Fig 1: Paquinimod disrupts the function of S100A8+ senescent immune cells and ameliorates age-related metabolic dysfunction.a, b Representative flow cytometry plots and quantification of frequencies of S100A8+ T cells (a) and myeloid cells (b) in SVFs of BAT of 15-month-old mice treated with paquinimod or vehicle (n = 4–5). c Representative images of TH and S100A8 staining in the BAT of 15-month-old mice treated with paquinimod or vehicle. Scale bar, 20 µm. d Relative mRNA levels of Th and Ucp1 in the BAT of 15-month-old mice treated with paquinimod or vehicle (n = 5). e Representative immunoblots of UCP1 and PGC-1a in the BAT of 15-month-old mice treated with paquinimod or vehicle (n = 6). f Representative immunoblots of P16 and P21 in the BAT of 15-month-old mice treated with paquinimod or vehicle (n = 3). g Food intake of mice treated with paquinimod or vehicle (n = 5). h Body weight changes of normal chow diet-fed 15-month-old mice treated with paquinimod or vehicle (n = 5–6). i Fasting glucose levels of normal chow diet-fed mice after treated with paquinimod or vehicle for 6 months (n = 5–6). j–n Body weight changes (j), glucose tolerance (k), insulin sensitivity (l), tissue weights ratio (m), HE staining of adipose tissue and liver (n) in HFD-fed mice treated with paquinimod or vehicle (n = 5). Scale bar, 50 µm. Data shown are representative of three independent experiments with similar results. n indicates the number of biologically independent samples examined. Data are shown as the mean ± SEM. Statistical differences were supposed to be significant when P < 0.05. Statistical analysis was performed by two-way ANOVA (h, j–l), or unpaired two-tailed Student’s t test (a, b, d–f, i, m). Source data are provided as a Source Data File.
Fig 2: Adipose RBM3 mediates the action of S100A8+ immune cells in sympathetic innervation.a Schematic diagram of experimental processes. This diagram was created with BioRender.com. b Representative images of TH staining in the BAT of mice injected with AAV-RBM3 or AAV-NC and transferred with S100A8+ immune cells. Scale bar, 100 µm. c Representative immunoblots of UCP1, TH, p-TH, TUBB3 and RBM3 in the BAT of mice injected with AAV-RBM3 or AAV-NC and transferred with S100A8+ immune cells (n = 3). d Relative mRNA levels of Rbm3, Th Ucp1, Ppargc1a, p16, and p21 in the BAT of mice injected with AAV-RBM3 or AAV-NC and transferred with S100A8+ immune cells (n = 5). e Oxygen consumption of mice injected with AAV-RBM3 or AAV-NC and transferred with S100A8+ immune cells or S100A8- immune cells (n = 6–7). f Core body temperature of mice injected with AAV-RBM3 or AAV-NC were transferred with S100A8+ immune cells and subjected to cold challenge (n = 5). g Venn diagram showing overlapped genes which are RBM3-bounded and differentially expressed upon Rbm3 knockdown. h KEGG analysis of the 909 overlapped genes shown in (g). i Heatmap of axon guidance-related gene expressions based on RNA-seq data of Rbm3 knockdown differentiated brown adipocytes. j Relative mRNA levels of Rbm3 and axon guidance-related genes Nrp1 and Epha7 in differentiated brown adipocytes transfected with si-Rbm3 or si-NC (n = 3). k Representative immunoblots of NRP1 and EPHA7 in differentiated brown adipocytes transfected with si-Rbm3 or si-NC (n = 4). l RNA immunoprecipitation (RIP) assay assessing RBM3 binding on 3´UTR of Nrp1 and Epha7 in differentiated brown adipocytes (n = 4). m Relative mRNA level of Nrp1 and Epha7 in si-Rbm3 or si-NC transfected brown adipocytes upon transcriptional inhibition with actinomycin D at indicated time (n = 3). n Representative images of TUBB3 staining in PC12 cells cocultured with differentiated brown adipocytes overexpressed with RBM3 and transfected with si-Nrp1 or si-Epha7 (n = 4). Scale bar, 100 µm. Data shown are representative of three independent experiments with similar results. n indicates the number of biologically independent samples examined. Data are shown as the mean ± SEM. Statistical differences were supposed to be significant when P < 0.05. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple-comparison test (n) or two-way ANOVA (f, m), ANCOVA with body weight as covariant (e) or unpaired two-tailed Student’s t test (c, d, j–l). Source data are provided as a Source Data File.
Fig 3: BAT-infiltrating S100A8+ immune cells originate from the bone marrow.a Schematic diagram for isolation, labeling and transferring bone marrow-derived S100A8+ immune cells into young mice. This diagram was created with BioRender.com. b Representative images of PKH26-labeled S100A8- or S100A8+ immune cells infiltrating BAT, iWAT, eWAT and liver (n = 3). Scale bar, 100 µm. c Schematic diagram for bone marrow reconstitution of mice with HSPCs isolated from S100a8-Cre-EGFP mice. This diagram was created with BioRender.com. d Gating strategy of bone marrow hematopoietic stem/progenitor cells (HSPCs). e Representative images of GFP+ cells in the BAT of bone marrow reconstitution mice during aging (n = 4). Scale bar, 100 µm. f, g Representative flow cytometry plots and quantification of the frequencies of GFP+ cells in CD45+ CD3 + T cells (f) (n = 5/group), and CD45+CD11b+ myeloid cells (g) (n = 5/group), in the SVFs of BAT from bone marrow reconstitution mice during aging. Data shown are representative of three independent experiments with similar results. n indicates the number of biologically independent samples examined. Data are shown as mean ± SEM. Statistical differences were supposed to be significant when P < 0.05. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple-comparison test (e–g). Source data are provided as a Source Data File.
Fig 4: Pro-inflammatory and senescent S100A8+ immune cells accumulate in brown adipose tissue with age.a Bioinformatics analysis of scRNA-seq of BAT from young (5-month-old) and aged (27-month-old) rats. b GO analysis of differentially expressed genes in T cells (left) and neutrophils (right) from aged rats. c Violin plots for gene expression of S100a8 in cell populations of BAT from young and aged rats. d Relative cell proportion of S100A8+ and S100A8− cell populations in the BAT of young and aged rats. e Violin plots for gene expression of S100a8 in T cell subclusters of young and aged rats. f UMAP plot shows clustering of macrophages based on gene expression. g Violin plots for gene expression of S100a8 in macrophage subclusters of young and old. h, i Representative flow cytometry plots and quantification of the frequencies of S100A8+ cells in CD45+ CD3+ T cells (h), and CD45+CD11b+ myeloid cells (i), in the stromal vascular fractions (SVFs) of BAT from 2-, 8- and 15-month-old mice (n = 3–5/group). j Representative images of CD3 (green) and S100A8 (red) and staining in the BAT of young (2 months) and aged (15 months) mice. Scale bar, 50 µm. k Representative images of CD11b (green) and S100A8 (red) staining in the BAT of young (2 months) and aged (15 months) mice. Scale bar, 50 µm. Data shown are representative of three independent experiments with similar results. n indicates the number of biologically independent samples examined. Quantitative data are shown as mean ± SEM. Statistical differences were supposed to be significant when P < 0.05. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple-comparison test (h, i). Source data are provided as a Source Data File.
Fig 5: S100A8+ immune cells, coupling with sympathetic nerves and adipocytes, form neuroimmune adipose interfaces that inhibit sympathetic innervation.a Heatmap of differentially expressed genes (DEGs) in differentiated brown adipocytes treated with S100A8 (P < 0.05 and |log2(Fold change)| >0.38). b GO analysis of DEGs in (a). c Representative images of age-associated changes in S100A8+ immune cells and tyrosine hydroxylase positive (TH+) sympathetic nerves in the BAT of 2-month-old and 15-month-old mice. Scale bar, 100 µm. d Representative images of the localization of S100A8+ immune cells and TH+ sympathetic nerves in the BAT of 2- and 15-month-old mice. Scale bar, 10 µm. e Pearson correlation analysis of the fluorescence intensity between S100A8 and TH in the BAT of 2-month-old and 15-month-old mice. f Representative immunoblots of TH, phospho-TH (Ser40) (p-TH) and TUBB3 in the BAT of mice transferred with S100A8+ immune cells or S100A8- immune cells (n = 3). g Schematic diagram of neuroimmune adipose interface in the BAT. This diagram was created with BioRender.com. h Venn diagram of downregulated DEGs in RNA-seq datasets of S100A8-treated brown adipocytes and BAT of aged mice. i Representative immunoblots of RBM3 in the BAT of 2- and 15-month-old mice (n = 3). j Representative immunoblots of RBM3 in the BAT of mice transferred with S100A8+ immune cells or S100A8- immune cells (n = 3). k Representative images of TH staining in the BAT of mice injected with AAV-ShRbm3 or AAV-Scramble. Scale bar, 100 µm. l Representative immunoblots of UCP1, TH, p-TH, TUBB3 and RBM3 in the BAT of mice injected with AAV-ShRbm3 or AAV-Scramble (n = 3). m Core body temperature of mice injected with AAV-ShRbm3 or AAV-Scramble under cold stimulation (n = 5–7). Data shown are representative of three independent experiments with similar results. n indicates the number of biologically independent samples examined. Data are shown as the mean ± SEM. Statistical differences were supposed to be significant when P < 0.05. Statistical analysis was performed by two-way ANOVA (m), or unpaired two-tailed Student’s t test (f, i–l). Source data are provided as a Source Data File.
Supplier Page from Sino Biological, Inc. for Mouse S100A8 / CAGA Protein (His Tag)