistent with the presence of recruitment, retention, or survival signals in adipose tissue (Fig. 4 a). Although present in Rag-deficient mice, VAT eosinophils were substantially and tissue-specifically reduced in Rag x γc-deficient mice that lack ILC2s (Fig. 4 b). Prolonged HFD results in a decline of VAT eosinophils, as previously described (Wu et al., 2011), which is associated with a loss of VAT ILC2s but increased numbers of total VAT macrophages and CD8+ T cells (Fig. 4 c). In contrast, lung ILC2s were not reduced after HFD (unpublished data). Indeed, VAT ILC2 cell numbers correlate strongly with VAT eosinophils across multiple mouse WT strains, genetic mutations, and dietary perturbations, whereas total CD4+ T cells show no corresponding correlation (Fig. 4 d). 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× γc–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 γc-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 2), a one-tailed ANOVA was performed with Tukey’s post-test correction. Supplementary Material Supplemental Material
