IL-6 signaling drives self-renewal and alternative activation of adipose tissue macrophages

Abstract

Introduction: Obesity is associated with chronic low-grade inflammation of adipose tissue (AT) and an increase of AT macrophages (ATMs) that is linked to the onset of type 2 diabetes. We have recently shown that neutralization of interleukin (IL)-6 in obese AT organ cultures inhibits proliferation of ATMs, which occurs preferentially in alternatively activated macrophage phenotype.

Methods: In this study, we investigated AT biology and the metabolic phenotype of mice with myeloid cell-specific IL-6Rα deficiency (Il6ra ^Δmyel) after normal chow and 20 weeks of high-fat diet focusing on AT inflammation, ATM polarization and proliferation. Using organotypical AT culture and bone marrow derived macrophages (BMDMs) of IL-4Rα knockout mice (Il4ra ^-/-) we studied IL-6 signaling.

Results: Obese Il6ra ^Δmyel mice exhibited no differences in insulin sensitivity or histological markers of AT inflammation. Notably, we found a reduction of ATMs expressing the mannose receptor 1 (CD206), as well as a decrease of the proliferation marker Ki67 in ATMs of Il6ra ^Δmyel mice. Importantly, organotypical AT culture and BMDM data of Il4ra ^-/- mice revealed that IL-6 mediates a shift towards the M2 phenotype independent from the IL-6/IL-4Rα axis.

Discussion: Our results demonstrate IL-4Rα-independent anti-inflammatory effects of IL-6 on macrophages and the ability of IL-6 to maintain proliferation rates in obese AT.

Keywords: IL-6; adipose tissue inflammation; alternative activation; diabetes; macrophages; mannose receptor; obesity; self-renewal.

Figure 1

Myeloid Il6ra knockout does not affect weight gain and disturbed metabolism of protracted diet-induced obesity. (A) Relative gene expression analysis of Il6ra ^fl/fl and Il6ra ^Δmyel BMDMs to verify the Il6ra knockout (n=7-8). (B) Comparison of body weight in either lean female, lean male and obese male Il6ra ^fl/fl and Il6ra ^Δmyel mice. (C) Organ weights of different fat depots, liver and pancreas in obese male Il6ra ^fl/fl and Il6ra ^Δmyel after 20 weeks of HFD (n=9-16). Blood glucose measurements by ipITT (D) and ipGTT (E) of Il6ra ^fl/fl and Il6ra ^Δmyel male mice after 20 weeks of HFD (n=17). Data are presented as mean ± SEM. ***p < 0.001.

Figure 2

Adipocyte expansion and ATM distribution are unaltered in obese mice lacking IL-6Rα. (A) Representative images of immunohistochemistry of paraffin-embedded AT from obese (left side) and lean (right side) Il6ra ^fl/fl and Il6ra ^Δmyel mice stained with Perilipin (adipocytes, green), pan macrophage marker Mac-2 (red) and DAPI (nuclei, blue). (B) Abundance of CLS in % of adipocyte number, (C) interstitial macrophages in % of adipocyte number and (D) measurements of adipocyte size in µm from paraffin-embedded AT sections of obese Il6ra ^Δmyel and Il6ra ^fl/fl controls (n=6-7). (E, F) Distribution of adipocytes concerning adipocyte size in (E) lean and (F) obese male Il6ra ^Δmyel and Il6ra ^fl/fl mice. Data are presented as mean ± SEM. *p < 0.05. Scale bar represents 100 µm.

Figure 3

Myeloid Il6ra knockout impairs alternative activation and proliferation of ATMs in diet-induced obesity. (A, B) Flow cytometry analysis of classically activated M1 (A; CD11c+CD206-) and alternatively activated M2 (B, CD11c-CD206+) given as percentage of overall ATMs (CD45+F4/80+) in AT of lean and obese female and obese male Il6ra ^fl/fl and Il6ra ^Δmyel mice (either NCD or 20 weeks of HFD; n=6-12). (C) Ratio of M1 to M2 macrophages in female and male NCD and HFD Il6ra ^fl/fl and Il6ra ^Δmyel mice measured by flow cytometry (n=4-12). (D) Representative flow cytometry plots of female and male Il6ra ^fl/fl and Il6ra ^Δmyel mice under NCD or HFD reflecting CD11c and CD206 expression in ATMs. (E) Representative flow cytometry plots for Ki67 expression on overall ATMs (CD45+F4/80+) in obese AT of Il6ra ^fl/fl and Il6ra ^Δmyel mice. (F) Percentage of Ki67+ ATMs (CD45+F4/80+) in AT of obese Il6ra ^fl/fl and Il6ra ^Δmyel mice (n=6-11). (G) Ki67+ nuclei within CLS as percentage of all nuclei in CLS using paraffin-embedded AT sections of obese male Il6ra ^fl/fl and Il6ra ^Δmyel mice (n=4). (H) Representative images of an obese CLS with staining of macrophages (CD68, green), nuclei (blue) and Ki67 (red). Scale bar represents 10 µm. Data are presented as mean ± SEM. *p < 0.05.

Figure 4

IL-6 elevates anti-inflammatory M2 macrophages partially independent of the IL-4Rα. (A-D) Flow cytometry data of AT explants from lean male Il4ra ^+/+ (wildtype) and Il4r ^-/- (knockout) mice after ex vivo induction of AT inflammation showing (A) CD11c+CD206- (M1) macrophages, (B) CD11c-CD206+ (M2) macrophages and (C) M1/M2 ratio after treatment with IL-6 or IL-13 (50 ng/ml) for 48h (n=4-5). (D) Representative flow cytometry plots for IL-6 and IL-13 treatment in lean inflammatory AT of male and female Il4ra ^+/+ and Il4r ^-/- mice. (E-H) Proliferation measurements using Ki67 staining and flow cytometry in lean male and female Il4ra ^+/+ inflammatory AT after IL-6 and IL-13 stimulation (50 ng/ml, 48h; n=4-5). (E) Representative flow cytometry plots of IL-6 stimulated AT explants after induction of inflammation (control black, IL-6 blue events). (F) Ki67+ cells as percentage of overall ATMs. (G) CD11c+CD206- und (H) CD11c-CD206+ proliferating ATMs as percentage of Ki67+ ATM. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01.

Figure 5

IL-6 signaling boosts BMDM Mrc1 expression and decreases Itgax expression independently of the IL-4Rα subunit. (A, B) Relative gene expression data of Mrc1 (A, Mannose receptor 1) and Itgax (B, Integrin alpha X) in BMDMs (M0) from non-obese male Il4ra ^-/- or control (Il4ra ^+/+) mice after stimulation with 20 ng/ml IL-13 or IL-6 for 48h (n=5-6). (C, D) Relative gene expression of Il10ra and Il10 of IL-6 treated (20 ng/ml) BMDMs from non-obese male Il4ra ^-/- or wildtype (Il4ra ^+/+) mice. PBS served as control. Gene expression data were related to Importin-8 (Ipo8) as internal control (n=6). (E) Heat map of RNA sequencing data depicting only significantly regulated genes of Il4ra ^-/- or control (Il4ra ^+/+) BMDMs treated with PBS, IL-13 or IL-6 (20 ng/ml) for 48h as log2FC (n=4). Genes of interest were assigned to alternatively activated (M2) and classically activated (M1) genes. Data are presented as mean ± SEM. *p < 0.05, ****p < 0.0001.