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Hepatic macrophages act as a central hub for relaxin-mediated alleviation of liver fibrosis

Abstract

Relaxin is an antifibrotic peptide hormone previously assumed to directly reverse the activation of hepatic stellate cells for liver fibrosis resolution. Using nanoparticle-mediated delivery, here we show that, although relaxin gene therapy reduces liver fibrosis in vivo, in vitro treatment fails to induce quiescence of the activated hepatic stellate cells. We show that hepatic macrophages express the primary relaxin receptor, and that, on relaxin binding, they switch from the profibrogenic to the pro-resolution phenotype. The latter releases exosomes that promote the relaxin-mediated quiescence of activated hepatic stellate cells through miR-30a-5p. Building on these results, we developed lipid nanoparticles that preferentially target activated hepatic stellate cells in the fibrotic liver and encapsulate the relaxin gene and miR-30a-5p mimic. The combinatorial gene therapy achieves synergistic antifibrosis effects in models of mouse liver fibrosis. Collectively, our findings highlight the key role that macrophages play in the relaxin-primed alleviation of liver fibrosis and demonstrate a proof-of-concept approach to devise antifibrotic strategies through the complementary application of nanotechnology and basic science.

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Fig. 1: Discrepancy between the in vivo and in vitro antifibrosis effects of RLN.
Fig. 2: RLN treatment drives the macrophage phenotype switch via Nur77 activation.
Fig. 3: Key role of macrophages in RLN-mediated aHSC deactivation.
Fig. 4: RLN-treated macrophages deactivate aHSCs via exosome release.
Fig. 5: MiR-30a-5p in exosomes derived from RLN-educated macrophages deactivates aHSCs by targeting ASK1.
Fig. 6: Combination of pRLN LPD with miR-30a-5p LPH achieves a synergistic antifibrosis effect in the CCl4-induced liver fibrosis model.
Fig. 7: The combination of pRLN LPD with miR-30a-5p LPH achieves a synergistic antifibrosis effect in a CDAHFD-induced steatohepatitis model.

Data availability

The MicroRNA Data Integration Portal was used to identify gene targets for exosomal miRNAs and can be accessed with http://ophid.utoronto.ca/mirDIP28. All data supporting the findings of this study are available within the article and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank S. Li for helpful discussions regarding the flow cytometry analysis on hepatic macrophages. The work in LH.’s lab was supported by NIH grants DK100664 and CA198999, and by a grant from Eshelman Institute for Innovation. Human sample collection and procession was done by J.T.’s lab and supported by Huamei Research Foundation (Grant 2017HMKY07), the Medicine and Health Sciences Research Foundation of Zhejiang Province (Grant 2019KY177).

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M.H. and L.H. conceived and designed the research. M.H., Y.W., Z.L., Z.Y., K.G., M.L. and M.W. performed the in vivo mouse experiments. J.T. provided paraffin-embedded liver tissues from patients. M.H. and Y.W. prepared the frozen sections, immunofluorescence and histological staining. M.H. and Y.W. analysed and quantified the microscopic images. M.H. and Y.W. purified the liver macrophage and mononuclear cells, did the flow cytometry assay and analysed the results. M.H. and Y.W. performed the western blot and qPCR assays on the cell and tissue samples. M.H. and Y.W. did the hydroxyproline assay on liver tissues. M.H. isolated and cultured the primary bone marrow-derived macrophages. M.H. designed the in vitro experiments to analyse the interaction between the macrophages and aHSCs. M.H., Y.W. and Z.L. isolated exosomes from Raw264.7 cells, hBMDMs, and differentiated THP-1 cells. M.H. performed miRNA array analyses. M.H. and Y.W. prepared lipid nanoparticles and characterized the exosomes and lipid nanoparticles. M.H., Y.W., K.G. and M.W. analysed the data. M.H. and L.H. wrote the manuscript.

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Correspondence to Leaf Huang.

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Peer review information Nature Nanotechnology thanks Frank Tacke and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–29 and Tables 1–3.

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Supplementary video 1

Bleeding time recording for PBS treatment group.

Supplementary video 2

Bleeding time recording for free recombinant RLN treatment group.

Supplementary video 3

Bleeding time recording for pRLN LPD treatment group.

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Hu, M., Wang, Y., Liu, Z. et al. Hepatic macrophages act as a central hub for relaxin-mediated alleviation of liver fibrosis. Nat. Nanotechnol. 16, 466–477 (2021). https://doi.org/10.1038/s41565-020-00836-6

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