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. 2010 Sep 3;107(5):650-8.
doi: 10.1161/CIRCRESAHA.110.218867. Epub 2010 Jul 15.

Interleukin-33 induces protective effects in adipose tissue inflammation during obesity in mice

Ashley M Miller  1 , Darren L Asquith, Axel J Hueber, Lesley A Anderson, William M Holmes, Andrew N McKenzie, Damo Xu, Naveed Sattar, Iain B McInnes, Foo Y Liew
Affiliations

Interleukin-33 induces protective effects in adipose tissue inflammation during obesity in mice

Ashley M Miller et al. Circ Res. .

Abstract

Rationale: Chronic low-grade inflammation involving adipose tissue likely contributes to the metabolic consequences of obesity. The cytokine interleukin (IL)-33 and its receptor ST2 are expressed in adipose tissue, but their role in adipose tissue inflammation during obesity is unclear.

Objective: To examine the functional role of IL-33 in adipose tissues and investigate the effects on adipose tissue inflammation and obesity in vivo.

Methods and results: We demonstrate that treatment of adipose tissue cultures in vitro with IL-33 induced production of Th2 cytokines (IL-5, IL-13, IL-10) and reduced expression of adipogenic and metabolic genes. Administration of recombinant IL-33 to genetically obese diabetic (ob/ob) mice led to reduced adiposity, reduced fasting glucose and improved glucose and insulin tolerance. IL-33 also induced accumulation of Th2 cells in adipose tissue and polarization of adipose tissue macrophages toward an M2 alternatively activated phenotype (CD206(+)), a lineage associated with protection against obesity-related metabolic events. Furthermore, mice lacking endogenous ST2 fed high-fat diet had increased body weight and fat mass and impaired insulin secretion and glucose regulation compared to WT controls fed high-fat diet.

Conclusions: In conclusion, IL-33 may play a protective role in the development of adipose tissue inflammation during obesity.

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Figures

Figure 1
Figure 1. IL-33 induces Th2 cytokines and chemokines in both macrophages and adipocytes in WAT cultures
Epididymal fat pads from 10 age-matched male BALB/c WT (filled bars) and BALB/c × ST2−/− (open bars) mice were pooled, dissected and separated into mature adipocyte and SVF populations. (A) Expression of ST2 by FACS in SVF cells from BALB/c mice stained with anti-F4/80 and either isotype control or anti-ST2 mAbs. SVF cells were then cultured for 7 days ± IL-33 (10-100 ng/ml). (A) Cytokines and (B) chemokines in cell supernatants (all pg/ml) collected on day 7 were measured by Luminex assay. Data shown are Means ± SEM, n=4-5 independent experiments. * p<0.05, ** p<0.01, *** p<0.001 One-way ANOVA with Dunnett’s post test comparing all values to respective control column or students unpaired t test. Cells with or without macrophages depleted (by F4/80+ cell sorting) were then cultured for 7 days ± IL-33 (10 ng/ml) and (C) cytokines and chemokines measured on day 7 were measured by Luminex assay, or (D) intracellular cytokine staining for IL-6 in day 7 SVF cells re-stimulated with PMA/Ionomycin/GolgiPlug. Numbers indicate mean % of CD45+ cells. Data are Means ± SEM, n=2-3 independent experiments. * p<0.05 students unpaired t test.
Figure 2
Figure 2. IL-33 induces changes in lipid storage and expression of metabolic genes in WAT cultures in vitro
SVF cells from BALB/c WT or BALB/c ST2−/− mice were cultured for 7 days ± PBS (open bars) or IL-33 (100 ng/ml, filled bars). (A) Oil Red O (ORO) staining for lipid storage analysis (20× magnification, inset 40×), and (B) mean number of ORO+ cells quantified (counted from 5 randomly selected microscope fields on duplicate slides) (n=3). (C) Quantitative RT-PCR analysis of total RNA extracted on day 7 for expression of adipogenic and metabolic genes compared to TBP endogenous control. (D-E) Mouse obesity array on SVF supernatants. Representative membrane images (D), and quantification of mean spot pixel density for genes >2 fold up- or down-regulated (E). Data are Means ± SEM, n=3-5 independent experiments. p<0.05, *** p<0.001 students unpaired t test.
Figure 3
Figure 3. IL-33 induces changes in adiposity and metabolic parameters in vivo
Ob/ob mice were treated with PBS (open symbols/bars) or IL-33 (filled symbols/bars) 3x/week for 3 weeks. (A) Body weight (g). (B) Food intake (g/mouse/day). (C) Representative epididymal fat pad (eWAT) images, and (D) eWAT weights. (E) Representative proton whole body MRS spectra and analysis (n=6) showing areas under the curve (AUC) for water (H2O) and lipid peaks, and (F) Percentages of body fat (%FAT) and fat free mass by MRS (%FFM). (G) Mean adipocyte size expressed in μm2 and representative histology of eWAT stained with hematoxylin and eosin (original magnification 20×). (H) Total serum cholesterol, HDL-c and triglyceride levels (mmol/L). (I) Weekly blood fasting glucose levels (mmol/L) after 16 hr overnight fast. (J) Fasting serum insulin levels (pg/ml) after 16 hr overnight fast at week 5 and 9. (K) Glucose and (L) insulin tolerance tests at week 7 or 9 in ob/ob mice. Data are Means ± SEM pooled from 2 independent experiments, n=10 mice/group. * p<0.05, ** p<0.01, *** p<0.001 students unpaired t test.
Figure 4
Figure 4. IL-33 induces accumulation of Th2 cytokines in serum and Th2 cells in WAT
(A) Circulating cytokines in serum of ob/ob mice treated with PBS or IL-33. (B-D) Epididymal fat pads from ob/ob mice treated with PBS or IL-33 were collagenase digested for SVF cell isolation and analyzed by FACS. Representative plots show that the majority of CD45+F480− cells are CD3+ T cells (B) and express ST2 (C). Intracellular cytokine staining for IL-5 in SVF cells re-stimulated with PMA/Ionomycin/GolgiPlug (D). All representative plots shown are gated on CD45+ and the numbers indicate mean % of CD45+ positive cells ± St. Dev. in each gate (n=6). Data are pooled from 2 independent experiments.
Figure 5
Figure 5. IL-33 induces accumulation of alternatively activated (M2) macrophages in WAT
Epididymal fat pads from ob/ob mice treated with PBS (open bars) or IL-33 (filled bars) were either formalin fixed and embedded in paraffin wax for immunohistochemistry or collagenase digested for SVF cell isolation and analyzed by FACS. (A) Representative photomicrographs of F4/80+ macrophages (brown) in eWAT (original magnification 40×). (B) Representative FACS plots of CD45+F4/80+ cells in eWAT with subsequent gating showing TLR2 marker expression of classically activated (M1) macrophages and the CD206 marker of alternatively activated (M2) macrophages. (C) Quantification of the % CD45+F4/80+ macrophage cells, and the % of those cells expressing either TLR2 or CD206, in eWAT. Data are Means ± SEM pooled from 2 independent experiments, n=8-10 mice/group. ** p<0.01, *** p<0.001 students unpaired t test vs. respective control.
Figure 6
Figure 6. IL-33 induces expression of M2 macrophage genes in liver
Ob/ob mice were treated with PBS (open bars) or IL-33 (filled bars) 3×/week for 3 weeks, and then sacrificed and liver removed for subsequent quantitative RT-PCR analysis of total RNA extracted for expression of (A) NOS2 and Arg1, (B) Chi313 and retnla and (C) PPARγ. Data are mean gene expression ± SEM compared to TBP endogenous control pooled from 2 independent experiments, n=10 mice/group. ** p<0.01, *** p<0.001 students unpaired t test.
Figure 7
Figure 7. Endogenous ST2 affects body weight, adipose tissue and glucose homeostasis
WT or ST2−/− mice were fed either normal diet or HFD for 24 weeks. (A) Representative WT and ST2−/− mice photographed at 24 weeks of age and body weight (g) of the mice over 24 weeks. (B) eWAT weights from WT and ST2−/− mice at 24 weeks of age. (C) Representative proton whole body MRS spectra and analysis of HFD-fed WT and ST2−/− mice at 24 weeks of age showing area under the curve (AUC) for water (H2O) and lipid peaks and (D) percentages of body fat (%FAT) and fat free mass by MRS (%FFM). (E) Fasting serum insulin levels (pg/ml) at week 24. (F) Fortnightly blood fasting glucose levels (mmol/L). Glucose (G) and insulin (H) tolerance tests in 24 week old HFD-fed WT and ST2−/− mice (n=5). Data are Means ± SEM pooled from 2 independent experiments, n=8-9 mice/group. * p<0.05, ** p<0.01, *** p<0.001 students unpaired t test.
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