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, 15 (5), 423-30

Signaling by IL-6 Promotes Alternative Activation of Macrophages to Limit Endotoxemia and Obesity-Associated Resistance to Insulin

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Signaling by IL-6 Promotes Alternative Activation of Macrophages to Limit Endotoxemia and Obesity-Associated Resistance to Insulin

Jan Mauer et al. Nat Immunol.

Abstract

Obesity and resistance to insulin are closely associated with the development of low-grade inflammation. Interleukin 6 (IL-6) is linked to obesity-associated inflammation; however, its role in this context remains controversial. Here we found that mice with an inactivated gene encoding the IL-6Rα chain of the receptor for IL-6 in myeloid cells (Il6ra(Δmyel) mice) developed exaggerated deterioration of glucose homeostasis during diet-induced obesity, due to enhanced resistance to insulin. Tissues targeted by insulin showed increased inflammation and a shift in macrophage polarization. IL-6 induced expression of the receptor for IL-4 and augmented the response to IL-4 in macrophages in a cell-autonomous manner. Il6ra(Δmyel) mice were resistant to IL-4-mediated alternative polarization of macrophages and exhibited enhanced susceptibility to lipopolysaccharide (LPS)-induced endotoxemia. Our results identify signaling via IL-6 as an important determinant of the alternative activation of macrophages and assign an unexpected homeostatic role to IL-6 in limiting inflammation.

Figures

Figure 1
Figure 1. IL-6 signaling in myeloid cells regulates glucose homeostasis
(a) Body weight of control (Ctrl) and Il6raΔmyel mice (n=12 Ctrl NCD; n=32 Ctrl HFD; n=16 Il6raΔmyel NCD; n=18 Il6raΔmyel HFD). (b) Glucose tolerance tests (GTT; n=32 vs 31; *p≤0.05; **p≤0.01) or (c) insulin tolerance tests (ITT; n=27 vs 17; *p≤0.05; **p≤0.01; Data are expressed as % of initial blood glucose) of HFD Ctrl and Il6raΔmyel mice. (d) Fasted serum insulin concentrations (n=9 in each group; *p≤0.05) and (e) homeostatic model assessment of insulin resistance (HOMA-IR; n=9 in each group; *p≤0.01)) indices were determined in HFD Ctrl and Il6raΔmyel mice. (f) Glucose infusion rate (GIR; n=8 vs 7; *p≤0.001) and (g) glucose uptake rate (GUR; n=8 vs 7; *p≤0.05) in skeletal muscle (SM), brown adipose tissue (BAT) and white adipose tissue (WAT; p=0.06) during euglycemic-hyperinsulinemic clamp analyses of HFD Ctrl and Il6raΔmyel mice. (h) Suppression of free fatty acid (FFA) release in the serum of HFD Ctrl and Il6raΔmyel mice during clamp analyses (n=8 vs 7; *p≤0.05). (i) qRT-PCR analyses of Lipe expression in WAT from Ctrl and Il6raΔmyel mice at the end of the clamp analyses (n=8 vs 7; *p≤0.01; Data are expressed as % of Ctrl). (j) Suppression of hepatic glucose production (HGP) during clamp analyses of HFD Ctrl and Il6raΔmyel mice (n=8 vs 7; *p≤0.05). (k) qRT-PCR analyses of G6pc expression in livers from Ctrl and Il6raΔmyel mice at the end of the clamp analyses (n=8 vs 7; *p≤0.05; Data are expressed as % of Ctrl). (Values are expressed as mean ± sem).
Figure 2
Figure 2. HFD Il6raΔmyel mice have increased systemic inflammation
(a) qRT-PCR analyses of white adipose tissue (WAT) from HFD control (Ctrl) and Il6raΔmyel mice (n=8–9 per genotype; *p≤0.05; **p≤0.01; ***p≤0.001; Data are expressed as % of Ctrl). (b) Representative immunohistochemical staining of F4/80-positive cells in WAT from HFD Ctrl and Il6raΔmyel mice and quantification of F4/80-positive crown-like structures (CLS) in WAT from HFD Ctrl and Il6raΔmyel mice (n=7 per genotype;; *p≤0.05; Data are expressed as % CLS of adipocytes; scale bars represent 100μm). (c) qRT-PCR analyses of brown adipose tissue (BAT) from HFD Ctrl and Il6raΔmyel mice (n=8–9 per genotype; *p≤0.05; **p≤0.01; Data are expressed as % of Ctrl). (d) qRT-PCR analyses of liver from HFD Ctrl and Il6raΔmyel mice (n=8–9 per genotype; *p≤0.05; **p≤0.01; Data are expressed as % of Ctrl). (e) C-Jun N-terminal kinase (JNK) activity in WAT, BAT and liver of HFD Ctrl and Il6raΔmyel mice was measured by performing immunoprecipitation (IP) of p-JNK and after subsequent in vitro phosphorylation assay, recombinant c-Jun was detected by immunoblot (IB). Total JNK and calnexin loading was used as input control; Representative immunoblots are shown (upper panel); Quantification of JNK activity in WAT, BAT and liver of HFD Ctrl and Il6raΔmyel mice (lower panel; n=5–9 per genotype; *p≤0.05; **p≤0.01; ***p≤0.001; Data are expressed as % of Ctrl). (Values are expressed as mean ± sem;).
Figure 3
Figure 3. IL-6 signaling promotes IL-4R expression in macrophages
(a) Heatmap of differentially regulated transcripts between IL-6-stimulated (4 hours) bone marrow-derived macrophages (BMDM) either from control (Ctrl) or Il6raΔmyel (Il6ra−/−) mice. (n=4 per group; fold-change cut off ± 1.25; p≤0.05). (b) qRT-PCR analyses of Il6ra and Il4ra expression in untreated (NT) and IL-6-stimulated (12 hour) Ctrl and Il6ra−/− BMDM (n=6 per group; *p≤0.001 vs Ctrl; **p≤0.001 vs NT; Data are expressed as % of NT Ctrl). (c) FACS analysis of untreated (NT) and IL-6-stimulated (24 hour) Ctrl BMDM (n=5; *p≤0.001 vs NT). (d) Ctrl BMDM were left untreated (NT) or stimulated with IL-6 (12 hours) in the absence (IgG) or presence of an IL-10-neutralizing antibody (αIL-10) and qRT-PCR analysis of Il4ra expression was performed (Data is representative of two independent experiments; Data are expressed as % of NT Ctrl). (e) Immunoblot of Ctrl BMDM that were left untreated (NT) or stimulated with IL-6 (12 hours) in the presence of a control (IgG) or IL-10-neutralizing antibody (αIL-10) (n=3). (f) qRT-PCR analyses of Stat3 and Il6ra expression in siRNA-transfected Ctrl BMDM (n=3; *p≤0.001; Data are expressed as % of Ctrl siRNA). (g) qRT-PCR analyses of Il4ra expression in untreated (NT) or IL-6 stimulated (4h) siRNA-transfected Ctrl BMDM (n=3; *p≤0.001 vs IL-6 stimulated Ctrl siRNA; Data are expressed as % of NT Ctrl siRNA). (Values are expressed as mean ± sem)
Figure 4
Figure 4. IL-6-induced STAT3 binds to distinct motifs in the Il4ra promoter
(a) Luciferase activity of the indicated reporter constructs in untreated (NT) or IL-6-treated (12h) immortalized macrophages. (n=3; *p≤0.001 vs NT −1,300 bp fragment). (b) qRT-PCR analyses of ChIP assay showing occupancy of p-STAT3 over the Il4ra promoter in control (Ctrl) and Il6ra−/− BMDM stimulated with IL-6 for the indicated time points (n=3 vs 3; *p≤0.001 vs Ctrl; **p≤0.05, ***p≤0.001 vs NT; Data are expressed as % of NT Ctrl). (Values are expressed as mean ± sem)
Figure 5
Figure 5. IL-6 signaling in myeloid cells augments the IL-4 response
(a) Immunoblot of control (Ctrl) and Il6ra−/− BMDM that were left untreated or pre-incubated with IL-6 (12 hours) before stimulation with IL-4 (30 minutes) (Representative immunoblot of 3 independent experiments shown). (b) qRT-PCR analyses of Ctrl and Il6ra−/− BMDM that were stimulated with IL-6 (12 hours) and subsequently exposed to IL-4 or IL-4 + IL-6 for 24 hours (n=6 vs 6; *p≤0.01 vs Ctrl; **p≤0.001 vs NT; Data are expressed as % of NT Ctrl). (c) FACS analysis of CD206/MRC1 and ARG1 expression in Ctrl BMDM that were left untreated or stimulated with IL-6, IL-4 or IL-4 + IL-6 (n=5; *p≤0.05 vs IL-6 + IL-4; **p≤0.001). (d) FACS analysis of CD206/MRC1 expression in WAT and BAT (n=5; *p≤0.05 vs IL-6+IL-4; **p≤0.01 ***p≤0.001) or (e) blood and peritoneum of Ctrl mice that were treated with IL-6, IL-4 or IL-4 + IL-6 (n=5; *p≤0.05 vs IL-6+IL-4; **p≤0.01). (f) GTT area under the curve (AUC) of HFD Ctrl and Il6raΔmyel mice before (NT) and after a 4-week treatment period with IL-4 (n=15 vs 17; *p≤0.05 vs Ctrl; **p≤0.01). (g) qRT-PCR analyses of HFD Ctrl and Il6raΔmyel mice treated with IL-4 for 4 weeks (n=7–8 vs 7; *p≤0.05; **p≤0.01 ***p≤0.001; Data are expressed as % of NT Ctrl). (h) Immunohistochemical staining of CD206/MRC1 in livers from HFD Ctrl and Il6raΔmyel mice that were left untreated (NT) or treated with IL-4 for 1 week (Representative images of 3 per group shown; scale bars represent 50μm).
Figure 6
Figure 6. Myeloid cell IL-6 signaling limits LPS-endotoxemia
(a) qRT-PCR analyses of control (Ctrl) and Il6ra−/− BMDM that were left untreated or stimulated with IL-6 (12 hours) and subsequently exposed to bacterial lipopolysaccharides (LPS) alone or LPS + IL-6 for an additional 24 hours (n=6 vs 6; *p≤0.05 vs LPS + IL-6 stimulated Ctrl; **p≤0.05, ***p≤0.01; Data are expressed as % of LPS stimulated Ctrl). (b) Body weight loss of Ctrl and Il6raΔmyel mice 24 hours after exposure to 1mg/kg LPS (n=7 vs 8; *p≤0.05). (c) Food intake of Ctrl and Il6raΔmyel mice before (basal) and 24 hours after exposure to LPS (n=7 vs 8; *p≤0.01). (d) Respiratory exchange ratio (RER) of Ctrl and Il6raΔmyel mice before (basal) and 24 hours after exposure 1mg/kg LPS (n=7 vs 8; *p≤0.01). (e) Cytokine concentration in circulation of Ctrl and Il6raΔmyel mice 6 hours after exposure to 1mg/kg LPS (n=7 vs 7; **p≤0.01, p=0.06 for IL-4). (f) qRT-PCR analyses of liver and WAT from Ctrl and Il6raΔmyel mice 48 hours after exposure to 1mg/kg LPS (n=7 vs 8; *p≤0.05; Data are expressed as % of Ctrl). (Values are expressed as mean ± sem).

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