Bone morphogenetic protein 8B promotes the progression of non-alcoholic steatohepatitis

Abstract

Non-alcoholic steatohepatitis (NASH) is characterized by lipotoxicity, inflammation and fibrosis, ultimately leading to end-stage liver disease. The molecular mechanisms promoting NASH are poorly understood, and treatment options are limited. Here, we demonstrate that hepatic expression of bone morphogenetic protein 8B (BMP8B), a member of the transforming growth factor beta (TGFβ)–BMP superfamily, increases proportionally to disease stage in people and animal models with NASH. BMP8B signals via both SMAD2/3 and SMAD1/5/9 branches of the TGFβ–BMP pathway in hepatic stellate cells (HSCs), promoting their proinflammatory phenotype. In vivo, the absence of BMP8B prevents HSC activation, reduces inflammation and affects the wound-healing responses, thereby limiting NASH progression. Evidence is featured in primary human 3D microtissues modelling NASH, when challenged with recombinant BMP8. Our data show that BMP8B is a major contributor to NASH progression. Owing to the near absence of BMP8B in healthy livers, inhibition of BMP8B may represent a promising new therapeutic avenue for NASH treatment.

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Fig. 1: BMP8B is overexpressed in human NASH according to disease stage, and is expressed in both hepatocytes and HSCs.
Fig. 2: BMP8B activates both SMAD2/3 and SMAD1/5/9 signalling pathways in BMP8B-KO HSC.
Fig. 3: Genetic ablation of BMP8B attenuates HSC trans-activation in vitro.
Fig. 4: Inflammation, stellate cell activation and hepatocyte proliferation are attenuated in BMP8B-KO livers injured with CCl4.
Fig. 5: Absence of BMP8B leads to defective liver regeneration in the PHx model.
Fig. 6: BMP8 stimulates TGFβ–BMP signalling in a human 3D in vitro NASH model promoting inflammation and proliferation.
Fig. 7: Attenuated inflammation and fibrosis in BMP8B-KO mice challenged with WD to model NASH/F1.
Fig. 8: NGS analysis confirms on a large scale that absence of BMP8B impacts proinflammatory, profibrotic and proliferative pathways in CCl4, PHx and WD models.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request. In vivo NGS data in this manuscript have been deposited in GEO, accession number GSE110404.

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Acknowledgements

The authors are indebted to the following colleagues and institutions: MRC Metabolic Diseases Unit (MC_UU_00014/5): M. Dale, M. Campbell, R. Dias, the Disease Model Core (DMC; H. Webber, D. Hart, S. Grocott, C. Beresford, D. Jessop, E. Rasijeff and A. Warner), the Biochemistry Assay Lab (K. Burling and collaborators), the Genomics and Transcriptomics Core (M. Ma), the Histology Core (J. Warner) and the Imaging Core (G. Strachan). MRC Human Nutrition Research and Department of Biochemistry, University of Cambridge: F. Sanders, Z. Hall and J. West. Histopathology/ISH core facility of Cancer Research UK—Cambridge Institute: J. Jones for assistance with in situ hybridization. The human NASH histological samples come from the Human Research Tissue Bank of the Cambridge University Hospitals, which is supported by the NIHR Cambridge Biomedical Research Centre. M.V., J.L.G. and A.V.P. are supported by MRC programs (MRC MDU Programme Grant. PO 4050281695 ‘Lipotoxicity and the Metabolic Syndrome’ and MRC DMC MC UU 12012/2 to A.V.P.; Lipid Profiling and Signalling, MC UP A90 1006 to J.L.G.) and MRC adjunct funding as part of the Cambridge Initiative in Metabolic Diseases (Lipid Dynamics and Regulation: MC_PC_13030). M.V., M.A. and A.V.P. are also supported by the Cambridge NIHR Biomedical Research Center (Gastroenterology); M.V. is a recipient of the BRC Gastroenterology Pump-Priming award 2018/2019 that funded part of this study. F.O. is supported by MRC program Grants MR/K0019494/1 and MR/R023026/1. J.L. is supported by Medical Research Council PhD studentship and a CRUK program grant (C18342/A23390). Q.M.A., M.V., A.V.P., V.R., M.A. and D.T. are contributing members of the European NAFLD Registry. Q.M.A. is supported by the Newcastle NIHR Biomedical Research Centre (BRC). M.V. has been fellow of the Fondazione Umberto Veronesi in 2014. M.A., A.V.P. and J.L.G. received funding from the Evelyn Trust. M.V., O.G., D.T., M.A., F.O., Q.M.A., M.J.N., D.J.L. and A.V.P. are members of the EPoS (Elucidating Pathways of Steatohepatitis) consortium, which is funded by the Horizon 2020 Framework Program of the European Union under Grant Agreement 634413.

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Authors

Contributions

M.V. and A.V.P. conceived and designed the study and wrote the manuscript. M.V., S.V. and V.P. designed and performed the WD experiment. J.L., F.O., M.V. and S.V. designed and performed the PHx and CCl4 experiments. M.V., S.S., T.K., Z.T. and K.P. performed the in vitro experiments. O.G., Q.M.A., V.R., M.E.D.A. and S.D. contributed with human data and samples. D.T. and S.D. scored the liver histology. Z.T. and W.L. contributed to the design of some of the in vitro experiments. M.J.N. and D.J.L. performed the collagen-3/MMP9 assays. Z.A. and J.L.G. performed liver lipidomics. B.Y.H.L. and M.V. analysed the NGS sequencing data. M.V. performed most of the analyses in all the models. All the authors provided useful criticism during the study, and critically reviewed the manuscript.

Corresponding authors

Correspondence to Michele Vacca or Antonio Vidal-Puig.

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Competing interests

At the time of this study, T.K. and S.S. were employees of CN Bio Innovations; M.J.N. and D.J.L. of Nordic Bioscience and are among original inventors and patent holders for the PRO-C3, C3M and MMP9 assays. F.O. is a director of Fibrofind. J.L. and F.O. are shareholders in Fibrofind. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 In liver disease, BMP8b is overexpressed in Albumin and αSma positive cells.

a, Relative mRNA expression levels of Bmp8b measured by quantitative real-time polymerase chain reaction (RTqPCR) in murine livers. The levels of BMP8b mRNA transcript increase in Western Diet (WD)-induced NASH [(CTRL) n: 8; (WD) n:8], but not in High Fat Diet (HFD)-induced NAFL [(CTRL) n:5; (HFD) n:6], and in acute [3 days; (CTRL) n: 6; (CCl4) n:4] (3 days; n:4-5/group) and chronic [8 weeks; (CTRL) n: 5; (CCl4) n:5] carbon tetrachloride (CCl4) -induced liver damage. All the results are shown as mean ± s.e.m.; expression data of biological replicates represented as dot plots; statistical significance (vs. control treatment) assessed by two-sided Student’s t-test. b-c, Representative IF of Bmp8b and Albumin (Alb) or αSma protein expression: in Western Diet (WD)-induced NASH and following acute (3 days) CCl4 challenge Bmp8b is expressed in the liver, and co-localizes with both αSma and Albumin thus suggesting that Bmp8b is expressed in hepatocytes and activated HSC following hepatic damage (2 replicates / condition; staining repeated twice).

Extended Data Fig. 2 Bmp8b is overexpressed by primary hepatocytes when cultured in vitro.

a, Relative mRNA gene expression levels of Bmp8b measured by quantitative real-time polymerase chain reaction (RTqPCR) in PH cultured at low (20,000 cells/cm2) or high (100,000 cells/cm2) confluence to model a highly proliferative (b; Cyclin E1, Ccne1) or differentiated (c; Albumin, Alb) behavior, respectively. Bmp8b mRNA expression levels are very low at baseline, and induced from 24h after culturing. All the results are shown as mean ± s.e.m. Statistical significance was assessed by Multivariate Analysis of Variance (MANOVA; 5 replicates/group). d, Microarray data from publicly available database GSE122660 of human primary hepatocytes (PH) and human hepatocytes cell lines (HepG2 and Huh7) cultured in 2D and challenged for 72h with DMSO or a mixture of oleic (OA) and palmitic (PA) fatty acids (with/without TNFα) suggest that also human hepatocytes express BMP8B and that the challenges with fatty acids and/or pro-inflammatory factors do not influence BMP8B expression. Expression data were retrieved using the tool Geo2R from NCBI, and statistical significance was assessed using One-Way Analysis of Variance (ANOVA; 3 replicates/group).

Extended Data Fig. 3 Bmp8b is overexpressed by Primary Hepatic Stellate Cells when cultured in vitro.

Relative mRNA gene expression levels measured by RNA sequencing (a-c) or quantitative real-time polymerase chain reaction (RTqPCR; d) in murine primary hepatic stellate cells cultured at a density of 35,000 cells/cm2 and harvested before (day 0) and after culture (days 1, 4, 8, 12). Average mRNA abundance (Log2CPM) of BMP/TGFβ receptors and effectors (a), and of TGFβ/BMP family members (b): Artn, Bmp 2/8a, and Inhb b/c/e were suppressed; Bmp3/5/7/9/10 and Gdf9/10 were suppressed after a transient upregulation at day 1 of culture; Tgfβ2/3, Gdf6 and Inhba were upregulated (Nodal and Gdf3 were not expressed by HSC). c, IPA “upstream regulator” analysis based on time-dependent GE changes (NGS) in HSC at different stages of the trans-activation program compared to Day 1 (D1) of culturing shows activation of multiple TGFβ-related effectors Detailed NGS analysis (significantly modulated genes, IPA analysis) available in Supplementary Table 5; statistical significance was assessed by GLM likelihood ratio (edgeR) and then adjusted by the Benjamini-Hochberg procedure to control the False Discovery Rate (FDR). “Upstream Regulators” shown are all significantly enriched (P<0.05 - and with a 2≤Z-Score≥2) in at least one comparison. d, Time-dependent changes of Bmp8b expression in HSC treated with drivers of HSC activation (PDGFB 10ng/mL; Oleic Acid 100 μM; Palmitic Acid 100 μM; TNFα 30ng/mL; LPS 50 ng/mL). Bmp8b mRNA expression levels are very low at baseline and induced 24h after culturing (Area Under the Curve, AUC, in the small panel); Palmitic Acid significantly induces Bmp8b, while LPS reduces its expression over the time. All the results are shown as mean ± s.e.m. or in a heatmap format (representing gene abundance expressed as Log2CPM, or the degree of “Upstream Regulator” activation -Z-score- at the IPA “Upstream Regulator” Analysis). Sample Size: 4 biological replicates/group for panels a-c (each biological replicate is a pool of 3 livers); 3 replicates/time point for panel (d). Statistical significance was assessed by one-way analysis of variance (ANOVA) plus Fisher’s least significant difference test (d). Lowercase letters indicate post hoc analysis significance: a, reference group; groups with different letters are statistically different per post hoc comparison; differences between groups with the same letter are statistically not significant per post hoc comparison.

Extended Data Fig. 4 Recombinant BMP8 rescues the defect in HSC activation observed in Bmp8b KO HSC cultured in vitro.

Freshly isolated HSC from Bmp8b KO mice and WT littermates were cultured for 4 and 6 days at a density of 35K cells/cm2 in presence/absence of recombinant BMP8 (30 mg/mL) to study the effect of BMP8b gain of function/recovery in both KO and WT cells. αSMA protein expression (a,b, IF; staining repeated twice in each biological replicate; magnification: 10X), and gene expression (RTqPCR) of multiple markers of HSC transactivation (c), inflammation (d) and TGFβ/BMP targets (e) were checked to study HSC’s activation status (4 biological replicates/group; each biological replicate is a pool of three livers). All the results are shown as mean ± s.e.m. Statistical significance was assessed by Multivariate Analysis of Variance (MANOVA).

Extended Data Fig. 5 BMP8 stimulates TGFβ/BMP signaling in a human 3D in vitro NASH model promoting inflammation and proliferation (continues from Figure 6).

a, BMP8b mRNA expression (RTqPCR) in the cells cultured without/with medium containing a mixture of saturated and unsaturated FFAs (Lean vs. Fat; n: 6 replicates/group). b, Targeted phospho-proteomics data of cells studied 30 min after BMP8 challenge (vs. Control; 3 biological replicates/group) in cells cultured in “Fat” medium; c, TGFβ/BMP targets studied by BMP8b mRNA expression (RTqPCR) studied after 2 challenges (every 24h) of BMP8, TGFβ, or BMP7 (in “Fat” medium; cells and media were harvested 48h after commencing the challenges; n: 4 replicates/group). d, Secreted proteins quantified in the culturing media of 48h treated cells (n: 4 replicates/group); e, IPA “upstream regulator” analysis of the RNA sequencing of cells treated with recombinant BMP8 for 5h vs 48h (4 replicates/group; full list of genes differentially regulated and statistical design are provided in Supplementary Table 7). All the results are shown as mean ± s.e.m. (expression data of biological replicates are represented as dot plots), or in a heatmap format [representing the degree of “Upstream Regulator” activation (-2 ≤ Z-score ≥2) at the IPA “Upstream Regulator” Analysis]. To provide a framework of interpretation of the sequencing data, we clustered the results in “early response” regulators (modulated at 5h; not modulated at 48h), “persistent response” regulators (modulated both at 5h and 48h with the same direction), “late response” regulators (mildly/not regulated at 5h and modulated at 48h), “biphasic” regulators (showing opposite direction of regulation between 5h and 48h data). Statistical significance was assessed by two-sided Student T-Test (a,b), or by One-Way analysis of variance (ANOVA; c, d) plus Fisher’s least significant difference test (n: 4 replicates/group). Lowercase letters indicate post hoc analysis significance: a, reference group; groups with different letters are statistically different per post hoc comparison; differences between groups with the same letter are statistically not significant per post hoc comparison.

Extended Data Fig. 6 Bmp8b KO mice challenged with CDHFD model (NASH F2 fibrosis).

BMP8b KO and wild-type littermates mice were treated for 14 weeks with a choline deficient high-fat diet (CDHFD – N: 9WT & 6KO). CDHFD -treated Bmp8b KO mice show no difference in BW (a), Liver to body weight percent ratio (b: LW/BW%), glucose and lipid metabolism (c), ALT (d), NASH activity (e, H&E; f, NASH activity score) and Fibrosis (g, Picro-Sirius Red, PSR; h, “Kleiner” Fibrosis Stage; i, PSR quantification (% of stained area quantified using HALO imaging software, Indica Lab) on the whole-tissue scanned slide; j, Procollagen C3, PRO-C3). However, the relative mRNA expression levels of key genes measured by RTqPCR in the livers (k), and in freshly isolated HSC (l; Sample size: 6WT & 3 KO HSC pools) show impaired activation of TGFβ/BMP signaling, reduced inflammation, and defective HSC activation in Bmp8b KO mice compared to WT littermates. a-l, All the results are shown as mean ± s.e.m. (biological replicates are represented as dot plots). Statistical significance was assessed by two-sided Student T-Test.

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Vacca, M., Leslie, J., Virtue, S. et al. Bone morphogenetic protein 8B promotes the progression of non-alcoholic steatohepatitis. Nat Metab 2, 514–531 (2020). https://doi.org/10.1038/s42255-020-0214-9

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