Multigenerational epigenetic adaptation of the hepatic wound-healing response

Abstract

We investigated whether ancestral liver damage leads to heritable reprogramming of hepatic wound healing in male rats. We found that a history of liver damage corresponds with transmission of an epigenetic suppressive adaptation of the fibrogenic component of wound healing to the male F1 and F2 generations. Underlying this adaptation was less generation of liver myofibroblasts, higher hepatic expression of the antifibrogenic factor peroxisome proliferator-activated receptor γ (PPAR-γ) and lower expression of the profibrogenic factor transforming growth factor β1 (TGF-β1) compared to rats without this adaptation. Remodeling of DNA methylation and histone acetylation underpinned these alterations in gene expression. Sperm from rats with liver fibrosis were enriched for the histone variant H2A.Z and trimethylation of histone H3 at Lys27 (H3K27me3) at PPAR-γ chromatin. These modifications to the sperm chromatin were transmittable by adaptive serum transfer from fibrotic rats to naive rats and similar modifications were induced in mesenchymal stem cells exposed to conditioned media from cultured rat or human myofibroblasts. Thus, it is probable that a myofibroblast-secreted soluble factor stimulates heritable epigenetic signatures in sperm so that the resulting offspring better adapt to future fibrogenic hepatic insults. Adding possible relevance to humans, we found that people with mild liver fibrosis have hypomethylation of the PPARG promoter compared to others with severe fibrosis.

Figure 1. Presence of liver injury in male ancestors reduces liver fibrogenesis in F₂ male offspring

a) Adult outbred male rats were divided into vehicle control or a chronically injured liver group. Five rats from each group were harvested at peak fibrosis while another 5 from each group were bred following complete recovery from liver injury. This procedure was repeated with F₁ generated males to produce F₂ males which were then divided in groups A (no injury in F₀ or F₁), B (Injured F₁, uninjured F₀), C (injured F₀, uninjured F₁) and D (injury in both F₀ and F₁). Groups A-D were chronically injured and tissues collected at peak fibrosis (control uninjured group also included). b) Representative images of Sirius Red (collagen deposition) stained peak fibrosis livers from groups A to D. Panel in each row shows three separate animals. Scale bar, 100μm. Right hand side panel- representative H&E stained livers showing comparable level of liver injury in all groups. c) Percentage area collagen I and d) mRNA level of collagen I expressed as relative level of transcription difference (RLTD) in peak fibrosis livers of groups A to D rats. Error bars are means ± s.e.m (n=5). Statistical analysis; one way parametric analysis of variance (ANOVA), Tukey-Kramer post test. (*) p<0.05, (***) p<0.001.

Figure 2. Paternally transmitted adaptation from fibrogenic response in chronic injury is mediated via a decrease in αSMA positive myofibroblasts in the liver

a) Representative images αSMA stained peak fibrosis livers from groups A to D. Panel in each row shows three separate animals Scale bar, 100μm. b) Average number of αSMA positive cells and c) mRNA level of αSMA expressed as relative level of transcription difference (RLTD) in peak fibrosis livers of groups A to D rats. Error bars are means ± s.e.m (n=5). Statistical analysis; one way parametric analysis of variance (ANOVA), Tukey-Kramer post test. (*) p<0.05, (**) p<0.01 and (***) p<0.001. d–e) Representative images of desmin stained olive oil control (d) and peak fibrosis (e) livers from groups A to D. Panel in each row shows two separate animals Scale bar, 50μm. f) Average number of desmin positive cells in control or injured groups A and D. Statistical analysis; two-tailed Student’s t-test, (**) p<0.01. g) mRNA level of TNFα expressed as relative level of transcription difference (RLTD) in peak fibrosis livers of groups A to D rats. Error bars are means ± s.e.m (n=5).

Figure 3. Protected animals have altered expression of profibrogenic and antifibrogenic genes in their livers

a) mRNA level of PPAR-γ, PPAR-α and TGF-β1 expressed as relative level of transcription difference (RLTD) in peak fibrosis livers of groups A to D rats. Error bars are means ± s.e.m (n=5). Statistical analysis- one way parametric ANOVA, Tukey-Kramer post test for A to D groups where (*) indicates p<0.05. b) Western blot detection of PPAR-γ and β actin in group A and D peak fibrosis livers c) mRNA level of Bambi and SMADs 1 to 7 expressed as relative level of transcription difference (RLTD) in peak fibrosis livers of groups A to D rats. Error bars are means ± s.e.m (n=5). Statistical analysis; Student’s t-test, (***) indicates p<0.001.

Figure 4. Ancestral injury influenced fibrogenic response is limited to liver

a) Adult outbred male rats were divided into vehicle control or a chronically injured liver group. Five rats from each group were bred following complete recovery from liver injury. Males produced from the breeding cycle (F1) were subjected to unilateral ureter obstruction (UUO) and fibrotic and control kidneys harvested 7 days after the surgery (n=10). b) and c) Sirius red (b) and αSMA (c) staining in contralateral uninjured or obstructed kidneys in control and ancestral liver injury groups of rats. d) and e) Percentage area Sirius Red (d) and αSMA (e) positive in contralateral uninjured or obstructed kidneys in control and ancestral liver injury groups. Error bars are means ± s.e.m (n=7). Statistical analysis; Student’s t-test, where (*) in d) indicates p=0.041, (**) p=0.028 and in e) (***) indicates p<0.001.

Figure 5. Altered expression of profibrogenic and antifibrogenic genes in the livers of protected animals is underpinned by differences in DNA methylation

a) DNA methylation at particular CG dinucleotides within PPAR-γ promoter in peak fibrosis livers from A to D groups of rats determined by pyrosequencing. Position of the differentially methylated CGs is shown in the schematic drawing above the graphs. Differences are expressed as percentage DNA methylation (n=5). Statistical analysis; one way parametric ANOVA, Tukey-Kramer post test. (*) p<0.05, (**) p<0.01 and (***) p<0.001. b) and c) Pyrosequencing as in a) determined DNA methyation in PPAR-α promoter (b) TGF-β1 promoter and intron 1 (c). Position of the differentially methylated CGs is shown in the schematic drawing above the graphs. Differences are expressed as percentage DNA methylation (n=5). Statistical analysis; one way parametric ANOVA, Tukey-Kramer post test. (*) p<0.05 and (***) p<0.001 for PPAR-α and two tailed Student’s t-test, (**) indicates p<0.01, for TGF-β1. d) ChIP analysis of acetylated H3 enrichment at the PPAR-γ gene promoter (left panel) and TGF-β1 promoter (right panel) in peak fibrosis livers of groups A to D rats. Results were expressed as fold control IgG (n=5). Statistical analysis; Student’s t-test, p=0.0159 (*) and p=0.0278 (*) for PPAR-γ and TGF-β1 respectively.

Figure 6. Extrahepatic transmission of epigenetic modifications and evidence for modifications in DNA methylation at fibrogenic regulator gene associated with liver disease progression in humans

a–b) ChIP analysis of trimethylated H3 lysine 27 (H3K27me3) and histone variant H2A.Z enrichment at the rat PPAR-γ gene promoter in mature sperm collected from male adult rats that were given chronic CCl₄ or olive oil (control) for 4 weeks, then recovered for 2 weeks (n=5). All ChIP results in a) to e) are expressed as fold control isotype matched antibody. b) ChIP analysis as in a) was carried out on mature sperm isolated from male adult rats that underwent bile duct ligation (BDL) or sham operation (control) 15 days previously. c) ChIP analysis as in a) was carried out on mature sperm isolated from rats that received twice weekly intravenous serum transfers (for four weeks total) from control or rats that were given CCl₄ for 4 weeks and serum collected 48hrs following last injection (n=6) d) ChIP analysis as in a) was carried out on primary rat mesenchymal stem cells which were treated with control or 48hrs conditioned activated HSC media for 72hrs (n=3). e) ChIP analysis as in a) was carried out on human PPAR-γ gene promoter in primary human mesenchymal stem cells which were treated with quiescent (day 1) or activated HSC (day 15) conditioned media for 72hrs (n=3). f) DNA methylation at particular CG dinucleotides within human PPAR-γ promoter in NAFLD patients liver biopsy tissues was determined by pyrosequencing. Position of the differentially methylated CGs is shown in the schematic drawing above the graphs. Differences are expressed as percentage DNA methylation Statistical analysis; Mann Whitney test, where p=0.0013 for CpG1 and p=0.0047 for CpG2.