Adipocyte-Secreted IL-6 Sensitizes Macrophages to IL-4 Signaling

Abstract

Complex bidirectional cross talk between adipocytes and adipose tissue immune cells plays an important role in regulating adipose function, inflammation, and insulin responsiveness. Adipocytes secrete the pleiotropic cytokine IL-6 in response to both inflammatory and catabolic stimuli. Previous studies have suggested that IL-6 secretion from adipocytes in obesity may promote adipose tissue inflammation. Here, we investigated catabolic stimulation of adipocyte IL-6 secretion and its impact on adipose tissue immune cells. In obesity, catecholamine resistance reduces cAMP-driven adipocyte IL-6 secretion in response to catabolic signals. By restoring adipocyte catecholamine sensitivity in obese adipocytes, amlexanox stimulates adipocyte-specific IL-6 secretion. We report that in this context, adipocyte-secreted IL-6 activates local macrophage STAT3 to promote Il4ra expression, thereby sensitizing them to IL-4 signaling and promoting an anti-inflammatory gene expression pattern. Supporting a paracrine adipocyte to macrophage mechanism, these effects could be recapitulated using adipocyte conditioned media to pretreat bone marrow-derived macrophages prior to polarization with IL-4. The effects of IL-6 signaling in adipose tissue are complex and context specific. These results suggest that cAMP-driven IL-6 secretion from adipocytes sensitizes adipose tissue macrophages to IL-4 signaling.

Graphical abstract

Figure 1

STAT3 phosphorylation in adipose cells after amlexanox treatment. Experiments were performed 4 h after gavage with 25 mg/kg amlexanox or vehicle control in obese male mice aged 20–24 weeks. A: Western blot analysis of STAT3 and pY705 STAT3 in epididymal (eWAT) and inguinal WAT (iWAT) of Stat3 adipocyte-specific KO (SAKO) mice and floxed littermate controls. B: Immunohistochemical analysis of pY705 STAT3 brown DAB staining. Slides were also stained with hematoxylin and eosin. Tissues were harvested and immediately fixed after 52 h of amlexanox treatment by daily oral gavage (n = 3 per treatment). Scale bar = 50 μm. C: Quantification of STAT3 over p38 levels in eWAT and iWAT. The effect of genotype is significant in both tissues at P < 0.01 by two-way ANOVA. D: Quantification of pY705 STAT3 over total STAT3 levels in eWAT and iWAT. *P < 0.05 vehicle vs. amlexanox within genotype; ∼P < 0.05 WT vs. KO within the treatment group. E–J: FACS analysis of the percent positivity for pY705 STAT3 in SVC populations (n = 5 per treatment): all Cd45⁺ immune cells (E); proinflammatory ATMs Cd45⁺, Cd64⁺, and Cd11c^High (F); anti-inflammatory ATMs Cd45⁺, Cd64⁺, and Cd11c^Low (G); neutrophilsCd45⁺ and Ly6G⁺ (H); dendritic cells Cd45⁺, Cd64⁻, and Cd11c⁺ (I); and T cells Cd45⁺ and Cd3⁺ (J). Statistical significance determined by post hoc analysis after significant two-way ANOVA. *P < 0.05 by Student t test vehicle vs. amlexanox. V, vehicle.

Figure 2

STAT3 activation in adipose tissue immune cells is IL-6 dependent. Experiments were performed 4 h after gavage with 25 mg/kg amlexanox or vehicle control in obese Il6 KO and WT littermate control male mice aged 20–24 weeks. A: Serum IL-6 levels (n = 8 per group). B–E: Immunohistochemical analysis of pY705 STAT3 (brown DAB staining). Slides also stained with hematoxylin and eosin. Tissues were harvested and immediately fixed 4 h after gavage. Liver is shown in B and C and WAT in D and E. Panels B and D show representative images from each genotype and treatment (scale bar = 100 μm), and panels C and E show the percentage of positive nuclei in sections from four animals per condition (three fields of view with ∼200 nuclei each were averaged for each animal). F: Relative expression of Socs3 in WAT (n = 8 per group). G–K: FACS analysis of the percent positivity of pY705 STAT3 in SVC populations (n = 4 per group): proinflammatory ATMs Cd45⁺, Cd64⁺, and Cd11c^High (G); anti-inflammatory ATMs Cd45⁺, Cd64⁺, and Cd11c^Low (H); neutrophils Cd45⁺ and Ly6G⁺ (I); dendritic cells Cd45⁺, Cd64⁻, and Cd11c⁺ (J); and T cells Cd45⁺ and Cd3⁺ (K). Statistical significance determined by post hoc analysis after significant two-way ANOVA. *P < 0.05 vehicle vs. amlexanox within genotype; ∼P < 0.05 WT vs. KO within treatment group; #P < 0.05 vehicle vs. amlexanox within genotype using Fisher exact test. AU, arbitrary unit.

Figure 3

Adipocyte-secreted IL-6 sensitizes macrophages to IL-4. Adipocyte conditioned media was generated by treating adipocytes with 100 μmol/L amlexanox in RPMI medium for 4 h. Direct treatment of BMDMs with amlexanox was also performed with 100 μmol/L amlexanox. A: Schematic of adipocyte media conditioning and treatment of BMDMs. B: Percent CD301⁺ staining of F4/80, CD11b dual-positive BMDM treated with amlexanox directly, or ACM (n = 3 per group). *P < 0.05, comparison indicated by line. C–E: Gene expression in BMDMs pretreated with NCM, VCM, or ACM for 24 h before the addition of IL-4 for another 24 h (n = 4 per group). *P < 0.05 ACM vs. VCM; ∼P < 0.05 ACM vs. NCM. F–H: Gene expression in BMDMs treated with 50 ng/mL IL-6 with and without amlexanox, normalized to the VCM (n = 6 per group). *P < 0.05 vehicle vs. amlexanox; ∼P < 0.05 control vs. IL-6. I: Schematic of adipocyte media conditioning with neutralizing antibodies and administration to BMDMs. J–M: BMDMs treated with VCM or ACM in which IL-6 was neutralized with IL-6NA or IgG control. *P < 0.05 IgG vs. IL-6NA; ∼P < 0.05 ACM vs. NCM. J: Western blot analysis of pY705 STAT3; β-tubulin serves as a loading control (n = 3 per group). Statistical significance determined by post hoc analysis after significant ANOVA. A, amlexanox; AU, arbitrary unit; V, vehicle; V-cont, vehicle control.

Figure 4

In vivo sensitization of macrophages to IL-4 by amlexanox requires STAT3. A–C: Obese male mice aged 20–24 weeks were treated with 25 mg/kg amlexanox or vehicle control. After 4 h, the eWAT was collected and digested with collagenase (n = 3 per group). A: Quantitative PCR analysis of Adrb3 expression in mature adipocytes from epididymal fat. B and C: Quantitative PCR analysis of gene expression in epididymal ATMs (Cd45⁺, F4/80⁺, Cd11b⁺, and Cd3⁻) isolated by FACS from SVCs. D and E: FACS analysis of SVCs isolated from the epididymal fat of obese male mice aged 20–24 weeks treated with 25 mg/kg amlexanox or vehicle control for 52 h (n = 6 per group). Macrophages defined as CD45⁺, F4/80⁺, CD11b⁺, and Cd3⁻ cells. D: CD11c⁺ macrophages as a percentage of CD45⁺ cells. E: CD11c⁺ macrophages as a percentage of total macrophages. F: Quantitative PCR analysis of Mcp1 expression in mature adipocytes from epididymal fat (n = 3 per group). G: Schematic of oral amlexanox treatment and activation of this adipocyte-to-macrophage communication axis. Statistical significance determined by post hoc analysis after significant two-way ANOVA. *P < 0.05 vehicle vs. amlexanox within genotype; ∼P < 0.05 WT vs. KO within treatment group. AU, arbitrary unit.