Activation of NF-κB drives the enhanced survival of adipose tissue macrophages in an obesogenic environment

Abstract

Objective: Macrophage accumulation in adipose tissue (AT) during obesity contributes to inflammation and insulin resistance. Recruitment of monocytes to obese AT has been the most studied mechanism explaining this accumulation. However, recent evidence suggests that recruitment-independent mechanisms may also regulate pro-inflammatory AT macrophage (ATM) numbers. The role of increased ATM survival during obesity has yet to be explored.

Results: We demonstrate that activation of apoptotic pathways is significantly reduced in ATMs from diet-induced and genetically obese mice. Concurrently, pro-survival Bcl-2 family member protein levels and localization to the mitochondria is elevated in ATMs from obese mice. This increased pro-survival signaling was associated with elevated activation of the transcription factor, NF-κB, and increased expression of its pro-survival target genes. Finally, an obesogenic milieu increased ATM viability only when NF-κB signaling pathways were functional.

Conclusions: Our data demonstrate that obesity promotes survival of inflammatory ATMs, possibly through an NF-κB-regulated mechanism.

Keywords: Adipose tissue; Apoptosis; Cleaved caspase-3; Macrophage; NF-κB; Survival.

Graphical abstract

Figure 1

HFD feeding decreases apoptosis of ATMs. Male C57Bl/6 mice were placed on LFD or HFD for 9 weeks. A) SVF was collected and cleaved caspase-3 was analyzed using Western blot. B) AT explants were collected and analyzed by confocal staining for the macrophage marker F4/80 (green), nuclear stain DAPI (blue), and apoptosis marker TUNEL (pink). Magnification: 40×. C–D) Quantification of TUNEL positive ATMs by percent of F4/80 positive cells (C) or by number per high-power field (D). E) Quantification of localization of apoptotic ATMs. Data are presented as mean ± SEM, A) n = 11–14/group, C–D) n = 4–8/group for confocal imaging.**p < 0.01, ***p < 0.001, ****p < 0.0001 between groups.

Figure 2

Apoptosis of ATMs is negatively correlated with body weight. Male C57Bl/6 mice were placed on a LFD or HFD for 16 weeks. A) SVF was collected and cleaved caspase-3 was analyzed using Western blot. B) Quantification of TUNEL positive ATMs by percent of F4/80 positive cells. C) Correlation of SVF cleaved caspase-3 with body weight of mice fed LFD and HFD for either 9 or 16 weeks. D) Correlation of SVF cleaved caspase-3 with body weight for HFD fed mice only. A–B) Data are presented as mean ± SEM, n = 5–6/group. *p < 0.05, ****p < 0.0001 between groups.

Figure 3

Genetic model of obesity decreases apoptosis of ATMs. Male C57Bl/6 lean or ob/ob mice were maintained on chow diet until 9–10 weeks of age. A) SVF was collected and cleaved caspase-3 was analyzed using Western blot. B) AT explants were collected and analyzed by confocal staining for the macrophage marker F4/80 (green), nuclear stain DAPI (blue), and apoptosis marker TUNEL (pink). Magnification: 40×. C–D) Quantification of TUNEL positive ATMs by percent of F4/80 positive cells (C) or by number per high-power field (D). E) Quantification of localization of apoptotic ATMs. Data are presented as mean ± SEM, A) n = 5/group, C–E) n = 4–6/group. *p < 0.05, **p < 0.01 between groups.

Figure 4

Pro-apoptotic/survival Bcl-2 family members are differentially regulated in the SVF of AT of obese mice. Male C57Bl/6 mice were placed on LFD or HFD for 9 weeks. A–D) SVF was collected and Bcl-2 family pro-apoptotic/survival gene expression was analyzed using real-time RT-PCR: A) Bax, B) Bak1, C) Bcl2, and D) Bcl2l1. E–H) SVF was collected and Bcl-2 family pro-apoptotic/survival protein levels were analyzed using Western blot: E) Bax, F) Bak, G) Bcl-2, and H) Bcl-xl. mRNA levels were normalized to housekeeping gene Rplpo and levels of specific proteins were normalized to β-actin. Data are presented as mean ± SEM, A-D) n = 7/group, E–F) n = 17–19/group, G-H), n = 4–8/group. **p < 0.01, ***p < 0.001, ****p < 0.0001 between groups.

Figure 5

Mitochondrial localization of the pro-survival protein Bcl-2 is increased in ATMs of obese mice. Male C57Bl/6 mice were placed on a LFD or HFD diet for 9 weeks. ATMs were obtained using a 2 h macrophage selection by adhesion assay and stained for quantification of Bax and Bcl-2 mitochondrial localized protein levels using Image Xpress Automated HTS Fluorescence Microscopy or visualization by confocal microscopy. A) Quantification of the co-localization of Bax to the mitochondria of ATMs. B) Quantification of the co-localization of Bcl-2 to the mitochondria of ATMs. Magnification for quantifications: 40×. C) Representative images of Bax (green) mitochondrial (red, MitoTracker (Mito)) localization by confocal microscopy. D) Representative images of Bcl-2 (green) mitochondrial (red, MitoTracker (Mito)) localization by confocal microscopy. Magnification for representative images: 60× with a 4.5 zoom. Data are presented as mean ± SEM, n = 6–7/group. *p < 0.05 between groups.

Figure 6

NF-κB activity and its pro-survival target genes are increased in ATMs of obese mice. Male C57Bl/6 mice were placed on a LFD or a HFD for 9 weeks. A) SVF was collected and phosphorylated p65 (P-p65) was analyzed using Western blot. B) Nuclear localization of the p65 subunit of NF-κB. ATMs were obtained using a 2 h macrophage selection by adhesion assay and stained for nuclear stain DAPI (blue), p65 (red), F4/80 (green). Magnification: 100× with a 4.5 zoom. C) Transcriptional activity of NF-κB in ATMs using NF-κB-GFP-Luciferase mice. D) Real-time RT-PCR analysis of NF-κB-driven pro-inflammatory and pro-survival target genes in ATMs (Tnf, Xiap, Birc3). Data are presented as mean ± SEM, A) n = 4/group, C) n = 9–10/group, and D) n = 7–8/group. *p < 0.05 between groups.

Figure 7

Inhibition of NF-κB activity decreases ATM survival in an obesogenic setting. Male C57Bl/6 mice were placed on a HFD for 3 weeks to obtain sufficient numbers of ATMs for ex vivo studies. A) Nuclear translocation of NF-κB. Adhesion-selected ATMs were treated with the metabolic cocktail (MetaC, 30 mM glucose, 10 nM insulin, 0.4 mM of palmitic acid) for 30 min and subsequently stained with DAPI (blue), p65 (red), and F4/80 (green). Magnification: 60× with a 1.5 zoom. B) Gene expression. ATMs were treated with the MetaC for 2 h and RNA isolated for real-time RT-PCR analysis of expression of lipid metabolism (Abca1 and Plin2) as well as NF-κB-driven pro-inflammatory (Tnf) and pro-survival (Bcl2, Xiap, Birc3) genes. C) Cell viability. ATMs were treated with DMEM (control), MetaC, BMS-34551 (BMS), or MetaC + BMS for 0–8 h. Cell viability was detected using the Cell-Titer Blue assay as described in the Methods. Data are presented as mean ± SEM, n = 4–5/group. *p < 0.05, **p < 0.01, ***p < 0.001 between groups.