Differential regulation of hepatic macrophage fate by Chi3l1 in metabolic dysfunction-associated steatotic liver disease
- Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, Yunnan University, China
- Bio-X Center for Interdisciplinary Innovation, Yunnan University, China
Figures
Figure 1 with 2 supplements
Hepatic macrophages express Chi3l1 and upregulate its expression post high-fat, high-cholesterol (HFHC) diet.
(A) Immunofluorescent staining of TIM4 (white), F4/80 (red), Chi3l1 (green), and nuclear DAPI (blue) in liver sections of mice fed with either normal chow diet (NCD) or HFHC diet for 16 weeks, illustrating Chi3l1 expression in hepatic macrophages. Scale bar = 20 µm and 10 µm (Inset). Chi3l1+ F4/80+ cells/F4/80+ cells were statistically analyzed. n=4 mice/group. (B) Representative immunofluorescence images of liver sections from WT and Chi3l1-/- mice stained for Chi3l1 (green), F4/80 (macrophages), and TIM4 (Kupffer cells [KCs]). DAPI (blue) marks nuclei. Scale bar = 20 µm and 10 µm (Insets). (C, D) Western blot analysis of Chi3l1 in either isolated KCs (C) or whole liver tissue (liver, D) from mice fed either NCD or HFHC diet. n=2–3 mice/group. (E) mRNA expression levels of Chi3l1 in liver tissues of patients with metabolic dysfunction-associated fatty liver (MAFL) or with metabolic dysfunction-associated steatohepatitis (MASH; GEO datasets: GSE167523, GSE207310, GSE130970). No-MAFLD or healthy individuals serve as controls. (F) The correlation between mRNA expression levels of Chi3l1 and MASLD activity score or fibrosis stage was analyzed (GEO datasets: GSE130970). Representative images were shown in A and B. Mann-Whitney test was performed in E. Pearson’s correlation was performed in F. p value and r value are as indicated.
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Figure 1—source data 1
Numerical data of Figure 1A and E–F.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig1-data1-v1.xlsx
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Figure 1—source data 2
PDF file containing original western blots for Figure 1C and D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig1-data2-v1.pdf
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Figure 1—source data 3
Original files for western blot analysis displayed in Figure 1C and D.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig1-data3-v1.zip
Figure 1—figure supplement 1
Metabolic dysfunction-associated steatotic liver disease progression in the high-fat, high-cholesterol (HFHC) diet-induced mouse model.
(A) Representative liver sections from wild-type C57BL/6 J mice fed either a normal chow diet (NCD) or an HFHC diet for 16 weeks. H&E and Sirius Red staining were used to assess lipid deposition, inflammation, and fibrosis. Scale bar: 20 µm. Quantification of Sirius Red–positive area is shown. (B) Western blot analysis of α-SMA expression in whole liver lysates from NCD- and HFHC-fed mice (n = 3 mice/group) to evaluate activation of hepatic stellate cells.
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Figure 1—figure supplement 1—source data 1
Numerical data of Figure 1—figure supplement 1A.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig1-figsupp1-data1-v1.xlsx
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Figure 1—figure supplement 1—source data 2
PDF file containing original western blots for Figure 1—figure supplement 1B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig1-figsupp1-data2-v1.pdf
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Figure 1—figure supplement 1—source data 3
Original files for western blot analysis displayed in Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig1-figsupp1-data3-v1.zip
Figure 1—figure supplement 2
Generation and validation of Chi3l1-/- mice.
(A) The construction, genotyping strategy, and genotyping results of Chi3l1-/- mice. P: positive control; WT: Wild-type; Neg: Blank control (ddH2O). (B) qRT-PCR analysis of mRNA expression levels of Chil1 in liver tissues of WT and Chi3l1-/- mice fed with HFHC for 0, 8, and 16 weeks. n=3–4 mice/group. Two-tailed, unpaired student t-test was performed in B. p value is as indicated.
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Figure 1—figure supplement 2—source data 1
Numerical data of Figure 1—figure supplement 2B.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig1-figsupp2-data1-v1.xlsx
Figure 2 with 2 supplements
Deficiency of Chi3l1 in Kupffer cells promotes insulin resistance and hepatic lipid accumulation.
Chi3l1fl/fl and Chi3l1-KpKO mice were fed either a normal chow diet (NCD) or a high-fat, high-cholesterol (HFHC) diet for 16 weeks. (A, B) Body weight was recorded during HFHC diet feeding (A) and expressed as a percentage of initial body mass (B). (C) H&E (Upper panel) and Oil Red O staining (Lower panel) was performed to examine liver histology and hepatic lipid accumulation in both genotypes after 16 weeks of NCD or HFHC diet. Scale bar = 20 µm. (D) Liver index (liver weight/body weight ×100%), ALT levels, and serum and liver cholesterol or triglyceride levels were measured in both genotypes after 16 weeks on NCD or HFHC diets. n=4–12 mice/group. (E, F) Intraperitoneal glucose tolerance test (IGTT) and insulin tolerance test (ITT) were performed after 16 weeks of NCD or HFHC feeding in both genotypes (n=4–12 mice per group). Representative images were shown in (C). One-way ANOVA was performed in (A, B, D–F). p value is as indicated.
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Figure 2—source data 1
Numerical data of Figure 1A–B and D–F.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
The construction and genotype of Chi3l1-KpKO mice (A) The construction, genotyping strategy, and genotyping results of Chi3l1-KpKO mice.
P: positive control; WT: Wild-type; Neg: Blank control (ddH2O). (B) qRT-PCR analysis of mRNA expression levels of Chil1 in KCs (CD45+ F4/80hi CD11blow TIM4hi) or MoMFs (CD45+ F4/80low CD11bhi Ly6G- TIM4-) FACS sorted from Chi3l1fl/fl and Chi3l1-KpKO mice at 0 and 4 weeks post HFHC diet. n=3 mice/group. (C) Western blot to detect Chi3l1 expression in isolated KCs of Chi3l1fl/fl and Chi3l1-KpKO mice. n=2 mice/group. (D) The expression specificity of Clec4f was examined in various tissues in Clec4fCreERT2/+; Rosa26LSL-tdTomato/+ mice, which is generated by crossing Clec4fCreERT2/+ with Rosa26LSL-tdTomato/+ mice.
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Figure 2—figure supplement 1—source data 1
Numerical data of Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig2-figsupp1-data1-v1.xlsx
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Figure 2—figure supplement 1—source data 2
PDF file containing original western blots for Figure 2—figure supplement 1C, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig2-figsupp1-data2-v1.pdf
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Figure 2—figure supplement 1—source data 3
Original files for western blot analysis displayed in Figure 2—figure supplement 1C.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig2-figsupp1-data3-v1.zip
Figure 2—figure supplement 2
Deficiency of Chi3l1 in Kupffer cells promotes insulin resistance and hepatic lipid accumulation.
Clec4f cre and Chi3l1-KpKO mice were fed with an HFHC diet for 16 weeks. (A, B) Body weight was recorded during HFHC diet feeding (A) and expressed as a percentage of initial body mass (B). (C, D) H&E (C) and Oil Red O staining (D) was performed to examine liver histology and hepatic lipid accumulation in both genotypes after 16 weeks of HFHC diet. Scale bar = 20 µm. (E) Liver index (liver weight/body weight × 100%), ALT levels, and serum and liver cholesterol or triglyceride levels were measured in both genotypes after 16 weeks of HFHC diet. n=3–6 mice/group. (F&G) Intraperitoneal glucose tolerance test (IGTT) and insulin tolerance test (ITT) were performed after 16 weeks of HFHC feeding in both genotypes. n=3–6 mice/group. Representative images were shown in C and D. Two-tailed, unpaired Student t-test was performed in A, B, and E–G. p value is as indicated.
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Figure 2—figure supplement 2—source data 1
Numerical data of Figure 2—figure supplement 2A–B and E-G.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig2-figsupp2-data1-v1.xlsx
Figure 3 with 2 supplements
Deficiency of Chi3l1 in Kupffer cells (KCs) promotes liver steatosis and fibrosis in metabolic dysfunction-associated steatohepatitis.
Male wild-type C57B/6 J mice were fed with normal chow diet (NCD) or methionine-choline deficient (MCD) diet for 6 weeks (A–B). Chi3l1fl/fl and Chi3l1-KpKO mice were fed with an MCD diet for 6 weeks (C–E). (A, B) qRT-PCR (A) and western blot (B) analysis of Chi3l1 expression in whole liver tissues under NCD and MCD diets. n=3 mice/group. (C) Body weight of mice with conditional deletion of Chi3l1 in KCs (Chi3l1-KpKO) and their control mice (Chi3l1fl/fl) was recorded during MCD diet. (D) Histological analyses were performed in liver tissue of Chi3l1-KpKO and Chi3l1fl/fl fed the MCD diet for 6 weeks. Scale bar = 20 μm. (E) Liver index (liver weight/body weight ×100%), ALT levels, and serum and liver cholesterol or triglyceride levels were measured in both genotypes fed the MCD diet for 6 weeks. n=4–6 mice/group. Representative images are shown in D. Two-tailed, unpaired student t-test was performed in A, C, D, and E. p value is as indicated.
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Figure 3—source data 1
Numerical data of Figure 3A, C–D and E.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig3-data1-v1.xlsx
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Figure 3—source data 2
PDF file containing original western blots for Figure 3B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig3-data2-v1.pdf
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Figure 3—source data 3
Original files for western blot analysis displayed in Figure 3B.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig3-data3-v1.zip
Figure 3—figure supplement 1
The construction and genotype of Chi3l1-MKO mice.
(A) The construction, genotyping strategy, and genotyping results of Chi3l1-MKO mice. pos: positive control; WT: Wild-type; Neg: Blank control (ddH2O). (B) qRT-PCR analysis of mRNA expression levels of Chi3l1 in KCs (CD45+ F4/80hi CD11blow TIM4hi) or MoMFs (CD45+ F4/80low CD11bhi Ly6G- TIM4-) FACS sorted from Chi3l1fl/fl and Chi3l1-MKO mice at 0 and 4 weeks post HFHC diet. n=3 mice/group. (C) Western blotting analysis of protein levels of Chi3l1 in bone-marrow-derived macrophage (BMDM) and primary KCs of Chi3l1fl/fl and Chi3l1-MKO mice. n=2–3 mice/group.
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Figure 3—figure supplement 1—source data 1
Numerical data of Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig3-figsupp1-data1-v1.xlsx
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Figure 3—figure supplement 1—source data 2
PDF file containing original western blots for Figure 3—figure supplement 1C, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig3-figsupp1-data2-v1.pdf
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Figure 3—figure supplement 1—source data 3
Original files for western blot analysis displayed in Figure 3—figure supplement 1C.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig3-figsupp1-data3-v1.zip
Figure 3—figure supplement 2
Deficiency of Chi3l1 in monocyte-derived macrophages barely affects insulin resistance and hepatic lipid accumulation.
Chi3l1fl/fl and Chi3l1-MKO mice were fed either a normal chow diet (NCD) or a high-fat, high-cholesterol (HFHC) diet for 16 weeks. (A, B) Body weight was recorded during HFHC diet feeding (A) and expressed as a percentage of initial body mass (B). (C) H&E (upper panel) and Oil Red O staining (lower panel) was performed to examine liver histology and hepatic lipid accumulation in both genotypes after 16 weeks of NCD or HFHC diet. Scale bar = 20 µm. (D) Liver index (liver weight/body weight ×100%), ALT levels, and serum and liver cholesterol or triglyceride levels were measured in both genotypes after 16 weeks on NCD or HFHC diets. n=4–9 mice/group. (E, F) Intraperitoneal glucose tolerance test (IGTT) and insulin tolerance test (ITT) were performed after 16 weeks of NCD or HFHC feeding in both genotypes (n=4–9 mice per group). Representative images were shown in (C). One-way ANOVA was performed in (A, B, D–F). p values are as indicated.
Figure 4 with 1 supplement
ScRNA-seq reveals upregulated glucose metabolism-related transcripts in kupffer cells (KCs), correlating with cell death signatures.
Wild-type (WT) C57BL/6 J mice were fed either a normal chow diet (NCD) or a high-fat, high-cholesterol (HFHC) for 16 weeks. Non-parenchymal cells (NPCs) were isolated and subjected to BD Rhapsody scRNA sequencing. (A) Uniform manifold approximation and projection (UMAP) plots illustrate the clustering of NPCs in the livers of mice fed NCD and HFHC. Cell clusters are color-coded, with monocytes/macrophages clusters outlined. (B) UMAP plots depict the clustering of monocytes/macrophages in the livers of mice fed NCD and HFHC. Cell clusters are color-coded. (C) Dot plot displays the scaled gene expression levels of lineage-specific marker genes in different cell clusters. (D) Quantification of each cell cluster is presented. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis reveals the top 12 enriched pathways for upregulated genes when comparing HFHC versus NCD in KCs, monocytes, and monocyte-derived macrophages (MoMFs), respectively. (F) Gene set variation analysis (GSVA) shows pathway activity for cell death, glucose metabolism, and cell proliferation in KCs, monocytes, and MoMFs of WT mice fed NCD or HFHC for 16 weeks, respectively. (G) The correlation between cell death and glucose metabolism pathways, based on GSVA score, is depicted.
Figure 4—figure supplement 1
Gene expression levels of lineage-specific marker genes in monocytes/macrophages clusters.
Scaled gene expression levels of each lineage-specific marker gene are shown in UMAP plots of monocytes/macrophages clusters. Colors indicate gene expression levels.
Figure 5 with 2 supplements
Chi3l1 deficiency promotes Kupffer cells (KCs) death during metabolic dysfunction-associated steatotic liver disease.
(A) GSVA analysis showed the enrichment of cell death-related pathways in KCs from wild-type (WT) mice fed with either normal chow diet (NCD) or high-fat, high-cholesterol (HFHC) or Chi3l1-/- mice fed with HFHC. (B) Dot plot showing the scaled gene expression levels of apoptosis-related genes and repressor genes in KCs from either WT or Chi3l1-/- fed with HFHC. (C) Strategy used to gate KCs (CD45+ F4/80hi CD11blow TIM4hi) and monocyte-derived macrophages (MoMFs; CD45+ F4/80low CD11bhi Ly6G- TIM4-) in the liver by flow cytometry. (D) Number of KCs and MoMFs /liver or gram (g) liver were statistically analyzed. n=3–4 mice per group. (E) Immunofluorescent staining to detect TIM4 (green), TUNEL (red), and nuclear DAPI (blue) in liver sections. Scale bar = 50 µm and 20 µm (insets). TUNEL+ TIM4+ cells/TIM4+ cells were statistically analyzed. n=4 mice/group. Representative images are shown in C and E. One-way ANOVA was performed in D. Two-tailed, unpaired student t-test was performed in E. p value is as indicated.
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Figure 5—source data 1
Numerical data of Figure 5D–E.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Chi3l1 deficiency promotes Kupffer cells (KCs) death during metabolic dysfunction-associated steatotic liver disease.
WT and Chi3l1-/- mice were fed with a high-fat, high-cholesterol (HFHC) diet for 0, 8, and 16 weeks. (A) Flow cytometry analysis of KCs (CD45+ F4/80hi CD11blow TIM4hi) and MoMFs (CD45+ F4/80low CD11bhi Ly6G- TIM4-) among non-parenchymal cells (NPCs) between WT and Chi3l1-/- mice. (B) Immunofluorescent staining to detect TIM4 (red), cleaved caspase-3 (green), and nuclear DAPI (blue) in liver sections. Scale bar = 20 μm and 5 μm (insets). Cleaved caspase-3+ TIM4+ cells/TIM4+ cells were statistically analyzed. n=4–6 mice/group. Representative images are shown in A and B. Student t-test was performed in B. p value is as indicated.
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Figure 5—figure supplement 1—source data 1
Numerical data of Figure 5—figure supplement 1B.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig5-figsupp1-data1-v1.xlsx
Figure 5—figure supplement 2
Deficiency of Chi3l1 in Kupffer cells (KCs) but not monocyte-derived macrophages (MoMFs) promotes KCs death during metabolic dysfunction-associated steatotic liver disease.
Chi3l1fl/fl and Chi3l1-KpKO mice were fed with a methionine-choline deficient (MCD) diet for 6 weeks. Chi3l1fl/fl and Chi3l1-MKO mice were fed with a high-fat, high-cholesterol (HFHC) diet for 20 weeks. (A) Immunofluorescent staining to detect TIM4 (green), TUNEL (red), and nuclear DAPI (blue) in liver sections of Chi3l1fl/fl and Chi3l1-KpKO mice. Scale bar = 50 µm and 20 µm (insets). TUNEL+ TIM4+ cells/TIM4+ cells were statistically analyzed. n=4–6 mice/group. (B) Immunofluorescent staining to detect TIM4 (green), TUNEL (red), and nuclear DAPI (blue) in liver sections of Chi3l1fl/fl and Chi3l1-MKO mice. Scale bar = 50 µm and 20 µm (insets). TUNEL+ TIM4+ cells/TIM4+ cells were statistically analyzed. n=4–5 mice/group. (C) Flow cytometry analysis of KCs (CD45+ F4/80hi CD11blow TIM4hi) and MoMFs (CD45+ F4/80low CD11bhi Ly6G- TIM4-) among non-parenchymal cells (NPCs) between Chi3l1fl/fl and Chi3l1-MKO mice. (D) Number of KCs or MoMFs/g liver were statistically analyzed. n=3 mice/group. Representative images are shown in A–C. Student t-test was performed in A and B. One-way ANOVA was performed in D. p value is as indicated.
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Figure 5—figure supplement 2—source data 1
Numerical data of Figure 5—figure supplement 2A-B and D.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig5-figsupp2-data1-v1.xlsx
Figure 6
Molecular interaction between Chi3l1 and glucose.
(A) A comparison of chemical structures between glucose and chitin. (B) Prediction of Chi3l1-glucose interaction using STITCH database (http://stitch.embl.de). (C) Strategy for pulling down glucose-binding proteins in murine serum. (D) Biotin-conjugated glucose was incubated with murine serum from mice fed with high-fat, high-cholesterol (HFHC) diet for 16 weeks. Proteins bound to glucose were precipitated by streptavidin beads. Biotin or biotin-conjugated glucose plus glucose were used as negative controls. Western blot was performed to examine Chi3l1 in the precipitate. (E) Microscale thermophoresis assay to detect the interaction between recombinant mouse Chi3l1 (rChi3l1) and glucose. Kd = 4.95 ± 0.66 mM. (F) Western blot to detect Chi3l1 expression in murine serum before and after HFHC feeding. n=3 mice/group.
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Figure 6—source data 1
Numerical data of Figure 6E.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig6-data1-v1.xlsx
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Figure 6—source data 2
PDF file containing original western blots for Figure 6D and F, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig6-data2-v1.pdf
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Figure 6—source data 3
Original files for western blot analysis displayed in Figure 6D and F.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig6-data3-v1.zip
Figure 7
Chi3l1 limits glucose uptake and protects hepatic macrophages from cell death.
(A) Following 12 hr of glucose starvation, isolated Kupffer cells (KCs) or bone-marrow-derived macrophages (BMDM) were divided into two groups: one treated with no 2-NBDG and the other with 2-NBDG. Within each group, KCs or BMDM were further treated without or with recombinant murine Chi3l1 (rChi3l1) for 6 hr. Glycogen aggregate formation labeled by 2-NBDG (Green) in KCs or BMDM was examined after counterstaining with nuclear DAPI (Blue). Scale bar = 2 μm. Area of 2-NBDG in KCs was quantified. (B) Following 12 hr of glucose starvation, BMDM were treated with either no glucose or high glucose (25 mM). Concurrently, BMDM were treated without or with rChi3l1 for 24 hr under each condition. Glycogen aggregate formation in BMDM was detected using immunofluorescence staining for Stbd1 (red) and nuclear DAPI (blue). Scale bar = 10 μm. (C and D) BMDM cells were treated without or with rChi3l1 for 24 hr and subjected to Seahorse metabolic analysis to measure the extracellular acidification rate (ECAR). (E and F) KCs were treated without (blank) or with either isopropyl alcohol (Iso) or 800 µM palmitic acid (PA) or 100 ng rChi3l1 with 800 µM PA for 24 hr. Western blot was performed to detect cleaved caspase-3 (Cl-Casp3) in E. Calcein/PI staining was quantified to detect cell viability in F. Scale bar = 50 μm. (G) Measurement of 2-NBDG (a fluorescent glucose analog) uptake by KCs in vivo. WT and Chi3l1-/- mice, either untreated or supplemented with rChi3l1, were injected intraperitoneally with 12 mg/kg 2-NBDG. After 45 min, KCs were isolated and glucose uptake assessed by spectrophotometry. (H) Representative immunofluorescence images of liver sections stained for TIM4 (red) and 2-NBDG uptake (green) to visualize glucose uptake by KCs in situ. Scale bar = 10 µm (Insets). Quantification is shown as the percentage of TIM4+ cells that are also 2-NBDG+. Representative images were shown in A, B, and H. One-way ANOVA was performed in A, F, G, and H. Two-tailed, unpaired Student t-test was performed in D. p value is as indicated.
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Figure 7—source data 1
Numerical data of Figure 7A, C–D and F–H.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig7-data1-v1.xlsx
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Figure 7—source data 2
PDF file containing original western blots for Figure 7E, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig7-data2-v1.pdf
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Figure 7—source data 3
Original files for western blot analysis displayed in Figure 7E.
- https://cdn.elifesciences.org/articles/107023/elife-107023-fig7-data3-v1.zip
Figure 8
Differential regulation of Kupffer cells (KCs) and monocyte-derived macrophages (MoMFs) fate by Chi3l1-glucose interaction.
KCs maintain a high-glucose activation state, while MoMFs exhibit a relatively low-glucose metabolic program. Chi3l1-glucose binding inhibits glucose uptake in KCs, thereby delaying KCs death and alleviating MASLD progression and metabolic dysfunction. In contrast, although Chi3l1-glucose binding similarly inhibits glucose uptake in MoMFs, their low basal glucose metabolism renders them resistant to this metabolic perturbation, resulting in minimal impact on MASLD pathogenesis.
Author response image 1
The expression of Chi3l1 in liver tissues of Chil1fl/fl, Lyz2∆Chil1and Clec4f∆Chil1mice.
Immunofluorescent staining to detect Chi3l1 (green) expression in liver sections of Chil1fl/fl, Lyz2∆Chil1and Clec4f∆Chil1mice under normal chow diet. TIM4 (KCs marker, white), F4/80 (macrophage marker, red), nuclei were counterstained with DAPI, Scale bar=20 µm and 10 µm (inset).
Author response image 2
Analysis of Chil1 expression in additional single-cell RNA sequencing datasets.
(A-C) Chil1 expression in a mouse model of NASH. (A) t-SNE projection of cell clusters from scRNA-seq data (GSE1283338) of livers from C57BL/6J mice fed a control or NASH diet for 30 weeks. (B) Dot plot showing scaled Chil1 expression across all identified cell clusters. (C) Dot plot of scaled Chil1 expression after excluding the neutrophil cluster, highlighting expression in macrophage populations. Analyzed cell clusters and cell numbers: KC_H (healthy, 1178); KC3_Control (1142); KC_N (NASH, 1045); KN_RM (recruited macrophage in KC niche, 950); Proliferating_KC (364); PDC_Control (356); Ly6CHi_RM (320); LSEC (299); NK_NKT (393); B_cell (244); DC_1 (107); DC_2 (118); Ly6CLo_RM (127); Hepatocyte (57); PDC_NASH (46); Neutrophil (21). (D-E) Chil1 expression during NAFLD progression in a mouse Western diet model. (D) t-SNE projection of cell clusters from scRNA-seq data (GSE156059) of livers from C57BL/6J mice fed a Western diet with fructose/sucrose for 12, 24, and 36 weeks. (E) Dot plot showing scaled Chil1 expression across all identified cell clusters. Analyzed cell clusters and cell numbers: capsule macs (250), LAMs (1419), Ly6chi monocytes (6912), mac1 (638), moKCs (767), Patrolling monocytes (690), Prolif.macs (521), Resident KCs (3629), Transitioning monocytes (3615). (F-H) Chil1 expression in human cirrhotic liver biopsies. (F) t-SNE projection of cell clusters from scRNA-seq data (GSE136103) of healthy and cirrhotic human liver samples. (G) Dot plot showing scaled Chil1 expression across major cell lineages. (H) Dot plot of scaled Chil1 expression specifically within the mononuclear phagocyte (MP) population. Analyzed cell clusters and cell numbers: B cell (1951); cycling (967); Epithelia (3751); ILC (10091); mast cell (2511); Mesenchyme (2382); MP (10874); pDC (317); Plasma cell (877); T cell (19076). (I-K) Chil1 expression in a human NAFLD explant. (I) t-SNE projection of cell clusters from scRNA-seq data (GSE190487) of a human NAFLD liver explant. (J) Dot plot showing scaled Chil1 expression across all identified cell clusters. (K) Dot plot of scaled Chil1 expression within the MP subpopulations. Analyzed cell clusters and cell numbers: B cell (1278); Cycling (152); MP (2897); pDC (391); Plasma cell (85); T cell (1551); KC (403); SAMac (scar-associated macrophages, 723); TM (tissue monocytes, 1265).
Author response image 3
Hepatic macrophages express Chi3l1.
(A-D) Wildtype C57BL/6J mice were fed either a normal chow diet (NCD) or HFHC for 16 weeks. NPCs were isolated and subjected to BD Rhapsody scRNA sequencing. (A) Uniform manifold approximation and projection (UMAP) plots illustrate the clustering of NPCs from the livers of mice fed NCD and HFHC. Major cell types are colored. (B) Heatmap showing the mean expression of top2 markers of each cell type. (C) Violin plots show the RNA expression of Chil1 between NCD and HFHC livers in each cell cluster. (D) UMAP plots depict the clustering of Monocytes/Macrophages in the livers of mice fed NCD and HFHC. Cell clusters are color-coded. (E) Dot plot displays the scaled gene expression levels of lineage-specific marker genes in different cell clusters. (F) Dot plot shows the scaled gene expression levels of Chil1 in the indicated cell clusters.
Author response image 4
The expression of Chi3l1 in serum of Clec4f cre mice.
(A) Western blot to detect Chi3l1 expression in murine serum of Clec4f cre mice before and after HFHC feeding. n=3 mice/group.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Mouse monoclonal anti-TIM4 (Alexa Fluor 647) antibody | Biolegend | Cat# 130008; RRID:AB_2271648 | IF (1:300) |
| Antibody | Mouse monoclonal anti-β-actin antibody | Proteintech | Cat# 66009–1-lg; RRID:AB_2687938 | WB (1:1000) |
| Antibody | Cleaved caspase-3 rabbit antibody | Cell Signalling Technology | Cat# 9664 S; RRID:AB_2070042 | IF (1:300), WB (1:1000) |
| Antibody | Caspase-3 rabbit antibody | Cell Signalling Technology | Cat# 9662 S; RRID:AB_331439 | WB (1:1000) |
| Antibody | Rabbit polyclonal anti-YKL-40/CHI3L1 antibody | Abcam | Cat# ab180569; RRID:AB_2891040 | IF (1:400) |
| Antibody | Anti-alpha-smooth muscle actin | Invitrogen | Cat# 50-9760-82; RRID:AB_2574362 | WB (1:1000) |
| Antibody | GAPDH monoclonal antibody | Proteintech | Cat# 60004–1; RRID:AB_2107436 | WB (1:1500) |
| Antibody | Albumin antibody | Cell Signaling | Cat# 4929 s; RRID:AB_2225785 | WB (1:1000) |
| Antibody | Alexa Fluor 594 anti-mouse F4/80 | BioLegend | Cat# 123140; RRID:AB_2563241 | IF (1:300) |
| Antibody | Anti-STBD1 rabbit | Proteintech | Cat# 11842–1-AP; RRID:AB_2197523 | IF (1:300) |
| Antibody | Rat monoclonal anti-F4/80 (APC) antibody | Invitrogen | Cat# 17-4801-82; RRID:AB_2784648 | Flow cytometry (1:100) |
| Antibody | Rat monoclonal anti-CD45 (eFluor450) antibody | Invitrogen | Cat# 48-0451-82; RRID:AB_1518806 | Flow cytometry (1:100) |
| Antibody | Rat monoclonal anti-TIM-4 (PE) antibody | Invitrogen | Cat# 12-5866-82; RRID:AB_1257163 | Flow cytometry (1:100) |
| Antibody | Rat monoclonal anti-CD16/CD32 | Invitrogen | Cat# 14-0161-86; RRID:AB_467135 | Flow cytometry (1:100) |
| Antibody | Rat monoclonal anti-CD11b (PerCP/Cyanine5.5) antibody | Biolegend | Cat# 101228; RRID:AB_893232 | Flow cytometry (1:100) |
| Antibody | Ly-6G monoclonal antibody (1A8-Ly6g) PE-Cyanine7 | Invitrogen | Cat# 25-9668-82; RRID:AB_2811793 | Flow cytometry (1:100) |
| Antibody | Peroxidase-conjugated affinipure goat anti-rabbit IgG (H+L) | Jackson | Cat# 111-035-003; RRID:AB_2313567 | WB (1:2000) |
| Antibody | Peroxidase-conjugated affinipure goat anti-mouse IgG (H+L) | Jackson | Cat# 115-035-003; RRID:AB_10015289 | WB (1:2000) |
| Antibody | Alexa Fluor 488-conjugated affinipure goat anti-mouse IgG +IgM(H+L) | Jackson | Cat# 115-545-044; RRID:AB_2338844 | IF (1:1000) |
| Antibody | Alexa fluor 568-goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody | Invitrogen | Cat# A11011; RRID:AB_143157 | IF (1:1000) |
| Chemical compound, drug | FBS | VivaCell | Cat# C04001-500 | |
| Chemical compound, drug | PBS | VivaCell | Cat# C3580-0500 | |
| Chemical compound, drug | DMEM (high glucose) | VivaCell | Cat# C3113-0500 | |
| Chemical compound, drug | DMEM (no glucose) | Sigma | Cat# D5030 | |
| Chemical compound, drug | Sodium pyruvate | Sangon Biotech | Cat# A501259-0100 | |
| Chemical compound, drug | Penicillin-streptomycin solution | VivaCell | Cat# C3421-0100 | |
| Chemical compound, drug | Cell dissociation solution | Sartorius | Cat# 03-079-1B | |
| Chemical compound, drug | β-Mercaptoethanol | Sigma | Cat# M3148 | |
| Chemical compound, drug | Eosin Y (water soluble) | Aladdin | Cat# E141405 | |
| Chemical compound, drug | Hematoxylin | BBI | Cat# A600701-0050 | |
| Chemical compound, drug | Oil Red O | Solarbio | Cat# IO1720 | |
| Chemical compound, drug | Sirius red | Sangon Biotech | Cat# A500684-0500 | |
| Chemical compound, drug | High effect paraffin cere sin | Shanghai Hualing Rehabilitation Equipment Manufacturing Plant | Cat# N/A | |
| Chemical compound, drug | 10% Neutral formalin fix solution | BBI | Cat# E672001-0500 | |
| Chemical compound, drug | Xylene | Tianjin Zhiyuan Chemical Reagents Co., Ltd | Cat# N/A | |
| Chemical compound, drug | Neutral balsam | Solarbio | Cat# G8590 | |
| Chemical compound, drug | Isopropanol | Sangon Biotech | Cat# A507048-0500 | |
| Chemical compound, drug | Tissue-tek OCT compound | SAKURA | Cat# REF:4583 | |
| Chemical compound, drug | Paraformaldehyde | Sangon Biotech | Cat# A500684-0500 | |
| Chemical compound, drug | Acetone | Chron Chemicals | Cat# N/A | |
| Chemical compound, drug | Sucrose | Sangon Biotech | Cat# A502792-0005 | |
| Chemical compound, drug | Triton X-100 | BBI | Cat# A600198-0500 | |
| Chemical compound, drug | Goat serum | VivaCell | Cat# C2530-0100 | |
| Chemical compound, drug | Tween20 | BBI | Cat# A600560-0500 | |
| Chemical compound, drug | DAPI staining solution | Beyotime | Cat# C1006 | |
| Chemical compound, drug | Omni-Easy one-step PAGE gel fast preparation kit | Epizyme | Cat# PG213 | |
| Chemical compound, drug | SDS | BBI | Cat# A600485-0500 | |
| Chemical compound, drug | Glycine | BBI | Cat# A502065-0005 | |
| Chemical compound, drug | Tris | Solarbio | Cat# T8060 | |
| Chemical compound, drug | Methanol | Ghtech | Cat# N/A | |
| Chemical compound, drug | Non-fat powdered milk | BBI | Cat# NON-Fat Powdered Milk | |
| Chemical compound, drug | Collagenase, type 1 | Diamond | Cat# A004194-0001 | |
| Chemical compound, drug | Cytiva Percoll Centrifugation Media | Cytiva | Cat# 17089101 | |
| Chemical compound, drug | Heparin sodium from porcine intestinal | Sangon Biotech | Cat# A603251-0001 | |
| Chemical compound, drug | 1 M HEPES | Solarbio | Cat# H1095 | |
| Chemical compound, drug | Optiprep | Serumwerk Bernburg | Cat# 1893 | |
| Chemical compound, drug | DNaseI, RNase-free | Thermo | Cat# EN0521 | |
| Chemical compound, drug | CaCl2 | Ghtech | Cat#10043-52-4 | |
| Chemical compound, drug | MgSO4·7H2O | Sangon Biotech | Cat# A610329-0500 | |
| Chemical compound, drug | Trizol reagent | Invitrogen | Cat# 15596018 | |
| Chemical compound, drug | UltraPure DNase/RNase-free distilled water | Invitrogen | Cat# 10977015 | |
| Chemical compound, drug | Trichloromethane | Chron Chemicals | Cat# N/A | |
| Chemical compound, drug | PowerUp SYBR Green Master Mix | Applied biosystems | Cat# A25742 | |
| Chemical compound, drug | DEPC水 | Biosharp | Cat# 701062 | |
| Chemical compound, drug | MgCl2 | Ghtech | Cat# N/A | |
| Chemical compound, drug | KCl | Sangon Biotech | Cat# A501159-0500 | |
| Chemical compound, drug | NaHCO3 | Sangon Biotech | Cat# A500873-0500 | |
| Chemical compound, drug | NaOH | BBI | Cat# A620617-0500 | |
| Chemical compound, drug | Palmitic acid | Sigma | Cat# P0500 | |
| Chemical compound, drug | DMSO | Sangon Biotech | Cat# A100231-0500 | |
| Chemical compound, drug | Proteinase K solution (20 mg/mL) | BBI | Cat# B600169-0002 | |
| Chemical compound, drug | Glycerol gelatin aqueous slide mounting medium | Solarbio | Cat# S2150 | |
| Chemical compound, drug | XF basal medium | Agilent | Cat#103334–100 | |
| Chemical compound, drug | XF 200 mmol/L glutamine solution | Agilent | Cat#103579–100 | |
| Chemical compound, drug | BD Pharmingen Stain Buer (FBS) | BD Biosciences | Cat# 554656 | |
| Chemical compound, drug | Draq7 | BD Biosciences | Cat# 564904 | |
| Chemical compound, drug | High-fat rodent diet with 1.25%cholesterol | Research diet | Cat# d12108c | |
| Chemical compound, drug | Methionine and choline-deficient diet | Research Diet | Cat# A02082002BR | |
| Chemical compound, drug | Proteinase K solution (20 mg/mL) | BBI | Cat# B600169-0002 | |
| Chemical compound, drug | Glycerol Gelatin aqueous slide mounting medium | Solarbio | Cat# S2150 | |
| Chemical compound, drug | XF basal medium | Agilent | Cat#103334–100 | |
| Chemical compound, drug | XF 200 mmol/L Glutamine solution | Agilent | Cat#103579–100 | |
| Chemical compound, drug | BD Pharmingen Stain Buer (FBS) | BD Biosciences | Cat# 554656 | |
| Chemical compound, drug | Draq7 | BD Biosciences | Cat# 564904 | |
| Chemical compound, drug | Dynabeads M-280 streptavidin | Invitrogen | Cat# 11205D | |
| Chemical compound, drug | 2-NBDG | Invitrogen | Cat# N13195 | |
| Peptide, recombinant protein | Recombinant mouse Chi3l1 | SB | Cat# 50929-M08H | |
| Commercial assay or kit | TMR (red) Tunel cell apoptosis detection kit | Servicebio | Cat# G1502-100T | |
| Commercial assay or kit | Calcein/PI cell viability /cytotoxicity assay kit | Beyotime | Cat# C2015M | |
| Commercial assay or kit | Alanine aminotransferase assay kit | Nanjing Jiancheng Bioengineering Institute | Cat# C009-2-1 | |
| Commercial assay or kit | Aspartate aminotransferase assay kit | Nanjing Jiancheng Bioengineering Institute | Cat# C010-2-1 | |
| Commercial assay or kit | Total cholesterol assay kit | Nanjing Jiancheng Bioengineering Institute | Cat# A111-1-1 | |
| Commercial assay or kit | Triglyceride assay kit | Nanjing Jiancheng Bioengineering Institute | Cat# A110-1-1 | |
| Commercial assay or kit | Seahorse XF glycolysis stress test kit | Agilent | Cat# 103020–100 | |
| Commercial assay or kit | PrimeScript II 1st strand cDNA synthesis kit | TaKaRa | Cat# 6210B | |
| Software, algorithm | GraphPad Prism | GraphPad Software | RRID:SCR_002798 | https://www.graphpad.com |
| Software, algorithm | Flowjo V10 | Flowjo Software | RRID:SCR_008520 | https://www.flowjo.com/ |
| Software, algorithm | ImageJ | National Institutes of Health | RRID:SCR_003070 | https://imagej.nih.gov/ij/ |
| Software, algorithm | SPSS | IBM SPSS software | RRID:SCR_002865 | https://www.ibm.com/ |
| Software, algorithm | Seahorse wave | Agilent Technologies | RRID:SCR_024491 | https://www.agilent.com/ |
| Software, algorithm | Zen microscope software | ZEISS | RRID:SCR_013672 | https://www.zeiss.com.cn/ |
| Cell line (Mus musculus, male) | NCTC clone 929 (L-929) | ATCC | CCL-1 RRID:CVCL_0462 | L929 was a gift from Dr. Guangxun Meng (Hainan Academy of Medical Sciences) |
| Strain, strain background (Mus musculus, male) | Chi3l1-/- | GemPharmatech Co., Ltd. | RRID:IMSR_GPT:T014402 | Genetic modification: constitutive knockout |
| Strain, strain background (Mus musculus, male) | Chi3l1flox/flox | GemPharmatech Co., Ltd. | RRID:IMSR_GPT:T013652 | Genetic modification: floxed allele (homozygous) |
| Strain, strain background (Mus musculus, male) | Clec4f cre | GemPharmatech Co., Ltd. | RRID:IMSR_GPT:T036801 | Genetic modification: Cre recombinase transgene under Clec4f promoter |
| Strain, strain background (Mus musculus, male) | Lyz2 cre | GemPharmatech Co., Ltd. | RRID:IMSR_GPT:T003822 | Genetic modification: Cre recombinase transgene under Lyz2 promoter |
| Strain, strain background (Mus musculus, male) | Rosa26LSL-tdTomato/+ | Jackson Laboratory | RRID:IMSR_JAX:007909 | Genetic modification: Conditional tdTomato reporter |
| Other | Bone-marrow-derived macrophage (BMDM) | This paper | PMID:42187013 | Strain: C57BL/6 J |
| Other | Kupffer cell (KC) | This paper | PMID:42187013 | Strain: C57BL/6 J |
Additional files
-
Supplementary file 1
PCR primer sequences for genotyping.
- https://cdn.elifesciences.org/articles/107023/elife-107023-supp1-v1.docx
-
Supplementary file 2
qPCR primers.
- https://cdn.elifesciences.org/articles/107023/elife-107023-supp2-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/107023/elife-107023-mdarchecklist1-v1.pdf
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Differential regulation of hepatic macrophage fate by Chi3l1 in metabolic dysfunction-associated steatotic liver disease
eLife 14:RP107023.
https://doi.org/10.7554/eLife.107023.4
