Journal Pre-proof Fibrogenic signals persist in DAA-treated HCV patients after sustained virological response Claudia Montaldo, Michela Terri, Veronica Riccioni, Cecilia Battistelli, Veronica Bordoni, Gianpiero D’Offizi, Maria Giulia Prado, Flavia Trionfetti, Tiziana Vescovo, Eleonora Tartaglia, Raffaele Strippoli, Chiara Agrati, Marco Tripodi PII: S0168-8278(21)01901-2 DOI: https://doi.org/10.1016/j.jhep.2021.07.003 Reference: JHEPAT 8358 To appear in: Journal of Hepatology Received Date: 21 December 2020 Revised Date: 22 June 2021 Accepted Date: 1 July 2021 Please cite this article as: Montaldo C, Terri M, Riccioni V, Battistelli C, Bordoni V, D’Offizi G, Prado MG, Trionfetti F, Vescovo T, Tartaglia E, Strippoli R, Agrati C, Tripodi M, Fibrogenic signals persist in DAA- treated HCV patients after sustained virological response, Journal of Hepatology (2021), doi: https:// doi.org/10.1016/j.jhep.2021.07.003. 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Journal Pre-proof Fibrogenic signals persist in DAA-treated HCV patients after sustained virological response Claudia Montaldo1,*, Michela Terri1,2,*, Veronica Riccioni2, Cecilia Battistelli2, Veronica Bordoni1, Gianpiero D’Offizi1, Maria Giulia Prado2, Flavia Trionfetti1,2, Tiziana Vescovo1, Eleonora Tartaglia1, Raffaele Strippoli1,2, Chiara Agrati1 and Marco Tripodi1,2. 1National Institute for Infectious Diseases L. Spallanzani, IRCCS, 2Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy. *These authors equally contributed Corresponding Author: Prof. Marco Tripodi, Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 324, Rome 00161, Italy. Tel. +390649918244; Fax: +39064462891; E-mail: [email protected]; Keywords: Fibrosis, HCV, DAA, Extracellular vesicles, miRNAs, Proteomics, nLC-MS/MS, DIAPH1, miR204-5p, miR181a-5p, miR93-5p, miR143-3p. Word count (main text): 6173 Figures and table number: 4 Figures, 4 Tables Supplementary data: 4 Figures, 3 Tables Supplementary material: 1 MS Office File Conflict of interests: The authors declare no potential conflicts of interest. Financial support: This study was funded by the EXO-DAA project, L.R. Lazio 13/08, Ministry for Health of Italy (Ricerca Corrente) and Sapienza University of Rome RG11916B6A9C42C7 (to M.T.), Italian Ministry of Health Ricerca Finalizzata GR-2013-02359524 to JournalCM and TV Pre-proof, Consorzio Interuniversitario Biotecnologie: fellowship to MGP. Authors’ contributions C. Montaldo: Investigation, conceptualization, data curation, formal analysis, validation, investigation, visualization, methodology. M. Terri: data curation, conceptualization, formal analysis, validation, investigation, visualization, methodology. V. Riccioni: data curation, formal analysis, 1 validation, investigation, visualization, methodology. C. Battistelli: data curation, formal analysis, validation, investigation, visualization, writing-original draft and editing. V. Bordoni: data curation, formal analysis, methodology. G.P D’Offizi: patients’ management, clinical data analysis. M.G. Prado: data curation, formal analysis, validation and methodology. F. Trionfetti: investigation, visualization, methodology. T. Vescovo: data curation, formal analysis, validation, investigation, visualization, methodology. E. Tartaglia: data curation, methodology. R. Strippoli: supervision, visualization, editing original draft. C. Agrati: conceptualization, resources, funding acquisition, visualization, project administration. M. Tripodi: conceptualization, resources, supervision, funding acquisition, visualization, writing-original draft, project administration and editing. Journal Pre-proof 2 Abstract Background & Aims: HCV SVR, achievable now by means of DAA therapy, identifies a new class of patients requiring medical surveillance to be designed in relation to the liver disease stage advancement. To this end, identification of both disease biomarkers and therapeutic targets appears necessary. Methods: Extracellular Vesicles (EVs) purified from plasma of 15 healthy donors (HD), and 16 HCV infected patients before (T0) and after (T6) DAA treatment have been utilised for functional and miRNA cargo analysis. EVs purified from plasma of 17 HD, 23 T0 and T6 patients have been employed for proteomic and western blot analysis. Functional analysis in LX2 cells measured fibrotic markers (mRNAs and proteins) in response to EVs. Structural analysis was performed by qPCR, label-free liquid chromatography-mass spectrometry (nLC-MS/MS) and Western blot. Results: On the basis of observations indicating functional differences (i.e. modulation of FN-1, ACTA2, Smad2/3 phosphorylation, collagen deposition) of plasma-derived EVs from HD, T0 and T6, we performed EVs structural analysis. We found consistent differences in terms of both miRNA and protein cargos: (i) antifibrogenic miR204-5p, miR181a-5p, miR143-3p, miR93-5p and miR122- 5p were found statistically underrepresented in T0 EVs with respect to HD whereas miR204-5p and miR143-3p were found statistically underrepresented between HD and T6 (p-value<0.05) (ii) proteomic analysis highlighted, in both T0 and T6, the modulation of several proteins with respect to HD; among them, the fibrogenic DIAPH1 was confirmed upregulated by western blot (4.4 Log2 fold change). Journal Pre-proof Conclusions: Taken together, these results highlight structural EVs modifications, conceivably causal for long-term liver disease progression in HCV patients that persists despite the DAA-mediated HCV SVR. 3 Introduction New direct antiviral agents (DAAs)-based therapy, available with first generation compounds since 2011, efficiently reaches the Hepatitis C Virus (HCV) eradication, thus providing a therapeutic opportunity for 71 million people (WHO estimated) affected by chronic HCV infection who eventually develop cirrhosis or liver cancer[1]. HCV elimination does not always result into a healing of liver disease, particularly in patients with advanced fibrosis or cirrhosis; emerging clinical studies with DAAs in patients with liver cirrhosis stirred a heated debate about the risk of HCC occurrence and recurrence after viral cure[2–4]. In this scenario, early non-invasive prognostic tools useful to highlight the long-term DAA therapy clinical output appear necessary in addressing patients’ management. Recently the scientific community has focused on circulating extracellular vesicles (EVs) for two reasons: i) EVs-delivered biological message may be causal to disease progression and ii) their analysis may provide prognostic and diagnostic evidence. Current technologies allow EV analysis for both informational content, (i.e. DNA, mRNAs, microRNAs, long non-coding RNAs and proteins) and functional analysis, assessed by means of target cell response. In fact, EV analysis has already provided an important contribution as in the case of EBV-associated tumours, where it was found that circulating EV cargo may include Epstein Barr Virus (EBV) miR-BART2-5p, a diagnostic and prognostic biomarker functionally able to protect latent cells from EBV reactivation[5]. Similarly, in antiretroviral therapy (ART)-treated HIV patients EVs were found to carry proteins related to immune activation and oxidative stress, functionally bringing immunomodulatory effects[6]. Specifically in the frame of DAA therapy, it has been previously described that the expression of specific miRNAs present in EVs is modulated by this therapyJournal as these changes Pre-proof correlate with the EV-mediated NK cell degranulation capability[7]. In this study, we address the DAA therapy impact on liver fibrosis progression analysing the functional abilities and the structure of circulating EVs derived from HCV-infected and DAA-cleared patients. 4 EVs functional analysis was performed on LX2 as recipient cells, an HSC/myofibroblast cell line partially mimicking the highly proliferative myofibroblast-like cells that derive from quiescent hepatic stellate cells (HSCs). Indeed, during chronic HCV infection, HSCs convert into highly proliferative myofibroblast-like cells expressing inflammatory and fibrogenic mediators responsible for ECM accumulation within the microenvironment, thus contributing to the fibrotic process leading to cirrhosis and liver failure in advanced stages[8,9]. EV structural analysis was performed for both miRNA and protein content. Data obtained show that EVs derived from HCV-infected patients, with respect to EVs derived from healthy donors (HD), increase the fibrogenic activity of LX2 cell line and that this functional data correlates with upregulation of HSC activators (e.g. of DIAPH1) and downregulation of some antifibrogenic miRNAs (e.g. miR204-5p, miR93-5p, miR143-3p, miR181a-5p and miR122-5p). Notably, longitudinal analysis highlights the persistent EV pro-fibrogenic activity in spite of the DAA- mediated HCV eradication,