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Group 2 Innate Lymphoid Cells Promote Beiging of White Adipose Tissue and Limit Obesity

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Group 2 Innate Lymphoid Cells Promote Beiging of White Adipose Tissue and Limit Obesity

Jonathan R Brestoff et al. Nature.

Abstract

Obesity is an increasingly prevalent disease regulated by genetic and environmental factors. Emerging studies indicate that immune cells, including monocytes, granulocytes and lymphocytes, regulate metabolic homeostasis and are dysregulated in obesity. Group 2 innate lymphoid cells (ILC2s) can regulate adaptive immunity and eosinophil and alternatively activated macrophage responses, and were recently identified in murine white adipose tissue (WAT) where they may act to limit the development of obesity. However, ILC2s have not been identified in human adipose tissue, and the mechanisms by which ILC2s regulate metabolic homeostasis remain unknown. Here we identify ILC2s in human WAT and demonstrate that decreased ILC2 responses in WAT are a conserved characteristic of obesity in humans and mice. Interleukin (IL)-33 was found to be critical for the maintenance of ILC2s in WAT and in limiting adiposity in mice by increasing caloric expenditure. This was associated with recruitment of uncoupling protein 1 (UCP1)(+) beige adipocytes in WAT, a process known as beiging or browning that regulates caloric expenditure. IL-33-induced beiging was dependent on ILC2s, and IL-33 treatment or transfer of IL-33-elicited ILC2s was sufficient to drive beiging independently of the adaptive immune system, eosinophils or IL-4 receptor signalling. We found that ILC2s produce methionine-enkephalin peptides that can act directly on adipocytes to upregulate Ucp1 expression in vitro and that promote beiging in vivo. Collectively, these studies indicate that, in addition to responding to infection or tissue damage, ILC2s can regulate adipose function and metabolic homeostasis in part via production of enkephalin peptides that elicit beiging.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Human and murine white adipose tissue contains group 2 innate lymphoid cells that are dysregulated in obesity
(a) Identification of lineage (Lin)-negative CD25+ CD127+ innate lymphoid cells (ILCs) in human abdominal subcutaneous white adipose tissue (WAT). Pre-gated on live CD45+ Lin cells that lack CD3, CD5, TCRαβ, CD19, CD56, CD11c, CD11b, CD16, and FcεRIα. (b) Histograms of GATA-3 and IL-33R expression by human WAT ILCs (line). Shaded histogram, isotype control. (c) Identification of Lin CD25+ CD127+ ILCs in murine epididymal (E)-WAT. Pre-gated on live CD45+ Lin cells that lack CD3, CD5, CD19, NK1.1, CD11c, CD11b and FcεRIα. (d) Histograms of GATA-3 and IL-33R expression by murine E-WAT ILCs (line). Shaded histogram, isotype control. (e) Representative plots and (f) frequencies of human WAT ILC2s from donors stratified into non-obese (body mass index [BMI]<30.0 kg/m2, n=7) and obese (BMI≥30.0 kg/m2, n=7) groups. (g) Representative plots and frequencies of murine E-WAT ILC2s from mice fed a control diet (CD, 10% kcal fat, n=5) or high fat diet (HFD, 45% kcal fat, n=4) for 12 weeks. (h) Numbers of murine ILC2s/gram E-WAT in mice fed a CD (n=8) or HFD (n=6) for 12 weeks. Student’s t-test, *P<0.05, **P<0.01, ***P<0.001.
Figure 2
Figure 2. IL-33 critically regulates ILC2 responses in white adipose tissue and limits adiposity
(af) Il33+/+ (n=6) or Il33−/− (n=5) mice were fed a low fat diet (10% kcal fat) for 12 weeks starting at 7 weeks of age. (a) Frequencies and (b) numbers of live CD45+ Lin CD25+ IL-33R+ ILC2s in epididymal (E)-WAT. Plots pre-gated on CD45+ Lin cells that lack CD3, CD5, CD19, NK1.1, CD11c, CD11b and FcεRIα. (c) Numbers of ILC2s in inguinal (i)WAT. (d) Body weight, first 10 weeks of feeding. (e) Absolute and relative E-WAT and iWAT weights. (f) Body composition. (gn) Wildtype mice were treated with phosphate buffered saline (PBS, n=10) or recombinant murine IL-33 (12.5 μg/kg/day, n=12) by intraperitoneal injection for 7 days. (g) Frequencies and (h) numbers of ILC2s in E-WAT. (i) Numbers of ILC2s in iWAT. (j) Body weight and (k) body composition. (l) Caloric expenditure over a 24-hour period, days 6-to-7 of treatment. Non-shaded area, lights on. Shaded area, lights off. (m) Food intake and (n) total activity (beam breaks) over the 24-hour period in (L). Student’s t-test or ANOVA with repeated measures. *P<0.05, **P<0.01, ***P<0.001.
Figure 3
Figure 3. IL-33 and ILC2s contribute to beiging of white adipose tissue
(ac) Il33+/+ (n=6) or Il33−/− (n=5) mice were fed a low fat diet (10% kcal fat) for 12 weeks starting at age 7 weeks. Uncoupling protein 1 (UCP1) immunohistochemistry (IHC) in iWAT from (a) Il33+/+ or (b) Il33−/− mice. Scale bars, 100 μm. (c) Ucp1 transcript levels in iWAT. (df) Wildtype mice were treated with PBS or recombinant murine IL-33 (12.5 μg/kg/day) by intraperitoneal injection for 7 days. (d) E-WAT and (e) iWAT UCP1 IHC. Scale bars, 100 μm. (f) Ucp1 transcript levels in E-WAT and iWAT. (gk) Sort-purified CD45.1+ ILC2s (105) from E-WAT of IL-33-treated mice were transferred into 12-week-old CD45.2+ wildtype recipients by subcutaneous and intraperitoneal injection daily for 4 days (PBS, n=8; ILC2, n=8 except panel K). (g) Representative plots identifying donor and recipient ILC2s. Plots pre-gated on live CD45+ Lin CD25+ IL33R+ cells. Lineage cocktail: CD3, CD5, CD19, NK1.1, CD11c, CD11b and FcεRIα. (h) Total numbers of ILC2s per gram iWAT. (i) iWAT UCP1 IHC, bar 100 μm. (j) Ucp1 expression in iWAT. (k) iWAT oxygen consumption. PBS, n=14; ILC2, n=15. (lm) Sort-purified congenic CD45.1+ ILC2s (105) from E-WAT of IL-33-treated mice were transferred into Rag2−/− γc−/− mice once by intraperitoneal injection. ILC2-sufficient Rag2−/− mice, ILC2-deficient Rag2−/− γc−/− mice and ILC2-reconstituted Rag2−/− γc−/− mice were treated with PBS or recombinant murine IL-33 (12.5 μg/kg/day) by intraperitoneal injection for 7 days (n=4 mice/group). (l) ILC2 numbers per gram E-WAT. (m) Ucp1 expression in E-WAT. Student’s t-test or two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001.
Figure 4
Figure 4. ILC2s produce methionine-enkephalin, a peptide that promotes beige fat formation
(a) Gene expression enrichment analyses of 69 obesity-associated genes in ILC2s (x-axis) versus ILC3s (y-axis). Genes significantly enriched in one cell type but not the other are red. (b) Differential expression of PCSK1 target genes in ILC2s vs ILC3s. (c) Intracellular staining of MetEnk (black line) or rabbit IgG isotype control (shaded histogram) in ILC2s sort-purified from E-WAT and re-stimulated in vitro with IL-2 and IL-7 (10 ng/mL) for 4 days. (d) MetEnk mean fluorescence intensity (MFI) in sort-purified E-WAT ILC2s re-stimulated in vitro with IL-2 and IL-7 (10 ng/mL) with or without IL-33 (30 ng/mL) for 4 days. Isotype control MFI for each group was subtracted before calculating relative expression. Shown are averages from 4 independent experiments, each representing pooled cells from n=3–5 mice and measured in duplicate or triplicate. (ej) Wildtype mice were treated with PBS (n=7) or MetEnk (n=9) by subcutaneous injection (10 mg/kg/day) for 5 days. (e) Uncoupling protein 1 (UCP1) immunohistochemistry (IHC) in inguinal white adipose tissue (iWAT). Scale bars, 100 μm. (f) iWAT Ucp1 expression, (g) oxygen consumption, (h) relative mass, (i) Il4 and Il13 expression and (j) numbers of eosinophils (Eos, live CD45+ SiglecF+) and alternatively activated macrophages (AAMacs, live CD45+ SiglecF F4/80+ CD206+) per gram of adipose. (k) Il33 and Penk mRNA and (l) Ogfr and Oprd1 mRNA in iWAT vs brown adipose tissue (BAT), n=8. (mn) Stromal vascular fraction (SVF) cells from (m) iWAT or (n) BAT of 4-week-old C57BL/6 mice were differentiated into adipocytes for 2 days, treated with PBS or 50 μM MetEnk from days 2–8 and harvested on day 8 (iWAT: n=7 PBS, n=8 MetEnk; BAT: n=6 PBS, n=6 MetEnk). Student’s t-test or ANOVA, *P<0.05, ***P<0.001.

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