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. 2019 Feb 1;129(2):834-849.
doi: 10.1172/JCI123069. Epub 2019 Jan 22.

Adipocyte-secreted exosomal microRNA-34a inhibits M2 macrophage polarization to promote obesity-induced adipose inflammation

Yong Pan  1   2 , Xiaoyan Hui  1   2 , Ruby Lai Chong Hoo  1   3 , Dewei Ye  4   5 , Cyrus Yuk Cheung Chan  1   2 , Tianshi Feng  1   3 , Yu Wang  1   3 , Karen Siu Ling Lam  1   2 , Aimin Xu  1   2   3
Affiliations

Adipocyte-secreted exosomal microRNA-34a inhibits M2 macrophage polarization to promote obesity-induced adipose inflammation

Yong Pan et al. J Clin Invest. .

Abstract

Persistent, unresolved inflammation in adipose tissue is a major contributor to obesity-associated metabolic complications. However, the molecular links between lipid-overloaded adipocytes and inflammatory immune cells in obese adipose tissues remain elusive. Here we identified adipocyte-secreted microRNA-34a (miR-34a) as a key mediator through its paracrine actions on adipose-resident macrophages. The expression of miR-34a in adipose tissues was progressively increased with the development of dietary obesity. Adipose-selective or adipocyte-specific miR-34a-KO mice were resistant to obesity-induced glucose intolerance, insulin resistance, and systemic inflammation, and this was accompanied by a significant shift in polarization of adipose-resident macrophages from proinflammatory M1 to antiinflammatory M2 phenotype. Mechanistically, mature adipocyte-secreted exosomes transported miR-34a into macrophages, thereby suppressing M2 polarization by repressing the expression of Krüppel-like factor 4 (Klf4). The suppressive effects of miR-34a on M2 polarization and its stimulation of inflammatory responses were reversed by ectopic expression of Klf4 in both bone marrow-derived macrophages and adipose depots of obese mice. Furthermore, increased miR-34a expression in visceral fat of overweight/obese subjects correlated negatively with reduced Klf4 expression, but positively with the parameters of insulin resistance and metabolic inflammation. In summary, miR-34a was a key component of adipocyte-secreted exosomal vesicles that transmitted the signal of nutrient overload to the adipose-resident macrophages for exacerbation of obesity-induced systemic inflammation and metabolic dysregulation.

Keywords: Adipose tissue; Inflammation; Macrophages; Metabolism; Obesity.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. MiR-34a is highly expressed in mature adipocytes of visceral adipose tissues and is elevated in obese mice.
(A) Copy numbers of miR-34a in epididymal white adipose tissue (epiWAT), subcutaneous white adipose tissue (scWAT), brown adipose tissue (BAT), liver, and heart of 12-week-old C57BL/6J mice fed with standard chow (STC) (n = 6). (B) The mRNA abundance of miR-34a in epiWAT of mice fed with STC or high-fat diet (HFD) for 0, 1, 8, 16, and 32 weeks (n = 6–8). (C) The mRNA abundance of miR-34b and miR-34c in the epiWAT of mice on STC or HFD for 16 weeks (n = 5). (D) Copy number of miR-34a in mature adipocytes and stromal vascular fraction (SVF) of epiWAT isolated from mice on STC or HFD for 16 weeks (n = 5). (E) The mRNA abundance of Tnfa in epiWAT of mice on STD or HFD for various periods (n = 6–8). (F) Correlation analysis between miR-34a and Tnfa levels in the same epiWAT of mice after HFD (n = 35). Correlation was assessed by nonparametric Spearman’s test (r = 0.8095, P < 0.001). (G and H) The mRNA abundance of miR-34a in mature adipocytes treated with vehicle, recombinant TNF-α protein (G), or palmitic acid (H) for 24 hours (n = 4). Data represent mean ± SEM. Differences between groups were determined by ANOVA (B–E, G, and H); *P < 0.05, **P < 0.01, ***P < 0.001. Copy numbers of miR-34a were calculated based on a standard curve generated using a synthetic lin-4. Relative levels of miR-34a, miR-34b, and miR-34c were normalized to sno202, and Tnfa mRNA levels were normalized to 18S RNA abundance.
Figure 2
Figure 2. Adipose tissue–specific ablation of miR-34a protects mice from obesity-induced glucose intolerance and insulin resistance.
(A) Representative photos of adipose-specific miR-34a–KO mice and their WT miR-34afl/fl littermates fed with either STC or HFD for 16 weeks. (B) Dynamic changes in body weight of WT and KO mice during 16 weeks of STC or HFD feeding (n = 6–8). (C and D) Fat mass of whole body (C) and individual tissues (D) (n = 6–8). (E) Glucose tolerance test after mice were fed with HFD for 0, 4, 8, and 16 weeks (n = 6–8). (F) Serum concentrations of fed insulin in mice on HFD for 0, 4, 8, and 16 weeks (n = 6–8). (G) Insulin tolerance test after mice were fed with HFD for 0, 4, 8, and 16 weeks (n = 6–8). (H and I) The KO mice or WT littermates on STC or HFD for 16 weeks were injected intraperitoneally with insulin (2 IU/kg body weight) for 10 minutes. (H) Immunoblot analyses of epiWAT samples using antibodies against phospho-AKT (p-AKT-S473), total AKT, and tubulin. (I) Densitometry analysis for the p-AKT/AKT ratio (n = 4–5). Data represent mean ± SEM. Differences between WT and KO mice were determined by ANOVA (B–G and I); *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3. Adipose miR-34a modulates infiltration and polarization of macrophages in mice with dietary obesity.
(A and B) Representative images of immunohistological staining of F4/80 (A) and quantification of crown-like structures (CLSs; B) in epiWAT of WT or KO mice on HFD for 0, 8, and 16 weeks (n = 6). Scale bar: 40 μm. (C and D) Cells isolated from SVFs of epiWAT in KO and WT mice fed with HFD for 0, 8, and 16 weeks were subjected to flow cytometry analysis for percentage of F4/80+ total macrophages (C), M1 (CD11c+CD206–), and M2 (CD206+CD11c–) within the macrophage population (D) (n = 4–6). (E and F) Immunoblot analysis for the expression of iNOS and arginase 1 (Arg1) in epiWAT of mice on STC or HFD for 16 weeks (E), and densitometric analysis for their abundance relative to tubulin (F) (n = 6–8). (G) Real-time PCR analysis for the mRNA levels of the M1 and M2 markers in epiWAT of mice on STD or HFD 16 weeks (n = 6). Data represent mean values ± SEM. Differences between groups were determined by ANOVA (B–D, F, and G); *P < 0.05, **P < 0.01, ***P < 0.001. Gene levels were normalized to 18S RNA abundance.
Figure 4
Figure 4. The metabolic benefits of adipose-selective miR-34a deficiency are mediated by macrophages.
Male adipose-specific miR-34a–KO mice or WT controls on HFD for 12 weeks were intraperitoneally injected with PBS-liposomes or clodronate-conjugated liposomes (CLOD-liposomes) for 4 weeks. (A) Representative flow cytometry dot plots showing F4/80+ total macrophages in SVFs of epiWAT from HFD-fed WT mice injected with PBS- or CLOD-liposomes for 4 weeks. (B) Representative images of immunohistological staining of F4/80 in epiWAT from WT and KO mice treated with PBS- or CLOD-liposomes. Scale bar: 40 μm. (C) Glucose tolerance test of HFD-fed WT and KO mice treated with PBS- or CLOD-liposomes (n = 8; *P < 0.05, **P < 0.01 compared with KO+PBS). (D) Area under the curve (AUC) of glucose tolerance test (n = 8). (E) Insulin tolerance test of mice (n = 8; *P < 0.05, **P < 0.01 compared with KO+PBS). (F) AUC of insulin tolerance test (n = 8). (G–L) Serum levels of insulin (G), TNF-α (H), IL-6 (I), IL-1β (J), MCP-1 (K), and adiponectin (L) determined with ELISA (n = 8). Data represent mean ± SEM. Differences were determined by ANOVA (C–L); *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5. MiR-34a modulates inflammation and macrophage polarization by targeting Klf4.
(A) The mRNA abundance of Klf4 in epiWAT of mice (n = 6–8). (B) Correlative analysis between the mRNA abundance of miR-34a and Klf4 levels (n = 35). Correlation was assessed by nonparametric Spearman’s test. (C) The protein levels of Klf4 in mature adipocytes and SVF of epiWAT isolated from mice on STC or HFD for 16 weeks. (D) Immunoblot analysis for Klf4 protein in SVF. (E and F) Analysis of miR-34a levels (E) and Klf4 protein levels (F) in bone marrow derived macrophages (BMDMs) infected with lentivirus encoding eGfp or miR-34a. (n = 4). (G and H) A schematic diagram showing the construction of the luciferase reporter vectors (G), and luciferase assays were performed in HEK293 cells (H, n = 4). Data are presented as ratio of Renilla luciferase (RL) to firefly luciferase (FL) activity. (I and J) BMDMs were infected with lentivirus for 24 hours, followed by stimulation with IL-4 (10 ng/ml) for 24 hours. Analysis of gene expression of Klf4 and Arg1 (I) and protein levels of Klf4 and Arg1 (J) (n = 4). (K–N) KO and WT littermates on HFD for 14 weeks were locally injected with lentivirus expressing siRNA against Klf4 (siKlf4) or scramble control (siCtrl) in epiWAT for 2 weeks. (K) Immunoblot analysis for KLF4 protein in SVFs. (L and M) Flow cytometric quantification for percentage of total macrophages (L), M1 and M2 macrophages in SVFs (M, n = 4–5). (N) The mRNA abundance of M1 macrophage–associated genes in epiWAT (n = 4–5). Data represent mean ± SEM. Differences were determined by ANOVA (A, H, I, and L–N) or Student’s t test (E); *P < 0.05, **P < 0.01, ***P < 0.001. MiR-34a abundance was normalized to sno202 level, and other gene levels were normalized to 18S RNA abundance.
Figure 6
Figure 6. Exosomes secreted from mature adipocytes transport miR-34a into macrophages to suppress M2 polarization via downregulation of KLF4.
(A) Electron microscopy (top panel) and Western blot analysis (bottom panel) of exosomes (EXO) secreted by adipocytes (scale bar: 100 nm). (B) Mature adipocytes (MA) in epiWAT were isolated from miR-34a KO or their WT littermates fed with HFD and cultured in 1 ml FBS-free DMEM for 48 hours. Analysis of miR-34a levels in isolated exosomes from conditional medium (n = 6–8). (C–E) Isolated exosome was prestained with Dil-C18 and applied to IL-4–treated BMDMs for 24 hours. Schematic depiction of the experiment (C). Flow cytometry analysis for the percentage of Dil-C18–positive BMDMs (D). Abundance of miR-34a in BMDMs stimulated with exosomes from KO mice and WT littermates (E). Blank DMEM (BLK) was used as control (n = 5). (F–H) BMDMs were pretreated without or with exosomes secreted from KO mice and WT littermates, followed by treatment without or with IL-4 for 24 hours. Percentage of M2 macrophages as determined by flow cytometry (F). Immunoblot analyses of Klf4 and Arg1 protein levels (G) and densitometry analysis (H) (n = 5). (I–K) BMDMs were infected with 1.5 × 106 viral particles of adeno-associated virus (AAV), followed by treatment with exosomes derived from mice on HFD or STC for 16 weeks in the presence of IL-4 for another 24 hours. Percentage of M2 macrophages as determined by flow cytometry (I). Immunoblot analyses of Klf4 and Arg1 protein levels (J), and densitometry analysis (K) (n = 5). Data represent mean ± SEM. Differences were determined by ANOVA (B, E, F, H, I, and K); *P < 0.05, **P < 0.01, ***P < 0.001. MiR-34a levels were normalized to sno202 for all experiments, and other gene levels were normalized to 18S RNA abundance.
Figure 7
Figure 7. Dysregulated miR-34a/KLF4 axis in adipose tissues is associated with inflammation and insulin resistance in overweight/obese Chinese subjects.
(A) Real-time PCR analysis for the expression levels of miR-34a in visceral adipose tissue (VAT) and subcutaneous white adipose tissue (scWAT) from 29 lean and 24 overweight/obese individuals undergoing elective abdominal surgery for benign, noninfective gynecological conditions. (B) Gene expression of Klf4. (C) Correlation between miR-34a and KLF4 levels in visceral fat of these study subjects. (D and E) The association between miR-34a and the mRNA abundance of TNFA (D) and IL6 (E) in visceral fat. (F and G) Correlation between the insulin resistance index (HOMA-IR) and the expression level of miR-34a (F) or KLF4 (G) in the visceral fat. Data represent mean ± SEM. Differences were determined by ANOVA (A) or Student’s t test (B), and correlation was assessed by nonparametric Spearman’s test (C–G); *P < 0.05, ***P < 0.001. MiR-34a expression was normalized to sno202 levels, and gene abundance of Klf4, TNFA, and IL6 were normalized to 18S RNA abundance.
Figure 8
Figure 8. Schematic summary of the role of adipocyte-secreted exosomal miR-34a in obesity-induced alterations in macrophage polarization, adipose inflammation, and insulin resistance.
Hypertrophic adipocytes in obese adipose tissue release exosomes, which carry miR-34a to adipose-resident macrophages, where it shifts macrophage polarization from M2 to M1 by downregulation of Klf4, thereby leading to induction of proinflammatory cytokines and local fibrosis and downregulation of adiponectin, thereby leading to systemic insulin resistance and glucose intolerance.
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