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