Macrophage phenotype and bioenergetics are controlled by oxidized phospholipids identified in lean and obese adipose tissue

Fig. 1.. ATMs from lean mice are metabolically inhibited compared with those from obese mice which are highly energetic. (A) Schematic showing experimental set-up. Briefly, gonadal adipose tissue was excised from mice fed a chow (lean) or HFD (obese) for 12 wk. SVF was isolated and either immediately subjected to bioenergetic profiling or further enriched for CD11b+ cells. (B) MST of SVF cells isolated from lean or obese (12 wk HFD-fed) mice. Bioenergetics plot relates the cells’ respiratory capacity (based on maximal OCR, y axis) to the glycolytic capacity (based on stressed ECAR, x axis) (n = 5). (C) MST and bioenergetic profile of SVF cells isolated from lean and obese mice, enriched for ATM using CD11b-magnetic bead purification (n = 5). Data are expressed as mean ± SEM. Statistical significance calculated by Welch’s t test (*P < 0.05; ***P < 0.001).

Fig. 2.. Resident CX3CR1- and F4/80lo ATMs remain present but are out-populated by recruited CX3CR1+ and F4/80hi macrophages during obesity. (A) Flow cytometry analysis of live CD45+ SVF cells, showing the spread of F4/80 expression and two distinct populations of macrophages discriminated by CX3CR1 expression. Representative sample from 8-wk HFD-fed mice shown. (B) Expression of F4/80 and CX3CR1 by live CD45+F4/80+CD11b+ cells isolated from SVF of lean, 8-wk, and 16-wk HFD-fed mice, showing two distinct macrophage populations (CX3CR1+ and CX3CR1−) (n ≥ 4). (C) Absolute number of total CD45+ F4/80+CD11b+ macrophages in SVF that are CX3CR1+ (purple, top) or CX3CR1− (orange, bottom) (n ≥ 4). (D) Expression of F4/80 and CD11b by live CD45+ F4/80+CD11b+ cells isolated from SVF of lean, 8-wk, and 16-wk HFD-fed mice, showing two distinct macrophage populations labeled F4/80hi and F4/80lo (n ≥ 4). (E) Absolute number of total live CD45+ cells in SVF that are F4/80hi (blue, top) and F4/80lo (red, bottom) (n ≥ 4). **P < 0.01; ***P < 0.001.

Fig. 3.. The majority of resident ATMs are positive for Mox markers and are out-populated by M1/M2 ATMs during obesity. (A) Characterization of F4/80hi and F4/80lo populations by Txnrd1, HO1, CD11c, CD206, and CX3CR1, as shown through histograms. Representative sample from 8-wk HFD-fed mice shown. (B) Absolute number of live CD45+F4/80+CD11b+ cells in SVF that are Txnrd1+HO1+ (Mox), CD11c+ (M1), CD11c+CD206+ (M1/M2), or CD206+ (M2) (n ≥ 4). (C) Relative levels of ATMs (CD45+ F4/80+CD11b+) that are Txnrd1+HO1+ (Mox), CD11c+ (M1), CD11c+CD206+ (M1/M2), CD206+ (M2), or total negative (population that CD45+ F4/80+CD11b+ but negative for Txnrd1, HO1, CD11c, and CD206) (n ≥ 4). (D) Correlation between abundance of individual ATM populations and total number of SVF cells per epididymal fat pad.

Fig. 4.. Increase of full-length at the expense of truncated OxPAPC species in blood of mice fed HFD. (A) Schematic outlining the oxidation products of PAPC, the dotted red line highlighting the arachidonic acid, which is susceptible to oxidation. The predicted oxidation products were first assigned into two broad groups, full-length or truncated, based on mass-to-charge ratio (m/z) that is larger or smaller than PAPC (782 m/z), respectively. Next, they were subdivided into four classes, based on chemical moieties and biological activity. Finally, the product of terminal oxidation or enzymatic cleavage is LysoPC (495 m/z). (B) Accumulation of 16:0 LysoPC normalized to blood volume of lean mice and 9- or 17-wk HFD-fed mice (n ≥ 4). Assessment of correlation between LysoPC in blood and murine body weight. (C) Accumulation of full-length OxPAPC classes HETE/HPETE-PC and isoprostane-PC normalized to blood volume of lean mice and 9- or 17-wk HFD-fed mice (n ≥ 4). Phospholipids were quantified on an individual species basis, using the integrated peak area of the ion count. The integrated peak area of each analyte was normalized to the internal standard, DNPC, and to the volume of blood used for…

Fig. 5.. HFD feeding leads to a greater increase of full-length compared with truncated OxPAPC species in adipose stromal vascular fraction. (A) Histology section of gonadal fat pad from a mouse fed a HFD for 12 wk. Immunohistochemistry using the E06 antibody, an IgM recognizing oxidized phosphatidylcholines, is shown in brown. The asterisk denotes an adipocyte at the center of a crown-like structure. (Magnification: 20×.) (B) Lean mice or mice fed a HFD for 9 or 17 wk had the epididymal fat pad removed, and the SVF purified. A Bligh–Dyer lipid extraction was performed on the SVF, and the ipids were subjected to LC-MS analysis. Total SVF levels of PAPC and its summed oxidation products were quantified on an individual species basis using the integrated peak area of the ion count, normalized to internal standard DNPC. (C) Assessment of the relationship between the ratio of OxPAPC to PAPC in SVF and body weight shows no correlation. Assessment of the relationship between the ratio of OxPAPC to PAPC in SVF and fat pad weight shows a positive correlation. (D) Distribution of OxPAPC classes in SVF of lean or HFD-fed mice. LysoPC (dark orange), carbonyl-PC (orange), γ-keto/hydroxy-PC (light…

Fig. 6.. Truncated OxPAPC species promote a quiescent metabolism and antioxidant gene expression while full-length stimulate metabolism and proinflammatory gene expression. (A) Schematic representing the generation of OxPAPC from native PAPC followed by solid-phase extraction-based separation of full-length (FL) and truncated (T) OxPAPC species. (B) Fraction FL, is shown using a pie chart for relative species abundance (Left) and a precursor ion scan for 184 (Right). (C) Fraction T, is shown using a pie chart for relative species abundance (Left) and a precursor ion scan for 184 (Right). (D) MST of BMDMs treated with vehicle (RPMI media), 10 μg/mL OxPAPC, Fraction FL, or Fraction T for 4 h. Maximal OCR (respiratory capacity) and stressed ECAR (glycolytic capacity) also presented, with the results summarized in a bioenergetics chart (Right). (E) mRNA expression of proinflammatory genes Il1β, Il6, Cxcl1, and Cox2 measured by qPCR in BMDMs treated with vehicle (RPMI media), 10 μg/mL OxPAPC, vehicle FL/T (combined eluents from column loaded with only chloroform, dried down and resuspended with RPMI media), 10 μg/mL Fraction FL, or Fraction T for 4 h. ns, nonsignificant. (F) mRNA expression of…