Kupffer cell death is a characteristic feature of MASLD progression

(A-D) Male Wild-type C57BL/6J mice were fed a high-fat high-cholesterol diet (HFHC) for 0, 4, or 16 weeks. (A) KCs death was assessed by immunostaining of liver sections for Clec4f (KCs marker, green), TUNEL (red), and DAPI (nuclei, blue). Scale bar: 20μm (main panels) and 5μm (Inset). (B) KCs death was quantified. n=4 mice/group. (C) Flow cytometry analysis of KCs (CD45+ F4/80hi CD11blow Timd4+) and MoMFs (CD45+ F4/80lowCD11bhi Timd4-) among isolated NPCs. (D) KCs counts were quantified. n=4-5 mice/group. (E-F) Male wild-type C57BL/6J mice were fed either: (E) Normal chow diet (NCD) or high-fat diet (HFD) for 16 weeks, or (F) NCD or methionine-choline-deficient (MCD) diet for 6 weeks. KCs death was assessed by immunostaining of liver sections for Clec4f (green), TUNEL (red), and DAPI (nuclei, blue). Scale bar: 20μm (main panels) and 5μm (Inset). KCs death was quantified. n=4 mice/group. Representative images are shown in A, C, E, F. One-way ANOVA (B, D). Unpaired Student’s t-test (E, F). P value as indicated.

Kupffer cells exhibit spatially-patterned vulnerability in MASLD.

(A-E) Male Wild-type C57BL/6J mice were fed a HFHC diet for 0, 4, or 16 weeks. (A-D) Hepatic cell death was assessed by co-staining TUNEL with: (A) HNF4α (hepatocytes), (B) Desmin (hepatic stellate cells, HSCs), (C) Iba1 (monocyte-derived macrophages, MoMFs), and DAPI (nuclei, blue). Scale bars: 20 µm (main panels), 5 µm (insets). Hepatic cell death was quantified (n = 4 mice/group). (D) Zonal distribution of KCs death was evaluated by co-staining Timd4 (KCs), TUNEL, Glutamine Synthetase (GS, central vein marker) and DAPI (nuclei, blue). Scale bars: 50 µm. Zonal distribution of KCs death was quantified (n = 4 mice/group). FOV: field of view. PV: portal vein. CV: central vein. Representative images are shown in A-D. One-way ANOVA (A-D). P value as indicated.

Kupffer cells exhibit metabolic reprogramming with increased glycolysis during early MASLD.

(A) Experimental design for metabolomic analysis of KCs isolated from male wild-type mice fed a HFHC diet for 0, 4 or 8 weeks. n=3 mice/group. (B) Principal component analysis (PCA) of enriched metabolites in KCs across different dietary durations. (C-D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of metabolic pathways upregulated in KCs at 4 weeks (C) or 8 weeks (D). The glucose metabolism pathway is highlighted by red rectangles. (E) Heatmap depicting significantly altered metabolites involved in glucose metabolism pathways in KCs across different dietary durations. (F) Heatmap depicting significantly altered metabolites involved in cell death in KCs across different dietary durations.

Excessive glucose metabolic activity contributes to Kupffer cell death.

(A-B) Isolated KCs were treated for 24 h with: 5.5 mM glucose + isopropanol (control), 5.5 mM glucose + 800 µM palmitic acid (PA), 10 mM glucose + 800 µM PA. Cell viability was assessed by Cleaved caspase-3 (Cl-Casp3) staining (Cl-Casp3+ cells = dead). Scale bars: 20 µm (main panels), 5 µm (insets). Cl-Casp3 was detected by Western blot. (C-D) Isolated Kupffer cells were treated for 24 h with: Blank (no treatment), DMSO (vehicle control), 20 µM PS48 (PDK1 activator). Scale bars: 20 µm (main panels), 5 µm (insets). Cell death were analyzed as above. (E-F) Isolated Kupffer cells were treated for 24 h with: Blank (no treatment), DMSO (vehicle control), 20 µM oligomycin (Oligo, ATP synthase inhibitor). Scale bars: 20 µm (main panels), 5 µm (insets). Cell death were analyzed as above. One-way ANOVA (A, C, E). P value as indicated.

Enhanced glycolytic flux in Chi3l1-/- macrophages.

(A) Schematic diagram depicting the fate of glucose-derived ribose carbons in WT mouse primary hepatocytes. (B) Principal component analysis (PCA) of metabolites in WT and Chil1-/- BMDMs cultured with [U-13C]glucose. (C) Heatmap depicting significantly altered Glycolysis and Pentose phosphate (PPP) metabolites in WT and Chil1-/- BMDMs. (D) Glucose metabolic flux analysis in WT and Chil1-/- BMDMs cultured with [U-13C]glucose showing mass isotopologue distributions of: Glycolytic intermediates (Glc, F6P, FBP, 3PGA, 2PGA, PEP, PA, LA, G6P). PPP intermediates (Ru5P, R5P, S7P, DHAP). Data represent n= 6 biological replicates/group. (E-F) Extracellular acidification rate (ECAR) analysis of WT or Chil1-/- BMDMs cells. BMDM were sequentially treated with Glucose, oligomycin and 2-DG as indicated during seahorse.

Enhanced glycolysis accelerated Kupffer cell death during MASLD.

(A) Cleaved caspase-3 (Cl-Casp3) staining to detect WT and Chil1-/- Kupffer cell death. Cells were under treatment without (Blank) or with either Isopropyl alcohol (Iso) or Palmitic Acid (PA) for 24 h. Scale bar: 20μm. (B) Cl-Casp3+ cells were quantified. (C) LDH release measurement in culture medium of KCs isolated from male WT and Chil1-/- mice was measured after treatment for 24 h with: Blank (no treatment), ISO (vehicle control), 800 µM PA. (D) Flow cytometry analysis of KCs (CD45+ F4/80hi CD11blow Timd4+) and MoMFs (CD45+ F4/80low CD11bhi Timd4-) among NPCs in Clec4f-cre and Clec4fΔChil1 mice fed HFHC diet for 0 or 16 weeks. (E) KCs counts were quantified. n= 4 mice/group. (F) Kupffer cell death was assessed by immunostaining of Timd4 (KCs marker, green), TUNEL (red), and DAPI (nuclei, blue) in liver sections from Clec4f-cre and Clec4fΔChil1 mice fed HFHC diet for 0 or 16 weeks. Scale bar: 20μm (main panels) and 5μm (Inset). (G) KCs death was quantified. n=4 mice/group. Representative images shown (A, D, F). Unpaired student t-test (B,C,E,G). P value as indicated.

Excessive glycolysis enhancement promotes Kupffer cell death in MASLD.

(Left) Under physiological conditions, Kupffer cells (KCs) maintain basal glucose metabolism supporting cellular homeostasis and survival. (Right) During MASLD progression, KCs undergo excessive glycolysis enhancement, which accelerates KCs death.