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

Ari B. Molofsky; Jesse C. Nussbaum; Hong-Erh Liang; Steven J. Van Dyken; Laurence E. Cheng; Alexander Mohapatra; Ajay Chawla; Richard M. Locksley

doi:10.1084/jem.20121964

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

Molofsky, Ari B.; Nussbaum, Jesse C.; Liang, Hong-Erh; Van Dyken, Steven J.; Cheng, Laurence E.; Mohapatra, Alexander; Chawla, Ajay; Locksley, Richard M. 2013-03-11 00:00:00 A r t i c l e Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages 3,4 2,3 1 Ari B. Molofsky, Jesse C. Nussbaum, Hong-Erh Liang, 1,3 5 3 Steven J. Van Dyken, Laurence E. Cheng, Alexander Mohapatra, 2,6 1,2,3 Ajay Chawla, and Richard M. Locksley 1 2 3 4 Howard Hughes Medical Institute, Departments of Medicine, Microbiology and Immunology, Laboratory Medicine, 5 6 Pediatrics and Physiology, University of California, San Francisco, San Francisco 94143 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 lym- phoid 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 signifi - cant reductions in VAT eosinophils and AAMs, and also impairs the expansion of VAT eosinophils after infection with Nippostrongylus brasiliensis, an intestinal parasite associ- ated 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. Diverse immune cells participate in the regu Prolonged VAT eosinophilia after helminth CORRESPONDENCE Richard M. Locksley: lation of visceral adipose tissue (VAT) and meta infection is also correlated with improved met [email protected] bolic homeostasis. With obesity, pro ina fl mmatory abolic parameters in animals challenged with + + macrophages, neutrophils, CD8 T cells, CD4 high fat diet (HFD; Wu et al., 2011). Eosino Abbreviations used: AAM, alternatively activated macro Th1 cells, and mast cells accumulate in VAT phil production, bone marrow release and tis phage; HFD, high fat diet; and contribute to local and systemic inflam sue recruitment and retention depend on several ILC2, innate lymphoid type 2 mation, ultimately promoting insulin resistance cytokines, chemokines, and integrins. IL 5 is cell; iNKT, invariant natural killer T cell; ND, normal diet; and the development of metabolic syndrome and integral at multiple levels, promoting eosino SVF, stromal vascular fraction; type 2 diabetes; in contrast, normal lean VAT phil bone marrow production, release, and tis VAT, visceral adipose tissue. contains eosinophils, alternatively activated mac sue recruitment, and is required for optimal rophages (AAM), invariant natural killer T cells systemic and local eosinophilia in diverse mod (iNKTs), and regulatory T (T reg) cells that can els of allergic inflammatory responses (Mould promote insulin sensitivity and metabolic homeo et al., 1997; Kopf et al., 1996; Foster et al., stasis (Chawla et al., 2011; Schipper et al., 2012; 1996). In contrast, IL 5 deficiency in unper Wu et al., 2011). How lean, healthy VAT recruits turbed animals leads to a modest reduction in and sustains these distinct immune cell types re bone marrow, blood, and gastrointestinal tract mains largely unknown. eosinophil levels, indicating eosinophil produc We previously reported that eosinophils tion and recruitment to certain tissues can occur reside in VAT and that eosinophil deficiency impairs Arginase 1 AAM accumulation. VAT © 2013 Molofsky et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after eosinophils are abundant in IL 5 transgenic mice the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share and promote AAM accumulation and insulin Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ sensitivity (Wu et al., 2011; Chawla et al., 2011). by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. 2013 Vol. 210 No. 3 535-549 www.jem.org/cgi/doi/10.1084/jem.20121964 The Journal of Experimental Medicine without IL5 (Mishra et al., 1999; Kopf et al., 1996). Eotax To control for potential genetic or microbiome contribu ins (CCL11 and CCL24) are chemokines that recruit eo tions to these phenotypes, we compared IL 5–decient fi Red5 sinophils, are central to eosinophil maintenance within the homozygote and IL5sufficient Red5 heterozygote mice. gastrointestinal tract, and can be upregulated by IL13 dur Eosinophildeficient and IL5deficient animals fed HFD for ing allergic inflammation (Mishra et al., 1999; Rothenberg 18–20 wk gained more weight (Fig. 1 a), with increased total and Hogan, 2006; Voehringer et al., 2007). Eosinophils also body adiposity (Fig. 1 b) and perigonadal VAT weight (Fig. 1 c), use endothelial cell integrins, which can be increased by IL4 as compared with IL5–sufficient mice. Fasting glucose levels and IL13, to traffic into tissues (Blanchard and Rothenberg, were elevated in both strains of mice (Fig. 1 d), and both had 2009). The relative dependence of VAT eosinophils on these impaired glucose (Fig. 1 e) and insulin tolerance (Fig. 1 f and factors, including IL4, IL5, and IL13, remains unknown. unpublished data). These findings support and extend our Innate lymphoid type 2 cells (ILC2s) are recently charac previous results (Wu et al., 2011) to implicate IL5 in meta terized innate cells widely distributed in mammalian tissues bolic homeostasis. (Spits and Di Santo, 2011). Also, designated innate helper To further understand the mechanisms by which eosino type 2 cells (Price et al., 2010), nuocytes (Neill et al., 2010), or phils and IL5 influence metabolism, we placed eosinophil natural helper cells (Moro et al., 2010), ILC2s share features dec fi ient and suc ffi ient animals on HFD for 12 wk in metabolic with other populations of innate lymphocytes, including NK cages. Although food and water intake and physical activity cells (ILC1) and ILC3, comprising the RORtdependent were not altered (unpublished data), total oxygen consump ILC: lymphoid tissueinducer cells (LTic), innate IL22 pro tion (VO ) and energy utilization (heat) were decreased in ducing cells (also referred to as NK22, ILC22, NCR22, and eosinophil deficient mice (Fig. 1, g and h); similar results NKR+ LTic) and innate IL 17 producing cells (Spits and occurred in IL5–deficient animals (unpublished data). Thus, Di Santo, 2011). ILCs all share a dependence on the transcrip eosinophils and IL5 do not alter caloric intake or caloric ex tion factor Id2 and the common  chain (c) cytokine re penditures by enhancing physical activity. Instead, they may ceptor (Spits and Di Santo, 2011). In response to the epithelial act in metabolically relevant tissue to promote increased oxi cytokines IL25 and IL33, ILC2s expand and produce large dative metabolism and limit inflammation. Consistent with amounts of type 2 cytokines, particularly IL 13 and IL 5 these findings, activation of iNKT IL4 production (Lynch (Hurst et al., 2002; Price et al., 2010; Moro et al., 2010; Neill et al., 2012; Ji et al., 2012a) or exogenous IL 4 administration et al., 2010), which can promote AAMs and eosinophils, re (RicardoGonzalez et al., 2010) each promoted loss of adi spectively (Blanchard and Rothenberg, 2009; Martinez et al., posity and insulin sensitivity. 2009). Although ILC2s are functionally similar to CD4 T helper type 2 (Th2) cells (Price et al., 2010), ILC2s are widely ILC2s are the major source of IL-5 and IL-13 in VAT distributed within tissues independent of antigenic stimula ILC2s have been implicated in promoting eosinophil influx tion and appear poised to respond to epithelial signals. One of into tissues such as the lung and intestines during allergic in the earliest descriptions of ILC2s identified them within lym flammation (Neill et al., 2010; Price et al., 2010; Liang et al., phoid structures in mouse and human mesenteric adipose tis 2012). We used flow cytometry to analyze perigonadal VAT sues (Moro et al., 2010). With this in mind, we sought to to ascertain a potential role for ILC2s in controlling eosino quantify ILC2s in metabolically active perigonadal VAT and phils in this tissue. Perigonadal adipose tissue was isolated and determine whether these cells and the cytokines they pro digested to yield the stromal vascular fraction (SVF) enriched duce, including IL 5 and IL 13, were responsible for the for hematopoietic cells, endothelial cells, and other stromal localization of eosinophils and AAMs to this tissue under basal components, but devoid of adipocytes. After using lineage conditions and after their activation by cytokines or in re markers to exclude B cells, T cells, and NK cells, we could sponse to intestinal helminth infection. readily identify a discrete population of lymphoid cells in the SVFexpressing receptors for IL2 (CD25), IL7, and IL33 RESULTS (Fig. 2, a and b), as well as intracellular Gata3 (Fig. 2 b). Eosinophils and IL-5 promote insulin These markers were previously demonstrated for ILC2s sensitivity and lean physiology (Moro et al., 2010; Neill et al., 2010; Price et al., 2010). Similar We previously reported metabolic consequences of eosino to other ILC2s, VAT ILC2s were present in Rag decient fi phil deficiency using dblGata1 mice (Wu et al., 2011). Be mice but absent in Rag x cdeficient and IL7R –deficient cause IL5 can promote local and systemic eosinophilia, we mice (Fig. 2, a–c), strains previously shown to lack ILC2s. compared metabolic parameters in eosinophildeficient and VAT ILC2s were present in male and female mice and in IL 5–dec fi ient C57BL/6 mice during HFD challenge. We used C57BL/6 and BALB/c mice in both WT and Ragdeficient Red5 mice, which contain a tandemdimer red fluorescent (T/B cell–dec fi ient) backgrounds, although consistently more protein (tdTomato) linked by an internal ribosomal entry site abundant in C57BL/6 mice (see also Fig. 4 d, bottom, and (IRES) to a Cre element, replacing the first exon of the il5 not depicted). Thus, the SVF of perigonadal adipose tissue gene (unpublished data), thus marking cells producing IL5; contains innate lymphoid cells with the phenotype of pre Red5 homozygous mice are IL5–deficient and the Cre ele viously described ILC2s (Moro et al., 2010; Neill et al., 2010; ment facilitates deletional studies based on IL5 expression. Price et al., 2010). 536 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e Figure 1. Deficiency of IL-5 or eosino - phils promotes obesity and insulin resis- tance 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 per- formed in mice on ND or HFD for 18–20 wk. Results are representative of three experi- +/ /+ ments. IL-5 , Red5 C57BL/6 R heterozy- / gotes; 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 pair- wise comparisons. Error bars are the mean ± SEM. P-values are shown. To assess the contribution of VAT ILC2s to the total IL 5 CD25 (IL2R), IL33R (T1/ST2), CD122 (IL2R), Thy1.2 and IL13 cytokine production in VAT, we used reporter (CD90.2), cKit, Sca1, and KRLG1, and were uniformly mice with knockin fluorescent alleles at various gene loci, negative for T cell markers, including CD4, CD8, CD3, thus allowing interrogation of the cytokine expression of these TCR , and TCR  (Fig. 2 e), consistent with previously cells without the need for restimulation ex vivo. Both adi described ILC2s (Moro et al., 2010; Neill et al., 2010; Price + +   pose SVF cells from Red5 mice, which mark IL 5–expressing et al., 2010). VAT B cells, CD8 T cells, CD3 CD4 CD8 cells with tdTomato expression, and YetCre13 x ROSA “double negative” T cells, macrophages, eosinophils, and YFP mice, which functionally mark cells that have ever ex  galactosylceramide (GC) reactive invariant NKT cells pressed IL 13 by establishing constitutive YFP expression from (iNKT) did not show IL5 fluorescence (Fig. 2 f and gating the ROSA26 locus (Price et al., 2010), each contained cells in Fig. S2), consistent with previous studies about lung IL5 marked by in situ IL5 and IL13 expression (Fig. 2 d). IL5– cells (Ikutani et al., 2012). Similar results were found for VAT expressing cells were negative for the myeloid marker CD11b, IL 13–expressing cells, although small percentages of eosino + + + + and included a small subset of CD4 CD3e IL33R (T1/ST2 ) phils (0.2–0.4%) and iNKT cells (3–5%) expressed IL 13 Th2 cells (5–15%) and a large population of lineage negative using lineagetracked expression (Fig. 2 g). After prolonged cells (85–95%). These VAT lineagenegative cells expressed IL 33 administration or helminth infection, ILC2s remain JEM Vol. 210, No. 3 537 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×  –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 CD3 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-13 538 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e the predominant IL5– and IL13–expressing cells, with no ILC2s are required to sustain adipose eosinophils and AAMs significant increased expression by macrophages, eosinophils, Eosinophils home to and are sustained in VAT, where they or other lymphocytes (Figs. S1 and S2 and unpublished data). promote AAM maintenance and systemic insulin sensitivity Together, these results establish that ILC2s are the predom (Wu et al., 2011). As assessed after mitotic labeling during bone marrow die ff rentiation, eosinophils had signic fi antly lower turn inant IL 5– and IL 13–expressing cells in VAT and that rare Th2 cells account for most of the remaining cytokine over in VAT as compared with spleen and lung, consistent with expressing cells. the presence of recruitment, retention, or survival signals in adi As assessed using these reporter alleles, significant pro pose tissue (Fig. 4 a). Although present in Rag dec fi ient mice, portions of VAT ILC2s spontaneously produced IL 5 and VAT eosinophils were substantially and tissue specic fi ally re IL13 ( Fig. 3, a and b), and this was particularly striking for duced in Rag x  dec fi ient mice that lack ILC2s (Fig. 4 b). IL5. We could identify no phenotypic differences between Prolonged HFD results in a decline of VAT eosinophils, as cytokinepositive and negative ILC2s, suggesting a uniform previously described (Wu et al., 2011), which is associated population with variable cytokine expression. IL 13 cytokine with a loss of VAT ILC2s but increased numbers of total VAT marked cells, the great majority of which are ILC2s (Fig. 2 d), macrophages and CD8 T cells (Fig. 4 c). In contrast, lung ILC2s were not reduced after HFD (unpublished data). Indeed, VAT were readily detected in close apposition to the adipose vas culature and dispersed within VAT (Fig. 3c). Unlike ILC2s ILC2 cell numbers correlate strongly with VAT eosinophils reported in mesenteric lymph nodes and mesenteric lymphoid across multiple mouse WT strains, genetic mutations, and di clusters (Moro et al., 2010), we were unable to identify dis etary perturbations, whereas total CD4 T cells show no cor crete lymphoid structures within perigonadal adipose tissue responding correlation (Fig. 4 d). (unpublished data). In contrast to VAT ILC2s, bone marrow To assess the ee ff cts of deleting IL 13–expressing ILC2s on  + + ILC2s (lineage IL7R T1/ST2 ; Brickshawana et al., 2011), adipose eosinophils, we crossed the YetCre13 mice to ROSA which were also described as ILC2 precursors (Hoyler et al., DTA deleter mice, which led to diphtheria toxin A–mediated 2012), did not express basal IL13 as assessed with IL13 lin death of cells that express IL 13 (Voehringer et al., 2008). These eage tracking (2.0 ± 0.3%, n = 8), although marrow ILC2s IL 13 deleter mice had an 40% loss of adipose ILC2s, consis tent with the IL 13 expression data (Fig. 4 e), and had a signic fi ant were predominantly IL4 competent, as assessed using cells from 4get mice (85.5 ± 7.4%, n = 3). Although a subset of reduction in adipose tissue eosinophils that was not apparent in + + VAT ILC2s were competent to make IL4 (4get ; Fig. 3, spleen or bone marrow (Fig. 4 f). Total VAT CD4 T cells and a and b), they were unmarked by reporter expression in KN2 macrophages were not ae ff cted by deletion of IL 13–producing + + mice (unpublished data), whose cells contain an IL4 replace cells (Fig. 4 e), although rare IL 13 CD4 T cells were likely ment allele and reveal cells actively producing IL4 in situ deleted. In contrast to the IL13 deleter mice, deficiency of (Mohrs et al., 2001; Wu et al., 2011), as previously described IL 4/IL 13 or STAT6 did not ae ff ct basal levels of VAT or (Price et al., 2010; Wu et al., 2011). spleen eosinophils (Fig. 4, d and g, unpublished data), indicat To conr fi m the d fi elity of the cytokine reporters and con ing that although VAT ILC2s can produce IL 13, other ILC2 firm additional cytokines secreted by these cells, VAT ILC2s derived factors are required to sustain VAT eosinophils. + + We performed similar studies using Red5 mice, which (lineagenegative Thy1.2 CD25 ) were purified by flow cy tometry and placed in vitro for 72 h with various cytokines. contain a disrupted il5 gene replaced with a fluorescent td Low amounts of IL5, IL6, IL13, and GMCSF spontane Tomato with an embedded Cre recombinase. Eosinophils in ously accumulated in the VAT ILC2 culture supernatants (Fig. 3, adipose were strongly dependent on IL5. Thus, Red5 het +/ d and e, and unpublished data). After addition of IL 33, greater erozygous mice (IL5 ) had fewer adipose eosinophils than amounts of IL 5, IL 6, IL 9, IL 13, and GM CSF accumulated did mice with two intact il5 alleles (WT), and IL5–deficient / (Fig. 3 d), and these cytokines increased further with the ad Red5 homozygous mice (IL5 ) containing two marker dition of IL 2 or IL 7, similar to results reported by ILC2s from alleles were drastically depleted of adipose eosinophils (Fig. 4 h). other tissues (Moro et al., 2010; Halim et al., 2012). Together, These ee ff cts of IL 5 dec fi iency were greater on adipose eosin these data suggest that VAT ILC2s spontaneously produce IL 5 ophils compared with systemic eosinophils, with a 12–14 fold reduction in VAT (Fig. 4 h) versus a 2–3fold reduction in and IL13, and can respond to IL33 with high levels of cyto kine production, as shown for other ILC2s. Although rare in spleen, blood, and small intestine (unpublished data). Although + + + VAT, IL 5 (Red5 ) CD4 T cells revealed a similar capacity to IL 5–deficient Red5 homozygous mice, similar to prior produce IL 2, IL 5, IL 6, IL 13, and GM CSF after in vitro cul studies in eosinophildeficient mice (Price et al., 2010), had ture with PMA/ionomycin (Fig. 3 e). These data indicate IL 5 normal numbers of ILC2s (unpublished data), Red5 mice ILC2s are numerically predominant within VAT, but otherwise containing a ROSADTA deleter allele exhibited significant + + + have a similar cytokine capacity to IL 5 Th2 cells. depletion of total adipose ILC2s (Fig. 4 i), IL5 (Red5 ) + + + expression on the following VAT populations: CD4 T cells (CD4), iNKT (aGC-loaded tetramer), CD8 T cells (CD8), NK cells (NK1.1), CD3 double-negative T cells (CD3), B cells (CD19), macrophages (CD11b), eosinophils (SiglecF), and lin cells (SSC). Cells were pregated as shown in Fig. S2. Data are represen- tative of two or more experiments. JEM Vol. 210, No. 3 539 ILC2s (Fig. 4 j), and almost complete ablation of adipose eosin could not conclusively identify which cells produced these cyto ophils (Fig. 4 h). Similar to IL 13 deleter animals, VAT kines (Wu et al., 2011). We used YARG mice containing a from IL 5 deleter mice had normal numbers of macrophages, u fl orescent arginase 1 knock in reporter allele to assess numbers + + CD8 T cells, and total CD4 T cells. When assessed specifi of adipose AAMs, as previously described (Reese et al., 2007; + + cally for IL 5 CD4 T cells, IL 5 deleter animals also ec ffi iently Wu et al., 2011). As assessed by o fl w cytom etry of dispersed deleted the rare IL5 Th2 cells (Fig. 4 j). However, as ILC2s SVF cells, adipose AAMs were depleted in  dec fi ient mice are the predominant IL5 VAT cell (85–95%), and VAT (Fig. 4 k) and in IL 13 deleter mice (Fig. 4 l), strains that have eosinophils are normal to elevated in T cell–deficient Rag absent or diminished ILC2s. Thus, adipose AAMs as assessed animals (Fig. 4 b), we conclude that ILC2expressing IL5 by arginase1 expression are dependent on ILC2s, and loss of are the primary cells required for the maintenance of visceral ILC2s based on their cytokine expression or dependence upon adipose eosinophils under basal conditions. the  cytokine chain results in a significant reduction in basal Adipose AAMs, like eosinophils, are present in the SVF adipose AAMs. of VATs in lean mice (Schipper et al., 2012). IL 13, like IL 4, can promote an AAM phenotype, which in mice can be re Exogenous IL-33 results in ILC2-dependent increases vealed by expression of signature target genes such as arginase 1. in adipose eosinophils and AAMs We previously determined a role for eosinophils and hema ILC2s were initially revealed by their capacity to release IL 13 topoietic IL 4/IL 13 in sustaining lean adipose AAMs, but and IL5 in response to IL25 and IL33, epithelial cytokines 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 IL7R 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 combina- +  + tions 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. 540 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e 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×  –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 c (Rag2 dec fi ient x c dec fi ient), 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  -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. JEM Vol. 210, No. 3 541 implicated in allergic immunity (Fort et al., 2001; Schmitz including the IL13 deleter and IL5 deleter ( Fig. 6, a, b, e, et al., 2005). IL33 has been identified in VAT and exoge and f ), and in IL7Rdeficient mice (Fig. 6 g, unpublished nous IL 33 was shown to promote Th2 associated cytokines data). In contrast, eosinophil accumulation is normal in re and to improve insulin sensitivity in obese mice (Miller et al., sponse to IL 33 in Rag1 deficient animals that lack B and 2010). After administering IL 33, we noted the rapid accumu T cells (Fig. 6 c) or in IL4/13–deficient animals (Fig. 6 a). lation of eosinophils in VAT that was accompanied by a de Similar to the percentage data (Fig. 6, a, b, and e–g), total crease in eosinophils in spleen and bone marrow, which is adipose eosinophils and AAM also accumulate after IL 33 ad consistent with a rapid redistribution of eosinophils from sys ministration in an ILC2 dependent manner, although the temic compartments to VAT (Fig. 5 a). Several types of adi increase is more robust in genetic strains on the C57BL/6 pose SVF cells expressed T1/ST2, a nonredundant component genetic background (unpublished data). We conclude that ILC2s of the IL33 receptor, including ILC2s, a proportion of CD4 are required for the IL33–mediated increases in VAT eosin T cells and, at low levels, eosinophils; VAT macrophages and ophils and AAM. CD8 T cells did not express the receptor under these condi VAT Arginase1 AAMs are dependent on eosinophils + + tions (Fig. 5 b, unpublished data). VAT T1/ST2 CD4 T cells and IL 4/ 13 (Wu et al., 2011); however, the precise cellu were primarily FoxP3 T reg cells with fewer FoxP3 Gata3 hi lar sources of these cytokines are unclear. Loss of VAT ILC2s Th2 cells (unpublished data). In response to IL 33, ILC2s decreases VAT YARG+ AAM (Fig. 4, k and l), but also rapidly increased their sidescatter and surface CD25 levels, leads to a loss of VAT eosinophils (Fig. 4, b, e, f, and i). as described for lung ILC2s (Bartemes et al., 2012), and de Therefore, it remained unclear if ILC2s have the capacity creased their surface levels of IL 7R (Fig. 5 d). To conr fi m that to promote AAM accumulation independent of eosinophils. these parameters indicate ILC2 cell activation, we assessed After IL 33 administration, YARG AAM can accumulate the cytokine response of ILC2s in IL13 and IL5 reporter in VAT independent of eosinophils as revealed in dblGata1 mice. In IL 13 lineage tracker mice, where only a subset of mutant mice (Fig. 6g), demonstrating that exogenous IL 33 ILC2s are IL 13 cytokine marked under basal conditions can induce IL 13 from VAT ILC2s sufficient to promote + + (Fig. 3, a and b), increased numbers of IL13 (YFP ) ILC2s adipose AAM. Nonetheless, our understanding of the rela were readily detected after administering IL33 (Fig. 5 d). tive contributions of VAT eosinophils and ILC2 derived In contrast, VAT CD4 T cells revealed minimal increases in IL 13 to AAM maintenance under homeostatic conditions + + + IL 13 (YFP ) CD4 T cells (Fig. 5 c). IL33 also caused in remains incomplete. creased fluorescence intensity in VAT ILC2s in IL5 (Red5) reporter mice (Fig. 5 d). Together, these findings are consis Intestinal helminth infection drives ILC2-dependent tent with direct activation of ILC2 ee ff ctor function by IL 33. increases in adipose eosinophils After three days of daily IL33 administration, total VAT eo We previously reported that infection of mice with Nippostron- sinophils and macrophages continued to accumulate, although gylus brasiliensis, a 10d selflimited migratory helminth infec ILC2 cell numbers did not significantly increase (Fig. 5 f ). tion, resulted in a prolonged elevation of visceral adipose Cell populations in the spleen were not significantly affected eosinophils that correlated with improved metabolic homeo during this timeframe (Fig. 5 e). With prolonged IL33 ad stasis when mice were placed on HFD (Wu et al., 2011). To ministration, ILC2s accumulate within VAT and systemically, assess whether ILC2 activation, which accompanies N. brasil- as previously described (Neill et al., 2010), accompanied by a iensis infection (Liang et al., 2012; Neill et al., 2010), is re systemic eosinophilia and macrophage accumulation (Fig. 5, quired for VAT eosinophil accumulation, we infected IL 13 and g and h). Even after prolonged IL33 administration, ILC2s IL 5 reporter mice. By 2 wk after infection, total numbers of + + + remain the predominant IL 5 cells in VAT, and IL 5 (Red5 ) VAT ILC2s were not increased, but IL 13– and IL 5–secreting + + CD4 T cells expand minimally (Fig. S2). In contrast, FoxP3 ILC2s were increased, as assessed using reporter mice (Fig. 7, T reg cells accumulate both systemically, as described pre a and b). As compared with control mice, which developed viously (Turnquist et al., 2011; Brunner et al., 2011), and within robust accumulations of VAT eosinophils, IL 5 deleter (Fig. 7c) VAT (unpublished data). We conclude that IL33 rapidly ac and IL13 deleter mice (Fig. 7d) developed little eosinophilia. tivates VAT ILC2s and promotes VAT eosinophils and AAM, IL13 cytokinemarked ILC2s remain the predominant IL and, over time, leads to additional local and systemic accu 13expressing cells, similar to basal conditions, although VAT mulations of ILC2s and T reg cells. IL13 expressing Th2 cells are modestly increased (Fig. 7a). To assess the requirement for ILC2 cell activation in me Rag1 dec fi ient animals accumulate VAT eosinophils similarly to diating the adipose cellular effects of exogenous IL33, we WT animals (Fig. 7e). Thus, as with IL 33 administration, hel administered IL33 or control PBS to IL5 and IL13 control minth infection activates VAT ILC2s to produce IL5 and or deleter mice. Similar experiments were performed in con IL 13, and loss of IL 5 and IL 13–producing cells, but not loss trol or deleter mice crossed onto the YARG arginase1 back of T cells, results in a failure to accumulate VAT eosinophils. ground to assess requirements for ILC2s in the accumulation of adipose AAMs. Although control mice rapidly increased DISCUSSION adipose eosinophils and AAMs in response to IL33, this ef ILC2s have been increasingly implicated in host type 2 im fect was abrogated after crossing onto strains dec fi ient in ILC2s, mune responses associated with asthma and intestinal helminth 542 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e infection in mice and humans (Brickshawana et al., 2011; IL13 and IL5 (Fort et al., 2001; Hurst et al., 2002; Schmitz Chang et al., 2011; Mjösberg et al., 2011; Moro et al., 2010; et al., 2005). During helminth infection or allergen challenge, Neill et al., 2010; Price et al., 2010; Halim et al., 2012; Ikutani ILC2s constitute the major innate cell source of these cyto et al., 2012; Klein Wolterink et al., 2012; Liang et al., 2012; kines, and loss of these cells can compromise the host type 2 Klein Wolterink et al., 2012). These cells were first identi immune response (Brickshawana et al., 2011; Chang et al., fied by their capacity to respond to the epithelial cytokines 2011; Mjösberg et al., 2011; Moro et al., 2010; Neill et al., IL25 and IL33 through the production of large amounts of 2010; Price et al., 2010; Halim et al., 2012; Ikutani et al., 2012; 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 cir- cles) 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 IL7R 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 IL7R 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 IL7R 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. JEM Vol. 210, No. 3 543 + + + 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 ad- ministration 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. Klein Wolterink et al., 2012; Liang et al., 2012). Despite subtle deficiency is also associated with a partial loss of B1 B cells die ff rences in surface markers used to characterize various lab (Kopf et al., 1996), and these cells might also contribute to oratories’ designation of these cells, their shared genetic and this phenotype. Loss of eosinophils or IL5 does not affect functional characteristics suggest a single cell type that is highly animal food intake or physical exertion, but instead causes associated with allergic and antihelminth immunity. Recent a decline in oxidative metabolism and energy expenditure studies have called attention to additional roles for ILC2 (heat), ultimately resulting in increased adiposity and meta like cells in inflammatory processes (Monticelli et al., 2011; bolic impairment. The precise molecular and cellular mech Chen et al., 2012), including limiting lung damage mediated anisms leading to these metabolic alterations remain to be by acute viral infection, potentially implicating ILC2s in re determined, but could reflect increased adipose inflamma parative responses to tissue and organ injury. These studies did tion secondary to the loss of adipose eosinophils and AAM. not find a role for IL 13 and IL 5, the canonical cytokines Whether eosinophils directly inhibit VAT inflammation or released in large abundance by ILC2s, in reestablishing organ promote a lean state with decreased VAT mass that indirectly homeostasis, leaving it unclear what the purpose of these cyto reduces ina fl mmation remain intriguing questions. Using o fl w kines might be in normal host physiology. cytometric phenotyping, in situ imaging, and genetic approaches, Using metabolic analysis, we demonstrate a role for IL 5 in we demonstrate that ILC2s are normal constituents of peri sustaining metabolic homeostasis. As in eosinophil deficient gonadal VAT of the mouse. ILC2s reside in the SVF where dblGata1 animals, IL5 deficiency promotes increased obesity eosinophils and AAMs are also present. Using cytokine re and insulin resistance with HFD. We demonstrate that loss of porter mice, we show that ILC2s are the primary producers IL 5 leads to a profound decrease in VAT eosinophils, with only of IL5 and IL13 under homeostatic conditions and, as dem modest alterations in systemic eosinophil pools, suggesting that onstrated by functional deletion, that these cells are required a loss of VAT eosinophils is responsible for the metabolic for the constitutive localization of eosinophils and AAMs to consequences of IL 5 dec fi iency. However, we note that IL 5 VAT. Further, IL33, shown to activate ILC2s systemically, 544 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e 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. induces rapid increases in adipose eosinophils and AAMs that to ascertain whether these or additional cytokines are neces are dependent on ILC2s. Similarly, helminth infection pro sary to sustain ILC2 homeostasis and activation in adipose motes VAT eosinophilia that is dependent on ILC2s. In con tissues. Additionally, ILC2s have been reported to produce a trast, HFD results in a decline in VAT ILC2s that is associated variety of factors in addition to IL 5 and IL 13; we have with declining eosinophils. These data extend our understand conr fi med that VAT ILC2s spontaneously produce IL 5, IL 6, ing of these innate lymphoid cells, and, in conjunction with IL 13, and GM CSF protein in culture, and can be induced previous studies, suggest a mechanism by which metabolic in vitro with exogenous IL 33 or PMA stimulation to pro needs of the organism might be regulated in response to chronic duce these cytokines, as well as IL 2 and IL 9. The impact of mucosal immune stimulation through activation of ILC2s. each of these additional ILC2 cytokines on VAT cellular com Based on the capacity of epithelial cytokines to activate position and metabolism requires further study. ILC2s, we administered IL 33 to mice and assessed the effects VAT eosinophils are highly dependent on IL 5. Deletion + + on adipose tissue. IL 33 rapidly activated ILC2s to increase of IL 5 cells or IL 13 cells, which are predominantly ILC2s, expression of IL 5 and IL 13, and this led to the accumula led to profound loss of VAT eosinophils and only minimally tion of eosinophils and AAMs in adipose tissue. As assessed by to eosinophils in other tissues. As such, the VAT eosinophil surface markers, side scatter characteristics, and cytokine re dependence upon IL 5 resembles many models of allergic porter expression, IL 33 directly activates adipose ILC2s. and helminth induced tissue eosinophilia, which can show Deletion of these cells led to loss of eosinophil and AAM ac strong IL 5 dependence (Foster et al., 1996; Kopf et al., 1996; cumulation in adipose. IL 33 is abundant in adipose tissues Mould et al., 1997). However, ILC2s are widely distributed where it may be produced and released by endothelial cells in tissues of unchallenged animals, and their presence alone is (Zeyda et al., 2012). Whether endothelial cells or other adi not associated with tissue eosinophils (Ikutani et al., 2012; pose cells undergo cell damage or can respond to environ Price et al., 2010). VAT ILC2s may be relatively more abun mental cues to release IL 33 remains unknown. Administration dant or activated than ILC2s from other tissues, sustaining of IL 33 has beneficial metabolic effects in mice, consistent VAT eosinophils and AAM. However, it is also likely that with those seen by increased eosinophils and AAMs (Miller other VAT cells and signals contribute to the maintenance et al., 2010), suggesting that IL 33–mediated ILC2 activation of these important cellular constituents. Indeed, VAT is an can promote insulin sensitivity. ample source of chemokines, including constitutive eotaxin 1 Interestingly, IL 7 is necessary to sustain ILC2s and has (Vasudevan et al., 2006), which could promote eosinophil also been localized to adipose (Lucas et al., 2012). TSLP is also trafficking into VAT. During helminth infections and allergic present in adipose tissue (Turcot et al., 2012) and can sustain challenge in the lung, IL 5 promotes eosinophil production, ILC2s in vitro (Halim et al., 2012). Further study is needed survival, and retention, whereas IL 13 mediates eosinophil JEM Vol. 210, No. 3 545 recruitment through the promotion of tissue eotaxins (Blanchard insulin sensitizing products, or through the activation of and Rothenberg, 2009). In contrast, VAT eosinophils are pres nonshivering thermogenesis (Nguyen et al., 2011; Chawla ent in the genetic absence of IL 13 or IL 13 signaling (STAT6), et al., 2011). suggesting that constitutive factors, including eotaxins or other Intestinal helminth infections are widespread in feral ani chemokines, may help recruit these cells. In addition, VAT mals, suggesting a long standing mutualism. The host response eosinophil trafficking requires  and  mediated integrin is characterized by chronic type 2 immune responses, includ L 4 signaling (Wu et al., 2011), indicating VAT endothelium con ing the presence of epithelial mucus changes, Th2 cells, ele tributes to eosinophil accumulation. Although VAT eosinophils vated IgE and mucosal mast cell hyperplasia, but also chronic are likely sustained by multiple pathways, ILC2s play an in eosinophilia and the accumulation of tissue AAMs. Similar dispensable role under our experimental conditions. responses, presumably dysregulated, accompany responses Adipose AAMs and eosinophils are present in lean adi to ubiquitous environmental antigens in people with allergy pose, sustaining resistance of VATs to the proinflammatory and asthma. ILC2s respond to epithelial cytokines such as IL 25, effects of HFD and obesity (Chawla et al., 2011). Here, we IL33, and TSLP, and have been implicated in both antihel demonstrate that ILC2 IL5 is necessary to maintain VAT minth and allergic immunity ((Brickshawana et al., 2011; eosinophils, which are themselves required to sustain popula Chang et al., 2011; Mjösberg et al., 2011; Moro et al., 2010; tions of Arginase1 AAMs (Wu et al., 2011). ILC2s, in con Neill et al., 2010; Price et al., 2010; Halim et al., 2012; Ikutani trast to eosinophils (Fig. 2 f), produce ample IL13 and could et al., 2012; Klein Wolterink et al., 2012; Liang et al., 2012). directly contribute to AAM polarization and maintenance. As shown here, adipose eosinophils, which accumulate after Indeed, with IL 33 stimulation ILC2s appear sufficient to N. brasiliensis infection (Wu et al., 2011), do not accumulate promote Arginase1 AAM, even in the absence of eosino in the absence of ILC2s, although accumulation is normal phils. However, under basal conditions, eosinophils also pro in T cell deficient Rag mice. Of interest, both IL 33 ad mote AAM (Wu et al., 2011), suggesting ILC2 IL13 may be ministration and helminth infection promote insulin sensi insufficient to fully polarize and maintain VAT AAM under tivity in mice fed HFD (Miller et al., 2010; Wu et al., 2011), resting conditions. Consistent with this hypothesis, only a suggesting ILC2 activation and/or accumulation may be subset of ILC2s is marked for IL13 expression, even using contributing to these metabolic effects. Based on prior stud mice with lineagetracked markers that reveal cells that have ies, release of IL25 and/or IL33 during tissueinvasive hel ever expressed IL13 through their history. Ultimately, the minth infection is likely (Moro et al., 2010; Neill et al., 2010), relative contributions of eosinophils and ILC2 IL13 produc but further investigation will be necessary to show defini tion to AAM maintenance in lean adipose remain to be de tively which cytokines activate VAT ILC2s. The potential finitively elucidated. interactions between VAT ILC2s, Th2 cells and T reg cells, The mechanisms by which eosinophils promote AAM and their relative contributions to metabolic pathways during remain poorly understood. Although IL4 remains a candi homeostasis and after helminth infection remain intriguing date cytokine, only a small subset of adipose eosinophils questions. Understanding the processes that sustain AAMs (<1%) were marked for IL4 protein expression as assessed and eosinophils in VATs may offer new insights toward using KN2 mice (Wu et al., 2011). Eosinophils produce therapeutic strategies attempting to block the adverse effects abundant secreted products, including TGF, proteases, and of adipose inflammation and protect against insulin resistance RNase, which could also participate in AAM maintenance and type 2 diabetes. Although further metabolic studies are (Blanchard and Rothenberg, 2009). Adipocytes are also re needed, investigations of the role of these unusual innate ported to be potential sources of IL4 and IL13 (Kang et al., lymphoid cells in adipose and other tissues are warranted, 2008), but we have not observed fluorescence in adipocytes and may provide novel insights into more global aspects of using IL4 or IL13 cytokine marker alleles (Wu et al., 2011; vertebrate biology. Fig. 3 c), and il4 and il13 transcripts are primarily found within the VAT SVF (Wu et al., 2011). VAT iNKT cells MATERIALS AND METHODS were recently proposed to mediate metabolic homeostasis Mice. Cytokine reporter mice previously described include 4get mice for tracking IL 4 competent cells (Mohrs et al., 2001; Wu et al., 2011), YetCre13 and produce abundant IL 4 after TCR stimulation that mice for tracking IL 13–producing cells (Price et al., 2010; Liang et al., 2012), might promote AAM (Ji et al., 2012a; b; Lynch et al., 2012); and KN2 mice for tracking IL 4–producing cells (Mohrs et al., 2005; Wu in our mouse colony, iNKT cells are rare in VAT (Fig. S2). et al., 2011). Where indicated, YetCre13 mice were crossed onto ROSA26 Finally, IL5– and IL13–expressing Th2 cells accumulate in eYFP (The Jackson Laboratory) or ROSA26 ZsGreen (Ai6; The Jackson VAT of older animals on normal diet (ND; unpublished Laboratory; Madisen et al., 2010). The eYFP Cre fusion protein downstream data), and similar to VAT T reg cells (Feuerer et al., 2009), of the IL 13 locus in YetCre13 mice mediates deletion of the stop cassette from the ROSA26 locus, resulting in constitutive u fl orophore expression in cells may provide an additional layer of adaptive regulation to that have expressed IL 13. Newly generated Red5 mice contain a tdTomato maintain metabolic homeostasis. How these cells cooperate IRES Cre replacement allele at the endogenous IL 5 start site, thus replacing to promote AAM maintenance remains to be determined. the endogenous il5 gene with tdTomato and revealing IL5–expressing cells Further, how AAMs themselves promote insulin sensitivity by red fluorescence. Homozygous Red5 mice (R/R) are IL5 deficient, as is also unclear, although proposed mechanisms include both alleles are replaced by the marker construct, whereas heterozygous mice through the production of antiinflammatory cytokines and have one functional IL 5 copy (R/+). YARG mice contain a YFP marker 546 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e allele in the arginase 1 gene, permitting identic fi ation of AAMs, as previously manufacturer’s instructions. When used with fluorescent reporter strains, a described (Reese et al., 2007; Wu et al., 2011). ROSA DTA mice contain a brief 2min “prefix” in 4% paraformaldehyde was performed before pro Cre a fl nked u fl x stop sequence upstream of diphtheria toxin  (DTA) in ceeding with eBioscience fix/perm instructions. Edu detection was achieved serted into the constitutively expressed ROSA26 locus, thus causing Cre with the Clickit Edu A647 kit, after first staining for extracellular surface expressing cells to be deleted, and have been previously described (Jackson; markers, per the manufacturer’s instructions (Life Technologies). Voehringer et al., 2008)). ROSA DTA mice were crossed with YetCre13 or Representative gating schemes for each population are shown in Fig. S1.     Red5 mice to create mice in which IL 13– or IL 5–expressing cells are deleted. ILC2s are identified as lineage negative (CD11b , F4/80 , DX5 , CD3 ,       Additional mice used in these studies include Rag dec fi ient mice (Rag1; CD4 , CD8 , CD19 , SiglecF , FcR1 , NK1.1 ), FSC/SSClowto + + + Jackson Laboratories; (Mombaerts et al., 1992) or Rag2 (Taconic Farms moderate, CD45 , CD127 (IL7Ra) or thy1.2 (CD90.2) , and T1/ST2 + + + + RAGN12), Rag2 x  deficient mice (Taconic Farms 4111M), eosinophil (IL33R) or CD25 (IL2Ra) or KLRG1 , as indicated. CD4 T cells are + + + + deficient dblGATA mice (Yu et al., 2002), IL 4/ 13–deficient mice identified as FSC/SSClo, CD45 , CD3 , CD4 . CD8 T cells are identi + + + (McKenzie et al., 1999), Stat6 deficient mice (The Jackson Laboratory; fied as FSC/SSClo, CD45 , CD3 , and CD8 . Eosinophils are identified + + + Kaplan et al., 1996), IL7R dec fi ient mice (The Jackson Laboratory; Peschon as CD45 , sidescatter high, DAPIlo, CD11b , and SiglecF . Adipose tissue + + + et al., 1994), and T1/ST2 dec fi ient mice (Hoshino et al., 1999), and were macrophages are identie fi d as CD45 , CD11b , F4/80 , SiglecF lo. All pop crossed onto cytokine reporter alleles where designated. Mice used for these ulations were routinely back gated to verify purity and gating. Samples were experiments were male animals fully backcrossed on C57BL/6 or BALB/c analyzed on an LSR II (BD). For cell sorting, a FACS AriaII was used. Live backgrounds, as designated. Mice were maintained in the University of Cali lymphocytes were gated by DAPI exclusion, size, and granularity based on fornia San Francisco specic fi pathogen–free animal facility, and all animal pro forward and sidescatter. Data were analyzed using FlowJo software (Tree tocols were approved by and in accordance with the guidelines established by Star) and compiled using Prism (GraphPad Software). As indicated, VAT the Institutional Animal Care and Use Committee and Laboratory Animal data were normalized per gram of adipose or as a percentage of total viable Resource Center. cells or percentage of CD45 hematopoietic cells, as indicated. Cell culture and cytokine analysis. VAT from 8–15 WT C57BL/6 or Tissue preparation. Perigonadal adipose tissue was used as representative Red5 (R/+) animals was pooled for cell sorting. Sorted ILC2s or CD4 VAT in all experiments. Testicles were removed and tissue was kept on ice in T cells were transferred to 96well plates in 100 l of cRMPI at 1,500 cells 0.5 ml of adipose digestion medium (low glucose DMEM, 0.2 M Hepes, and per well. Cytokines were added to culture media at 10 ng/ml, as indicated. 10 mg/ml fatty acid poor BSA [Sigma]). VAT was n fi ely minced with a mul PMA was used at 40 ng/ml and ionomycin was used at 500 ng/ml. Human tiple razor blades, dispersed by shaking into 10 ml of adipose digestion me IL2 was used at 10 U/ml, and all other cytokines were purchased from dium containing 0.2 mg/ml Liberase Tm (Roche) and 25 µg/ml DNase I R&D Systems. After 72 h of culture, supernatant cytokine levels were ana (Roche) at 37°C for 30–40 min with gentle agitation, and passed through lyzed by cytokine bead arrays (BD) per the manufacturer’s instructions. 100 µm filters to generate single cell suspensions. Filters were washed with 10 ml FACS bue ff r (PBS, 3% FCS, 0.05% NaN ) and supernatants pooled. Cells were centrifuged at 1,000 g for 10 min and the cell pellets were resus Metabolic assays and diet. Male mice were fed normal chow diet (Mouse pended in 5 ml FACS bue ff r, transferred to fresh tubes and centrifuged at diet 20; PicoLab) and used between 8 and 15 wk of age, unless otherwise 1,500 rpm for 5 min. The red blood cells were lysed using PharmLyse (BD) noted. Where indicated, C57BL/6 WT mice were fed HFD D12492 (60% for 1 min, and the remaining cells were washed with FACS bue ff r, incubated kcal fat; Research Diets, Inc.) for 12–24 wk as noted. To measure animal with FcBlock, and stained with the indicated antibodies. adiposity and lean mass, MRI was performed using an EchoMRI 3 in1 Spleen was prepared by mashing tissue through 70 µm lfi ters without tis machine according to the manufacturer’s instructions (Echo Medical Sys sue digestion, and processing similar to VAT. Bone marrow was prepared by tems LTD). Glucose tolerance testing was performed after fasting mice carefully dissecting one femur and tibia, liberating hematopoietic cells with overnight for 14 h and challenging with 1.5 g/kg glucose by i.p. injection. mortar and pestle into 10 ml FACS bue ff r, passing through a 70 µm lfi ter, and Fasting blood glucose was measured after a 4h morning fast. Insulin toler processing similar to VAT. Whole lung was prepared by harvesting both lung ance tests were performed after a 4–5 h morning fast, injecting insulin i.p. lobes into 5 ml DMEM media with 0.2 mg/ml Liberase Tm and 25 g/ml (0.75 mU/g human insulin; Eli Lilly), and measuring blood glucose at the DNase 1, followed by tissue dissociation (GentleMacs; Miltenyi Biotec) using times indicated. Blood glucose was measured at indicated times using a glu the “lung1 program,” followed by tissue digestion for 30 min at 37°C with cometer (Bayer). Wholeanimal metabolic analysis was performed using gentle agitation. Samples were processed on the GentleMacs using the “lung2” CLAMS cages (Comprehensive Laboratory Animals Monitoring System) per program, passed through 70 µm lfi ters, and processed as described for VAT. the manufacturer’s instructions (Columbus Instruments). In brief, animals were singly housed and measurements were taken every 12 min for 4 d, including oxygen consumption, carbon dioxide output, food consumption, Flow cytometry. Monoclonal antibodies used for flow cytometry were as water consumption, and three unique measures of movement. Respiratory follows: allophycocyanin (APC) eFluor 780 anti CD4 (RM4 5; eBioscience); exchange ratio and heat were calculated as VCO /VO (RER) and VO (3.815 + 2 2 2 Qdot605antiCD4 (S3.5, Invitrogen); phycoerythrin (PE)antiSiglecF 1.232 × RER; heat), respectively. Heat, VO , and VCO were all normal 2 2 (E502440; BD); APC or Brilliant Violet 650– or Pacific Blue (PB)– anti 0.75 ized to effective body mass Vxx = Vxx/[(weight(g)/mass unit)] , per the CD11b (M1/70; BioLegend); PECy7 or PerCPCy5.5 antiF4/80 (BM8; manufacturer’s recommendations. eBioscience); biotinantipanNK (CD49b; DX5; eBioscience), PB anti NK1.1 (PK136; BioLegend), biotin anti FcRI (MAR 1; eBioscience); PB Helminth infection and cytokine administration. 500 third stage larvae or Alexa Fluor 488– or PerCPCy5.5 anti CD3e (17A2; BioLegend); of N. brasiliensis, purie fi d as described, were injected subcutaneously into mice PB anti CD8 (53–6.7; BioLegend); PerCPCy5.5 anti CD19 (1D3; BD); PB (Voehringer et al., 2006). Mice were killed at the indicated time points and anti CD19 (6D5; BioLegend), APC or PE anti CD25 (IL2R, PC61; tissues were harvested and analyzed as previously described (Reese et al., 2007; BioLegend); PerCPCy5.5 or PECy7antiCD127 (IL7R , A7R34; eBio Wu et al., 2011). IL 33 (R&D Systems) was given as 500 ng in 0.2 ml PBS i.p. science); Biotin anti T1/ST2 (DT8; MD Biosciences); APC or APC Cy7 anti CD45 (30 F11; BioLegend). Secondary u fl orophore for biotin conjugated antibodies were eF605 (eBioscience), APC (BD), or FITC (BD). CD1d aGC Microscopy. 5 min before sacrifice, animals were injected with 20 µg loaded and unloaded tetramer (PE or APC) were obtained from the National CD31APC (clone 390; eBioscience) to label vasculature. After VAT re Institutes of Health tetramer facility. PECy7 FoxP3 (FJK16S; eBioscience) moval, the tissue was immediately mounted and examined by laserscanning and A647 Gata3 (TWAJ; eBioscience) were used after first using a fixable confocal microscopy (Nikon C1si). Images were resolved to 1.2 µm/pixel in live/dead stain (Invitrogen), and then fixing and permeabilizing cells per the the xy plane and 1.0 µm in the z plane. JEM Vol. 210, No. 3 547 Statistical analysis. Unless otherwise noted, significance was determined allergeninduced airway inflammation. Immunity. 36:451–463. http:// by the Student’s t test, with P < 0.05 considered significant. *, P < 0.05; dx.doi.org/10.1016/j.immuni.2011.12.020 Hoshino, K., S. Kashiwamura, K. Kuribayashi, T. Kodama, T. Tsujimura, K. **, P < 0.01; ***, P < 0.001. Error bars represent standard error of the mean. Nakanishi, T. Matsuyama, K. Takeda, and S. Akira. 1999. The absence Each data point represents one animal. When possible, data from multiple of interleukin 1 receptorrelated T1/ST2 does not affect T helper cell independent experiments were pooled, as indicated. In cases with multiple type 2 development and its effector function. J. Exp. Med. 190:1541– comparisons within an experiment (>2), a onetailed ANOVA was per 1548. http://dx.doi.org/10.1084/jem.190.10.1541 formed with Tukey’s posttest correction. Hoyler, T., C.S.N. Klose, A. Souabni, A. TurquetiNeves, D. Pfeifer, E.L. Rawlins, D. Voehringer, M. Busslinger, and A. Diefenbach. 2012. We thank Drs. A. August, A. McKenzie, M. Steinhoff, and S. Wirtz for mice, The transcription factor GATA3 controls cell fate and maintenance A. DeFranco, C. Lowell and A. Ma for helpful comments on the manuscript, of type 2 innate lymphoid cells. Immunity. 37:634–648. http://dx.doi Zhi-En Wang for technical assistance, the Diabetes Endocrinology Research Core .org/10.1016/j.immuni.2012.06.020 for assistance with CLAMS analysis, and N. Flores and M. Consengco for Hurst, S.D., T. Muchamuel, D.M. Gorman, J.M. Gilbert, T. Clifford, S. animal support. Kwan, S. Menon, B. Seymour, C. Jackson, T.T. Kung, et al. 2002. New This work was supported by AI026918, AI030663, AI078869, HL107202, and IL17 family members promote Th1 or Th2 responses in the lung: in P30 DK063720 from the National Institutes of Health, the UCSF Diabetes Family vivo function of the novel cytokine IL25. J. Immunol. 169:443–453. Fund (ABM), the Department of Laboratory Medicine discretionary fund (ABM), the Ikutani, M., T. Yanagibashi, M. Ogasawara, K. Tsuneyama, S. Yamamoto, Larry Hillblom Foundation, the Sandler Asthma Basic Research Center at UCSF and Y. Hattori, T. Kouro, A. Itakura, Y. Nagai, S. Takaki, and K. Takatsu. the Howard Hughes Medical Institute. 2012. Identification of innate IL5producing cells and their role in lung The authors have no conflicting financial interests. eosinophil regulation and antitumor immunity. J. Immunol. 188:703– 713. http://dx.doi.org/10.4049/jimmunol.1101270 Submitted: 30 August 2012 Ji, Y., S. Sun, A. Xu, P. Bhargava, L. Yang, K.S.L. Lam, B. Gao, C.H. Accepted: 30 January 2013 Lee, S. Kersten, and L. Qi. 2012a. Activation of natural killer T cells promotes M2 Macrophage polarization in adipose tissue and improves systemic glucose tolerance via interleukin4 (IL4)/STAT6 protein sig REFERENCES naling axis in obesity. J. Biol. Chem. 287:13561–13571. http://dx.doi Bartemes, K.R., K. Iijima, T. Kobayashi, G.M. Kephart, A.N. McKenzie, .org/10.1074/jbc.M112.350066 and H. Kita. 2012. IL33responsive lineage CD25+ CD44(hi) lym Ji, Y., S. Sun, S. Xia, L. Yang, X. Li, and L. Qi. 2012b. Short term high phoid cells mediate innate type 2 immunity and allergic inflammation fat diet challenge promotes alternative macrophage polarization in adi in the lungs. J. Immunol. 188:1503–1513. http://dx.doi.org/10.4049/ pose tissue via natural killer T cells and interleukin4. J. Biol. Chem. jimmunol.1102832 287:24378–24386. http://dx.doi.org/10.1074/jbc.M112.371807 Blanchard, C., and M.E. Rothenberg. 2009. Biology of the eosinophil. Adv. Kang, K., S.M. Reilly, V. Karabacak, M.R. Gangl, K. Fitzgerald, B. Immunol. 101:81–121. http://dx.doi.org/10.1016/S0065 2776(08)01003 1 Hatano, and C. H. Lee. 2008. Adipocyte derived Th2 cytokines Brickshawana, A., V.S. Shapiro, H. Kita, and L.R. Pease. 2011. Lineage and myeloid PPARdelta regulate macrophage polarization and in ( )Sca1+c Kit( )CD25+ cells are IL 33 responsive type 2 innate cells in sulin sensitivity. Cell Metab. 7:485–495. http://dx.doi.org/10.1016/ the mouse bone marrow. J. Immunol. 187:5795–5804. http://dx.doi.org/ j.cmet.2008.04.002 10.4049/jimmunol.1102242 Kaplan, M.H., U. Schindler, S.T. Smiley, and M.J. Grusby. 1996. Stat6 is Brunner, S.M., G. Schiechl, W. Falk, H.J. Schlitt, E.K. Geissler, and S. required for mediating responses to IL4 and for development of Th2 FichtnerFeigl. 2011. Interleukin33 prolongs allograft survival during cells. Immunity. 4:313–319. http://dx.doi.org/10.1016/S10747613(00) chronic cardiac rejection. Transpl. Int. 24:1027–1039. http://dx.doi 80439 2 .org/10.1111/j.14322277.2011.01306. x Klein Wolterink, R.G., A. Kleinjan, M. van Nimwegen, I. Bergen, M. de Chang, Y.J., H.Y. Kim, L.A. Albacker, N. Baumgarth, A.N.J. McKenzie, Bruijn, Y. Levani, and R.W. Hendriks. 2012. Pulmonary innate lym D.E. Smith, R.H. Dekruyff, and D.T. Umetsu. 2011. Innate lymphoid phoid cells are major producers of IL5 and IL13 in murine models cells mediate influenzainduced airway hyperreactivity independently of allergic asthma. Eur. J. Immunol. 42:1106–1116. http://dx.doi.org/ of adaptive immunity. Nat. Immunol. 12:631–638. http://dx.doi.org/10 10.1002/eji.201142018 .1038/ni.2045 Kopf, M., F. Brombacher, P.D. Hodgkin, A.J. Ramsay, E.A. Milbourne, Chawla, A., K.D. Nguyen, and Y.P.S. Goh. 2011. Macrophagemediated W.J. Dai, K.S. Ovington, C.A. Behm, G. Köhler, I.G. Young, and inflammation in metabolic disease. Nat. Rev. Immunol. 11:738–749. K.I. Matthaei. 1996. IL5deficient mice have a developmental de http://dx.doi.org/10.1038/nri3071 fect in CD5+ B1 cells and lack eosinophilia but have normal anti Chen, F., Z. Liu, W. Wu, C. Rozo, S. Bowdridge, A. Millman, N. Van body and cytotoxic T cell responses. Immunity. 4:15–24. http://dx.doi Rooijen, J.F. Urban Jr., T.A. Wynn, and W.C. Gause. 2012. An essen .org/10.1016/S10747613(00)80294 0 tial role for TH2type responses in limiting acute tissue damage during Liang, H.E., R.L. Reinhardt, J.K. Bando, B.M. Sullivan, I.C. Ho, and experimental helminth infection. Nat. Med. 18:260–266. http://dx.doi R.M. Locksley. 2012. Divergent expression patterns of IL4 and IL13 .org/10.1038/nm.2628 define unique functions in allergic immunity. Nat. Immunol. 13:58–66. Feuerer, M., L. Herrero, D. Cipolletta, A. Naaz, J. Wong, A. Nayer, J. Lee, http://dx.doi.org/10.1038/ni.2182 A.B. Goldfine, C. Benoist, S. Shoelson, and D. Mathis. 2009. Lean, but Lucas, S., S. Taront, C. Magnan, L. Fauconnier, M. Delacre, L. Macia, not obese, fat is enriched for a unique population of regulatory T cells A. Delanoye, C. Verwaerde, C. Spriet, P. Saule, et al. 2012. Interleukin7 that affect metabolic parameters. Nat. Med. 15:930–939. http://dx.doi regulates adipose tissue mass and insulin sensitivity in high fat diet .org/10.1038/nm.2002 fed mice through lymphocyte dependent and independent mecha Fort, M.M., J. Cheung, D. Yen, J. Li, S.M. Zurawski, S. Lo, S. Menon, T. nisms. PLoS ONE. 7:e40351. http://dx.doi.org/10.1371/journal.pone Clifford, B. Hunte, R. Lesley, et al. 2001. IL25 induces IL4, IL5, and .0040351 IL13 and Th2associated pathologies in vivo. Immunity. 15:985–995. Lynch, L., M. Nowak, B. Varghese, J. Clark, A.E. Hogan, V. Toxavidis, http://dx.doi.org/10.1016/S10747613(01)00243 6 S.P. Balk, D. O’Shea, C. O’Farrelly, and M.A. Exley. 2012. Adipose Foster, P.S., S.P. Hogan, A.J. Ramsay, K.I. Matthaei, and I.G. Young. 1996. tissue invariant NKT cells protect against dietinduced obesity and Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, metabolic disorder through regulatory cytokine production. Immunity. and lung damage in a mouse asthma model. J. Exp. Med. 183:195–201. 37:574–587. http://dx.doi.org/10.1016/j.immuni.2012.06.016 http://dx.doi.org/10.1084/jem.183.1.195 Madisen, L., T.A. Zwingman, S.M. Sunkin, S.W. Oh, H.A. Zariwala, H. Halim, T.Y.F., R.H. Krauss, A.C. Sun, and F. Takei. 2012. Lung natural Gu, L.L. Ng, R.D. Palmiter, M.J. Hawrylycz, A.R. Jones, et al. 2010. A helper cells are a critical source of Th2 celltype cytokines in protease robust and highthroughput Cre reporting and characterization system 548 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e for the whole mouse brain. Nat. Neurosci. 13:133–140. http://dx.doi in type 2 immunity. Proc. Natl. Acad. Sci. USA. 107:11489–11494. .org/10.1038/nn.2467 http://dx.doi.org/10.1073/pnas.1003988107 Martinez, F.O., L. Helming, and S. Gordon. 2009. Alternative activa Reese, T.A., H.E. Liang, A.M. Tager, A.D. Luster, N. Van Rooijen, D. tion of macrophages: an immunologic functional perspective. Annu. Voehringer, and R.M. Locksley. 2007. Chitin induces accumulation in Rev. Immunol. 27:451–483. http://dx.doi.org/10.1146/annurev.immunol tissue of innate immune cells associated with allergy. Nature. 447:92–96. .021908.132532 http://dx.doi.org/10.1038/nature05746 McKenzie, G.J., P.G. Fallon, C.L. Emson, R.K. Grencis, and A.N. McKenzie. Ricardo Gonzalez, R.R., A. Red Eagle, J.I. Odegaard, H. Jouihan, C.R. 1999. Simultaneous disruption of interleukin (IL) 4 and IL 13 de Morel, J.E. Heredia, L. Mukundan, D. Wu, R.M. Locksley, and A. fines individual roles in T helper cell type 2–mediated responses. Chawla. 2010. IL 4/STAT6 immune axis regulates peripheral nutri J. Exp. Med. 189:1565–1572. http://dx.doi.org/10.1084/jem.189.10 ent metabolism and insulin sensitivity. Proc. Natl. Acad. Sci. USA. .1565 107:22617–22622. http://dx.doi.org/10.1073/pnas.1009152108 Miller, A.M., D.L. Asquith, A.J. Hueber, L.A. Anderson, W.M. Holmes, Rothenberg, M.E., and S.P. Hogan. 2006. The eosinophil. Annu. Rev. Immunol. A.N. McKenzie, D. Xu, N. Sattar, I.B. McInnes, and F.Y. Liew. 2010. 24:147–174. http://dx.doi.org/10.1146/annurev.immunol.24.021605 Interleukin33 induces protective effects in adipose tissue inflamma .090720 tion during obesity in mice. Circ. Res. 107:650–658. http://dx.doi.org/ Schipper, H.S., B. Prakken, E. Kalkhoven, and M. Boes. 2012. Adipose 10.1161/CIRCRESAHA.110.218867 tissueresident immune cells: key players in immunometabolism. Trends Mishra, A., S.P. Hogan, J.J. Lee, P.S. Foster, and M.E. Rothenberg. 1999. Endocrinol. Metab. 23:407–415. http://dx.doi.org/10.1016/j.tem.2012 Fundamental signals that regulate eosinophil homing to the gastrointes .05.011 tinal tract. J. Clin. Invest. 103:1719–1727. http://dx.doi.org/10.1172/ Schmitz, J., A. Owyang, E. Oldham, Y. Song, E. Murphy, T.K. McClanahan, G. JCI6560 Zurawski, M. Moshrefi, J. Qin, X. Li, et al. 2005. IL33, an interleukin Mjösberg, J.M., S. Trifari, N.K. Crellin, C.P. Peters, C.M. van Drunen, B. 1like cytokine that signals via the IL1 receptorrelated protein ST2 Piet, W.J. Fokkens, T. Cupedo, and H. Spits. 2011. Human IL25 and and induces T helper type 2associated cytokines. Immunity. 23:479–490. IL33responsive type 2 innate lymphoid cells are defined by expression http://dx.doi.org/10.1016/j.immuni.2005.09.015 of CRTH2 and CD161. Nat. Immunol. 12:1055–1062. http://dx.doi Spits, H., and J.P. Di Santo. 2011. The expanding family of innate lymphoid .org/10.1038/ni.2104 cells: regulators and effectors of immunity and tissue remodeling. Nat. Mohrs, M., K. Shinkai, K. Mohrs, and R.M. Locksley. 2001. Analysis of Immunol. 12:21–27. http://dx.doi.org/10.1038/ni.1962 type 2 immunity in vivo with a bicistronic IL4 reporter. Immunity. Turcot, V., L. Bouchard, G. Faucher, V. Garneau, A. Tchernof, Y. Deshaies, 15:303–311. http://dx.doi.org/10.1016/S10747613(01)00186 8 L. Pérusse, S. Marceau, S. Biron, O. Lescelleur, et al. 2012. Thymic Mohrs, K., A.E. Wakil, N. Killeen, R.M. Locksley, and M. Mohrs. 2005. stromal lymphopoietin: an immune cytokine gene associated with the A two step process for cytokine production revealed by IL 4 dual metabolic syndrome and blood pressure in severe obesity. Clin. Sci. reporter mice. Immunity. 23:419–429. http://dx.doi.org/10.1016/j 123:99–109. http://dx.doi.org/10.1042/CS20110584 .immuni.2005.09.006 Turnquist, H.R., Z. Zhao, B.R. Rosborough, Q. Liu, A. Castellaneta, K. Mombaerts, P., J. Iacomini, R.S. Johnson, K. Herrup, S. Tonegawa, and Isse, Z. Wang, M. Lang, D.B. Stolz, X.X. Zheng, et al. 2011. IL33 ex V.E. Papaioannou. 1992. RAG 1 dec fi ient mice have no mature B and pands suppressive CD11b+ Gr1(int) and regulatory T cells, including T lymphocytes. Cell. 68:869–877. http://dx.doi.org/10.1016/0092 8674 ST2L+ Foxp3+ cells, and mediates regulatory T celldependent promo (92)90030 G tion of cardiac allograft survival. J. Immunol. 187:4598–4610. http:// Monticelli, L.A., G.F. Sonnenberg, M.C. Abt, T. Alenghat, C.G.K. dx.doi.org/10.4049/jimmunol.1100519 Ziegler, T.A. Doering, J.M. Angelosanto, B.J. Laidlaw, C.Y. Yang, T. Vasudevan, A.R., H. Wu, A.M. Xydakis, P.H. Jones, E.O. Smith, J.F. Sathaliyawala, et al. 2011. Innate lymphoid cells promote lungtissue Sweeney, D.B. Corry, and C.M. Ballantyne. 2006. Eotaxin and obe homeostasis after infection with influenza virus. Nat. Immunol. 12:1045– sity. J. Clin. Endocrinol. Metab. 91:256–261. http://dx.doi.org/10.1210/ 1054. http://dx.doi.org/10.1038/ni.2131 jc.2005128 0 Moro, K., T. Yamada, M. Tanabe, T. Takeuchi, T. Ikawa, H. Kawamoto, Voehringer, D., T.A. Reese, X. Huang, K. Shinkai, and R.M. Locksley. J.I. Furusawa, M. Ohtani, H. Fujii, and S. Koyasu. 2010. Innate pro 2006. Type 2 immunity is controlled by IL 4/IL 13 expression in he duction of T(H)2 cytokines by adipose tissueassociated cKit(+)Sca matopoietic noneosinophil cells of the innate immune system. J. Exp. 1(+) lymphoid cells. Nature. 463:540–544. http://dx.doi.org/10.1038/ Med. 203:1435–1446. http://dx.doi.org/10.1084/jem.20052448 nature08636 Voehringer, D., N. van Rooijen, and R.M. Locksley. 2007. Eosinophils Mould, A.W., K.I. Matthaei, I.G. Young, and P.S. Foster. 1997. Relationship develop in distinct stages and are recruited to peripheral sites by alter between interleukin5 and eotaxin in regulating blood and tissue eo natively activated macrophages. J. Leukoc. Biol. 81:1434–1444. http:// sinophilia in mice. J. Clin. Invest. 99:1064–1071. http://dx.doi.org/ dx.doi.org/10.1189/jlb.1106686 10.1172/JCI119234 Voehringer, D., H. E. Liang, and R.M. Locksley. 2008. Homeostasis and Neill, D.R., S.H. Wong, A. Bellosi, R.J. Flynn, M. Daly, T.K.A. effector function of lymphopenia induced “memory like” T cells in Langford, C. Bucks, C.M. Kane, P.G. Fallon, R. Pannell, et al. constitutively T cell depleted mice. J. Immunol. 180:4742–4753. 2010. Nuocytes represent a new innate effector leukocyte that me Wu, D., A.B. Molofsky, H.E. Liang, R.R. RicardoGonzalez, H.A. Jouihan, diates type 2 immunity. Nature. 464:1367–1370. http://dx.doi.org/ J.K. Bando, A. Chawla, and R.M. Locksley. 2011. Eosinophils sustain 10.1038/nature08900 adipose alternatively activated macrophages associated with glucose homeo Nguyen, K.D., Y. Qiu, X. Cui, Y.P.S. Goh, J. Mwangi, T. David, L. stasis. Science. 332:243–247. http://dx.doi.org/10.1126/science.1201475 Mukundan, F. Brombacher, R.M. Locksley, and A. Chawla. 2011. Yu, C., A.B. Cantor, H. Yang, C. Browne, R.A. Wells, Y. Fujiwara, and Alternatively activated macrophages produce catecholamines to sus S.H. Orkin. 2002. Targeted deletion of a high an ffi ity GATA binding tain adaptive thermogenesis. Nature. 480:104–108. http://dx.doi.org/10 site in the GATA1 promoter leads to selective loss of the eosinophil .1038/nature10653 lineage in vivo. J. Exp. Med. 195:1387–1395. http://dx.doi.org/10 Peschon, J.J., P.J. Morrissey, K.H. Grabstein, F.J. Ramsdell, E. Maraskovsky, .1084/jem.20020656 B.C. Gliniak, L.S. Park, S.F. Ziegler, D.E. Williams, C.B. Ware, et al. Zeyda, M., B. Wernly, S. Demyanets, C. Kaun, M. Hämmerle, B. Hantusch, M. 1994. Early lymphocyte expansion is severely impaired in interleukin 7 Schranz, A. Neuhofer, B.K. Itariu, M. Keck, et al. 2012. Severe obesity in receptordeficient mice. J. Exp. Med. 180:1955–1960. http://dx.doi creases adipose tissue expression of interleukin33 and its receptor ST2, .org/10.1084/jem.180.5.1955 both predominantly detectable in endothelial cells of human adipose Price, A.E., H. E. Liang, B.M. Sullivan, R.L. Reinhardt, C.J. Eisley, D.J. Erle, and tissue. Int J Obes (Lond). Epub before print. http://www.nature.com/ijo/ R.M. Locksley. 2010. Systemically dispersed innate IL 13 expressing cells journal/vaop/ncurrent/full/ijo2012118a.html. JEM Vol. 210, No. 3 549 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central http://www.deepdyve.com/lp/pubmed-central/innate-lymphoid-type-2-cells-sustain-visceral-adipose-tissue-KShivCv7eL

Loading next page...

References (63)

J. Exp. Med
L. Madisen, T. Zwingman, S. Sunkin, S. Oh, Hatim Zariwala, Hong Gu, Lydia Ng, R. Palmiter, M. Hawrylycz, Allan Jones, E. Lein, Hongkui Zeng (2009)
A robust and high-throughput Cre reporting and characterization system for the whole mouse brain
Nature neuroscience, 13
A. Mishra, S. Hogan, James Lee, P. Foster, M. Rothenberg (1999)
Fundamental signals that regulate eosinophil homing to the gastrointestinal tract.
The Journal of clinical investigation, 103 12
H. Turnquist, Zhen-Min Zhao, B. Rosborough, Quan Liu, A. Castellaneta, K. Isse, Zhiliang Wang, Megan Lang, D. Stolz, X. Zheng, A. Demetris, Fooyew Liew, Kathryn Wood, Angus Thomson (2011)
IL-33 Expands Suppressive CD11b+ Gr-1int and Regulatory T Cells, including ST2L+ Foxp3+ Cells, and Mediates Regulatory T Cell-Dependent Promotion of Cardiac Allograft Survival
The Journal of Immunology, 187
T. Halim, Ramona Krauss, A. Sun, F. Takei (2012)
Lung natural helper cells are a critical source of Th2 cell-type cytokines in protease allergen-induced airway inflammation.
Immunity, 36 3
Hong-Erh Liang, R. Reinhardt, Jennifer Bando, B. Sullivan, I. Ho, R. Locksley (2011)
Divergent expression patterns of IL-4 and IL-13 define unique functions in allergic immunity
Nature immunology, 13
Roel Wolterink, A. KleinJan, M. Nimwegen, I. Bergen, Marjolein Bruijn, Yelvi Levani, R. Hendriks (2012)
Pulmonary innate lymphoid cells are major producers of IL‐5 and IL‐13 in murine models of allergic asthma
European Journal of Immunology, 42
M. Mohrs, K. Shinkai, K. Mohrs, R. Locksley (2001)
Analysis of type 2 immunity in vivo with a bicistronic IL-4 reporter.
Immunity, 15 2
M. Kaplan, U. Schindler, S. Smiley, M. Grusby (1996)
Stat6 is required for mediating responses to IL-4 and for development of Th2 cells.
Immunity, 4 3
Blanchard (2009)
Biology of the eosinophil
Adv. Immunol., 101
F. Martínez, L. Helming, S. Gordon (2009)
Alternative activation of macrophages: an immunologic functional perspective.
Annual review of immunology, 27
(2012)
IL33responsive lineage CD25+ CD44(hi) lym
茂呂和世 (2010)
Innate production of T[H]2 cytokines by adipose tissue-associated c-Kit[+]Sca-1[+] lymphoid cells
C. Blanchard, M. Rothenberg (2014)
The Eosinophil : from an hematopoietic stem cell to a mature eosinophil
Yewei Ji, Shengyi Sun, A. Xu, Prerna Bhargava, Liu Yang, K. Lam, B. Gao, Chih‐Hao Lee, S. Kersten, Ling Qi (2012)
Activation of Natural Killer T Cells Promotes M2 Macrophage Polarization in Adipose Tissue and Improves Systemic Glucose Tolerance via Interleukin-4 (IL-4)/STAT6 Protein Signaling Axis in Obesity*
The Journal of Biological Chemistry, 287
April Price, Hong-Erh Liang, B. Sullivan, R. Reinhardt, C. Eisley, D. Erle, R. Locksley (2010)
Systemically dispersed innate IL-13–expressing cells in type 2 immunity
Proceedings of the National Academy of Sciences, 107
Philip Morrissey, K. Grabstein, Fred Ramsde, Eugene Maraskovsky, Brian Gliniak, Linda Park, Steven Ziegler, Doughs Williams, Carol Ware, Jeff Meyer, Barry Davison (1994)
Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice
The Journal of Experimental Medicine, 180
A. Miller, D. Asquith, A. Hueber, L. Anderson, W. Holmes, A. Mckenzie, Damo Xu, N. Sattar, I. McInnes, F. Liew (2010)
Interleukin-33 Induces Protective Effects in Adipose Tissue Inflammation During Obesity in Mice
Circulation Research, 107
L. Monticelli, Gregory Sonnenberg, Michael Abt, T. Alenghat, Carly Ziegler, Travis Doering, Jill Angelosanto, B. Laidlaw, Cliff Yang, Taheri Sathaliyawala, Masaru Kubota, D. Turner, J. Diamond, A. Goldrath, D. Farber, R. Collman, E. Wherry, D. Artis (2011)
Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus
Nature Immunology, 12
R. Ricardo-Gonzalez, Alex Eagle, J. Odegaard, H. Jouihan, Christine Morel, J. Heredia, Lata Mukundan, Davina Wu, R. Locksley, A. Chawla (2010)
IL-4/STAT6 immune axis regulates peripheral nutrient metabolism and insulin sensitivity
Proceedings of the National Academy of Sciences, 107
S. Hurst, T. Muchamuel, D. Gorman, J. Gilbert, T. Clifford, Sylvia Kwan, S. Menon, B. Seymour, Craig Jackson, T. Kung, J. Brieland, S. Zurawski, R. Chapman, G. Zurawski, R. Coffman (2002)
New IL-17 Family Members Promote Th1 or Th2 Responses in the Lung: In Vivo Function of the Novel Cytokine IL-251
The Journal of Immunology, 169
A. Chawla, K. Nguyen, Y. Goh (2011)
Macrophage-mediated inflammation in metabolic disease
Nature Reviews Immunology, 11
D. Voehringer, Hong-Erh Liang, R. Locksley (2008)
Homeostasis and Effector Function of Lymphopenia-Induced “Memory-Like” T Cells in Constitutively T Cell-Depleted Mice1
The Journal of Immunology, 180
K. Mohrs, A. Wakil, N. Killeen, R. Locksley, M. Mohrs (2005)
A two-step process for cytokine production revealed by IL-4 dual-reporter mice.
Immunity, 23 4
J. Mjösberg, S. Trifari, N. Crellin, C. Peters, C. Drunen, B. Piet, W. Fokkens, T. Cupedo, H. Spits (2011)
Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161
Nature Immunology, 12
Adipong Brickshawana, V. Shapiro, H. Kita, L. Pease (2011)
Lineage−Sca1+c-Kit−CD25+ Cells Are IL-33–Responsive Type 2 Innate Cells in the Mouse Bone Marrow
The Journal of Immunology, 187
Tiffany Reese, Hong-Erh Liang, A. Tager, A. Luster, N. Rooijen, D. Voehringer, R. Locksley (2007)
Chitin induces accumulation in tissue of innate immune cells associated with allergy
Nature, 447
P. Mombaerts, J. Iacomini, R. Johnson, K. Herrup, S. Tonegawa, V. Papaioannou (1992)
RAG-1-deficient mice have no mature B and T lymphocytes
Cell, 68
K. Bartemes, K. Iijima, Takao Kobayashi, G. Kephart, A. McKenzie, H. Kita (2012)
IL-33–Responsive Lineage−CD25+CD44hi Lymphoid Cells Mediate Innate Type 2 Immunity and Allergic Inflammation in the Lungs
The Journal of Immunology, 188
Ya‐Jen Chang, Hye Kim, L. Albacker, N. Baumgarth, A. McKenzie, Dirk Smith, R. DeKruyff, D. Umetsu (2011)
Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity
Nature Immunology, 12
S. Brunner, G. Schiechl, W. Falk, H. Schlitt, E. Geissler, S. Fichtner-Feigl (2011)
Interleukin‐33 prolongs allograft survival during chronic cardiac rejection
Transplant International, 24
D. Voehringer, N. Rooijen, R. Locksley (2007)
Eosinophils develop in distinct stages and are recruited to peripheral sites by alternatively activated macrophages
Journal of Leukocyte Biology, 81
M. Zeyda, B. Wernly, S. Demyanets, C. Kaun, M. Hämmerle, B. Hantusch, Michael Schranz, A. Neuhofer, B. Itariu, Maike Keck, Gerald Prager, Johann Wojta, T. Stulnig (2013)
Severe obesity increases adipose tissue expression of interleukin-33 and its receptor ST2, both predominantly detectable in endothelial cells of human adipose tissue
International Journal of Obesity, 37
Rothenberg (2006)
The eosinophil
Annu. Rev. Immunol., 24
Channing Yu, A. Cantor, Haidi Yang, C. Browne, R. Wells, Y. Fujiwara, S. Orkin (2002)
Targeted Deletion of a High-Affinity GATA-binding Site in the GATA-1 Promoter Leads to Selective Loss of the Eosinophil Lineage In Vivo
The Journal of Experimental Medicine, 195
in type 2 immunity. Proc. Natl. Acad. Sci. USA
A. Vasudevan, Huaizhu Wu, Antonios Xydakis, Peter Jones, E. Smith, J. Sweeney, D. Corry, C. Ballantyne (2006)
Eotaxin and obesity.
The Journal of clinical endocrinology and metabolism, 91 1
airway inflammation. Immunity
L. Lynch, Michael Nowak, B. Varghese, Justice Clark, A. Hogan, Vasillis Toxavidis, S. Balk, D. O'Shea, C. O’Farrelly, M. Exley (2012)
Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production.
Immunity, 37 3
S. Lucas, S. Taront, C. Magnan, Laurence Fauconnier, M. Delacre, L. Macia, A. Delanoye, C. Verwaerde, C. Spriet, Pasquine Saule, Gautier Goormachtigh, L. Héliot, A. Ktorza, J. Movassat, R. Polakowska, C. Auriault, O. Poulain-Godefroy, J. Santo, P. Froguel, I. Wolowczuk (2012)
Interleukin-7 Regulates Adipose Tissue Mass and Insulin Sensitivity in High-Fat Diet-Fed Mice through Lymphocyte-Dependent and Independent Mechanisms
PLoS ONE, 7
Davina Wu, Ari Molofsky, Hong-Erh Liang, R. Ricardo-Gonzalez, H. Jouihan, Jennifer Bando, A. Chawla, R. Locksley (2011)
Eosinophils Sustain Adipose Alternatively Activated Macrophages Associated with Glucose Homeostasis
Science, 332
Fei Chen, Zhugong Liu, Wenhui Wu, C. Rozo, S. Bowdridge, Ariel Millman, N. Rooijen, J. Urban, T. Wynn, W. Gause (2011)
An essential role for the Th2-type response in limiting tissue damage during helminth infection
Nature medicine, 18
H. Schipper, B. Prakken, E. Kalkhoven, M. Boes (2012)
Adipose tissue-resident immune cells: key players in immunometabolism
Trends in Endocrinology & Metabolism, 23
Khoa Nguyen, Yifu Qiu, Xiaojin Cui, Y. Goh, J. Mwangi, Tovo David, Lata Mukundan, F. Brombacher, R. Locksley, A. Chawla (2011)
Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis
Nature, 480
V. Turcot, L. Bouchard, G. Faucher, V. Garneau, A. Tchernof, Y. Deshaies, L. Pérusse, S. Marceau, S. Biron, O. Lescelleur, L. Biertho, M. Vohl (2012)
Thymic stromal lymphopoietin: an immune cytokine gene associated with the metabolic syndrome and blood pressure in severe obesity.
Clinical science, 123 2
J. Schmitz, A. Owyang, E. Oldham, Yaoli Song, E. Murphy, T. Mcclanahan, G. Zurawski, M. Moshrefi, J. Qin, Xiaoxia Li, D. Gorman, J. Bazan, R. Kastelein, Schering-Plough Biopharma (2005)
IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines.
Immunity, 23 5
M. Ikutani, Tsutomu Yanagibashi, M. Ogasawara, K. Tsuneyama, Seiji Yamamoto, Y. Hattori, T. Kouro, A. Itakura, Y. Nagai, S. Takaki, K. Takatsu (2012)
Identification of Innate IL-5–Producing Cells and Their Role in Lung Eosinophil Regulation and Antitumor Immunity
The Journal of Immunology, 188
K. Kang, Shannon Reilly, Volkan Karabacak, Matthew Gangl, Kelly Fitzgerald, B. Hatano, Chih‐Hao Lee (2008)
Adipocyte-derived Th2 cytokines and myeloid PPARdelta regulate macrophage polarization and insulin sensitivity.
Cell metabolism, 7 6
H. Spits, J. Santo (2011)
The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling
Nature Immunology, 12
G. McKenzie, P. Fallon, C. Emson, R. Grencis, A. McKenzie (1999)
Simultaneous Disruption of Interleukin (IL)-4 and IL-13 Defines Individual Roles in T Helper Cell Type 2–mediated Responses
The Journal of Experimental Medicine, 189
T. Hoyler, Christoph Klose, A. Souabni, Adriana Turqueti‐Neves, D. Pfeifer, E. Rawlins, D. Voehringer, M. Busslinger, A. Diefenbach (2012)
The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells.
Immunity, 37 4
P. Foster, S. Hogan, A. Ramsay, K. Matthaei, I. Young (1996)
Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model
The Journal of Experimental Medicine, 183
K. Hoshino, S. Kashiwamura, K. Kuribayashi, T. Kodama, T. Tsujimura, K. Nakanishi, T. Matsuyama, K. Takeda, S. Akira (1999)
The Absence of Interleukin 1 Receptor–Related T1/St2 Does Not Affect T Helper Cell Type 2 Development and Its Effector Function
The Journal of Experimental Medicine, 190
M. Kopf, F. Brombacher, Philip Hodgkin, Alistair Ramsay, Elizabeth Milbourne, W. Dai, Karen Ovington, Carolyn Behm, G. Köhler, Ian Young, Klaus Matthaei (1996)
IL-5-deficient mice have a developmental defect in CD5+ B-1 cells and lack eosinophilia but have normal antibody and cytotoxic T cell responses.
Immunity, 4 1
M. Fort, Jeanne Cheung, David Yen, Joana Li, S. Zurawski, S. Lo, S. Menon, T. Clifford, B. Hunte, R. Lesley, T. Muchamuel, S. Hurst, G. Zurawski, M. Leach, D. Gorman, D. Rennick (2001)
IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo.
Immunity, 15 6
D. Neill, S. Wong, A. Bellosi, R. Flynn, M. Daly, Theresa Langford, C. Bucks, C. Kane, P. Fallon, R. Pannell, H. Jolin, A. McKenzie (2010)
Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity
Nature, 464
K. Moro, Taketo Yamada, M. Tanabe, T. Takeuchi, T. Ikawa, H. Kawamoto, J. Furusawa, M. Ohtani, H. Fujii, S. Koyasu (2009)
Innate production of TH2 cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells
Nature, 463
A. Mould, K. Matthaei, I. Young, P. Foster (1997)
Relationship between interleukin-5 and eotaxin in regulating blood and tissue eosinophilia in mice.
The Journal of clinical investigation, 99 5
M. Peters (1987)
The eosinophil.
Advances in dermatology, 2
R. Reinhardt, Hong-Erh Liang, B. Sullivan, R. Locksley (2011)
Divergent expression patterns of IL-4 and IL-13 define unique functions in type 2 immunity (56.22)
The Journal of Immunology
M. Feuerer, Laura Herrero, D. Cipolletta, A. Naaz, J. Wong, A. Nayer, Jongsoon Lee, A. Goldfine, C. Benoist, S. Shoelson, D. Mathis (2009)
Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters
Nature Medicine, 15
D. Voehringer, Tiffany Reese, Xiaozhu Huang, K. Shinkai, R. Locksley (2006)
Type 2 immunity is controlled by IL-4/IL-13 expression in hematopoietic non-eosinophil cells of the innate immune system
The Journal of Experimental Medicine, 203
Yewei Ji, Shengyi Sun, Sheng Xia, Liu Yang, Xiaoqing Li, Ling Qi (2012)
Short Term High Fat Diet Challenge Promotes Alternative Macrophage Polarization in Adipose Tissue via Natural Killer T Cells and Interleukin-4*
The Journal of Biological Chemistry, 287

Publisher: Pubmed Central
Copyright: © 2013 Molofsky et al.
ISSN: 0022-1007
eISSN: 1540-9538
DOI: 10.1084/jem.20121964
Publisher site: See Article on Publisher Site

Abstract

A r t i c l e Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages 3,4 2,3 1 Ari B. Molofsky, Jesse C. Nussbaum, Hong-Erh Liang, 1,3 5 3 Steven J. Van Dyken, Laurence E. Cheng, Alexander Mohapatra, 2,6 1,2,3 Ajay Chawla, and Richard M. Locksley 1 2 3 4 Howard Hughes Medical Institute, Departments of Medicine, Microbiology and Immunology, Laboratory Medicine, 5 6 Pediatrics and Physiology, University of California, San Francisco, San Francisco 94143 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 lym- phoid 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 signifi - cant reductions in VAT eosinophils and AAMs, and also impairs the expansion of VAT eosinophils after infection with Nippostrongylus brasiliensis, an intestinal parasite associ- ated 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. Diverse immune cells participate in the regu Prolonged VAT eosinophilia after helminth CORRESPONDENCE Richard M. Locksley: lation of visceral adipose tissue (VAT) and meta infection is also correlated with improved met [email protected] bolic homeostasis. With obesity, pro ina fl mmatory abolic parameters in animals challenged with + + macrophages, neutrophils, CD8 T cells, CD4 high fat diet (HFD; Wu et al., 2011). Eosino Abbreviations used: AAM, alternatively activated macro Th1 cells, and mast cells accumulate in VAT phil production, bone marrow release and tis phage; HFD, high fat diet; and contribute to local and systemic inflam sue recruitment and retention depend on several ILC2, innate lymphoid type 2 mation, ultimately promoting insulin resistance cytokines, chemokines, and integrins. IL 5 is cell; iNKT, invariant natural killer T cell; ND, normal diet; and the development of metabolic syndrome and integral at multiple levels, promoting eosino SVF, stromal vascular fraction; type 2 diabetes; in contrast, normal lean VAT phil bone marrow production, release, and tis VAT, visceral adipose tissue. contains eosinophils, alternatively activated mac sue recruitment, and is required for optimal rophages (AAM), invariant natural killer T cells systemic and local eosinophilia in diverse mod (iNKTs), and regulatory T (T reg) cells that can els of allergic inflammatory responses (Mould promote insulin sensitivity and metabolic homeo et al., 1997; Kopf et al., 1996; Foster et al., stasis (Chawla et al., 2011; Schipper et al., 2012; 1996). In contrast, IL 5 deficiency in unper Wu et al., 2011). How lean, healthy VAT recruits turbed animals leads to a modest reduction in and sustains these distinct immune cell types re bone marrow, blood, and gastrointestinal tract mains largely unknown. eosinophil levels, indicating eosinophil produc We previously reported that eosinophils tion and recruitment to certain tissues can occur reside in VAT and that eosinophil deficiency impairs Arginase 1 AAM accumulation. VAT © 2013 Molofsky et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after eosinophils are abundant in IL 5 transgenic mice the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share and promote AAM accumulation and insulin Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ sensitivity (Wu et al., 2011; Chawla et al., 2011). by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. 2013 Vol. 210 No. 3 535-549 www.jem.org/cgi/doi/10.1084/jem.20121964 The Journal of Experimental Medicine without IL5 (Mishra et al., 1999; Kopf et al., 1996). Eotax To control for potential genetic or microbiome contribu ins (CCL11 and CCL24) are chemokines that recruit eo tions to these phenotypes, we compared IL 5–decient fi Red5 sinophils, are central to eosinophil maintenance within the homozygote and IL5sufficient Red5 heterozygote mice. gastrointestinal tract, and can be upregulated by IL13 dur Eosinophildeficient and IL5deficient animals fed HFD for ing allergic inflammation (Mishra et al., 1999; Rothenberg 18–20 wk gained more weight (Fig. 1 a), with increased total and Hogan, 2006; Voehringer et al., 2007). Eosinophils also body adiposity (Fig. 1 b) and perigonadal VAT weight (Fig. 1 c), use endothelial cell integrins, which can be increased by IL4 as compared with IL5–sufficient mice. Fasting glucose levels and IL13, to traffic into tissues (Blanchard and Rothenberg, were elevated in both strains of mice (Fig. 1 d), and both had 2009). The relative dependence of VAT eosinophils on these impaired glucose (Fig. 1 e) and insulin tolerance (Fig. 1 f and factors, including IL4, IL5, and IL13, remains unknown. unpublished data). These findings support and extend our Innate lymphoid type 2 cells (ILC2s) are recently charac previous results (Wu et al., 2011) to implicate IL5 in meta terized innate cells widely distributed in mammalian tissues bolic homeostasis. (Spits and Di Santo, 2011). Also, designated innate helper To further understand the mechanisms by which eosino type 2 cells (Price et al., 2010), nuocytes (Neill et al., 2010), or phils and IL5 influence metabolism, we placed eosinophil natural helper cells (Moro et al., 2010), ILC2s share features dec fi ient and suc ffi ient animals on HFD for 12 wk in metabolic with other populations of innate lymphocytes, including NK cages. Although food and water intake and physical activity cells (ILC1) and ILC3, comprising the RORtdependent were not altered (unpublished data), total oxygen consump ILC: lymphoid tissueinducer cells (LTic), innate IL22 pro tion (VO ) and energy utilization (heat) were decreased in ducing cells (also referred to as NK22, ILC22, NCR22, and eosinophil deficient mice (Fig. 1, g and h); similar results NKR+ LTic) and innate IL 17 producing cells (Spits and occurred in IL5–deficient animals (unpublished data). Thus, Di Santo, 2011). ILCs all share a dependence on the transcrip eosinophils and IL5 do not alter caloric intake or caloric ex tion factor Id2 and the common  chain (c) cytokine re penditures by enhancing physical activity. Instead, they may ceptor (Spits and Di Santo, 2011). In response to the epithelial act in metabolically relevant tissue to promote increased oxi cytokines IL25 and IL33, ILC2s expand and produce large dative metabolism and limit inflammation. Consistent with amounts of type 2 cytokines, particularly IL 13 and IL 5 these findings, activation of iNKT IL4 production (Lynch (Hurst et al., 2002; Price et al., 2010; Moro et al., 2010; Neill et al., 2012; Ji et al., 2012a) or exogenous IL 4 administration et al., 2010), which can promote AAMs and eosinophils, re (RicardoGonzalez et al., 2010) each promoted loss of adi spectively (Blanchard and Rothenberg, 2009; Martinez et al., posity and insulin sensitivity. 2009). Although ILC2s are functionally similar to CD4 T helper type 2 (Th2) cells (Price et al., 2010), ILC2s are widely ILC2s are the major source of IL-5 and IL-13 in VAT distributed within tissues independent of antigenic stimula ILC2s have been implicated in promoting eosinophil influx tion and appear poised to respond to epithelial signals. One of into tissues such as the lung and intestines during allergic in the earliest descriptions of ILC2s identified them within lym flammation (Neill et al., 2010; Price et al., 2010; Liang et al., phoid structures in mouse and human mesenteric adipose tis 2012). We used flow cytometry to analyze perigonadal VAT sues (Moro et al., 2010). With this in mind, we sought to to ascertain a potential role for ILC2s in controlling eosino quantify ILC2s in metabolically active perigonadal VAT and phils in this tissue. Perigonadal adipose tissue was isolated and determine whether these cells and the cytokines they pro digested to yield the stromal vascular fraction (SVF) enriched duce, including IL 5 and IL 13, were responsible for the for hematopoietic cells, endothelial cells, and other stromal localization of eosinophils and AAMs to this tissue under basal components, but devoid of adipocytes. After using lineage conditions and after their activation by cytokines or in re markers to exclude B cells, T cells, and NK cells, we could sponse to intestinal helminth infection. readily identify a discrete population of lymphoid cells in the SVFexpressing receptors for IL2 (CD25), IL7, and IL33 RESULTS (Fig. 2, a and b), as well as intracellular Gata3 (Fig. 2 b). Eosinophils and IL-5 promote insulin These markers were previously demonstrated for ILC2s sensitivity and lean physiology (Moro et al., 2010; Neill et al., 2010; Price et al., 2010). Similar We previously reported metabolic consequences of eosino to other ILC2s, VAT ILC2s were present in Rag decient fi phil deficiency using dblGata1 mice (Wu et al., 2011). Be mice but absent in Rag x cdeficient and IL7R –deficient cause IL5 can promote local and systemic eosinophilia, we mice (Fig. 2, a–c), strains previously shown to lack ILC2s. compared metabolic parameters in eosinophildeficient and VAT ILC2s were present in male and female mice and in IL 5–dec fi ient C57BL/6 mice during HFD challenge. We used C57BL/6 and BALB/c mice in both WT and Ragdeficient Red5 mice, which contain a tandemdimer red fluorescent (T/B cell–dec fi ient) backgrounds, although consistently more protein (tdTomato) linked by an internal ribosomal entry site abundant in C57BL/6 mice (see also Fig. 4 d, bottom, and (IRES) to a Cre element, replacing the first exon of the il5 not depicted). Thus, the SVF of perigonadal adipose tissue gene (unpublished data), thus marking cells producing IL5; contains innate lymphoid cells with the phenotype of pre Red5 homozygous mice are IL5–deficient and the Cre ele viously described ILC2s (Moro et al., 2010; Neill et al., 2010; ment facilitates deletional studies based on IL5 expression. Price et al., 2010). 536 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e Figure 1. Deficiency of IL-5 or eosino - phils promotes obesity and insulin resis- tance 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 per- formed in mice on ND or HFD for 18–20 wk. Results are representative of three experi- +/ /+ ments. IL-5 , Red5 C57BL/6 R heterozy- / gotes; 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 pair- wise comparisons. Error bars are the mean ± SEM. P-values are shown. To assess the contribution of VAT ILC2s to the total IL 5 CD25 (IL2R), IL33R (T1/ST2), CD122 (IL2R), Thy1.2 and IL13 cytokine production in VAT, we used reporter (CD90.2), cKit, Sca1, and KRLG1, and were uniformly mice with knockin fluorescent alleles at various gene loci, negative for T cell markers, including CD4, CD8, CD3, thus allowing interrogation of the cytokine expression of these TCR , and TCR  (Fig. 2 e), consistent with previously cells without the need for restimulation ex vivo. Both adi described ILC2s (Moro et al., 2010; Neill et al., 2010; Price + +   pose SVF cells from Red5 mice, which mark IL 5–expressing et al., 2010). VAT B cells, CD8 T cells, CD3 CD4 CD8 cells with tdTomato expression, and YetCre13 x ROSA “double negative” T cells, macrophages, eosinophils, and YFP mice, which functionally mark cells that have ever ex  galactosylceramide (GC) reactive invariant NKT cells pressed IL 13 by establishing constitutive YFP expression from (iNKT) did not show IL5 fluorescence (Fig. 2 f and gating the ROSA26 locus (Price et al., 2010), each contained cells in Fig. S2), consistent with previous studies about lung IL5 marked by in situ IL5 and IL13 expression (Fig. 2 d). IL5– cells (Ikutani et al., 2012). Similar results were found for VAT expressing cells were negative for the myeloid marker CD11b, IL 13–expressing cells, although small percentages of eosino + + + + and included a small subset of CD4 CD3e IL33R (T1/ST2 ) phils (0.2–0.4%) and iNKT cells (3–5%) expressed IL 13 Th2 cells (5–15%) and a large population of lineage negative using lineagetracked expression (Fig. 2 g). After prolonged cells (85–95%). These VAT lineagenegative cells expressed IL 33 administration or helminth infection, ILC2s remain JEM Vol. 210, No. 3 537 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×  –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 CD3 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-13 538 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e the predominant IL5– and IL13–expressing cells, with no ILC2s are required to sustain adipose eosinophils and AAMs significant increased expression by macrophages, eosinophils, Eosinophils home to and are sustained in VAT, where they or other lymphocytes (Figs. S1 and S2 and unpublished data). promote AAM maintenance and systemic insulin sensitivity Together, these results establish that ILC2s are the predom (Wu et al., 2011). As assessed after mitotic labeling during bone marrow die ff rentiation, eosinophils had signic fi antly lower turn inant IL 5– and IL 13–expressing cells in VAT and that rare Th2 cells account for most of the remaining cytokine over in VAT as compared with spleen and lung, consistent with expressing cells. the presence of recruitment, retention, or survival signals in adi As assessed using these reporter alleles, significant pro pose tissue (Fig. 4 a). Although present in Rag dec fi ient mice, portions of VAT ILC2s spontaneously produced IL 5 and VAT eosinophils were substantially and tissue specic fi ally re IL13 ( Fig. 3, a and b), and this was particularly striking for duced in Rag x  dec fi ient mice that lack ILC2s (Fig. 4 b). IL5. We could identify no phenotypic differences between Prolonged HFD results in a decline of VAT eosinophils, as cytokinepositive and negative ILC2s, suggesting a uniform previously described (Wu et al., 2011), which is associated population with variable cytokine expression. IL 13 cytokine with a loss of VAT ILC2s but increased numbers of total VAT marked cells, the great majority of which are ILC2s (Fig. 2 d), macrophages and CD8 T cells (Fig. 4 c). In contrast, lung ILC2s were not reduced after HFD (unpublished data). Indeed, VAT were readily detected in close apposition to the adipose vas culature and dispersed within VAT (Fig. 3c). Unlike ILC2s ILC2 cell numbers correlate strongly with VAT eosinophils reported in mesenteric lymph nodes and mesenteric lymphoid across multiple mouse WT strains, genetic mutations, and di clusters (Moro et al., 2010), we were unable to identify dis etary perturbations, whereas total CD4 T cells show no cor crete lymphoid structures within perigonadal adipose tissue responding correlation (Fig. 4 d). (unpublished data). In contrast to VAT ILC2s, bone marrow To assess the ee ff cts of deleting IL 13–expressing ILC2s on  + + ILC2s (lineage IL7R T1/ST2 ; Brickshawana et al., 2011), adipose eosinophils, we crossed the YetCre13 mice to ROSA which were also described as ILC2 precursors (Hoyler et al., DTA deleter mice, which led to diphtheria toxin A–mediated 2012), did not express basal IL13 as assessed with IL13 lin death of cells that express IL 13 (Voehringer et al., 2008). These eage tracking (2.0 ± 0.3%, n = 8), although marrow ILC2s IL 13 deleter mice had an 40% loss of adipose ILC2s, consis tent with the IL 13 expression data (Fig. 4 e), and had a signic fi ant were predominantly IL4 competent, as assessed using cells from 4get mice (85.5 ± 7.4%, n = 3). Although a subset of reduction in adipose tissue eosinophils that was not apparent in + + VAT ILC2s were competent to make IL4 (4get ; Fig. 3, spleen or bone marrow (Fig. 4 f). Total VAT CD4 T cells and a and b), they were unmarked by reporter expression in KN2 macrophages were not ae ff cted by deletion of IL 13–producing + + mice (unpublished data), whose cells contain an IL4 replace cells (Fig. 4 e), although rare IL 13 CD4 T cells were likely ment allele and reveal cells actively producing IL4 in situ deleted. In contrast to the IL13 deleter mice, deficiency of (Mohrs et al., 2001; Wu et al., 2011), as previously described IL 4/IL 13 or STAT6 did not ae ff ct basal levels of VAT or (Price et al., 2010; Wu et al., 2011). spleen eosinophils (Fig. 4, d and g, unpublished data), indicat To conr fi m the d fi elity of the cytokine reporters and con ing that although VAT ILC2s can produce IL 13, other ILC2 firm additional cytokines secreted by these cells, VAT ILC2s derived factors are required to sustain VAT eosinophils. + + We performed similar studies using Red5 mice, which (lineagenegative Thy1.2 CD25 ) were purified by flow cy tometry and placed in vitro for 72 h with various cytokines. contain a disrupted il5 gene replaced with a fluorescent td Low amounts of IL5, IL6, IL13, and GMCSF spontane Tomato with an embedded Cre recombinase. Eosinophils in ously accumulated in the VAT ILC2 culture supernatants (Fig. 3, adipose were strongly dependent on IL5. Thus, Red5 het +/ d and e, and unpublished data). After addition of IL 33, greater erozygous mice (IL5 ) had fewer adipose eosinophils than amounts of IL 5, IL 6, IL 9, IL 13, and GM CSF accumulated did mice with two intact il5 alleles (WT), and IL5–deficient / (Fig. 3 d), and these cytokines increased further with the ad Red5 homozygous mice (IL5 ) containing two marker dition of IL 2 or IL 7, similar to results reported by ILC2s from alleles were drastically depleted of adipose eosinophils (Fig. 4 h). other tissues (Moro et al., 2010; Halim et al., 2012). Together, These ee ff cts of IL 5 dec fi iency were greater on adipose eosin these data suggest that VAT ILC2s spontaneously produce IL 5 ophils compared with systemic eosinophils, with a 12–14 fold reduction in VAT (Fig. 4 h) versus a 2–3fold reduction in and IL13, and can respond to IL33 with high levels of cyto kine production, as shown for other ILC2s. Although rare in spleen, blood, and small intestine (unpublished data). Although + + + VAT, IL 5 (Red5 ) CD4 T cells revealed a similar capacity to IL 5–deficient Red5 homozygous mice, similar to prior produce IL 2, IL 5, IL 6, IL 13, and GM CSF after in vitro cul studies in eosinophildeficient mice (Price et al., 2010), had ture with PMA/ionomycin (Fig. 3 e). These data indicate IL 5 normal numbers of ILC2s (unpublished data), Red5 mice ILC2s are numerically predominant within VAT, but otherwise containing a ROSADTA deleter allele exhibited significant + + + have a similar cytokine capacity to IL 5 Th2 cells. depletion of total adipose ILC2s (Fig. 4 i), IL5 (Red5 ) + + + expression on the following VAT populations: CD4 T cells (CD4), iNKT (aGC-loaded tetramer), CD8 T cells (CD8), NK cells (NK1.1), CD3 double-negative T cells (CD3), B cells (CD19), macrophages (CD11b), eosinophils (SiglecF), and lin cells (SSC). Cells were pregated as shown in Fig. S2. Data are represen- tative of two or more experiments. JEM Vol. 210, No. 3 539 ILC2s (Fig. 4 j), and almost complete ablation of adipose eosin could not conclusively identify which cells produced these cyto ophils (Fig. 4 h). Similar to IL 13 deleter animals, VAT kines (Wu et al., 2011). We used YARG mice containing a from IL 5 deleter mice had normal numbers of macrophages, u fl orescent arginase 1 knock in reporter allele to assess numbers + + CD8 T cells, and total CD4 T cells. When assessed specifi of adipose AAMs, as previously described (Reese et al., 2007; + + cally for IL 5 CD4 T cells, IL 5 deleter animals also ec ffi iently Wu et al., 2011). As assessed by o fl w cytom etry of dispersed deleted the rare IL5 Th2 cells (Fig. 4 j). However, as ILC2s SVF cells, adipose AAMs were depleted in  dec fi ient mice are the predominant IL5 VAT cell (85–95%), and VAT (Fig. 4 k) and in IL 13 deleter mice (Fig. 4 l), strains that have eosinophils are normal to elevated in T cell–deficient Rag absent or diminished ILC2s. Thus, adipose AAMs as assessed animals (Fig. 4 b), we conclude that ILC2expressing IL5 by arginase1 expression are dependent on ILC2s, and loss of are the primary cells required for the maintenance of visceral ILC2s based on their cytokine expression or dependence upon adipose eosinophils under basal conditions. the  cytokine chain results in a significant reduction in basal Adipose AAMs, like eosinophils, are present in the SVF adipose AAMs. of VATs in lean mice (Schipper et al., 2012). IL 13, like IL 4, can promote an AAM phenotype, which in mice can be re Exogenous IL-33 results in ILC2-dependent increases vealed by expression of signature target genes such as arginase 1. in adipose eosinophils and AAMs We previously determined a role for eosinophils and hema ILC2s were initially revealed by their capacity to release IL 13 topoietic IL 4/IL 13 in sustaining lean adipose AAMs, but and IL5 in response to IL25 and IL33, epithelial cytokines 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 IL7R 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 combina- +  + tions 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. 540 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e 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×  –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 c (Rag2 dec fi ient x c dec fi ient), 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  -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. JEM Vol. 210, No. 3 541 implicated in allergic immunity (Fort et al., 2001; Schmitz including the IL13 deleter and IL5 deleter ( Fig. 6, a, b, e, et al., 2005). IL33 has been identified in VAT and exoge and f ), and in IL7Rdeficient mice (Fig. 6 g, unpublished nous IL 33 was shown to promote Th2 associated cytokines data). In contrast, eosinophil accumulation is normal in re and to improve insulin sensitivity in obese mice (Miller et al., sponse to IL 33 in Rag1 deficient animals that lack B and 2010). After administering IL 33, we noted the rapid accumu T cells (Fig. 6 c) or in IL4/13–deficient animals (Fig. 6 a). lation of eosinophils in VAT that was accompanied by a de Similar to the percentage data (Fig. 6, a, b, and e–g), total crease in eosinophils in spleen and bone marrow, which is adipose eosinophils and AAM also accumulate after IL 33 ad consistent with a rapid redistribution of eosinophils from sys ministration in an ILC2 dependent manner, although the temic compartments to VAT (Fig. 5 a). Several types of adi increase is more robust in genetic strains on the C57BL/6 pose SVF cells expressed T1/ST2, a nonredundant component genetic background (unpublished data). We conclude that ILC2s of the IL33 receptor, including ILC2s, a proportion of CD4 are required for the IL33–mediated increases in VAT eosin T cells and, at low levels, eosinophils; VAT macrophages and ophils and AAM. CD8 T cells did not express the receptor under these condi VAT Arginase1 AAMs are dependent on eosinophils + + tions (Fig. 5 b, unpublished data). VAT T1/ST2 CD4 T cells and IL 4/ 13 (Wu et al., 2011); however, the precise cellu were primarily FoxP3 T reg cells with fewer FoxP3 Gata3 hi lar sources of these cytokines are unclear. Loss of VAT ILC2s Th2 cells (unpublished data). In response to IL 33, ILC2s decreases VAT YARG+ AAM (Fig. 4, k and l), but also rapidly increased their sidescatter and surface CD25 levels, leads to a loss of VAT eosinophils (Fig. 4, b, e, f, and i). as described for lung ILC2s (Bartemes et al., 2012), and de Therefore, it remained unclear if ILC2s have the capacity creased their surface levels of IL 7R (Fig. 5 d). To conr fi m that to promote AAM accumulation independent of eosinophils. these parameters indicate ILC2 cell activation, we assessed After IL 33 administration, YARG AAM can accumulate the cytokine response of ILC2s in IL13 and IL5 reporter in VAT independent of eosinophils as revealed in dblGata1 mice. In IL 13 lineage tracker mice, where only a subset of mutant mice (Fig. 6g), demonstrating that exogenous IL 33 ILC2s are IL 13 cytokine marked under basal conditions can induce IL 13 from VAT ILC2s sufficient to promote + + (Fig. 3, a and b), increased numbers of IL13 (YFP ) ILC2s adipose AAM. Nonetheless, our understanding of the rela were readily detected after administering IL33 (Fig. 5 d). tive contributions of VAT eosinophils and ILC2 derived In contrast, VAT CD4 T cells revealed minimal increases in IL 13 to AAM maintenance under homeostatic conditions + + + IL 13 (YFP ) CD4 T cells (Fig. 5 c). IL33 also caused in remains incomplete. creased fluorescence intensity in VAT ILC2s in IL5 (Red5) reporter mice (Fig. 5 d). Together, these findings are consis Intestinal helminth infection drives ILC2-dependent tent with direct activation of ILC2 ee ff ctor function by IL 33. increases in adipose eosinophils After three days of daily IL33 administration, total VAT eo We previously reported that infection of mice with Nippostron- sinophils and macrophages continued to accumulate, although gylus brasiliensis, a 10d selflimited migratory helminth infec ILC2 cell numbers did not significantly increase (Fig. 5 f ). tion, resulted in a prolonged elevation of visceral adipose Cell populations in the spleen were not significantly affected eosinophils that correlated with improved metabolic homeo during this timeframe (Fig. 5 e). With prolonged IL33 ad stasis when mice were placed on HFD (Wu et al., 2011). To ministration, ILC2s accumulate within VAT and systemically, assess whether ILC2 activation, which accompanies N. brasil- as previously described (Neill et al., 2010), accompanied by a iensis infection (Liang et al., 2012; Neill et al., 2010), is re systemic eosinophilia and macrophage accumulation (Fig. 5, quired for VAT eosinophil accumulation, we infected IL 13 and g and h). Even after prolonged IL33 administration, ILC2s IL 5 reporter mice. By 2 wk after infection, total numbers of + + + remain the predominant IL 5 cells in VAT, and IL 5 (Red5 ) VAT ILC2s were not increased, but IL 13– and IL 5–secreting + + CD4 T cells expand minimally (Fig. S2). In contrast, FoxP3 ILC2s were increased, as assessed using reporter mice (Fig. 7, T reg cells accumulate both systemically, as described pre a and b). As compared with control mice, which developed viously (Turnquist et al., 2011; Brunner et al., 2011), and within robust accumulations of VAT eosinophils, IL 5 deleter (Fig. 7c) VAT (unpublished data). We conclude that IL33 rapidly ac and IL13 deleter mice (Fig. 7d) developed little eosinophilia. tivates VAT ILC2s and promotes VAT eosinophils and AAM, IL13 cytokinemarked ILC2s remain the predominant IL and, over time, leads to additional local and systemic accu 13expressing cells, similar to basal conditions, although VAT mulations of ILC2s and T reg cells. IL13 expressing Th2 cells are modestly increased (Fig. 7a). To assess the requirement for ILC2 cell activation in me Rag1 dec fi ient animals accumulate VAT eosinophils similarly to diating the adipose cellular effects of exogenous IL33, we WT animals (Fig. 7e). Thus, as with IL 33 administration, hel administered IL33 or control PBS to IL5 and IL13 control minth infection activates VAT ILC2s to produce IL5 and or deleter mice. Similar experiments were performed in con IL 13, and loss of IL 5 and IL 13–producing cells, but not loss trol or deleter mice crossed onto the YARG arginase1 back of T cells, results in a failure to accumulate VAT eosinophils. ground to assess requirements for ILC2s in the accumulation of adipose AAMs. Although control mice rapidly increased DISCUSSION adipose eosinophils and AAMs in response to IL33, this ef ILC2s have been increasingly implicated in host type 2 im fect was abrogated after crossing onto strains dec fi ient in ILC2s, mune responses associated with asthma and intestinal helminth 542 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e infection in mice and humans (Brickshawana et al., 2011; IL13 and IL5 (Fort et al., 2001; Hurst et al., 2002; Schmitz Chang et al., 2011; Mjösberg et al., 2011; Moro et al., 2010; et al., 2005). During helminth infection or allergen challenge, Neill et al., 2010; Price et al., 2010; Halim et al., 2012; Ikutani ILC2s constitute the major innate cell source of these cyto et al., 2012; Klein Wolterink et al., 2012; Liang et al., 2012; kines, and loss of these cells can compromise the host type 2 Klein Wolterink et al., 2012). These cells were first identi immune response (Brickshawana et al., 2011; Chang et al., fied by their capacity to respond to the epithelial cytokines 2011; Mjösberg et al., 2011; Moro et al., 2010; Neill et al., IL25 and IL33 through the production of large amounts of 2010; Price et al., 2010; Halim et al., 2012; Ikutani et al., 2012; 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 cir- cles) 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 IL7R 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 IL7R 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 IL7R 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. JEM Vol. 210, No. 3 543 + + + 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 ad- ministration 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. Klein Wolterink et al., 2012; Liang et al., 2012). Despite subtle deficiency is also associated with a partial loss of B1 B cells die ff rences in surface markers used to characterize various lab (Kopf et al., 1996), and these cells might also contribute to oratories’ designation of these cells, their shared genetic and this phenotype. Loss of eosinophils or IL5 does not affect functional characteristics suggest a single cell type that is highly animal food intake or physical exertion, but instead causes associated with allergic and antihelminth immunity. Recent a decline in oxidative metabolism and energy expenditure studies have called attention to additional roles for ILC2 (heat), ultimately resulting in increased adiposity and meta like cells in inflammatory processes (Monticelli et al., 2011; bolic impairment. The precise molecular and cellular mech Chen et al., 2012), including limiting lung damage mediated anisms leading to these metabolic alterations remain to be by acute viral infection, potentially implicating ILC2s in re determined, but could reflect increased adipose inflamma parative responses to tissue and organ injury. These studies did tion secondary to the loss of adipose eosinophils and AAM. not find a role for IL 13 and IL 5, the canonical cytokines Whether eosinophils directly inhibit VAT inflammation or released in large abundance by ILC2s, in reestablishing organ promote a lean state with decreased VAT mass that indirectly homeostasis, leaving it unclear what the purpose of these cyto reduces ina fl mmation remain intriguing questions. Using o fl w kines might be in normal host physiology. cytometric phenotyping, in situ imaging, and genetic approaches, Using metabolic analysis, we demonstrate a role for IL 5 in we demonstrate that ILC2s are normal constituents of peri sustaining metabolic homeostasis. As in eosinophil deficient gonadal VAT of the mouse. ILC2s reside in the SVF where dblGata1 animals, IL5 deficiency promotes increased obesity eosinophils and AAMs are also present. Using cytokine re and insulin resistance with HFD. We demonstrate that loss of porter mice, we show that ILC2s are the primary producers IL 5 leads to a profound decrease in VAT eosinophils, with only of IL5 and IL13 under homeostatic conditions and, as dem modest alterations in systemic eosinophil pools, suggesting that onstrated by functional deletion, that these cells are required a loss of VAT eosinophils is responsible for the metabolic for the constitutive localization of eosinophils and AAMs to consequences of IL 5 dec fi iency. However, we note that IL 5 VAT. Further, IL33, shown to activate ILC2s systemically, 544 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e 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. induces rapid increases in adipose eosinophils and AAMs that to ascertain whether these or additional cytokines are neces are dependent on ILC2s. Similarly, helminth infection pro sary to sustain ILC2 homeostasis and activation in adipose motes VAT eosinophilia that is dependent on ILC2s. In con tissues. Additionally, ILC2s have been reported to produce a trast, HFD results in a decline in VAT ILC2s that is associated variety of factors in addition to IL 5 and IL 13; we have with declining eosinophils. These data extend our understand conr fi med that VAT ILC2s spontaneously produce IL 5, IL 6, ing of these innate lymphoid cells, and, in conjunction with IL 13, and GM CSF protein in culture, and can be induced previous studies, suggest a mechanism by which metabolic in vitro with exogenous IL 33 or PMA stimulation to pro needs of the organism might be regulated in response to chronic duce these cytokines, as well as IL 2 and IL 9. The impact of mucosal immune stimulation through activation of ILC2s. each of these additional ILC2 cytokines on VAT cellular com Based on the capacity of epithelial cytokines to activate position and metabolism requires further study. ILC2s, we administered IL 33 to mice and assessed the effects VAT eosinophils are highly dependent on IL 5. Deletion + + on adipose tissue. IL 33 rapidly activated ILC2s to increase of IL 5 cells or IL 13 cells, which are predominantly ILC2s, expression of IL 5 and IL 13, and this led to the accumula led to profound loss of VAT eosinophils and only minimally tion of eosinophils and AAMs in adipose tissue. As assessed by to eosinophils in other tissues. As such, the VAT eosinophil surface markers, side scatter characteristics, and cytokine re dependence upon IL 5 resembles many models of allergic porter expression, IL 33 directly activates adipose ILC2s. and helminth induced tissue eosinophilia, which can show Deletion of these cells led to loss of eosinophil and AAM ac strong IL 5 dependence (Foster et al., 1996; Kopf et al., 1996; cumulation in adipose. IL 33 is abundant in adipose tissues Mould et al., 1997). However, ILC2s are widely distributed where it may be produced and released by endothelial cells in tissues of unchallenged animals, and their presence alone is (Zeyda et al., 2012). Whether endothelial cells or other adi not associated with tissue eosinophils (Ikutani et al., 2012; pose cells undergo cell damage or can respond to environ Price et al., 2010). VAT ILC2s may be relatively more abun mental cues to release IL 33 remains unknown. Administration dant or activated than ILC2s from other tissues, sustaining of IL 33 has beneficial metabolic effects in mice, consistent VAT eosinophils and AAM. However, it is also likely that with those seen by increased eosinophils and AAMs (Miller other VAT cells and signals contribute to the maintenance et al., 2010), suggesting that IL 33–mediated ILC2 activation of these important cellular constituents. Indeed, VAT is an can promote insulin sensitivity. ample source of chemokines, including constitutive eotaxin 1 Interestingly, IL 7 is necessary to sustain ILC2s and has (Vasudevan et al., 2006), which could promote eosinophil also been localized to adipose (Lucas et al., 2012). TSLP is also trafficking into VAT. During helminth infections and allergic present in adipose tissue (Turcot et al., 2012) and can sustain challenge in the lung, IL 5 promotes eosinophil production, ILC2s in vitro (Halim et al., 2012). Further study is needed survival, and retention, whereas IL 13 mediates eosinophil JEM Vol. 210, No. 3 545 recruitment through the promotion of tissue eotaxins (Blanchard insulin sensitizing products, or through the activation of and Rothenberg, 2009). In contrast, VAT eosinophils are pres nonshivering thermogenesis (Nguyen et al., 2011; Chawla ent in the genetic absence of IL 13 or IL 13 signaling (STAT6), et al., 2011). suggesting that constitutive factors, including eotaxins or other Intestinal helminth infections are widespread in feral ani chemokines, may help recruit these cells. In addition, VAT mals, suggesting a long standing mutualism. The host response eosinophil trafficking requires  and  mediated integrin is characterized by chronic type 2 immune responses, includ L 4 signaling (Wu et al., 2011), indicating VAT endothelium con ing the presence of epithelial mucus changes, Th2 cells, ele tributes to eosinophil accumulation. Although VAT eosinophils vated IgE and mucosal mast cell hyperplasia, but also chronic are likely sustained by multiple pathways, ILC2s play an in eosinophilia and the accumulation of tissue AAMs. Similar dispensable role under our experimental conditions. responses, presumably dysregulated, accompany responses Adipose AAMs and eosinophils are present in lean adi to ubiquitous environmental antigens in people with allergy pose, sustaining resistance of VATs to the proinflammatory and asthma. ILC2s respond to epithelial cytokines such as IL 25, effects of HFD and obesity (Chawla et al., 2011). Here, we IL33, and TSLP, and have been implicated in both antihel demonstrate that ILC2 IL5 is necessary to maintain VAT minth and allergic immunity ((Brickshawana et al., 2011; eosinophils, which are themselves required to sustain popula Chang et al., 2011; Mjösberg et al., 2011; Moro et al., 2010; tions of Arginase1 AAMs (Wu et al., 2011). ILC2s, in con Neill et al., 2010; Price et al., 2010; Halim et al., 2012; Ikutani trast to eosinophils (Fig. 2 f), produce ample IL13 and could et al., 2012; Klein Wolterink et al., 2012; Liang et al., 2012). directly contribute to AAM polarization and maintenance. As shown here, adipose eosinophils, which accumulate after Indeed, with IL 33 stimulation ILC2s appear sufficient to N. brasiliensis infection (Wu et al., 2011), do not accumulate promote Arginase1 AAM, even in the absence of eosino in the absence of ILC2s, although accumulation is normal phils. However, under basal conditions, eosinophils also pro in T cell deficient Rag mice. Of interest, both IL 33 ad mote AAM (Wu et al., 2011), suggesting ILC2 IL13 may be ministration and helminth infection promote insulin sensi insufficient to fully polarize and maintain VAT AAM under tivity in mice fed HFD (Miller et al., 2010; Wu et al., 2011), resting conditions. Consistent with this hypothesis, only a suggesting ILC2 activation and/or accumulation may be subset of ILC2s is marked for IL13 expression, even using contributing to these metabolic effects. Based on prior stud mice with lineagetracked markers that reveal cells that have ies, release of IL25 and/or IL33 during tissueinvasive hel ever expressed IL13 through their history. Ultimately, the minth infection is likely (Moro et al., 2010; Neill et al., 2010), relative contributions of eosinophils and ILC2 IL13 produc but further investigation will be necessary to show defini tion to AAM maintenance in lean adipose remain to be de tively which cytokines activate VAT ILC2s. The potential finitively elucidated. interactions between VAT ILC2s, Th2 cells and T reg cells, The mechanisms by which eosinophils promote AAM and their relative contributions to metabolic pathways during remain poorly understood. Although IL4 remains a candi homeostasis and after helminth infection remain intriguing date cytokine, only a small subset of adipose eosinophils questions. Understanding the processes that sustain AAMs (<1%) were marked for IL4 protein expression as assessed and eosinophils in VATs may offer new insights toward using KN2 mice (Wu et al., 2011). Eosinophils produce therapeutic strategies attempting to block the adverse effects abundant secreted products, including TGF, proteases, and of adipose inflammation and protect against insulin resistance RNase, which could also participate in AAM maintenance and type 2 diabetes. Although further metabolic studies are (Blanchard and Rothenberg, 2009). Adipocytes are also re needed, investigations of the role of these unusual innate ported to be potential sources of IL4 and IL13 (Kang et al., lymphoid cells in adipose and other tissues are warranted, 2008), but we have not observed fluorescence in adipocytes and may provide novel insights into more global aspects of using IL4 or IL13 cytokine marker alleles (Wu et al., 2011; vertebrate biology. Fig. 3 c), and il4 and il13 transcripts are primarily found within the VAT SVF (Wu et al., 2011). VAT iNKT cells MATERIALS AND METHODS were recently proposed to mediate metabolic homeostasis Mice. Cytokine reporter mice previously described include 4get mice for tracking IL 4 competent cells (Mohrs et al., 2001; Wu et al., 2011), YetCre13 and produce abundant IL 4 after TCR stimulation that mice for tracking IL 13–producing cells (Price et al., 2010; Liang et al., 2012), might promote AAM (Ji et al., 2012a; b; Lynch et al., 2012); and KN2 mice for tracking IL 4–producing cells (Mohrs et al., 2005; Wu in our mouse colony, iNKT cells are rare in VAT (Fig. S2). et al., 2011). Where indicated, YetCre13 mice were crossed onto ROSA26 Finally, IL5– and IL13–expressing Th2 cells accumulate in eYFP (The Jackson Laboratory) or ROSA26 ZsGreen (Ai6; The Jackson VAT of older animals on normal diet (ND; unpublished Laboratory; Madisen et al., 2010). The eYFP Cre fusion protein downstream data), and similar to VAT T reg cells (Feuerer et al., 2009), of the IL 13 locus in YetCre13 mice mediates deletion of the stop cassette from the ROSA26 locus, resulting in constitutive u fl orophore expression in cells may provide an additional layer of adaptive regulation to that have expressed IL 13. Newly generated Red5 mice contain a tdTomato maintain metabolic homeostasis. How these cells cooperate IRES Cre replacement allele at the endogenous IL 5 start site, thus replacing to promote AAM maintenance remains to be determined. the endogenous il5 gene with tdTomato and revealing IL5–expressing cells Further, how AAMs themselves promote insulin sensitivity by red fluorescence. Homozygous Red5 mice (R/R) are IL5 deficient, as is also unclear, although proposed mechanisms include both alleles are replaced by the marker construct, whereas heterozygous mice through the production of antiinflammatory cytokines and have one functional IL 5 copy (R/+). YARG mice contain a YFP marker 546 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e allele in the arginase 1 gene, permitting identic fi ation of AAMs, as previously manufacturer’s instructions. When used with fluorescent reporter strains, a described (Reese et al., 2007; Wu et al., 2011). ROSA DTA mice contain a brief 2min “prefix” in 4% paraformaldehyde was performed before pro Cre a fl nked u fl x stop sequence upstream of diphtheria toxin  (DTA) in ceeding with eBioscience fix/perm instructions. Edu detection was achieved serted into the constitutively expressed ROSA26 locus, thus causing Cre with the Clickit Edu A647 kit, after first staining for extracellular surface expressing cells to be deleted, and have been previously described (Jackson; markers, per the manufacturer’s instructions (Life Technologies). Voehringer et al., 2008)). ROSA DTA mice were crossed with YetCre13 or Representative gating schemes for each population are shown in Fig. S1.     Red5 mice to create mice in which IL 13– or IL 5–expressing cells are deleted. ILC2s are identified as lineage negative (CD11b , F4/80 , DX5 , CD3 ,       Additional mice used in these studies include Rag dec fi ient mice (Rag1; CD4 , CD8 , CD19 , SiglecF , FcR1 , NK1.1 ), FSC/SSClowto + + + Jackson Laboratories; (Mombaerts et al., 1992) or Rag2 (Taconic Farms moderate, CD45 , CD127 (IL7Ra) or thy1.2 (CD90.2) , and T1/ST2 + + + + RAGN12), Rag2 x  deficient mice (Taconic Farms 4111M), eosinophil (IL33R) or CD25 (IL2Ra) or KLRG1 , as indicated. CD4 T cells are + + + + deficient dblGATA mice (Yu et al., 2002), IL 4/ 13–deficient mice identified as FSC/SSClo, CD45 , CD3 , CD4 . CD8 T cells are identi + + + (McKenzie et al., 1999), Stat6 deficient mice (The Jackson Laboratory; fied as FSC/SSClo, CD45 , CD3 , and CD8 . Eosinophils are identified + + + Kaplan et al., 1996), IL7R dec fi ient mice (The Jackson Laboratory; Peschon as CD45 , sidescatter high, DAPIlo, CD11b , and SiglecF . Adipose tissue + + + et al., 1994), and T1/ST2 dec fi ient mice (Hoshino et al., 1999), and were macrophages are identie fi d as CD45 , CD11b , F4/80 , SiglecF lo. All pop crossed onto cytokine reporter alleles where designated. Mice used for these ulations were routinely back gated to verify purity and gating. Samples were experiments were male animals fully backcrossed on C57BL/6 or BALB/c analyzed on an LSR II (BD). For cell sorting, a FACS AriaII was used. Live backgrounds, as designated. Mice were maintained in the University of Cali lymphocytes were gated by DAPI exclusion, size, and granularity based on fornia San Francisco specic fi pathogen–free animal facility, and all animal pro forward and sidescatter. Data were analyzed using FlowJo software (Tree tocols were approved by and in accordance with the guidelines established by Star) and compiled using Prism (GraphPad Software). As indicated, VAT the Institutional Animal Care and Use Committee and Laboratory Animal data were normalized per gram of adipose or as a percentage of total viable Resource Center. cells or percentage of CD45 hematopoietic cells, as indicated. Cell culture and cytokine analysis. VAT from 8–15 WT C57BL/6 or Tissue preparation. Perigonadal adipose tissue was used as representative Red5 (R/+) animals was pooled for cell sorting. Sorted ILC2s or CD4 VAT in all experiments. Testicles were removed and tissue was kept on ice in T cells were transferred to 96well plates in 100 l of cRMPI at 1,500 cells 0.5 ml of adipose digestion medium (low glucose DMEM, 0.2 M Hepes, and per well. Cytokines were added to culture media at 10 ng/ml, as indicated. 10 mg/ml fatty acid poor BSA [Sigma]). VAT was n fi ely minced with a mul PMA was used at 40 ng/ml and ionomycin was used at 500 ng/ml. Human tiple razor blades, dispersed by shaking into 10 ml of adipose digestion me IL2 was used at 10 U/ml, and all other cytokines were purchased from dium containing 0.2 mg/ml Liberase Tm (Roche) and 25 µg/ml DNase I R&D Systems. After 72 h of culture, supernatant cytokine levels were ana (Roche) at 37°C for 30–40 min with gentle agitation, and passed through lyzed by cytokine bead arrays (BD) per the manufacturer’s instructions. 100 µm filters to generate single cell suspensions. Filters were washed with 10 ml FACS bue ff r (PBS, 3% FCS, 0.05% NaN ) and supernatants pooled. Cells were centrifuged at 1,000 g for 10 min and the cell pellets were resus Metabolic assays and diet. Male mice were fed normal chow diet (Mouse pended in 5 ml FACS bue ff r, transferred to fresh tubes and centrifuged at diet 20; PicoLab) and used between 8 and 15 wk of age, unless otherwise 1,500 rpm for 5 min. The red blood cells were lysed using PharmLyse (BD) noted. Where indicated, C57BL/6 WT mice were fed HFD D12492 (60% for 1 min, and the remaining cells were washed with FACS bue ff r, incubated kcal fat; Research Diets, Inc.) for 12–24 wk as noted. To measure animal with FcBlock, and stained with the indicated antibodies. adiposity and lean mass, MRI was performed using an EchoMRI 3 in1 Spleen was prepared by mashing tissue through 70 µm lfi ters without tis machine according to the manufacturer’s instructions (Echo Medical Sys sue digestion, and processing similar to VAT. Bone marrow was prepared by tems LTD). Glucose tolerance testing was performed after fasting mice carefully dissecting one femur and tibia, liberating hematopoietic cells with overnight for 14 h and challenging with 1.5 g/kg glucose by i.p. injection. mortar and pestle into 10 ml FACS bue ff r, passing through a 70 µm lfi ter, and Fasting blood glucose was measured after a 4h morning fast. Insulin toler processing similar to VAT. Whole lung was prepared by harvesting both lung ance tests were performed after a 4–5 h morning fast, injecting insulin i.p. lobes into 5 ml DMEM media with 0.2 mg/ml Liberase Tm and 25 g/ml (0.75 mU/g human insulin; Eli Lilly), and measuring blood glucose at the DNase 1, followed by tissue dissociation (GentleMacs; Miltenyi Biotec) using times indicated. Blood glucose was measured at indicated times using a glu the “lung1 program,” followed by tissue digestion for 30 min at 37°C with cometer (Bayer). Wholeanimal metabolic analysis was performed using gentle agitation. Samples were processed on the GentleMacs using the “lung2” CLAMS cages (Comprehensive Laboratory Animals Monitoring System) per program, passed through 70 µm lfi ters, and processed as described for VAT. the manufacturer’s instructions (Columbus Instruments). In brief, animals were singly housed and measurements were taken every 12 min for 4 d, including oxygen consumption, carbon dioxide output, food consumption, Flow cytometry. Monoclonal antibodies used for flow cytometry were as water consumption, and three unique measures of movement. Respiratory follows: allophycocyanin (APC) eFluor 780 anti CD4 (RM4 5; eBioscience); exchange ratio and heat were calculated as VCO /VO (RER) and VO (3.815 + 2 2 2 Qdot605antiCD4 (S3.5, Invitrogen); phycoerythrin (PE)antiSiglecF 1.232 × RER; heat), respectively. Heat, VO , and VCO were all normal 2 2 (E502440; BD); APC or Brilliant Violet 650– or Pacific Blue (PB)– anti 0.75 ized to effective body mass Vxx = Vxx/[(weight(g)/mass unit)] , per the CD11b (M1/70; BioLegend); PECy7 or PerCPCy5.5 antiF4/80 (BM8; manufacturer’s recommendations. eBioscience); biotinantipanNK (CD49b; DX5; eBioscience), PB anti NK1.1 (PK136; BioLegend), biotin anti FcRI (MAR 1; eBioscience); PB Helminth infection and cytokine administration. 500 third stage larvae or Alexa Fluor 488– or PerCPCy5.5 anti CD3e (17A2; BioLegend); of N. brasiliensis, purie fi d as described, were injected subcutaneously into mice PB anti CD8 (53–6.7; BioLegend); PerCPCy5.5 anti CD19 (1D3; BD); PB (Voehringer et al., 2006). Mice were killed at the indicated time points and anti CD19 (6D5; BioLegend), APC or PE anti CD25 (IL2R, PC61; tissues were harvested and analyzed as previously described (Reese et al., 2007; BioLegend); PerCPCy5.5 or PECy7antiCD127 (IL7R , A7R34; eBio Wu et al., 2011). IL 33 (R&D Systems) was given as 500 ng in 0.2 ml PBS i.p. science); Biotin anti T1/ST2 (DT8; MD Biosciences); APC or APC Cy7 anti CD45 (30 F11; BioLegend). Secondary u fl orophore for biotin conjugated antibodies were eF605 (eBioscience), APC (BD), or FITC (BD). CD1d aGC Microscopy. 5 min before sacrifice, animals were injected with 20 µg loaded and unloaded tetramer (PE or APC) were obtained from the National CD31APC (clone 390; eBioscience) to label vasculature. After VAT re Institutes of Health tetramer facility. PECy7 FoxP3 (FJK16S; eBioscience) moval, the tissue was immediately mounted and examined by laserscanning and A647 Gata3 (TWAJ; eBioscience) were used after first using a fixable confocal microscopy (Nikon C1si). Images were resolved to 1.2 µm/pixel in live/dead stain (Invitrogen), and then fixing and permeabilizing cells per the the xy plane and 1.0 µm in the z plane. JEM Vol. 210, No. 3 547 Statistical analysis. Unless otherwise noted, significance was determined allergeninduced airway inflammation. Immunity. 36:451–463. http:// by the Student’s t test, with P < 0.05 considered significant. *, P < 0.05; dx.doi.org/10.1016/j.immuni.2011.12.020 Hoshino, K., S. Kashiwamura, K. Kuribayashi, T. Kodama, T. Tsujimura, K. **, P < 0.01; ***, P < 0.001. Error bars represent standard error of the mean. Nakanishi, T. Matsuyama, K. Takeda, and S. Akira. 1999. The absence Each data point represents one animal. When possible, data from multiple of interleukin 1 receptorrelated T1/ST2 does not affect T helper cell independent experiments were pooled, as indicated. In cases with multiple type 2 development and its effector function. J. Exp. Med. 190:1541– comparisons within an experiment (>2), a onetailed ANOVA was per 1548. http://dx.doi.org/10.1084/jem.190.10.1541 formed with Tukey’s posttest correction. Hoyler, T., C.S.N. Klose, A. Souabni, A. TurquetiNeves, D. Pfeifer, E.L. Rawlins, D. Voehringer, M. Busslinger, and A. Diefenbach. 2012. We thank Drs. A. August, A. McKenzie, M. Steinhoff, and S. Wirtz for mice, The transcription factor GATA3 controls cell fate and maintenance A. DeFranco, C. Lowell and A. Ma for helpful comments on the manuscript, of type 2 innate lymphoid cells. Immunity. 37:634–648. http://dx.doi Zhi-En Wang for technical assistance, the Diabetes Endocrinology Research Core .org/10.1016/j.immuni.2012.06.020 for assistance with CLAMS analysis, and N. Flores and M. Consengco for Hurst, S.D., T. Muchamuel, D.M. Gorman, J.M. Gilbert, T. Clifford, S. animal support. Kwan, S. Menon, B. Seymour, C. Jackson, T.T. Kung, et al. 2002. New This work was supported by AI026918, AI030663, AI078869, HL107202, and IL17 family members promote Th1 or Th2 responses in the lung: in P30 DK063720 from the National Institutes of Health, the UCSF Diabetes Family vivo function of the novel cytokine IL25. J. Immunol. 169:443–453. Fund (ABM), the Department of Laboratory Medicine discretionary fund (ABM), the Ikutani, M., T. Yanagibashi, M. Ogasawara, K. Tsuneyama, S. Yamamoto, Larry Hillblom Foundation, the Sandler Asthma Basic Research Center at UCSF and Y. Hattori, T. Kouro, A. Itakura, Y. Nagai, S. Takaki, and K. Takatsu. the Howard Hughes Medical Institute. 2012. Identification of innate IL5producing cells and their role in lung The authors have no conflicting financial interests. eosinophil regulation and antitumor immunity. J. Immunol. 188:703– 713. http://dx.doi.org/10.4049/jimmunol.1101270 Submitted: 30 August 2012 Ji, Y., S. Sun, A. Xu, P. Bhargava, L. Yang, K.S.L. Lam, B. Gao, C.H. Accepted: 30 January 2013 Lee, S. Kersten, and L. Qi. 2012a. Activation of natural killer T cells promotes M2 Macrophage polarization in adipose tissue and improves systemic glucose tolerance via interleukin4 (IL4)/STAT6 protein sig REFERENCES naling axis in obesity. J. Biol. Chem. 287:13561–13571. http://dx.doi Bartemes, K.R., K. Iijima, T. Kobayashi, G.M. Kephart, A.N. McKenzie, .org/10.1074/jbc.M112.350066 and H. Kita. 2012. IL33responsive lineage CD25+ CD44(hi) lym Ji, Y., S. Sun, S. Xia, L. Yang, X. Li, and L. Qi. 2012b. Short term high phoid cells mediate innate type 2 immunity and allergic inflammation fat diet challenge promotes alternative macrophage polarization in adi in the lungs. J. Immunol. 188:1503–1513. http://dx.doi.org/10.4049/ pose tissue via natural killer T cells and interleukin4. J. Biol. Chem. jimmunol.1102832 287:24378–24386. http://dx.doi.org/10.1074/jbc.M112.371807 Blanchard, C., and M.E. Rothenberg. 2009. Biology of the eosinophil. Adv. Kang, K., S.M. Reilly, V. Karabacak, M.R. Gangl, K. Fitzgerald, B. Immunol. 101:81–121. http://dx.doi.org/10.1016/S0065 2776(08)01003 1 Hatano, and C. H. Lee. 2008. Adipocyte derived Th2 cytokines Brickshawana, A., V.S. Shapiro, H. Kita, and L.R. Pease. 2011. Lineage and myeloid PPARdelta regulate macrophage polarization and in ( )Sca1+c Kit( )CD25+ cells are IL 33 responsive type 2 innate cells in sulin sensitivity. Cell Metab. 7:485–495. http://dx.doi.org/10.1016/ the mouse bone marrow. J. Immunol. 187:5795–5804. http://dx.doi.org/ j.cmet.2008.04.002 10.4049/jimmunol.1102242 Kaplan, M.H., U. Schindler, S.T. Smiley, and M.J. Grusby. 1996. Stat6 is Brunner, S.M., G. Schiechl, W. Falk, H.J. Schlitt, E.K. Geissler, and S. required for mediating responses to IL4 and for development of Th2 FichtnerFeigl. 2011. Interleukin33 prolongs allograft survival during cells. Immunity. 4:313–319. http://dx.doi.org/10.1016/S10747613(00) chronic cardiac rejection. Transpl. Int. 24:1027–1039. http://dx.doi 80439 2 .org/10.1111/j.14322277.2011.01306. x Klein Wolterink, R.G., A. Kleinjan, M. van Nimwegen, I. Bergen, M. de Chang, Y.J., H.Y. Kim, L.A. Albacker, N. Baumgarth, A.N.J. McKenzie, Bruijn, Y. Levani, and R.W. Hendriks. 2012. Pulmonary innate lym D.E. Smith, R.H. Dekruyff, and D.T. Umetsu. 2011. Innate lymphoid phoid cells are major producers of IL5 and IL13 in murine models cells mediate influenzainduced airway hyperreactivity independently of allergic asthma. Eur. J. Immunol. 42:1106–1116. http://dx.doi.org/ of adaptive immunity. Nat. Immunol. 12:631–638. http://dx.doi.org/10 10.1002/eji.201142018 .1038/ni.2045 Kopf, M., F. Brombacher, P.D. Hodgkin, A.J. Ramsay, E.A. Milbourne, Chawla, A., K.D. Nguyen, and Y.P.S. Goh. 2011. Macrophagemediated W.J. Dai, K.S. Ovington, C.A. Behm, G. Köhler, I.G. Young, and inflammation in metabolic disease. Nat. Rev. Immunol. 11:738–749. K.I. Matthaei. 1996. IL5deficient mice have a developmental de http://dx.doi.org/10.1038/nri3071 fect in CD5+ B1 cells and lack eosinophilia but have normal anti Chen, F., Z. Liu, W. Wu, C. Rozo, S. Bowdridge, A. Millman, N. Van body and cytotoxic T cell responses. Immunity. 4:15–24. http://dx.doi Rooijen, J.F. Urban Jr., T.A. Wynn, and W.C. Gause. 2012. An essen .org/10.1016/S10747613(00)80294 0 tial role for TH2type responses in limiting acute tissue damage during Liang, H.E., R.L. Reinhardt, J.K. Bando, B.M. Sullivan, I.C. Ho, and experimental helminth infection. Nat. Med. 18:260–266. http://dx.doi R.M. Locksley. 2012. Divergent expression patterns of IL4 and IL13 .org/10.1038/nm.2628 define unique functions in allergic immunity. Nat. Immunol. 13:58–66. Feuerer, M., L. Herrero, D. Cipolletta, A. Naaz, J. Wong, A. Nayer, J. Lee, http://dx.doi.org/10.1038/ni.2182 A.B. Goldfine, C. Benoist, S. Shoelson, and D. Mathis. 2009. Lean, but Lucas, S., S. Taront, C. Magnan, L. Fauconnier, M. Delacre, L. Macia, not obese, fat is enriched for a unique population of regulatory T cells A. Delanoye, C. Verwaerde, C. Spriet, P. Saule, et al. 2012. Interleukin7 that affect metabolic parameters. Nat. Med. 15:930–939. http://dx.doi regulates adipose tissue mass and insulin sensitivity in high fat diet .org/10.1038/nm.2002 fed mice through lymphocyte dependent and independent mecha Fort, M.M., J. Cheung, D. Yen, J. Li, S.M. Zurawski, S. Lo, S. Menon, T. nisms. PLoS ONE. 7:e40351. http://dx.doi.org/10.1371/journal.pone Clifford, B. Hunte, R. Lesley, et al. 2001. IL25 induces IL4, IL5, and .0040351 IL13 and Th2associated pathologies in vivo. Immunity. 15:985–995. Lynch, L., M. Nowak, B. Varghese, J. Clark, A.E. Hogan, V. Toxavidis, http://dx.doi.org/10.1016/S10747613(01)00243 6 S.P. Balk, D. O’Shea, C. O’Farrelly, and M.A. Exley. 2012. Adipose Foster, P.S., S.P. Hogan, A.J. Ramsay, K.I. Matthaei, and I.G. Young. 1996. tissue invariant NKT cells protect against dietinduced obesity and Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, metabolic disorder through regulatory cytokine production. Immunity. and lung damage in a mouse asthma model. J. Exp. Med. 183:195–201. 37:574–587. http://dx.doi.org/10.1016/j.immuni.2012.06.016 http://dx.doi.org/10.1084/jem.183.1.195 Madisen, L., T.A. Zwingman, S.M. Sunkin, S.W. Oh, H.A. Zariwala, H. Halim, T.Y.F., R.H. Krauss, A.C. Sun, and F. Takei. 2012. Lung natural Gu, L.L. Ng, R.D. Palmiter, M.J. Hawrylycz, A.R. Jones, et al. 2010. A helper cells are a critical source of Th2 celltype cytokines in protease robust and highthroughput Cre reporting and characterization system 548 Innate lymphoid type 2 cells in visceral adipose tissue | Molofsky et al. A r t i c l e for the whole mouse brain. Nat. Neurosci. 13:133–140. http://dx.doi in type 2 immunity. Proc. Natl. Acad. Sci. USA. 107:11489–11494. .org/10.1038/nn.2467 http://dx.doi.org/10.1073/pnas.1003988107 Martinez, F.O., L. Helming, and S. Gordon. 2009. Alternative activa Reese, T.A., H.E. Liang, A.M. Tager, A.D. Luster, N. Van Rooijen, D. tion of macrophages: an immunologic functional perspective. Annu. Voehringer, and R.M. Locksley. 2007. Chitin induces accumulation in Rev. Immunol. 27:451–483. http://dx.doi.org/10.1146/annurev.immunol tissue of innate immune cells associated with allergy. Nature. 447:92–96. .021908.132532 http://dx.doi.org/10.1038/nature05746 McKenzie, G.J., P.G. Fallon, C.L. Emson, R.K. Grencis, and A.N. McKenzie. Ricardo Gonzalez, R.R., A. Red Eagle, J.I. Odegaard, H. Jouihan, C.R. 1999. Simultaneous disruption of interleukin (IL) 4 and IL 13 de Morel, J.E. Heredia, L. Mukundan, D. Wu, R.M. Locksley, and A. fines individual roles in T helper cell type 2–mediated responses. Chawla. 2010. IL 4/STAT6 immune axis regulates peripheral nutri J. Exp. Med. 189:1565–1572. http://dx.doi.org/10.1084/jem.189.10 ent metabolism and insulin sensitivity. Proc. Natl. Acad. Sci. USA. .1565 107:22617–22622. http://dx.doi.org/10.1073/pnas.1009152108 Miller, A.M., D.L. Asquith, A.J. Hueber, L.A. Anderson, W.M. Holmes, Rothenberg, M.E., and S.P. Hogan. 2006. The eosinophil. Annu. Rev. Immunol. A.N. McKenzie, D. Xu, N. Sattar, I.B. McInnes, and F.Y. Liew. 2010. 24:147–174. http://dx.doi.org/10.1146/annurev.immunol.24.021605 Interleukin33 induces protective effects in adipose tissue inflamma .090720 tion during obesity in mice. Circ. Res. 107:650–658. http://dx.doi.org/ Schipper, H.S., B. Prakken, E. Kalkhoven, and M. Boes. 2012. Adipose 10.1161/CIRCRESAHA.110.218867 tissueresident immune cells: key players in immunometabolism. Trends Mishra, A., S.P. Hogan, J.J. Lee, P.S. Foster, and M.E. Rothenberg. 1999. Endocrinol. Metab. 23:407–415. http://dx.doi.org/10.1016/j.tem.2012 Fundamental signals that regulate eosinophil homing to the gastrointes .05.011 tinal tract. J. Clin. Invest. 103:1719–1727. http://dx.doi.org/10.1172/ Schmitz, J., A. Owyang, E. Oldham, Y. Song, E. Murphy, T.K. McClanahan, G. JCI6560 Zurawski, M. Moshrefi, J. Qin, X. Li, et al. 2005. IL33, an interleukin Mjösberg, J.M., S. Trifari, N.K. Crellin, C.P. Peters, C.M. van Drunen, B. 1like cytokine that signals via the IL1 receptorrelated protein ST2 Piet, W.J. Fokkens, T. Cupedo, and H. Spits. 2011. Human IL25 and and induces T helper type 2associated cytokines. Immunity. 23:479–490. IL33responsive type 2 innate lymphoid cells are defined by expression http://dx.doi.org/10.1016/j.immuni.2005.09.015 of CRTH2 and CD161. Nat. Immunol. 12:1055–1062. http://dx.doi Spits, H., and J.P. Di Santo. 2011. The expanding family of innate lymphoid .org/10.1038/ni.2104 cells: regulators and effectors of immunity and tissue remodeling. Nat. Mohrs, M., K. Shinkai, K. Mohrs, and R.M. Locksley. 2001. Analysis of Immunol. 12:21–27. http://dx.doi.org/10.1038/ni.1962 type 2 immunity in vivo with a bicistronic IL4 reporter. Immunity. Turcot, V., L. Bouchard, G. Faucher, V. Garneau, A. Tchernof, Y. Deshaies, 15:303–311. http://dx.doi.org/10.1016/S10747613(01)00186 8 L. Pérusse, S. Marceau, S. Biron, O. Lescelleur, et al. 2012. Thymic Mohrs, K., A.E. Wakil, N. Killeen, R.M. Locksley, and M. Mohrs. 2005. stromal lymphopoietin: an immune cytokine gene associated with the A two step process for cytokine production revealed by IL 4 dual metabolic syndrome and blood pressure in severe obesity. Clin. Sci. reporter mice. Immunity. 23:419–429. http://dx.doi.org/10.1016/j 123:99–109. http://dx.doi.org/10.1042/CS20110584 .immuni.2005.09.006 Turnquist, H.R., Z. Zhao, B.R. Rosborough, Q. Liu, A. Castellaneta, K. Mombaerts, P., J. Iacomini, R.S. Johnson, K. Herrup, S. Tonegawa, and Isse, Z. Wang, M. Lang, D.B. Stolz, X.X. Zheng, et al. 2011. IL33 ex V.E. Papaioannou. 1992. RAG 1 dec fi ient mice have no mature B and pands suppressive CD11b+ Gr1(int) and regulatory T cells, including T lymphocytes. Cell. 68:869–877. http://dx.doi.org/10.1016/0092 8674 ST2L+ Foxp3+ cells, and mediates regulatory T celldependent promo (92)90030 G tion of cardiac allograft survival. J. Immunol. 187:4598–4610. http:// Monticelli, L.A., G.F. Sonnenberg, M.C. Abt, T. Alenghat, C.G.K. dx.doi.org/10.4049/jimmunol.1100519 Ziegler, T.A. Doering, J.M. Angelosanto, B.J. Laidlaw, C.Y. Yang, T. Vasudevan, A.R., H. Wu, A.M. Xydakis, P.H. Jones, E.O. Smith, J.F. Sathaliyawala, et al. 2011. Innate lymphoid cells promote lungtissue Sweeney, D.B. Corry, and C.M. Ballantyne. 2006. Eotaxin and obe homeostasis after infection with influenza virus. Nat. Immunol. 12:1045– sity. J. Clin. Endocrinol. Metab. 91:256–261. http://dx.doi.org/10.1210/ 1054. http://dx.doi.org/10.1038/ni.2131 jc.2005128 0 Moro, K., T. Yamada, M. Tanabe, T. Takeuchi, T. Ikawa, H. Kawamoto, Voehringer, D., T.A. Reese, X. Huang, K. Shinkai, and R.M. Locksley. J.I. Furusawa, M. Ohtani, H. Fujii, and S. Koyasu. 2010. Innate pro 2006. Type 2 immunity is controlled by IL 4/IL 13 expression in he duction of T(H)2 cytokines by adipose tissueassociated cKit(+)Sca matopoietic noneosinophil cells of the innate immune system. J. Exp. 1(+) lymphoid cells. Nature. 463:540–544. http://dx.doi.org/10.1038/ Med. 203:1435–1446. http://dx.doi.org/10.1084/jem.20052448 nature08636 Voehringer, D., N. van Rooijen, and R.M. Locksley. 2007. Eosinophils Mould, A.W., K.I. Matthaei, I.G. Young, and P.S. Foster. 1997. Relationship develop in distinct stages and are recruited to peripheral sites by alter between interleukin5 and eotaxin in regulating blood and tissue eo natively activated macrophages. J. Leukoc. Biol. 81:1434–1444. http:// sinophilia in mice. J. Clin. Invest. 99:1064–1071. http://dx.doi.org/ dx.doi.org/10.1189/jlb.1106686 10.1172/JCI119234 Voehringer, D., H. E. Liang, and R.M. Locksley. 2008. Homeostasis and Neill, D.R., S.H. Wong, A. Bellosi, R.J. Flynn, M. Daly, T.K.A. effector function of lymphopenia induced “memory like” T cells in Langford, C. Bucks, C.M. Kane, P.G. Fallon, R. Pannell, et al. constitutively T cell depleted mice. J. Immunol. 180:4742–4753. 2010. Nuocytes represent a new innate effector leukocyte that me Wu, D., A.B. Molofsky, H.E. Liang, R.R. RicardoGonzalez, H.A. Jouihan, diates type 2 immunity. Nature. 464:1367–1370. http://dx.doi.org/ J.K. Bando, A. Chawla, and R.M. Locksley. 2011. Eosinophils sustain 10.1038/nature08900 adipose alternatively activated macrophages associated with glucose homeo Nguyen, K.D., Y. Qiu, X. Cui, Y.P.S. Goh, J. Mwangi, T. David, L. stasis. Science. 332:243–247. http://dx.doi.org/10.1126/science.1201475 Mukundan, F. Brombacher, R.M. Locksley, and A. Chawla. 2011. Yu, C., A.B. Cantor, H. Yang, C. Browne, R.A. Wells, Y. Fujiwara, and Alternatively activated macrophages produce catecholamines to sus S.H. Orkin. 2002. Targeted deletion of a high an ffi ity GATA binding tain adaptive thermogenesis. Nature. 480:104–108. http://dx.doi.org/10 site in the GATA1 promoter leads to selective loss of the eosinophil .1038/nature10653 lineage in vivo. J. Exp. Med. 195:1387–1395. http://dx.doi.org/10 Peschon, J.J., P.J. Morrissey, K.H. Grabstein, F.J. Ramsdell, E. Maraskovsky, .1084/jem.20020656 B.C. Gliniak, L.S. Park, S.F. Ziegler, D.E. Williams, C.B. Ware, et al. Zeyda, M., B. Wernly, S. Demyanets, C. Kaun, M. Hämmerle, B. Hantusch, M. 1994. Early lymphocyte expansion is severely impaired in interleukin 7 Schranz, A. Neuhofer, B.K. Itariu, M. Keck, et al. 2012. Severe obesity in receptordeficient mice. J. Exp. Med. 180:1955–1960. http://dx.doi creases adipose tissue expression of interleukin33 and its receptor ST2, .org/10.1084/jem.180.5.1955 both predominantly detectable in endothelial cells of human adipose Price, A.E., H. E. Liang, B.M. Sullivan, R.L. Reinhardt, C.J. Eisley, D.J. Erle, and tissue. Int J Obes (Lond). Epub before print. http://www.nature.com/ijo/ R.M. Locksley. 2010. Systemically dispersed innate IL 13 expressing cells journal/vaop/ncurrent/full/ijo2012118a.html. JEM Vol. 210, No. 3 549

Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Mar 11, 2013

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

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

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

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

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

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

References (63)

Abstract

Journal

Recommended Articles

There are no references for this article.

Our policy towards the use of cookies