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LIFR regulates cholesterol-driven bidirectional hepatocyte–neutrophil cross-talk to promote liver regeneration

Nature Metabolism volume 6, pages 1756–1774 (2024)Cite this article

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Abstract

Liver regeneration is under metabolic and immune regulation. Despite increasing recognition of the involvement of neutrophils in regeneration, it is unclear how the liver signals to the bone marrow to release neutrophils after injury and how reparative neutrophils signal to hepatocytes to reenter the cell cycle. Here we report that loss of the liver tumour suppressor Lifr in mouse hepatocytes impairs, whereas overexpression of leukaemia inhibitory factor receptor (LIFR) promotes liver repair and regeneration after partial hepatectomy or toxic injury. In response to physical or chemical damage to the liver, LIFR from hepatocytes promotes the secretion of cholesterol and CXCL1 in a STAT3-dependent manner, leading to the efflux of bone marrow neutrophils to the circulation and damaged liver. Cholesterol, via its receptor ERRα, stimulates neutrophils to secrete hepatocyte growth factor to accelerate hepatocyte proliferation. Altogether, our findings reveal a LIFR–STAT3–CXCL1–CXCR2 axis and a LIFR–STAT3–cholesterol–ERRα–hepatocyte growth factor axis that form bidirectional hepatocyte–neutrophil cross-talk to repair and regenerate the liver.

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Fig. 1: Loss of Lifr in hepatocytes impairs liver regeneration.
Fig. 2: Hepatocytic Lifr promotes neutrophil recruitment in liver regeneration models.
Fig. 3: Hepatocytic Lifr promotes HGF production through neutrophils.
Fig. 4: LIFR upregulates hepatocyte-derived cholesterol, which acts on neutrophils to boost HGF production.
Fig. 5: Cholesterol promotes HGF transcription through ERRα in neutrophils.
Fig. 6: LIFR accelerates partial hepatectomy-induced liver injury repair and regeneration in a neutrophil-dependent manner.
Fig. 7: Hepatocytic Lifr promotes neutrophil recruitment through Cxcl1.
Fig. 8: LIFR regulates CXCL1 and cholesterol levels through STAT3, and treatment with a STAT3 inhibitor reverses LIFR-accelerated liver regeneration.

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Source data are provided with this paper. All other data supporting the findings of this study are available within the article, extended data figures or Supplementary Information.

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Acknowledgements

We thank MD Anderson’s Flow Cytometry and Cellular Imaging Core, Metabolomics Core, Functional Genomics Core, Cytogenetics and Cell Authentication Core, and Advanced Technology Genome Core for technical assistance. We are grateful to all members of the laboratory of L.M. for the discussion and to C. F. Wogan (MD Anderson’s Division of Radiation Oncology) for the critical reading of the paper. L.M. is supported by US National Institutes of Health (NIH) grants R01CA166051 and R01CA269140, an American Cancer Society grant (award DBG-22-161-01-MM) and the Nylene Eckles Distinguished Professorship of MD Anderson Cancer Center. H.Z. is supported by the NIH (R01AA028791, R01DK125396), Cancer Prevention and Research Institute of Texas (CPRIT, RP220614), the Emerging Leader Award from the Mark Foundation for Cancer Research (award 21-003-ELA) and the Nancy B. and Jake L. Hamon Distinguished Chair of University of Texas Southwestern Medical Center. The core facilities are supported by MD Anderson’s Cancer Center Support Grant P30CA016672 from the NIH. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Author notes

  1. Fan Yao

    Present address: Hubei Hongshan Laboratory, College of Biomedicine and Health, Huazhong Agricultural University, Wuhan, China

  2. These authors contributed equally: Yalan Deng, Zilong Zhao.

Authors and Affiliations

  1. Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Yalan Deng, Marisela Sheldon, Yang Zhao, Hongqi Teng, Consuelo Martinez, Jie Zhang, Fan Yao & Li Ma

  2. Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Zilong Zhao, Chunru Lin & Yutong Sun

  3. Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Michael A. Curran

  4. The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA

    Michael A. Curran & Li Ma

  5. Children’s Research Institute, Departments of Pediatrics and Internal Medicine, Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Hao Zhu

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Contributions

Y.D., Y.S. and L.M. conceived and designed the study. Y.D. and Z.Z. performed most experiments and data analyses. M.S., Y.Z., H.T., C.M. and J.Z. performed some experiments. C.L. and F.Y. provided technical assistance and consultation. Y.S. generated some reagents and contributed protocols. M.A.C. and H.Z. reviewed the data and provided substantial intellectual input and guidance. Y.D. and L.M. wrote the paper with input from all other authors. L.M. provided scientific direction, established collaborations and allocated funding for this study.

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Correspondence to Li Ma.

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Competing interests

H.Z. consults for Flagship Pioneering, Alnylam Pharmaceuticals, Jumble Therapeutics and Chroma Medicines, and serves on the Scientific Advisory Board of Ubiquitix. H.Z. has research support from Chroma Medicines. H.Z. owns stock in Ionis and Madrigal Pharmaceuticals. M.A.C. reports grants, personal fees and an ownership interest in ImmunoGenesis and personal fees from AstraZeneca. The above interests are not directly related to the contents of this paper. The other authors declare no competing interests.

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Nature Metabolism thanks Udayan Apte, Paul Kubes and Jan Tchorz for their contribution to the peer review of this work. Primary Handling Editors: Revati Dewal and Isabella Samuelson, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Loss of Lifr in hepatocytes impairs liver injury repair and injury-induced upregulation of proliferative genes.

a. Immunoblotting of Lifr, cyclin D1, cyclin A2, and Gapdh in mouse livers at different time points after 2/3 partial hepatectomy (PHx). b. qPCR of mRNA of Lifr, cyclin D1, cyclin A2, cyclin B1, and cyclin E1 in the livers of Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. n = 6, 5, 6, and 6 mice. c. Immunoblotting of Lifr, cyclin D1, and Gapdh in the livers of Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. d, e. TUNEL staining (d) and the number of TUNEL-positive hepatocytes per high-power field (HPF; e) at 72 hours after CCl4 treatment. Scale bars, 50 μm. n = 4 mice. f. qPCR of mRNA of cyclin D1, cyclin A2, and Pcna in the livers of Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 48 and 72 hours after CCl4 treatment. n = 7, 7, 4, 4, 6, 6, 8, and 8 mice. g, h. Serum ALT (g) and AST (h) levels in Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 6 and 12 hours after CCl4 treatment. n = 8, 6, 6, 7, 6, and 8 mice. i. Immunoblotting of Cyp2e1 and Gapdh in mouse livers at 6 and 12 hours after CCl4 treatment. Representative results from one of three independent experiments are shown. Statistical significance in b and e-h was determined by a two-tailed unpaired t-test. Error bars are s.e.m.

Source data

Extended Data Fig. 2 Overexpression of LIFR promotes liver injury repair and regeneration.

a-i. C57BL/6J mice received control or LIFR-expressing adenovirus 5 days before CCl4 or vehicle treatment. Analyses were done at 48 hours after treatment. a. Experimental design. b, c. Serum ALT (b) and AST (c) levels in mice after CCl4 or vehicle treatment. n = 6 mice. d, e. H&E staining (d) and percentage of necrotic areas (e) in mouse livers after CCl4 or vehicle treatment. Scale bars, 500 μm. n = 6 mice. f, g. DAPI and TUNEL staining (f) and the number of TUNEL-positive hepatocytes per high-power field (HPF; g) in mouse livers after CCl4 or vehicle treatment. Scale bars, 100 μm. n = 6 mice. h, i. Immunofluorescence staining of Ki67 (h; overlay with DAPI staining) and percentage of Ki67-positive hepatocytes (i) in mouse livers after CCl4 or vehicle treatment. Scale bars, 50 μm. n = 3 mice. j. DAPI and TUNEL staining of mouse livers 10 days after injection of control or LIFR-expressing adenovirus. Scale bars, 100 μm. Representative results from one of three independent experiments are shown. k-m. C57BL/6J mice received control or LIFR-expressing adenovirus 10 days before CCl4 treatment. Analyses were done at 48 hours after treatment. k. Experimental design. l, m. Immunofluorescence staining of Ki67 (l; overlay with DAPI staining) and percentage of Ki67-positive hepatocytes (m) after CCl4 treatment. Scale bars, 50 μm. n = 6 mice. Statistical significance in b, c, e, g, i, and m was determined by a two-tailed unpaired t-test. Error bars are s.e.m.

Source data

Extended Data Fig. 3 LIFR deficiency or overexpression does not affect hepatocyte proliferation ex vivo or in vitro.

a-e. Primary hepatocytes isolated from Lifrfl/fl and Lifrfl/fl;Alb-Cre mice were cultured for 3 hours, followed by treatment with 100 ng/mL of Hgf and/or 20 ng/mL of Egf for 48 hours. a-c. qPCR of mRNA of Lifr (a), cyclin D1 (b), and Pcna (c) in Hgf- and/or Egf-treated hepatocytes. n = 4, 4, 5, 5, 5, 5, 4, and 4 biological replicates. d, e. Immunofluorescence staining of Ki67 (d; overlay with DAPI staining) and percentage of Ki67-positive cells (e) in Hgf- and/or Egf-treated hepatocytes. Scale bars, 100 μm. n = 4 biological replicates. f-j. Primary hepatocytes isolated from C57BL/6J mice 10 days after injection with control adenovirus or LIFR-expressing adenovirus were cultured for 3 hours, followed by treatment with 100 ng/mL of Hgf and/or 20 ng/mL of Egf for 48 hours. f-h. qPCR of mRNA of Lifr (f), cyclin D1 (g), and Pcna (h) in Hgf- and/or Egf-treated hepatocytes. n = 5, 5, 4, 4, 4, 4, 5, and 5 biological replicates. i, j. Immunofluorescence staining of Ki67 (i; overlay with DAPI staining) and percentage of Ki67-positive cells (j) in Hgf- and/or Egf-treated hepatocytes. Scale bars, 100 μm. n = 5 biological replicates. k-m. qPCR of mRNA of Lifr (k), cyclin D1 (l), and Pcna (m) in Hgf- and/or Egf-treated hepatocytes isolated from C57BL/6J mice. The cells were infected with control adenovirus or LIFR-expressing adenovirus for 24 hours before Hgf and/or Egf treatment. n = 5, 5, 4, 4, 4, 4, 5, and 5 biological replicates. Statistical significance in a-c, e-h, and j-m was determined by a two-tailed unpaired t-test. Error bars are s.e.m.

Source data

Extended Data Fig. 4 Effects of LIFR on neutrophil recruitment after liver injury.

a, b. Quantification of liver-infiltrating immune cell populations in Lifrfl/fl and Lifrfl/fl;Alb-Cre mice 72 hours after PHx (a; n = 5 and 4 mice) or CCl4 treatment (b; n = 4 mice). NK: natural killer cells. KC: Kupffer cells. MoMa: monocyte-derived macrophages. PMN: polymorphonuclear neutrophils. DC: dendritic cells. c. Immunohistochemical staining of neutrophil elastase (NE) in the livers of Lifrfl/fl mice at 72 hours after PHx. Scale bars, 200 μm (top left) and 50 μm (top right and bottom). Representative results from one of three independent experiments are shown. d. Number of CD45+ cells per gram of liver in Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. n = 4 and 5 mice. e-g. C57BL/6J mice received control or LIFR-expressing adenovirus 10 days before CCl4 treatment. Analyses were done at 48 hours after CCl4 treatment. e. Experimental design. f, g. Flow cytometry plots and percentage of neutrophils in liver (e) and blood (f) CD45+ cells from mice at 48 hours after CCl4 treatment. n = 6 and 5 mice. Statistical significance in a, b, d, f, and g was determined by a two-tailed unpaired t-test. Error bars are s.e.m.

Source data

Extended Data Fig. 5 The neutrophils from Lifrfl/fl;Alb-Cre mice have a lower ability to promote hepatocyte proliferation.

a. Immunofluorescence staining of neutrophil elastase (NE, green) and Hgf (red) on liver sections from Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. Scale bars, 100 μm. Representative results from one of three independent experiments are shown. b. Immunofluorescence staining of Ki67 (overlay with DAPI staining) and percentage of Ki67-positive cells in primary mouse hepatocytes cultured for 48 hours with the conditioned medium of liver-infiltrating neutrophils purified from Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. Scale bars, 100 μm. n = 5 biological replicates. c. Immunofluorescence staining of Ki67 (overlay with DAPI staining) and percentage of Ki67-positive cells in primary mouse hepatocytes cultured for 48 hours with the conditioned medium of blood neutrophils purified from Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. Scale bars, 100 μm. n = 5 biological replicates. d. Immunoblotting of p-Met, Met, p-Erk, Erk, and Gapdh in the livers of Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. Representative results from one of three independent experiments are shown. Statistical significance in b and c was determined by a two-tailed unpaired t-test. Error bars are s.e.m.

Source data

Extended Data Fig. 6 Knockdown of HGF in AdHL-60 cells.

a. Giemsa staining of HL-60 and all-trans retinoic acid (ATRA)-differentiated HL-60 (AdHL-60) cells. Scale bars, 50 μm. b. Flow cytometry plots of CD11b in HL-60 and AdHL-60 cells. c. Giemsa staining of control and HGF-knockdown HL-60 cells with or without ATRA-induced differentiation. Scale bars, 50 μm. d. Flow cytometry plots of CD11b in control and HGF-knockdown HL-60 cells with or without ATRA-induced differentiation. e. qPCR of HGF in control and HGF-knockdown AdHL-60 cells. n = 3 biological replicates. f. Flow cytometry plots and quantification of HGF in control and HGF-knockdown AdHL-60 cells. n = 3 biological replicates. Statistical significance in e and f was determined by a two-tailed unpaired t-test. Error bars are s.e.m.

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Extended Data Fig. 7 LIFR accelerates CCl4-induced liver injury repair and regeneration in a neutrophil-dependent manner.

a-n. C57BL/6J mice received control or LIFR-expressing adenovirus 10 days before CCl4 treatment. Six hours after CCl4 treatment, the mice were treated with control IgG or anti-Ly6G. Analyses were done at 48 hours after CCl4 treatment. a. Experimental design. b, c. Flow cytometry plots (b) and percentage (c) of neutrophils in liver CD45+ cells. n = 5 mice. d, e. Flow cytometry plots (d) and percentage (e) of neutrophils in blood CD45+ cells. n = 5 mice. f, g. Flow cytometry plots (f) and percentage (g) of neutrophils in bone marrow (BM) CD45+ cells. n = 5 mice. h, i. Serum ALT (h) and AST (i) levels in control and LIFR-expressing adenovirus-infected C57BL/6J mice injected with control IgG or anti-Ly6G after CCl4 treatment. n = 5 mice. j, k. H&E staining (j) and percentage of necrotic areas (k). Scale bars, 300 μm. n = 5 mice. l, m. TUNEL staining (l) and the number of TUNEL-positive hepatocytes per high-power field (HPF; m). Scale bars, 100 μm. n = 5 mice. n, o. Immunohistochemical staining of Ki67 (n) and percentage of Ki67-positive hepatocytes (o). Scale bars, 50 μm. n = 5 mice. Statistical significance in c, e, g, h, i, k, m, and o was determined by a two-tailed unpaired t-test. Error bars are s.e.m.

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Extended Data Fig. 8 Loss of hepatic Lifr impairs neutrophil recruitment, neutrophilic Hgf production, and liver regeneration in female mice.

a. Experimental design for panels b-i. All mice used were females. b. Liver-to-body weight ratio of Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. n = 6 mice. c. Immunofluorescence staining of Ki67 and percentage of Ki67-positive hepatocytes in the livers of Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. LPF: low-power field; HPF: high-power field. Scale bars, 100 μm (left) and 20 μm (right). n = 6 mice. d, e. Flow cytometry plots (d) and percentage (e) of neutrophils in liver, blood, and bone marrow (BM) CD45+ cells from Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. n = 5 mice. f. Serum cholesterol levels in Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. n = 5 mice. g. Serum Hgf levels in Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. n = 6 mice. h. Flow cytometry plots and quantification of Hgf in liver neutrophils from Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. n = 5 mice. i. Flow cytometry plots and quantification of Hgf in blood neutrophils from Lifrfl/fl and Lifrfl/fl;Alb-Cre mice at 72 hours after PHx. n = 5 mice. Statistical significance in b, c, and e-i was determined by a two-tailed unpaired t-test. Error bars are s.e.m.

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Extended Data Fig. 9 Cxcl1 facilitates the recruitment of neutrophils to the liver after hepatectomy.

a. Immunofluorescence staining of neutrophil elastase (NE, red), Cxcr2 (green), and Hgf (cyan) in blood neutrophils from wild-type mice at 72 hours after PHx. Scale bars, 5 μm. Representative results from one of three independent experiments are shown. b, c. Schematic of the experimental design (b): at 40 hours after PHx, CD45.2 mice were treated with isotype IgG or anti-Cxcl1. At 62 hours after PHx, CD45.1 neutrophils were adoptively transferred to the antibody-treated mice. The liver infiltration of CD45.1 neutrophils was analyzed at 4 hours after adoptive transfer (c). n = 6 mice. Statistical significance in c was determined by a two-tailed unpaired t-test. Error bars are s.e.m.

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Extended Data Fig. 10 Model for LIFR-mediated regulation of neutrophil recruitment and cholesterol-driven hepatocyte-neutrophil crosstalk during liver injury repair and regeneration.

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Supplementary Table 1

Normalized z-scores of lipids in plasma samples collected from control and Lifr conditional knockout mice at 72 h after partial hepatectomy.

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Deng, Y., Zhao, Z., Sheldon, M. et al. LIFR regulates cholesterol-driven bidirectional hepatocyte–neutrophil cross-talk to promote liver regeneration. Nat Metab 6, 1756–1774 (2024). https://doi.org/10.1038/s42255-024-01110-y

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