Fig 2: Injection of sub-anaesthetic doses of ketamine modulates circulating myeloid compartment and promotes the differentiation of anti-inflammatory M2 macrophages in vivo. 72 hours after the intraperitoneal injection of sub-anaesthetic doses of ketamine (10 mg/kg) or saline solution, peripheral blood cells, bone marrow, spleen and total leukocytes from CNS were analysed. Peripheral circulating monocytes were analysed by gating on CD11b+Ly6C+Ly6G by flow cytometry. Ketamine administration significantly reduces (a) the inflammatory Ly6Chigh monocytes and increases (b) the Ly6Clow monocyte fraction. (c) The absolute number of bone marrow cells as well as (d) the total number of splenocytes was significantly increased 72 hours after the administration of sub-anaesthetic doses of ketamine compared with control mice. (e) The percentage of tissue resident CD45lowCD11b+ cells in the CNS was similar between groups, but (f) the percentage of hematopoietic CD45highCD11b+F4/80+macrophages was significantly reduced in CNS tissue from ketamine-treated mice. Furthermore, (g) the percentage of Arginase-1+ macrophages (CD45highCD11b+F4/80+) was significantly increased in the CNS and (h) spleens of ketamine-treated mice. (i) and (j) BMDM from ketamine-treated or control mice, were differentiated for 7 days in the presence of M-CSF. These BMDMs were harvested and then challenged with 100 ng/ml of LPS for 24 hours. The percentage of (i) CD86+CD206 cells and (j) the mean fluorescence intensity (MFI) of CD36+ pro-inflammatory markers were significantly reduced in BMDM obtained from ketamine-treated mice. (k) The percentage of CD206+CD86 macrophages was significantly increased in ketamine-treated compared with untreated mice. In-vivo experiments were performed with at least 4–5 mice per each condition, and repeated twice. Pooled data were graphed and statistical significance is denoted as *p < 0.05; **p < 0.01; ***p < 0.001. Student's t test or one-way ANOVA test were performed accordingly.

Fig 3: Cell Surface LRP6 Is Reduced in Bone Marrow MSCs under Microenvironment of High oxPLs. a–c Representative images (a) and the quantified CFU-F frequency (b), and CFU-Ob frequency (c) of bone marrow MSCs from 10-week-old C57BL/6 mice fed Western high fat diets (HFD) or standard chow diets (CHD) for 4 weeks. d Bone marrow MSCs from 10-week-old C57BL/6 mice fed HFD or CHD for 4 weeks were incubated with osteogenic medium for 7 days. mRNA expressions of RUNX2 and osteocalcin (OCN) were detected by Real-time PCR analysis. n = 5 mice per treatment group from six independent experiments, data are represented as mean ± s.e.m. *p < 0.05, vs. CHD group, as determined by Student’s t-tests. e–h Representative images of the flow cytometry analysis (e) and the percentage of LRP6+ (f), CD36+ cells (g), and LOX-1 (h) in LepR+CD45–CD31–Ter119- cell population from 10-week-old C57BL/6 mice fed Western high fat diets (HFD) or standard chow diets (CHD) for 2 weeks. n = 5, data are represented as mean ± s.e.m. *p < 0.01 vs. CHD group, as determined by Student’s t-tests. i–l Representative images of the flow cytometry analysis (i) and the percentage of LRP6+ (j), CD36+ cells (k), and LOX-1 (l) in CD11b+ cell population from 10-week-old C57BL/6 mice fed Western high fat diets (HFD) or standard chow diets (CHD) for 2 weeks. n = 5, data are represented as mean ± s.e.m. *p < 0.01 vs. CHD group, as determined by Student’s t-tests. m–o 10-week-old C57BL/6 mice fed CHD or HFD together with D-4F peptide (HFD + D-4F) or scrambled D-4F peptide (HFD + SD-4F) for 2 weeks. The peptides were administered orally in drinking water at 0.4 mg·mL-1, equivalent to 1.6 mg/d. Flow cytometry analysis of LRP6+ cells in bone marrow LepR+CD45–CD31–Ter119- MSCs (i). Representative histograms showing relative intensity of LRP6 antibody-conjugated fluorescence in the three groups (j). Quantitative analysis of the relative fluorescence intensity in the three groups (k). n = 5, data are represented as mean ± s.e.m. *p < 0.01 vs. CHD group, #p < 0.01, vs. HFD + SD-4F group, as determined by Student’s t-tests

Fig 4: BA and DF surface marker validation.a Dot plot showing expression level of Pdgfra, Twist2, Thy1, Fabp4, and Cd36 in LP, DF and BA. b FACS plot of YFP+ cells from E9.5 Pax7-traced mouse embryos on E12.5, E13.5, E14.5, E15.5, and E16.5. Cells were stained with Thy1 and Pdgfra antibodies to validate the presence of surface markers. X-axis indicates the signal from Thy1 antibody while y-axis indicates the signal from Pdgfra antibody. c FACS plot of YFP+ cells from E14.5 and E16.5 that were stained with Thy1 and Cd36 antibodies. X-axis indicates signal from Thy1 antibody while y-axis indicates signal from Cd36 antibody. d Correlation plot of bulk and pseudobulk samples. After FACS sorting with the following surface marker combination: Pdgfra+/Thy1+, Pdgfra+/Thy1−, and Pdgfra−/Cd36+, samples were either prepared for sequencing immediately or cultured. Each row represents one bulk sample. Rows annotated with the same color indicate replicates within the same condition. Gene expression was Z-score normalized. e Volcano plots of differentially expressed genes between sorted samples. Genes with adjusted P-value < 0.01 were colored by corresponding cluster color (log-fold change > 1.5 or < −1.5), whereas ≥ 0.01 were colored as gray. Lineage markers and surface markers were labeled adjacent to corresponding dots.

Fig 5: Fibroblast growth factor 21 (FGF21) modulates hepatic macrophage pool and protects against COL1A1 accumulation, as predicted by the reduction of CD36hi Kupffer cells (KCs) and CD9hi KCs.(A) Uniform manifold approximation and projection for dimension reduction (UMAP) of immune cell subsets from livers after 23 weeks of intervention. (B) The number of resident KCs (ResKCs), the proportion of CD11c+ ResKCs, and the expression of CD36 and CD9 in ResKCs were quantified. (C) The amount of monocyte-derived KCs (MoKCs) was assessed, the percentage of CD11c+ MoKCs was determined, the CD36 and CD9 expression levels in MoKCs were quantified. (D) Hepatic inflammation was evaluated by pro-inflammatory gene expression and the formation of crown-like structures (CLSs) within the liver. The mRNA expression of liver fibrogenesis markers was quantified, and the protein expression of collagen type 1α 1 (COL1A1) was determined. (E) The expression of CD36 in ResKCs, and the expression of CD9 and CD36 in MoKCs were plotted against COL1A1-positive area in the liver. (F) Mechanistic model. Data are shown as mean ± SEM (A–B and E, n=4–5 per group; D, n=16–18 per group). Linear regression analyses were performed. Differences were assessed using one-way ANOVA followed by a Fisher’s LSD test. *p<0.05, **p<0.01, ***p<0.001, compared with the low-fat low-cholesterol diet (LFCD) group. #p<0.05, ##p<0.01, ###p<0.001, compared with the high-fat high-cholesterol diet (HFCD) group. Acta2, actin α2; Ctgf, connective tissue growth factor; FA, fatty acid; Tgfb1, transforming growth factor-β. Figure 6—source data 1.Fibroblast growth factor 21 (FGF21) modulates hepatic macrophage pool and protects against COL1A1 accumulation, as predicted by the reduction of CD36hi Kupffer cells (KCs) and CD9hi KCs.