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Obese patients with NASH have increased hepatic expression of SARS-CoV-2 critical entry points

  • Author Footnotes
    † Contributed equally.
    Marcos F. Fondevila
    Footnotes
    † Contributed equally.
    Affiliations
    Department of Physiology, CIMUS, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela, Spain

    CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERobn), Spain
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  • Author Footnotes
    † Contributed equally.
    Maria Mercado-Gómez
    Footnotes
    † Contributed equally.
    Affiliations
    Liver Disease Laboratory, Center for Cooperative Research in Biosciences (CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160 Derio, and CIBER de Enfermedades Hepaticas y Digestivas (CIBERehd), Spain
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  • Author Footnotes
    † Contributed equally.
    Amaia Rodríguez
    Footnotes
    † Contributed equally.
    Affiliations
    CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERobn), Spain

    Obesity Area, Clínica Universidad de Navarra and IdiSNA, Pamplona, Spain
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  • Author Footnotes
    † Contributed equally.
    Maria J. Gonzalez-Rellan
    Footnotes
    † Contributed equally.
    Affiliations
    Department of Physiology, CIMUS, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela, Spain

    CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERobn), Spain
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  • Paula Iruzubieta
    Affiliations
    Department of Gastroenterology and Hepatology, Marqués de Valdecilla University Hospital, Research Institute Marqués de Valdecilla (IDIVAL), Santander, Spain
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  • Víctor Valentí
    Affiliations
    CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERobn), Spain

    Obesity Area, Clínica Universidad de Navarra and IdiSNA, Pamplona, Spain
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  • Javier Escalada
    Affiliations
    CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERobn), Spain

    Obesity Area, Clínica Universidad de Navarra and IdiSNA, Pamplona, Spain
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  • Markus Schwaninger
    Affiliations
    Institute for Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, Lübeck, Germany
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  • Vincent Prevot
    Affiliations
    Univ. Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience & Cognition, UMR-S 1172, European Genomic Institute for Diabetes (EGID), F-59000 Lille, France
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  • Carlos Dieguez
    Affiliations
    Department of Physiology, CIMUS, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela, Spain

    CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERobn), Spain
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  • Javier Crespo
    Affiliations
    Department of Gastroenterology and Hepatology, Marqués de Valdecilla University Hospital, Research Institute Marqués de Valdecilla (IDIVAL), Santander, Spain
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  • Gema Frühbeck
    Affiliations
    CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERobn), Spain

    Obesity Area, Clínica Universidad de Navarra and IdiSNA, Pamplona, Spain
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  • Maria L. Martinez-Chantar
    Correspondence
    Corresponding authors. Addresses: Liver Disease Laboratory, Center for Cooperative Research in Biosciences (CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160 Derio, Spain (M.L. Martinez-Chantar) or
    Affiliations
    Liver Disease Laboratory, Center for Cooperative Research in Biosciences (CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160 Derio, and CIBER de Enfermedades Hepaticas y Digestivas (CIBERehd), Spain
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  • Ruben Nogueiras
    Correspondence
    Department of Physiology, Research Centre of Molecular Medicine and Chronic Diseases (CIMUS), Instituto de Investigación Sanitaria de Santiago de Compostela, Universidad de Santiago de Compostela (USC), Santiago de Compostela, Spain (R. Nogueiras).
    Affiliations
    Department of Physiology, CIMUS, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela, Spain

    CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERobn), Spain

    Galician Agency of Innovation (GAIN), Santiago de Compostela, Spain
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  • Author Footnotes
    † Contributed equally.
Published:October 19, 2020DOI:https://doi.org/10.1016/j.jhep.2020.09.027

      Linked Article

      • No evidence for an increased liver uptake of SARS-CoV-2 in metabolic-associated fatty liver disease
        Journal of HepatologyVol. 73Issue 3
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          We read with interest the research article published by Ji and colleagues, in the Journal of Hepatology, showing that patients with metabolic-associated fatty liver disease (MAFLD) have a higher risk of COVID-19 disease progression and higher likelihood of abnormal liver blood tests from admission to discharge than patients without MAFLD.1 Given the absence of data on medical history of these patients, this persistence of liver blood test abnormalities could be either a mere reflection of pre-existing abnormalities related to MAFLD or could alternatively be due to a higher susceptibility of the fatty liver to SARS-CoV-2 infection.
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      To the Editor:
      We read with great interest the article published by Biquard and colleagues showing that, according to public transcriptomic data, the hepatic expression of angiotensin converting enzyme 2 (ACE2) and the cellular transmembrane protease serine 2 (TMPRSS2) remains unchanged in patients with metabolic-associated fatty liver disease (MAFLD).
      • Biquard L.
      • Valla D.
      • Rautou P.E.
      No evidence for an increased liver uptake of SARS-CoV-2 in metabolic-associated fatty liver disease.
      SARS-CoV-2 attaches to cells by binding to its receptor ACE2. TMPRSS2 then cleaves the SARS-CoV-2 spike protein, allowing fusion of cellular and viral membranes.
      • Hoffmann M.
      • Kleine-Weber H.
      • Schroeder S.
      • Kruger N.
      • Herrler T.
      • Erichsen S.
      • et al.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      Despite this retrospective study, there is growing evidence that patients with MAFLD are at higher risk of COVID-19 disease progression.
      • Ji D.
      • Qin E.
      • Xu J.
      • Zhang D.
      • Cheng G.
      • Wang Y.
      • et al.
      Non-alcoholic fatty liver diseases in patients with COVID-19: a retrospective study.
      • Zhou Y.J.
      • Zheng K.I.
      • Wang X.B.
      • Yan H.D.
      • Sun Q.F.
      • Pan K.H.
      • et al.
      Younger patients with MAFLD are at increased risk of severe COVID-19 illness: a multicenter preliminary analysis.
      • Zhou Y.J.
      • Zheng K.I.
      • Wang X.B.
      • Sun Q.F.
      • Pan K.H.
      • Wang T.Y.
      • et al.
      Metabolic-associated fatty liver disease is associated with severity of COVID-19.
      Given the ongoing discussion, we have assessed the expression of SARS-CoV-2 cell entry molecules in the liver of obese patients with non-alcoholic fatty liver disease (NAFLD) and/or type 2 diabetes T2D (see Table S1 for detailed characteristics), since this information seems crucial to understand and prevent cell infection. Considering that T2D has been associated with a worse prognosis in patients with COVID-19 and that well-controlled glycemia was associated with a markedly improved outcome,
      • Zhu L.
      • She Z.G.
      • Cheng X.
      • Qin J.J.
      • Zhang X.J.
      • Cai J.
      • et al.
      Association of blood glucose control and outcomes in patients with COVID-19 and pre-existing type 2 diabetes.
      we first focused on patients with T2D. Liver mRNA expression of ACE2 was significantly lower in patients with T2D while TMPRSS2 also tended to decrease but was not statistically significant (Fig. 1A). Then, we analysed separately men and women. In men, hepatic ACE2 and TMPRSS2 expression remained unchanged between the 2 groups (Fig. 1B). However, in women with T2D, ACE2 was significantly lower while TMPRSS2 gene expression tended to decrease compared to women without T2D (Fig. 1B). These results indicate that while the cell entry machinery of SARS-CoV-2 is not majorly altered in the liver of obese men with T2D, its downregulation in women might indicate a lower susceptibility to liver injury. These findings are in consonance with the well-established protective role of estrogens in dysmetabolism.
      • Mauvais-Jarvis F.
      • Clegg D.J.
      • Hevener A.L.
      The role of estrogens in control of energy balance and glucose homeostasis.
      Figure thumbnail gr1
      Fig. 1ACE2 and TMPRSS2 hepatic mRNA levels in patients with T2D or NAFLD.
      (A) ACE2 and TMPRSS2 expression in obese patients with T2D (n = 43) or NG (n = 51); and (B) separately by men and women with T2D or NG. (C) ACE2 and TMPRSS2 expression in obese patients without NAFLD (n = 17), steatosis (n = 57), NASH (n = 20). (D) Correlation between ACE2 and TMPRSS2 with NAS score. ∗p <0.05, ∗∗p <0.01, Mann-Whitney U test (A, B), Krustal-Wallis followed by Dunn post-hoc test (C). ACE2, angiotensin converting enzyme 2; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NG, normoglycemia; T2D, type 2 diabetes; TMPRSS2, transmembrane protease serine 2.
      Next, we measured the expression of these genes in the liver according to the presence of NAFLD. Liver mRNA expression of both ACE2 and TMPRSS2 did not show differences between individuals without liver injury and patients with only steatosis, but these genes were upregulated in obese patients with non-alcoholic steatohepatitis (NASH) (Fig. 1C). Moreover, ACE2 and TMPRSS2 were positively correlated with NAFLD activity score (Fig. 1D). Of note, TMPRSS2 was also positively correlated with weight, BMI and cholesterol (data not shown). These results are apparently different to those described in a previous study performing transcriptomics in individuals with and without MAFLD
      • Biquard L.
      • Valla D.
      • Rautou P.E.
      No evidence for an increased liver uptake of SARS-CoV-2 in metabolic-associated fatty liver disease.
      ,
      • Ahrens M.
      • Ammerpohl O.
      • von Schonfels W.
      • Kolarova J.
      • Bens S.
      • Itzel T.
      • et al.
      DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery.
      and also in animal models of diet-induced NASH,
      • Ahrens M.
      • Ammerpohl O.
      • von Schonfels W.
      • Kolarova J.
      • Bens S.
      • Itzel T.
      • et al.
      DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery.
      where no changes were detected for either ACE2 or TMPRSS2. However, it is important to highlight that there are important differences in the characteristics of the cohorts. In contrast to the previous report that analyzed lean and obese patients with MAFLD or NASH,
      • Biquard L.
      • Valla D.
      • Rautou P.E.
      No evidence for an increased liver uptake of SARS-CoV-2 in metabolic-associated fatty liver disease.
      our cohort is exclusively composed of obese patients. Moreover, whereas in the previous analysis T2D is not mentioned, in our cohort a significant percentage of patients had T2D, which commonly coexists with MAFLD.
      • Byrne C.D.
      • Targher G.
      NAFLD: a multisystem disease.
      Lastly, methodological differences might also explain the discrepant results, since we used real-time PCR to specifically measure gene expression of ACE2 and TMPRSS2, while in the previous reports
      • Ahrens M.
      • Ammerpohl O.
      • von Schonfels W.
      • Kolarova J.
      • Bens S.
      • Itzel T.
      • et al.
      DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery.
      results were obtained by less quantitative techniques namely microarray or RNA sequencing. Further studies using larger cohorts of patients with liver damage need to be meticulously evaluated to understand whether the SARS-CoV-2 receptor ACE2 and the serine protease TMPRSS2 are indeed affected in advanced stages of NAFLD and to what extent their expression affects the incidence of complications, severity and mortality.
      In summary, our results indicate that in the livers of obese patients, SARS-CoV-2 entry factors are differently affected by T2D and NAFLD. While obese women with T2D have unexpectedly lower levels of ACE2 and TMPRSS2 than obese normoglycemic women, obese patients with NASH show markedly higher expression of these genes, suggesting that advanced stages of NAFLD might predispose individuals to COVID-19.

      Financial support

      This work has been supported by grants from FEDER / Ministerio de Ciencia , Innovación y Universidades-Agencia Estatal de Investigación (CD: BFU2017-87721 ; RN: RTI2018-099413-B-I00 ; MLMC: SAF2017-87301-R ; Xunta de Galicia (RN: 2015-CP080 and 2016-PG057 ) and ED431G 2019/02 , Fundación BBVA (RN and MLM), Fundación Atresmedia (RN), and European Foundation for the Study of Diabetes (RN). FIS: PI18/0130. Immunomediatd Nonalcoholic SteaTohepatItis; prevalence and CharacTerization. INSTInCT study (PI: Javier Crespo). Intensificación IDIVAL I-1 (Javier Crespo). The research leading to these results has also received funding from the European Community’s H2020 Framework Programme under the following grant: ERC Synergy Grant-2019-WATCH- 810331 to MS, VP and RN. CIBERobn and CIBERehd are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain which is supported by FEDER funds. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644).

      Authors’ contributions

      MFF, MMG, AR, MJGR, PI, VV, JE, MS, VP, CD, JC, GF, MLMC, RN contributed to conception and design, acquisition of data, analysis and interpretation of data. PI, MS, VP, CD, JC, GF, MLMC, RN contributed drafting the article and revising it critically for important intellectual content.

      Data availability statement

      All the data used to support the findings of this study are included within the article. Reagents, resources and protocols are included in Supplementary methods.

      Conflict of interest

      The authors declare no conflict of interest.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

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