Metabolic dysfunction drives a mechanistically distinct proinflammatory phenotype in adipose tissue macrophages

Abstract

Adipose tissue macrophage (ATM)-driven inflammation plays a key role in insulin resistance; however, factors activating ATMs are poorly understood. Using a proteomics approach, we show that markers of classical activation are absent on ATMs from obese humans but are readily detectable on airway macrophages of patients with cystic fibrosis, a disease associated with chronic bacterial infection. Moreover, treating macrophages with glucose, insulin, and palmitate-conditions characteristic of the metabolic syndrome-produces a "metabolically activated" phenotype distinct from classical activation. Markers of metabolic activation are expressed by proinflammatory ATMs in obese humans/mice and are positively correlated with adiposity. Metabolic activation is driven by independent proinflammatory and anti-inflammatory pathways, which regulate balance between cytokine production and lipid metabolism. We identify PPARγ and p62/SQSTM1 as two key proteins that promote lipid metabolism and limit inflammation in metabolically activated macrophages. Collectively, our data provide important mechanistic insights into pathways that drive the metabolic-disease-specific phenotype of macrophages.

Fig. 1. Plasma membrane proteomics identifies markers of M1 macrophages

Panel A: Conditions for differentiating and activating human monocyte-derived macrophages. Panel B: Proteins consistently up- (or down-) regulated in M1 relative to M0 and M2 macrophages were identified using a combination of the G-test and t-test. Panel C: The consensus markers of M1 macrophages; heatmap depicts regulation of protein expression across all cells types (blue = down-regulated, yellow = up-regulated). Panel D: Validation of M1 markers by flow cytometry. Panel E: Cell surface levels of CD319 and CD38 in macrophages exposed to M1 or M1/M2 (LPS, IFNγ, and IL4); results are signal-to-noise ratio in mean fluorescence intensity (MFI). Panel F: Cell surface CD319, CD274, and CD38 levels in airway macrophages (defined as CD14⁺CD15⁻) of a CF patient. Where applicable, results are means and SEMs; *, denotes p<0.05, t-test relative to MM0; N=6. See also Figs. S1, S2; Table S1.

Fig. 2. ATMs of obese humans and mice are not classically activated

Panels A-C: Omental (OM) and subcutaneous (SQ) adipose tissue was collected from obese subjects (BMI>30kg/m²) undergoing bariatric surgery (N=7). For comparative purposes, subcutaneous adipose tissue was collected from non-obese subjects (BMI<30kg/m²) undergoing abdominoplasty (N=7). Panel A: Pro-inflammatory cytokine expression. Panel B: Cell surface CD319 levels in ATMs (defined as CD14⁺CD15⁻CD206⁺CD1c⁻) from a representative obese subject. Panel C: Cell surface levels of CD38, CD274, and CD319 in ATMs from non-obese and obese subjects and airway macrophages from CF patients; results are signal-to-noise ratio in mean fluorescence intensity (MFI). Panels D-F: Male C57BL/6 mice were place on a low-fat diet (LFD) or high-fat diet (HFD) for 16 weeks and ATMs from epididymal fat were isolated. Panel D: Pro-inflammatory cytokine expression. Panel E: ATM cell surface CD319, CD274, and CD40 levels. Panel F: ATM cell surface CD319, CD274, and CD40 levels in the presence and absence of M1 stimuli. Where applicable, results are means and SEMs; *, denotes p<0.05, t-test relative to non-obese or LFD; N=4–7. See also Fig. S3.

Fig. 3. Metabolically and classically activated macrophages are distinct

Panel A: Conditions for metabolic (MMe) or classical (MM1) activation of human monocyte-derived macrophages. Panel B: Pro-inflammatory cytokine expression. Panel C: Flow cytometric analysis of cell surface CD38, CD319, and CD274 levels in MM0, MM1, and MMe macrophages. ISO= isotype control. Panel D: Plasma membrane proteomics analysis of MM0, MM1, and MMe macrophages. Panels E-F: Quantification of cell surface proteins in MM0, MM1, and MMe macrophages by mass spectrometry (spectral count, SC). Where applicable, results are means and SEMs; *, denotes p<0.05, t-test relative to MM0; N=6. See also Table S2.

Fig. 4. ATMs display evidence of metabolic activation

Panel A: qRT-PCR analysis of human MM0 macrophages treated with media conditioned by human omental fat (Ad-CM) or sputum collected from a CF patient. Inset: Surface levels of CD38 and CD274. Panel B: qRT-PCR analysis of murine macrophages (MM0) treated with media conditioned by epididymal adipose tissue collected from obese (Ad-CM-HFD) or lean (Ad-CM-LFD) mice. Panels C-E: Omental (OM) and subcutaneous (SUBQ) adipose tissue was collected from obese subjects (BMI>30kg/m²) undergoing bariatric surgery (N=7). For comparative purposes, subcutaneous adipose tissue was collected from non-obese subjects (BMI<30kg/m²) undergoing abdominoplasty (N=7). Panel C: Cell surface levels of CD36 and ABCA1 in ATMs; results are signal-to-noise ratio in mean fluorescence intensity (MFI). Panel D: Relationships between BMI and cell surface CD36 or CD319 levels on ATMs (N=14). Panel E: Summary of linear regression analyses of cell surface protein levels on ATMs and BMI. Panels F-G: Male C57BL/6 mice were place on a low-fat diet (LFD) or high-fat diet (HFD) for 16 weeks and ATMs from epididymal fat were purified and analyzed by qRT-PCR. Where applicable, results are means and SEMs; *, denotes p<0.05, t-test relative to MM0 or non-obese or LFD; N=4–7. See also Fig. S3.

Fig. 5. Metabolic activation fails to induce the type I interferon response

Panel A: Heatmap depicts regulation of interferon-regulated proteins across human MM0, MM1, and MMe cells (blue = down-regulated, yellow = up-regulated). Panel B: Comparison of M1 cell surface marker levels in MM1 macrophages made from wt and Ifnar−/− mice. Panels C-F: Human MM0 macrophages were classically activated or treated with Pam2CSK4, a selective agonist for TLR2. Panel C: Pro-inflammatory cytokine expression. Panel D: IRF7 expression. Panel E: M1 cell surface markers were quantified by flow cytometry. Panel F: qRT-PCR analysis of MMe markers. Panels G-H: Human MM0 macrophages were classically or metabolically activated. Panel G: Immunoblotting for total and phosphorylated (P-Ser¹⁷²) IKKε. Panel H: IRF7 expression. Panels I-J: Male C57BL/6 mice were place on a low-fat diet (LFD) or high-fat diet (HFD) for 16 weeks and ATMs from epididymal fat were isolated. Panel I: Immunoblotting for total and phosphorylated (P-Ser¹⁷²) IKKε. Panel J: IRF7 expression in purified ATMs. Where applicable, results are means and SEMs; *, denotes p<0.05, t-test relative to MM0; N=4–6. Flow cytometry results are expressed as signal-to-noise ratios in the mean fluorescence intensity (MFI). See also Fig. S5.

Fig. 6. p62 and PPARγ promote MMe marker expression and limit inflammation

Panel A: Comparison of gene expression in MMe macrophages made from wild-type (wt), Tlr2−/−, and Tlr4−/− mice. Panel B: Heatmap depicts regulation of PPARγ-regulated proteins across human MM0, MM1, and MMe cells (blue = down-regulated, yellow = upregulated). Panel C: PPARγ binding to its target gene promoters is increased in MMe relative to MM0 macrophages, and correlated with abundance of the open chromatin mark H3K4m3. Relative abundance of MM0 ChIP levels were arbitrarily set to 1. Panel D: Protein levels of p62 in human macrophages in vitro (spectral counts, SC) and in vivo (arbitrary units, AU) in murine ATMs isolated from mice fed a low-fat (LFD) or high-fat diet (HFD). Panel E: Comparison of gene expression in MMe and MM1 macrophages from wild-type and p62−/− mice. Panel F: Kinetics of p62 accumulation and LC3-I/-II levels in human MMe macrophages. Panel G: p62 mRNA levels. Panel H: p62 levels in MM0 and MMe macrophages made from wild-type (wt), Tlr2−/−, and Tlr4−/− mice. Panel I: Kinetics of p62 accumulation and FFA uptake by human MMe macrophages. Panel J: Varying numbers of human macrophages were metabolically activated and palmitate uptake and p62 levels were determined. Where applicable, results are means and SEMs; *, denotes p<0.05, t-test relative to MM0 or LFD; N=3–6. See also Figs. S6, S7.

Fig. 7. A molecular model for metabolic and classical activation of macrophages

See text for details.