Extended Data Fig. 10: Chronic CD169⁺ NAM depletion impairs lipolysis and promotes fatty acid oxidation during aging.

(a) Relative protein quantification of Hormone-Sensitive Lipase (HSL), HSL phosphorylated at Ser563 (p-HSL_S563), HSL phosphorylated at Ser660 (p-HSL_S660), Adipose triglyceride lipase (ATGL), and Monoamine oxidase A (MAOA) in visceral white adipose tissue (VAT) lysates from ad libitum-fed 24-month-old wild-type (WT) and CD169-DTR female mice treated with diphtheria toxin (DT) and sacrificed on day 25 (D25) (n = 4 per genotype). Data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t-test (α = 0.05); p < 0.05 was considered significant. (b) Change in body weight curves for 24-month-old WT and CD169-DTR female mice (n = 5-7 per genotype) treated with DT and sacrificed on D34.(c) Relative mRNA expression of Adrb3, Pnpla2, and Hsl in the VAT of ad libitum-fed 24-month-old WT and CD169-DTR female mice treated with DT and sacrificed on D34 (n = 5-7 per genotype). Data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t-test (α = 0.05); p < 0.05 was considered significant. (d) Relative mRNA expression of Cpt1a in the VAT of ad libitum-fed 24-month-old WT and CD169-DTR female mice treated with DT and sacrificed on D34 (n = 5-7 per genotype). Data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t-test (α = 0.05); p < 0.05 was considered significant. (e) Flow cytometry data from the VAT of 24-month-old WT and CD169-DTR female mice treated with DT and sacrificed on D24. Bar plots represent the proportion of CD4⁺ regulatory T- cells (T-regs), gamma-delta T cells (gd T cells), CD4⁺ T helper 1 cells (T_H1 cells), CD4⁺ T helper 2 cells (T_H2 cells), and CD4⁺ T helper 17 cells (T_H17 cells) as a fraction of the total live CD45⁺ subset. Populations were defined using the cell surface markers and gating strategy outlined in Supplementary Figure 5. Data is represented as a mean +/- SEM. Statistical significance was assessed using a two-tailed Student’s t-test (α = 0.05); p < 0.05 was considered significant. (f) Flow cytometry data from the VAT of 24-month-old WT and CD169-DTR female mice treated with DT and sacrificed on D34. Bar plots represent the proportion of B cells, CD8⁺ T cells, CD4⁺ T cells, Monocytes, Neutrophils, Eosinophils, and MHCII⁺CD11c⁺ ATMs or dendritic cells (DCs) as a fraction of the total live CD45⁺ subset. Populations were defined using the cell surface markers and gating strategy outlined in Supplementary Figure 5. Data is represented as a mean +/- SEM. Statistical significance was assessed using a two-tailed Student’s t-test (α = 0.05); p < 0.05 was considered significant. (g) Flow cytometry data from the VAT of 24-month-old WT and CD169-DTR female mice treated with DT and sacrificed on D34. Bar plots represent the proportion of CD4⁺ regulatory T- cells (T-regs), gamma-delta T cells (gd T cells), CD4⁺ T helper 1 cells (T_H1 cells), CD4⁺ T helper 2 cells (T_H2 cells), and CD4⁺ T helper 17 cells (T_H17 cells) as a fraction of the total live CD45⁺ subset. Populations were defined using the cell markers and gating strategy outlined in Supplementary Figure 5. Data is represented as a mean +/- SEM. Statistical significance was assessed using a two-tailed Student’s t-test (α = 0.05); p < 0.05 was considered significant. (h) Tissue wet weight of the spleen from 24-month-old wild-type (WT) and CD169-DTR female mice treated with DT and sacrificed on D25 (n = 8–19 per genotype). Data is represented as a mean +/- SEM. Statistical significance was assessed using a two-tailed Student’s t-test (α = 0.05); p < 0.05 was considered significant. (i) Flow cytometry data from the spleen from 24-month-old WT and CD169-DTR female mice treated with DT and sacrificed on D25. Bar plots represent the proportion of nerve-associated macrophages (NAMs), B cells, CD8⁺ T cells, CD4⁺ T cells, Monocytes, Neutrophils, Eosinophils, and MHCII⁺CD11c⁺ ATMs or dendritic cells (DCs) as a fraction of the total live CD45⁺ subset. Populations were defined using the cell surface markers and gating strategy outlined in Supplementary Figure 5. Data is represented as a mean +/- SEM. Statistical significance was determined via Student’s two-tailed t-test with α = 0.05 and p < 0.05. (j) Relative mRNA expression of Nlrp3, Asc, Casp1, Il1b, Il18, Il10, Il6, and Tnf in the VAT from ad libitum-fed 24-month-old WT and CD169-DTR female mice treated with DT and sacrificed on D34 (n = 5-7 per genotype). Data is represented as a mean +/- SEM. Statistical significance was determined via Student’s two-tailed t-test with α = 0.05 and p < 0.05.