Research ArticleLiver disease

TGFβ inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence

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Science Translational Medicine  15 Aug 2018:
Vol. 10, Issue 454, eaan1230
DOI: 10.1126/scitranslmed.aan1230
  • Fig. 1 Human liver necrosis causes acute hepatocellular senescence.

    (A) Representative images of sections of explanted human liver after liver transplantation for severe acetaminophen overdose (n = 8) compared to control healthy human liver. Explanted livers injured by acetaminophen overdose show expression of the senescence marker p21 detected by immunohistochemistry in residual hepatocytes surrounding areas of necrosis. Necrosis interface, dashed white line; CV, central vein; black asterisk indicates area of necrosis. As a control, human liver with normal histology was used (n = 50). Scale bars, 50 μm. (B) A case series (n = 74) of patients with submassive liver necrosis divided into subgroups according to the extent of hepatocellular necrosis is presented. <25%, n = 8; 25 to 50%, n = 16; 50 to 75%, n = 22, >75%, n = 28. The extent of hepatocellular submassive necrosis (defined histologically by globalized confluent necrosis) was quantified by immunohistochemistry for the hepatocellular senescence marker p16 and the proliferation marker Ki67. *P < 0.05, one-way analysis of variance (ANOVA). Mean ± SEM.

  • Fig. 2 Toxin-mediated liver injury causes p21-dependent hepatocellular senescence in mice.

    (A and B) In murine toxin–induced acute liver injury models, mice were treated with either CCl4 (A) or acetaminophen (B). Treatment with these toxins resulted in pericentral necrosis 2 days after administration as shown by immunohistochemistry for expression of the senescence marker p21 (green); expression of the proliferation marker BrdU (magenta) and the hepatocyte marker hepatocyte nuclear factor 4 α (magenta) is also shown. (C) Immunohistochemistry for expression of the proliferation marker Ki67 is shown 2 days after acetaminophen treatment. Staining indicates hepatocyte proliferation away from but not next to the area of necrosis; red arrows indicate proliferating hepatocytes. (D) Quantification of p21+ hepatocytes after injury; n ≥ 3 for each time point, P < 0.0001 versus time 0, two-way ANOVA. (E) Gene set enrichment analysis (GSEA) plot showing enrichment of the early (24 hours) acetaminophen injury gene expression signature in liver compared to an oncogene-induced senescence (OIS) signature. Gene set: IMR90 ER:RAS OIS cell model (15). Enrichment score is 0.2564; normalized enrichment score is 2.466; nominal P < 0.001. (F) Perinecrotic hepatocytes (brown nuclei) were quantified for p21 expression 2 days after acetaminophen treatment; 74.9% of total perinecrotic hepatocytes expressed p21 (n = 8 mice). (G) Immunohistochemistry for expression of the proliferation marker Ki67 in p21-deficient (p21KO) mice 2 days after acetaminophen-induced liver injury. Ki67 expression indicates proliferating hepatocytes in the perinecrotic area of the injured mouse liver. (H) Quantification of perinecrotic hepatocytes shown in (G). (I) The number of Ki67+ hepatocytes in relation to serum alanine transaminase (ALT; units per liter), a marker of liver injury (n = 5 versus 8 mice; 20 high-power fields were quantified per liver). P = 0.0074, two-tailed t test. Linear regression for wild-type (WT) and p21KO mice, R2 = 0.54 and 0.92, with slope 95% confidence intervals of −0.10 to −0.0045 and 0.082 to 0.28 and probability slope ≠ 0, P = 0.037 and 0.010, respectively. Scale bars, 50 μm. CV, central vein. Dashed white lines, necrosis boundary; asterisk, area of necrosis.

  • Fig. 3 Non–cell-autonomous senescence in hepatocyte-specific mouse senescence models.

    (A) Plots of GSEA normalized enrichment scores comparing gene sets over time observed in the acetaminophen-treated mouse model to the unbiased top 15 ranked hallmark gene sets and the OIS signature from the IMR90 ER:RAS cell model (15). Black borders of data points highlight P < 0.05; raw data are shown in tables S2 and S3. Top and bottom panels show inflammatory and cell cycle arrest gene expression signatures. (B) Diagram showing the use of genetic induction of transgenes in hepatocytes to induce cell-autonomous senescence and assessment of senescence using a combination of markers—p53, p21, and p16. Presence of senescence markers, p21 or p16, in the absence of markers of genetic recombination, p53 or Tomato reporter (Tom), identifies non–cell-autonomous senescence. (C) p53 accumulates in a subpopulation of hepatocytes in the partial ΔMdm2 Hep mouse model where βNF (20 mg/kg) is given to AhCre+ Mdm2f l/f l mice. Immunohistochemical staining for p21/p53 and for p53/p16INK4As was assessed by confocal microscopy. (D) Immunohistochemical staining and confocal microscopic analysis of mouse liver sections for p53 and p21 after deletion of Mdm2 using AAV8-TBG-Cre [2.5 × 1011 genetic copies (GC) per mouse]. (E) Immunohistochemical staining and confocal microscopic analysis of mouse liver sections for p21 expression and GFP staining in a hepatocyte transplant mouse model 94 days after transplantation of GFP-tagged hepatocyte progenitor cells. AhCre+ Mdm2fl/fl mouse recipients were given wild-type (WT) donor cells tagged with GFP and iterative doses of βNF to induce hepatocyte recombination of Mdm2. Dashed white line, border of the engrafted cells. The magnified area is shown in individual color images on the right. (F) Immunohistochemical staining and confocal microscopic analysis of mouse liver sections for p21 expression after hepatocellular TGFβR1 activation by AAV8-TBG-Cre in LSL-TGFβR1-CA mice. (G) Immunohistochemical staining and confocal microscopic analysis of mouse liver sections for p21 expression and red fluorescent protein (RFP) staining to detect tdTomato reporter after reduced dosing of the AAV8-TBG-Cre vector (6.4 × 108 GC per animal) in LSL-TGFβR1-CA R26-LSL-tdTomato mice. (H) After partial ΔMdm2Hep, mice were given the TGFβR1 inhibitor SB525334 or vehicle control. Immunohistochemical staining and confocal microscopic analysis of mouse liver sections for p53 and p21 with quantification of non–cell-autonomous p21 expression; P = 0.0023, two-tailed Mann-Whitney test; n = 6 vehicle control versus n = 7 for SB525334-treated mice. Mean ± SEM. Scale bars, 50 μm. Open arrow, cell-autonomous senescence; closed arrow, non–cell-autonomous senescence; arrowhead, unaffected. TNFα, tumor necrosis factor–α; NFκB, nuclear factor κB; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3.

  • Fig. 4 TGFβ signaling is activated in acetaminophen-induced hepatocellular senescence.

    (A) Representative images showing immunohistochemistry for expression of p21 and pSMAD2/3 in healthy human liver and in liver from patients with fulminant hepatic failure secondary to acetaminophen overdose. White arrows indicate senescent hepatocytes. (B) Representative images showing in situ hybridization for TGFβ1 in the livers of acetaminophen-treated (350 mg/kg) and untreated C57BL/6J mice. TGFβ1 ligand is expressed by nonparenchymal cells with a monocyte-like appearance. CV, central vein. Black asterisk indicates area of necrosis. (C) Enzyme-linked immunosorbent assay (ELISA) of mouse liver TGFβ1 for untreated mice and acetaminophen-treated mice 12 hours after exposure. (n = 6 versus 7, respectively). Mean ± SEM. P = 0.0047, two-tailed Mann-Whitney test. (D) Quantification by in situ hybridization of SMAD7 expression in the perinecrotic region of mouse liver 2 days after acetaminophen treatment. P = 0.0286, compared to equivalent area in uninjured mouse liver, one-tailed Mann-Whitney test. (E) Mouse liver serial sections assessed for expression of SMAD7, TGFβR1, and TGFβ1 ligand by in situ hybridization and for p21 expression by immunohistochemistry 12 hours after acetaminophen treatment. Scale bars, 50 μm.

  • Fig. 5 Macrophage recruitment and TGFβ1 production drive hepatocellular senescence and impair hepatocellular regeneration in mice.

    (A) A representative mouse liver section assessed for hepatic TGFβ1 ligand production (red) and F4/80+ macrophages (pale blue) by in situ hybridization and F4/80 immunohistochemistry, respectively, 2 days after acetaminophen (350 mg/kg) treatment. CV, central vein. (B) In situ hybridization staining for expression of the CCL2 chemokine. Dashed white line, necrotic interface; black asterisk, area of necrosis. (C) Immunohistochemical staining for F4/80+ macrophages (green) and p21+ hepatocytes (magenta). Scale bars, 50 μm. (D) Quantification of peripheral monocytes in mice after acetaminophen treatment versus fasted untreated mice as baseline (dashed black line). n = 5 mice for each time point. P = 0.0001, one-way ANOVA with Dunnett’s multiple comparison baseline versus day 1. (E) Quantification of immunohistochemical staining for p53 and p21 expression or for BrdU in mouse livers 4 days after partial deletion of Mdm2 (partial ΔMdm2Hep), where βNF (20 mg/kg) is given to AhCre+ Mdm2fl/fl mice, followed by twice daily antibody-mediated CCL2 inhibition (with isotype antibody as the control). Non–cell-autonomous hepatocyte p21 expression (without p53 expression) and proliferation (BrdU) were quantified. P = 0.05, Mann-Whitney (n = 3 mice per group). (F) Liposomal clodronate depletion of macrophages 3 days after partial ΔMdm2Hep compared to phosphate-buffered saline (PBS) control. TGFβ and p21 expression in whole mouse liver were quantified by quantitative reverse transcription polymerase chain reaction (PCR). P = 0.000063, 0.237, and 0.126 for TGFβ1, TGFβ2, and TGFβ3, respectively, and P = 0.025 for p21, t test (n = 4 mice per group). Non–cell-autonomous p21+ hepatocytes were quantified after immunohistochemical staining for p53 and p21. P = 0.035, t test (n = 4 mice per group). (G) Acetaminophen (350 mg/kg) was administered to LysMCre+ TGFβfl/fl or LysMCreWT TGFβfl/fl mouse littermates. Hepatocyte proliferation was assessed by BrdU immunohistochemistry. P = 0.0006, two-tailed t test (n = 10 versus 8 mice). Mean ± SEM.

  • Fig. 6 Inhibition of TGFβR1 signaling reduces hepatocellular senescence and restores a proportional regenerative response after acetaminophen treatment in mice.

    (A) Cohorts of male C57BL/6J mice were given vehicle control or were treated with the TGFβR1 inhibitor AZ12601011, starting when acetaminophen (525 mg/kg) was administered. Mice were closely monitored throughout the experiment until death or the humane end point was reached, typically between 16 and 18 hours. Initially, the mice treated with the TGFβR1 inhibitor (n = 14) were sacrificed when the control animals reached the end point irrespective of clinical condition (total biological replicates, n = 14 with AZ12601011 and n = 16 with vehicle control; performed over three separate experiments). (B) Separate survival cohorts (n = 5 in each of two experiments) treated with the TGFβR1 inhibitor were compared to simultaneous vehicle controls to examine longer-term survival; P < 0.0001, Gehan-Breslow-Wilcoxon test. (C) At matched end point, the TGFβR1 inhibitor and vehicle control groups were compared for serum bilirubin. P = 0.0162, two-tailed Mann-Whitney test. (D) In an experiment examining delayed TGFβR1 inhibition commencing 12 hours after acetaminophen treatment in male C57BL/6J mice, the TGFβR1 inhibitor SB525334, or vehicle, was given twice daily. (E) Serum bilirubin over time from (D); P > 0.05 and P < 0.01 at days 2 and 4 for SB525334 treatment compared to vehicle control, respectively; two-way ANOVA with Bonferroni correction (n = 8 mice each group). (F) Immunohistochemical staining for hepatocellular p21 expression was quantified; P = 0.049, t test, 30 high-power fields in mouse liver sections were analyzed (n = 8 mice per group). (G) Immunohistochemical staining for BrdU (representative images for mouse liver sections 2 days after acetaminophen treatment and administration of either SB525334 or vehicle control). Effect of treatment upon BrdU+ hepatocytes was quantified in both whole liver (days 2 and 4) and perinecrotic hepatocytes (day 2 only). P = 0.0075 and 0.30 for total BrdU+ hepatocytes at days 2 and 4, respectively, and P < 0.0001 for BrdU+ perinecrotic hepatocytes comparing SB525334 treatment to vehicle control, t test (n = 8 per group, except day 2 vehicle control where n = 6 per group). Scale bars, 50 μm. (H) In individual mice, 2 days after acetaminophen treatment, hepatocytes were analyzed for serum ALT and BrdU staining, and linear regression was performed. R2 = 0.15 and 0.71, with slope 95% confidence intervals of −0.0094 to 0.0038 and 0.0049 to 0.085 and probability slope ≠ 0, P = 0.34 and 0.036, respectively. (I) A nonfatal dose of acetaminophen (450 mg/kg) was administered to male C57BL/6J mice, followed by treatment with AZ12601011 or vehicle control 12 hours later. Serum bilirubin was measured, and p21 expression in hepatocytes was quantified by immunohistochemistry. P = 0.0029 and 0.0017, respectively, comparing AZ12601011 treatment to vehicle control, two-tailed t test, n = 9 per group. Data presented as mean ± SEM.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/454/eaan1230/DC1

    Fig. S1. Senescence markers in human acute liver disease.

    Fig. S2. Senescence in the acute CCl4 model.

    Fig. S3. Senescence in the acute acetaminophen model.

    Fig. S4. Senescence in acute dietary models of liver injury.

    Fig. S5. Hepatocyte Mdm2 deletion model.

    Fig. S6. Non–cell-autonomous senescence marker induction.

    Fig. S7. Hepatocyte TGFβ pathway activation model.

    Fig. S8. Hepatocyte TGFβ pathway promotes hepatic TGFβ ligand production.

    Fig. S9. TGFβ pathway activity in the acetaminophen model.

    Fig. S10. Serial sections of the TGFβ pathway and senescent hepatocytes.

    Fig. S11. Macrophage recruitment, TGFβ secretion, and induced senescence.

    Fig. S12. TGFβR1 inhibition in acute and chronic CCl4 models.

    Fig. S13. Genetic deletion of hepatocyte TGFβR1 in the acetaminophen model.

    Fig. S14. Therapeutic TGFβR1 inhibition in the acetaminophen model.

    Fig. S15. Schematic representation of paracrine-induced senescence in acute liver injury.

    Fig. S16. Ductular reaction responses in murine models and human disease.

    Table S1. Source data for Fig. 2.

    Table S2. RNA-seq gene: Hallmarks.

    Table S3. RNA-seq GSEA: Selected ranked hallmarks and OIS signature.

    Table S4. Source data for Fig. 3.

    Table S5. Source data for Fig. 4.

    Table S6. Source data for Fig. 5.

    Table S7. Source data for Fig. 6.

  • The PDF file includes:

    • Fig. S1. Senescence markers in human acute liver disease.
    • Fig. S2. Senescence in the acute CCl4 model.
    • Fig. S3. Senescence in the acute acetaminophen model.
    • Fig. S4. Senescence in acute dietary models of liver injury.
    • Fig. S5. Hepatocyte Mdm2 deletion model.
    • Fig. S6. Non–cell-autonomous senescence marker induction.
    • Fig. S7. Hepatocyte TGFβ pathway activation model.
    • Fig. S8. Hepatocyte TGFβ pathway promotes hepatic TGFβ ligand production.
    • Fig. S9. TGFβ pathway activity in the acetaminophen model.
    • Fig. S10. Serial sections of the TGFβ pathway and senescent hepatocytes.
    • Fig. S11. Macrophage recruitment, TGFβ secretion, and induced senescence.
    • Fig. S12. TGFβR1 inhibition in acute and chronic CCl4 models.
    • Fig. S13. Genetic deletion of hepatocyte TGFβR1 in the acetaminophen model.
    • Fig. S14. Therapeutic TGFβR1 inhibition in the acetaminophen model.
    • Fig. S15. Schematic representation of paracrine-induced senescence in acute liver injury.
    • Fig. S16. Ductular reaction responses in murine models and human disease.
    • Table S2. RNA-seq gene: Hallmarks.
    • Table S3. RNA-seq GSEA: Selected ranked hallmarks and OIS signature.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Source data for Fig. 2.
    • Table S4 (Microsoft Excel format). Source data for Fig. 3.
    • Table S5 (Microsoft Excel format). Source data for Fig. 4.
    • Table S6 (Microsoft Excel format). Source data for Fig. 5.
    • Table S7 (Microsoft Excel format). Source data for Fig. 6.

    [Download Tables S1, S4 to S7]

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