Age‐associated adipose tissue inflammation promotes monocyte chemotaxis and enhances atherosclerosis

Figures

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  FIGURE 1. With aging, viseral fat exhibits increased macrophage numbers and secretes more inflammatory cytokines and chemokines. (a) The total body fat of young (Y) and aged (A) mice was measured by body composition analyzer, EchoMRI. The gonadal white adipose tissue (GWAT) was collected and weighed after euthanizing the mice. The fat mass (n = 13‐14/group) and GWAT mass (n = 21/group) to body weight (BW) ratio were calculated. (b, c) Representative images of Mac2‐stained visceral (GWAT), subcutaneous (inguinal white adipose tissue, IWAT) and perivascular adipose tissue (PVAT) sections of Y and A mice are shown (c), and the percentage of Mac2 positive areas was quantified and is shown in (b), n = 9–13/group. (d) Representative images of F4/80‐stained visceral fat (GWAT) sections of young and aged mice. Quantification of the percentage of F4/80 positive areas is shown on the right (n = 10/group). (e, f) Flow cytometric analysis of total adipose tissue macrophage (MΦ) and CD11c+ adipose tissue macrophages in visceral fat (GWAT) of Y and A mice. Representative FACS contour plots of CD64+ and CD64+CD11c+ adipose tissue macrophages are shown in (e), and the quantification of total (CD64+) and…

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  FIGURE 2. Visceral fat from aged donor mice increases recipient atherosclerotic lesion size and necrotic core size after transplantation. (a) Schematic for fat transplant model: visceral fat from young (Y) or aged (A) WT donor mice was transplanted onto the right common carotid artery (RCCA) of young Ldlr −/− recipient mice. The recipients were then fed a low fat chow diet for a week followed by a western diet for 10 weeks. (b–d) Representative images of H&E stained (b) right carotid artery, (c) aortic root and (d) brachiocephalic artery (BCA) sections of Ldlr −/− mice that received sham surgery or visceral fat transplants from young (Donor‐Y) or aged (Donor‐A) donor mice. The quantifications of lesion area (b–d) and necrotic area (c, d) in each section are shown on the right (n = 8‐13/group). Results are presented as means ± SEM. Two‐way ANOVA followed by Tukey's post hoc test was used for statistical analysis. p values indicate the main effect of the comparison between Donor‐Y vs. Donor‐A unless specifically indicated.

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  FIGURE 3. Visceral fat transplants from aged mice increase macrophage numbers within athersclerotic lesions and fat transplants after transplantation. (a–c) Representative images of Mac2‐stained (a) right carotid artery, (b) aortic root and (c) brachiocephalic artery (BCA) sections of Ldlr −/− mice that received sham surgery or visceral fat transplants from young (Donor‐Y) or aged (Donor‐A) mice. The quantification of Mac2 positive area in each section is shown on the right (n = 7‐10/group). (d, f) Representative images of (d) Mac2 or (f) F4/80 stained visceral fat (GWAT) sections of young (Y) or aged (A) donors, before (bef) and after (aft) fat transplant (FT) surgery, are shown. The quantification of Mac2 (n = 14‐17/group) or F4/80 (n = 7‐9/group) positive area is shown on the right. (e, g) The change in the percentage of (e) Mac2 or (g) F4/80 positive area after FT, compared to before FT, in young (Y) or aged (A) visceral fat is shown (n = 7 or 14 / group). Results are presented as means ± SEM. Two‐way ANOVA followed by Tukey's post hoc test was used for statistical analysis in (a–c). p values indicate the main effect between Donor‐Y vs. Donor‐A groups. Unpaired two‐tailed Student's…

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  FIGURE 4. Macrophages are major contributors to the inflammatory profile in visceral adipose tissue with aging. Aged mice were treated with control liposome or liposome‐encapsulated clodronate by intraperitoneal injection, and then the macrophage depletion efficiency in visceral fat (GWAT) was determined, as well as the secretion of inflammatory cytokines and chemokines. (a) Representative FACS contour plots of macrophages (F4/80+CD11b+) in visceral fat, and the numbers of macrophages per milligram tissue are shown (n = 9‐10/group). (b) Representative images of Mac2‐stained visceral fat sections are shown, and the percentage of Mac2 positive areas was quantified and is shown on the right (n = 8/group). (c) Levels of TNFα, IL‐6, IL‐1β, CCL2, CCL3, and CXCL2 in the tissue culture medium (CM) of visceral fat were determined by multiplex assay (n = 9‐10/group). Results are presented as means ± SEM. Unpaired two‐tailed Student's t‐test was used for statistical analysis.

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  FIGURE 5. Macrophages within the aged fat transplant contribute to the inflammatory profile of the fat transplant and systemically within the recipient. (a) Experimental schematic: aged WT donor mice were treated with control liposome (L) or liposome‐encapsulated clodronate (C) through intraperitoneal injection. 2 days later, visceral fat (GWAT) transplants were collected from the donor mice and transplanted onto the RCCA of young Ldlr −/− recipient mice, followed by a low fat chow diet for a week and then a western diet for 10 weeks. (b, c) The transplanted visceral fat (tGWAT) from the recipients and plasma from sham mice and the recipients were collected at the end of the experiment, and levels of TNFα, IL‐6, IL‐1β, CCL2, CCL3, and CXCL2 in (b) tissue culture medium (CM) of tGWAT and (c) plasma were determined by multiplex assay (n = 8/group). Results are presented as means ± SEM. Unpaired two‐tailed Student's t‐test was used in (b) and one‐way ANOVA followed by Tukey's post hoc test was used in (c) for statistical analysis.

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  FIGURE 6. Macrophages within the aged fat transplant contribute to atherogenesis. Aged WT donor mice were treated with clodronate liposomaes according to the experimental schema described in Figure 5a. (a) Representative images of H&E or Mac2 stained right carotid artery, aortic root and BCA sections of Ldlr −/− mice that received sham surgery or visceral fat transplants from control liposome treated (Donor‐L) or clodronate‐liposome treated (Donor‐C) aged donor mice. (b–d) The quantifications of lesion area, necrotic area, Mac2 positive area, and the percentage of Mac2 positive area in each section of (b) right carotid artery, (c) aortic root, and (d) BCA are shown (n = 8/group). Note: no necrosis was observed in carotid artery. Results are presented as means ± SEM. Two‐way ANOVA followed by Tukey's post hoc test was used for statistical analysis. p values indicate the main effect between groups of Donor‐L and Donor‐C.

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  FIGURE 7. Tissue culture media of aged visceral fat transplants enhance monocyte chemotaxis and monocyte expression of inflammatory and migratory genes. (a) The ability of monocytes to migrate toward the tissue culture media of explanted young (Donor‐Y) or aged (Donor‐A) visceral fat transplants (tGWAT) was determined via chemotaxis assay (n = 7/group). RFU = relative fluorescence. (b–d) Migrated monocytes were collected after migration toward tissue culture media of young (Donor‐Y) or aged (Donor‐A) visceral fat transplants. mRNA levels of (b) inflammatory cytokines including TNFα, IL‐6, IL‐1β; and the chemokines CCL2, CCL3, and CXCL2; and (C) chemokine receptors CCR2, CCR5, and CX3CR1; and (d) genes that are known to associate with cell migration including MMP2, MMP12, TGFβ1, and VEGFc were measured by qRT‐PCR (n = 6 / group). Gene expression was normalized to ribosomal gene L32 (fold change shown on Y axis of plots of b–d). Results are presented as means ± SEM. Unpaired two‐tailed Student's t‐test was used for statistical analysis.

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  FIGURE 8. Aged visceral fat transplants induce local and systemic release of inflammatory mediators promoting monocyte chemotaxis. (a) Protein levels of cytokines, TNFα, IL‐6, IL‐1β, and chemokines CCL2, CCL3, and CXCL2 in tissue culture medium (CM) of transplanted visceral fat (tGWAT) from young (Donor‐Y) or aged (Donor‐A) mice were determined by multiplex assay (n = 7‐8/group). (b) Protein levels of inflammatory mediators TNFα, IL‐6, IL‐1β, CCL2, CCL3, and CXCL2 in plasma of Ldlr −/− mice that received sham surgery or visceral fat (GWAT) transplants from young (Donor‐Y) or aged (Donor‐A) mice were determined by multiplex assay (n = 8‐10/group). (c) Monoclonal antibodies against CCL2, TNFα, and/or CXCL2 or isotype control were added into the tissue culture media of aged visceral fat transplants, and then monocyte chemotaxis was determined (n = 7/group). Serum‐free medium was used as negative control (Neg). p values above histograms without brackets are p values in comparison to isotype control group, far left histogram. Results are presented as means ± SEM. Unpaired two‐tailed Student's t‐test was used in (a) and one‐way ANOVA followed by Tukey's post hoc test was used in (b, c) for…

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  FIGURE 9. Combined blockade of TNFα, CCL2, and CXCL2 reduces the inflammatory profile of fat transplant and also reduces the systemic inflammatory profile in Ldlr −/− recipients. (a) Experimental schematic: aged WT donor mice were treated with IgG control (Donor‐IgG) or combined monoclonal antibodies against TNFα, CCL2, and CXCL2 (Donor‐B), 2 days later, visceral fat (GWAT) transplants were collected from the donor mice and transplanted onto the RCCA of young Ldlr −/− recipient mice, followed by a low fat chow diet for a week and then a western diet for 10 weeks. IgG control or TNFα, CCL2, and CXCL2 blockade were continuously injected into the recipients that received the corresponding transplants twice per week after transplantation till the end of the experiment. (b, c) The transplanted visceral fat (tGWAT) from the recipients and plasma from sham mice and the recipients were collected at the end of the experiment, and protein levels of TNFα, IL‐6, IL‐1β, CCL2, CCL3, and CXCL2 in (b) tissue culture medium (CM) of tGWAT and (c) plasma were determined by multiplex assay (n = 7–8). Results are presented as means ± SEM. Unpaired two‐tailed Student's t‐test was used in (b) and one‐way ANOVA…

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  FIGURE 10. Combined blockade of TNFα, CCL2, and CXCL2 reduces atherogenesis after visceral fat transplantation from aged donors to Ldlr −/− recipients. (a) Representative images of H&E or Mac2 stained right carotid artery, aortic root and BCA sections of Ldlr −/− mice that received sham surgery or visceral fat transplants from IgG control (Donor‐IgG) or TNFα, CCL2, and CXCL2 blockade (Donor‐B) treated aged donor mice. (b–d) The quantifications of lesion area, necrotic area, Mac2 positive area and the percentage of Mac2 positive area in each section of (b) right carotid artery, (c) aortic root and (d) BCA are shown (n = 7‐8/group). Note: no necrosis was observed in carotid artery. Results are presented as means ± SEM. Two‐way ANOVA followed by Tukey's post hoc test was used for statistical analysis. p values indicate the main effect between groups of Donor‐IgG vs. Donor‐B.