Activated type 2 innate lymphoid cells regulate beige fat biogenesis

Abstract

Type 2 innate lymphoid cells (ILC2s), an innate source of the type 2 cytokines interleukin (IL)-5 and -13, participate in the maintenance of tissue homeostasis. Although type 2 immunity is critically important for mediating metabolic adaptations to environmental cold, the functions of ILC2s in beige or brown fat development are poorly defined. We report here that activation of ILC2s by IL-33 is sufficient to promote the growth of functional beige fat in thermoneutral mice. Mechanistically, ILC2 activation results in the proliferation of bipotential adipocyte precursors (APs) and their subsequent commitment to the beige fat lineage. Loss- and gain-of-function studies reveal that ILC2- and eosinophil-derived type 2 cytokines stimulate signaling via the IL-4Rα in PDGFRα(+) APs to promote beige fat biogenesis. Together, our results highlight a critical role for ILC2s and type 2 cytokines in the regulation of adipocyte precursor numbers and fate, and as a consequence, adipose tissue homeostasis. PAPERCLIP:

Figure 1. IL-33 promotes growth of functional beige fat in thermoneutral mice

(A) Schematic for cytokine administration and metabolic analysis in thermoneutral mice. (B) Immunoblotting for UCP1 in the scWAT of thermoneutral C57BL/6J administered various doses of IL-33 over 8 days (n=2–3 per treatment dose). (C) Immunoblotting for UCP1 in the scWAT and BAT of thermoneutral mice administered IL-33, IL-13 or IL-4 for 8 days (n=3 per cytokine treatment). (D, E) Representative sections of scWAT from thermoneutral C57BL/6J mice administered Vehicle (Veh) or IL-33 were stained with hematoxylin and eosin. (D) 100× magnification, (E) 400× magnification. (F) Cold-induced changes in oxygen consumption in thermoneutral C57BL/6J mice administered Vehicle (Veh) or IL-33 over 8 days (n=4–5 per treatment). (G) Oxygen consumption rate at various temperatures of C57BL/6J mice treated with Veh or IL-33 (n=4–5 per treatment). (H, I) Norepinephrine stimulated changes in oxygen consumption (VO₂) in conscious, thermoneutral C57BL/6J (H) and Ucp1^−/− mice that were pretreated with vehicle (Veh) or IL-33 for 8 days (n=5 per genotype and treatment). Data are represented as mean ± SEM.

Figure 2. IL-33 stimulates proliferation and commitment of adipocyte precursors to the beige fat lineage

(A, B) Quantification of ILC2 numbers (A) and activation status (B) in the scWAT of thermoneutral, heterozygous Red5 (Il5^Red5/+) mice that were administered vehicle (Veh) or IL-33 for 8 days. Expression of IL-5 (td Tomato) from the Red5 allele was used as a marker of ILC2 activation (n=9–10 per treatment). (C) Quantification of eosinophils in the scWAT of thermoneutral heterozygous Red5 (Il5^Red5/+) mice that were administered Veh or IL-33 for 8 days (n=8–10 per treatment). (D) Quantification of adipocyte precursor (AP) proliferation in the scWAT of thermoneutral Il5^Red5/+ mice administered Veh or IL-33 for 8 days, as assessed by intracellular staining for Ki67 (D) and AP cell number per fat pad (n=8–10 per treatment). (F, G) Expression of beige adipocyte markers TMEM26 and CD137 on the scWAT APs of thermoneutral Il5^Red5/+ mice administered Veh or IL-33 for 8 days (n=8–10 per treatment). Representative histograms for TMEM26 (F) and CD137 (G) are shown; clear histogram-Veh, shaded histogram-IL-33. (H–J) Expression of IL1RLI (H), IL-5Rα (I), and IL-4Rα (J) on scWAT APs of mice. For IL1RL1 (H), the clear histogram represents WT APs, while the shaded represents Il1rl1^−/− APs. For IL-5Rα (I), the dashed line histogram represents isotype, the solid line represents APs stained for IL-5Rα, and the shaded histogram represents eosinophils stained for IL-5Rα. For IL-4Rα (J), the solid line histogram represents isotype and the shaded histogram represents APs stained for IL-4Rα. (K, L) Quantification of IL-33 induced AP proliferation in the scWAT of 114/13^−/− (K) and Il4ra^−/− (L) mice (n=4–8 per genotype and treatment). (M) Immunoblotting for UCP1 in the scWAT and BAT of thermoneutral WT and Il4ra^−/− mice administered IL-33 for 8 days (n=2–3 per genotype and treatment). (N–P) Quantification of AP proliferation (N), TMEM26 (O) and CD137 (P) expression in Rag2^−/− and Rag2^−/− Il2rgc^−/− mice treated with IL-33 (n=6–8 per genotype and treatment). Data are represented as mean ± SEM. See also Figure S1 and S2.

Figure 3. Type 2 cytokine signaling controls physiologic expansion of adipocyte precursors

(A) Age-dependent proliferation of adipocyte precursors (APs) in the scWAT of C57BL/6J mice as assessed by intracellular staining for Ki67 (n=4–5 per age). (B) Age-dependent incorporation of BrdU by scWAT APs in C57BL/6J mice (n=4–5 per age). (C) Quantification of pSTAT6 levels in scWAT APs of C57BL/6J mice at different ages (n=10–l1 per age). (D) Quantification of IL-4-producing cells in the scWAT of 4get mice at different ages. GFP expression marks cells competent for production of IL-4 (n=5 per age). (E–J) Quantification of Ki67⁺ (E, G, I) and total APs (F, H, J) in scWAT of 5 week-old ΔdblGATA (E, F), Il4/13^−/− (G, H), and Il4rα^−/− (I, J) mice (n=5–10 per genotype). Data are represented as mean ± SEM. See also Figure S3.

Figure 4. IL-4Rα signaling in PDGFRα⁺ cells promotes expansion of adipocyte precursors

(A) Quantification of scWAT adipocyte precursor (AP) proliferation in 5 week-old Il4ra^f/f and Il4ra^f/f/Lyz2^Cre mice (n=8–10 per genotype). (B) Analysis of IL-4-induced phosphorylation of STAT6 (pSTAT6) in scWAT APs of Il4ra^f/f and Il4ra^f/f Pdgfra^Cre mice (n=3–4 per genotype). (C–D) Quantification of scWAT AP proliferation by Ki67 staining (C) and total number (D) in 5 week-old Il4ra^f/f and Il4ra^f/f Pdgfra^Cre mice (n=7–12). (E) Quantification of scWAT AP proliferation by Ki67 staining in 8–10 week old C57BL/6J mice at 24 and 48 hours after injection with vehicle or IL-4 (n=5 per time point). (F, H) BrdU incorporation by the scWAT APs of wild type and Il4ra^−/− mice (F) or Il4ra^f/f and Il4ra^f/f/Pdgfra^Cre mice (H) 48 hours after administration of IL-4 (n=6–8 per genotype and treatment). (G) Quantification of scWAT AP proliferation by Ki67 staining 48 hours after administration of vehicle or IL-13 (n=6–8 per genotype and treatment). (I) Quantification of scWAT AP number in Il4ra^f/f and Il4ra^f/f/Pdgfra^Cre mice 48 hours after administration of vehicle or IL-4 (n=7 per genotype). (J) APs purified from wild type and Il4ra^−/− mice were stimulated with IL-4, and cellular proliferation was quantified by intracellular staining for Ki67 48 hours later (n=4 per genotype and treatment). (K) Quantification of scWAT AP proliferation in Il4ra^f/f and Il4ra^f/f/Pdgfra^Cre mice 48 hours after administration of vehicle or IL-33 (n=8–10 per genotype and treatment). Data are represented as mean ± SEM. See also Figure S4.

Figure 5. IL-4 and IL-13 direct commitment of PDGFRα⁺ adipocyte precursors to beige adipogenic precursors

(A) Quantitative RT-PCR analysis of beige adipocyte precursor markers in APs purified from the scWAT of C57BL/6J stimulated with vehicle or IL-4 (n=3 per condition and time point; data presented as mean ± SD). (B) Quantitative RT-PCR analysis of beige adipocyte precursor markers in APs purified from Balb/cJ or Il4ra^−/− mice that were stimulated with vehicle or IL-4 for 48 hours (n=3 per genotype and treatment; data presented as mean ± SD). (C–E) Flow cytometric analysis of IL-4Rα (C), CD137 (D) and TMEM26 (E) expression in scWAT APs of mice injected with vehicle or IL-4. Clear histogram: vehicle; shaded histogram: IL-4. (F–H) Quantification of IL-4Rα, CD137, and TMEM26 expression in scWAT APs 48 hours after administration of vehicle or IL-4 (n=5 per treatment). (I, J) Quantification of CD137 and TMEM26 expression in the scWAT APs of Il4ra^f/f and Il4ra^f/f/Pdgfra^Cre mice 48 hours after administration of IL-4 (n=4–6 per genotype and treatment). (K, L) Quantification of CD137 and TMEM26 expression in the scWAT APs of 5 week-old Il4ra^f/f and Il4ra^f/f/Pdgfra^Cre mice (n=8–12 per genotype). (M) Quantitative RT-PCR analysis of beige/brown adipocyte genes after in vitro differentiation of scWAT APs purified from Balb/cJ or Il4ra^−/− mice (n=3 per genotype and treatment; data presented as mean ± SD). (N) Immunoblot analysis for UCP1 and IL-4Rα in in vitro-differentiated APs. MDI refers to stimulation of differentiation by adipogenic cocktail, (n=3 genotype and treatment). Unless otherwise indicated, data are represented as mean ± SEM. See also Figure S5 and S6.

Figure 6. IL-4Rα signaling in adipocyte precursors directs growth of beige fat

(A) Representative sections of scWAT from 5 week-old Il4ra^f/f and Il4ra^f/f/Pdgfra^Cre mice were stained with hematoxylin and eosin. 100× magnification (top panels), 400× magnification (bottom panels). (B) UCP1 protein expression in scWAT and BAT of 5 week-old Il4ra^f/f and Il4ra^f/f/Pdgfra^Cre mice housed at 22°C (n=3 per genotype). (C–F) Assessment of metabolic rate, food intake, and activity in 5-week-old Il4ra^f/f and Il4ra^f/f/Pdgfra^Cre mice housed at 22°C (n=8 per genotype); (C) oxygen consumption (VO₂), (D) RER, (E) food intake, and (F) total activity. Data are represented as mean ± SEM.

Figure 7. IL-4Rα signaling in mature adipocytes is dispensable for growth of beige fat

(A) Histological analysis of scWAT of 5-week-old Il4ra^f/f and Il4ra^f/f/Adipoq^Cre mice housed at 22°C. Representative sections were stained with hematoxylin and eosin, and images are shown at 100× magnification (top panels) and 400× magnification (bottom panels). (B) Immunoblot analysis of UCP1 protein expression in scWAT and BAT of 5-week-old Il4ra^f/f and Il4ra^f/f/Adipoq^Cre mice. (C–F) Assessment of energy expenditure in 5 week-old Il4ra^f/f and Il4ra^f/f/Adipoq^Cre mice was performed using CLAMS; (C) oxygen consumption (VO₂), (D) RER, (E) food intake, and (F) total activity. Data are represented as mean ± SEM.