Original Research

Interferon Regulatory Factor 4 Regulates Obesity-Induced Inflammation Through Regulation of Adipose Tissue Macrophage Polarization

  1. Jun Eguchi1,2,
  2. Xingxing Kong1,
  3. Masafumi Tenta2,
  4. Xun Wang1,
  5. Sona Kang1 and
  6. Evan D. Rosen1,3⇑
  1. 1Division of Endocrinology, Beth Israel Deaconess Medical Center, Boston, Massachusetts
  2. 2Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
  3. 3Harvard Medical School, Boston, Massachusetts.
  1. Corresponding author: Evan D. Rosen, erosen{at}bidmc.harvard.edu.

  1. J.E. and X.K. contributed equally to this work.

Diabetes 2013 Oct; 62(10): 3394-3403. https://doi.org/10.2337/db12-1327

Article Figures & Tables

Figures

  • FIG. 1.
    FIG. 1.

    TLR4 activation and IL-4 both induce IRF4 expression in macrophages. A: Expression of Il1b and Tnfa is induced by LPS and palmitate and repressed by IL-4 in activated PMs and BMDMs. B: Expression of Arg1 and Chi3l3 is repressed by LPS and palmitate and induced by IL-4 in activated primary PMs and BMDMs. C: Expression of Irf4 is induced by LPS, palmitate, and IL-4 in activated primary PMs and BMDMs. D: Expression of Irf4 peaks after the rise in inflammatory gene expression in response to LPS in BMDMs. A–C: RNA was harvested 16 h after treatment with LPS (10 ng/mL), palmitate (C16; 200 μmol/L), and IL-4 (10 ng/mL). D: Treatment with LPS was carried out for the indicated times. Data are normalized to Gapdh expression and are expressed as fold induction relative to BSA control. Results expressed as mean ± SD (n = 5). *P < 0.05.

  • FIG. 2.
    FIG. 2.

    Hyper-responsiveness of Irf4−/− macrophages to C16 stimulation in vitro. A: mRNA expression of Il1b and Tnfa in control (Irf4flox/flox: FLOX) and Irf4−/− (KO) PMs. QPCR analysis of Il1b and Tnfa levels after 16-h stimulation with BSA or 200 μmol/L palmitate (C16). Data are normalized to Gapdh expression and are expressed as fold induction relative to BSA. Results expressed as mean ± SD (n = 5). *P < 0.05. B: Supernatant from FLOX and IRF4 KO PMs was collected 16 h after palmitate stimulation, and TNF-α levels were measured by ELISA. Results expressed as mean ± SD. *P < 0.05. C: Quantification of NF-κB promoter activity in PMs transfected with pNF-κB-Luc reporter plasmids after 16-h treatment with BSA or 200 μmol/L palmitate (C16). Data are normalized to β-gal expression. Results expressed as mean ± SD (n = 5). *P < 0.05. D: Western blot of RAW264.7 cells after transduction with lentivirus expressing green fluorescent protein (GFP) or IRF4. E: mRNA expression of Irf4, Il1b, and Tnfa in RAW264.7 cells expressing GFP or IRF4. QPCR analysis of Irf4, Il1b, and Tnfa after 16-h stimulation with BSA or 200 μmol/L palmitate (C16). Data are normalized to Gapdh expression and expressed as fold induction relative to BSA. Results expressed as mean ± SD (n = 5). *P < 0.05. F: Quantification of NF-κB promoter activity in RAW264.7 cells expressing GFP or IRF4 and transfected with pNF-κB-Luc reporter plasmids after 16-h treatment with BSA or 200 μmol/L palmitate (C16). Data are normalized to β-gal expression. Results expressed as mean ± SD (n = 5). *P < 0.05. G: Chemotaxis of FLOX and Irf4 KO BMDMs was analyzed using a transwell migration assay. Results expressed as mean ± SD (n = 6) from three independent experiments. AU, arbitrary unit.

  • FIG. 3.
    FIG. 3.

    IRF4 deficiency in macrophages induces insulin resistance in cocultured adipocytes in control (Irf4flox/flox; FLOX) or Irf4−/− (KO) mice. A: Insulin (100 nmol/L) stimulation of 3H-2-deoxyglucose (2DG) uptake was determined in 3T3-L1 adipocytes incubated in direct contact with FLOX or KO PMs. Results expressed as mean ± SD (n = 6) from three independent experiments. *P < 0.05. B: Insulin (100 nmol/L) stimulation of 2DG uptake was determined in 3T3-L1 adipocytes incubated with conditioned medium from FLOX or KO PMs. Results expressed as mean ± SD (n = 6) from three independent experiments. *P < 0.05. C: Insulin (100 nmol/L) stimulation of 2DG uptake was determined in 3T3-L1 adipocytes incubated in contact with FLOX or KO PMs, with and without the addition of exogenous IRF4. Results expressed as mean ± SD (n = 6) from three independent experiments. *P < 0.05. D: Insulin-stimulated phosphorylation of IRS1 and Akt in 3T3-L1 adipocytes incubated in direct contact with FLOX or KO PMs, with and without the addition of exogenous IRF4.

  • FIG. 4.
    FIG. 4.

    Characterization of MI4KO (KO) mice compared with control Irf4flox/flox (FLOX) mice. A: Irf4 mRNA expression in B cells, T cells, macrophages, and mature adipocytes of male control FLOX and KO mice (n = 3). Data are normalized to Gapdh. Results are expressed as mean ± SD. *P < 0.05 relative to FLOX mice. B: IRF4 protein expression in peritoneal macrophages of male FLOX and KO mice. C: Body weights of male WT, LysM-Cre (Cre), FLOX, and KO mice on chow diet (n = 10). Results are expressed as mean ± SD. D: Body composition analysis in 18-week-old male mice by MRI (n = 10). Results are expressed as mean ± SD. E: Body weights of male WT, Cre, FLOX, and KO mice on high-fat diet (n = 10). Results are expressed as mean ± SD. F: Body composition analysis by MRI in 18-week-old male mice fed the high-fat diet (n = 10). Results are expressed as mean ± SD. G: GTT on chow. Blood glucose in 18-week-old male Cre, FLOX, and KO mice fed the chow diet was determined at the indicated times after intraperitoneal injection with a bolus of glucose (n = 10). Results are expressed as mean ± SD. H: Insulin tolerance test on chow diet. Blood glucose in 20-week-old male Cre, FLOX, and KO mice fed the chow diet was determined at the indicated times after intraperitoneal injection with a bolus of insulin (n = 10). Results are expressed as mean ± SD. I: GTT on the high-fat diet. Blood glucose in 10-week-old male Cre, FLOX, and KO mice fed a high-fat diet was determined at the indicated times after intraperitoneal injection with a bolus of glucose (n = 7–8). Results are expressed as mean ± SD. *P < 0.05. J: Evoked insulin levels during the GTT shown in I (n = 7–8). Results are expressed as mean ± SD. K: Insulin tolerance test on high-fat diet. Blood glucose in 16-week-old male Cre, FLOX, and KO mice fed a high-fat diet was determined at the indicated times after intraperitoneal injection with a bolus of insulin (n = 10). Results are expressed as mean ± SD. *P < 0.05. L: Pyruvate tolerance test on high-fat diet. Blood glucose in 12-week-old male Cre, FLOX, and KO mice fed a high-fat diet was determined at the indicated times after intraperitoneal injection with a bolus of pyruvate (n = 7–8). Results are expressed as mean ± SD. *P < 0.05. M: Fasting glucose levels in chow and high-fat fed male mice (n = 10). Results are expressed as mean ± SD. N: Fasting insulin levels in chow and high-fat fed male mice (n = 10). Results are expressed as mean ± SD. *P < 0.05.

  • FIG. 5.
    FIG. 5.

    Systemic insulin resistance in MI4KO (KO) mice compared with control Irf4flox/flox (FLOX) mice is associated with increased inflammation. A: Increased macrophage infiltration into WAT of KO mice as assessed by F4/80 staining. B: Quantification of F4/80-positive cells (n = 4 mice per group). *P < 0.05. C: QPCR analysis of genes related to inflammation in WAT (n = 7 mice per group). Data are normalized to 36B4 expression and are expressed as fold induction relative to FLOX. Results expressed as mean ± SD. *P < 0.05. D: Hematoxylin and eosin staining of liver. E: QPCR analysis of genes related to inflammation in liver (n = 7 mice per group). Data are normalized to Gapdh expression and are expressed as fold induction relative to FLOX. Results expressed as mean ± SD. *P < 0.05. F: QPCR analysis of genes related to inflammation in skeletal muscle (n = 7 mice per group). Data are normalized to 36B4 expression and are expressed as fold induction relative to FLOX. Results expressed as mean ± SD. *P < 0.05. G: Insulin-stimulated Akt phosphorylation (Ser473) in liver, skeletal muscle, and WAT of FLOX and KO mice. The graph to the right of the blots shows quantification. Data are shown as mean ± SD. *P < 0.05. H: In vitro phosphorylation of c-jun in liver, skeletal muscle, and WAT of FLOX and KO mice. The graph to the right of the blots shows quantification. Data are mean ± SD. *P < 0.05. AU, arbitrary units.

  • FIG. 6.
    FIG. 6.

    IRF4 expression is associated with M2 ATM polarization in control Irf4flox/flox (FLOX) and MI4KO (KO) mice. A: SVF cells of epididymal adipose tissue were incubated with antibodies to CD11c and CD206 and analyzed using flow cytometry. B: mRNA expression of Ccl2, Ccr2, Il1b, Il6, Nos2, and Tnfa in ATMs (n = 5 mice per group). Data are expressed as mean ± SD. *P < 0.05. C: mRNA expression of Arg1, Chi3l3, Clec10a, Il10, Mgl2, and Pparg in ATMs (n = 5 mice per group). Data are expressed as mean ± SD. *P < 0.05. D: mRNA expression of Il1b and Tnfa in GM-CSF (50 ng/mL)-induced M1 BMDMs and M-CSF (50 ng/mL)-induced M2 BMDMs. QPCR analysis of Il1b and Tnfa levels after 16-h stimulation with BSA, 200 μmol/L palmitate (C16), or IL-4 (10 ng/mL). Data are normalized to Gapdh expression and expressed as fold induction relative to BSA. Results expressed as mean ± SD (n = 5). *P < 0.05.

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Interferon Regulatory Factor 4 Regulates Obesity-Induced Inflammation Through Regulation of Adipose Tissue Macrophage Polarization
Jun Eguchi, Xingxing Kong, Masafumi Tenta, Xun Wang, Sona Kang, Evan D. Rosen
Diabetes Oct 2013, 62 (10) 3394-3403; DOI: 10.2337/db12-1327
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