RESUMEN
Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Receptores Virales , Ácido Ursodesoxicólico , Animales , Humanos , Ratones , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/prevención & control , Receptores Virales/genética , Receptores Virales/metabolismo , Estudios Retrospectivos , SARS-CoV-2/metabolismo , Tratamiento Farmacológico de COVID-19 , Cricetinae , Transcripción Genética , Ácido Ursodesoxicólico/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Organoides/efectos de los fármacos , Organoides/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/metabolismo , Sistema de Registros , Reproducibilidad de los Resultados , Trasplante de HígadoRESUMEN
The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.
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COVID-19/patología , COVID-19/virología , Fusión de Membrana , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Serina Endopeptidasas/metabolismo , Internalización del Virus , Adulto , Anciano , Anciano de 80 o más Años , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/virología , Chlorocebus aethiops , Convalecencia , Femenino , Humanos , Sueros Inmunes/inmunología , Intestinos/patología , Intestinos/virología , Pulmón/patología , Pulmón/virología , Masculino , Persona de Mediana Edad , Mutación , Mucosa Nasal/patología , Mucosa Nasal/virología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Técnicas de Cultivo de Tejidos , Virulencia , Replicación ViralRESUMEN
The burden of liver diseases is increasing worldwide, with liver transplantation remaining the only treatment option for end-stage liver disease. Regenerative medicine holds great potential as a therapeutic alternative, aiming to repair or replace damaged liver tissue with healthy functional cells. The properties of the cells used are critical for the efficacy of this approach. The advent of liver organoids has not only offered new insights into human physiology and pathophysiology, but also provided an optimal source of cells for regenerative medicine and translational applications. Here, we discuss various historical aspects of 3D organoid culture, how it has been applied to the hepatobiliary system, and how organoid technology intersects with the emerging global field of liver regenerative medicine. We outline the hepatocyte, cholangiocyte, and nonparenchymal organoids systems available and discuss their advantages and limitations for regenerative medicine as well as future directions.
Asunto(s)
Gastroenterología , Humanos , Organoides , Hígado , Medicina Regenerativa , HepatocitosRESUMEN
BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disorder without effective medical treatment which is characterized by inflammation and fibrotic structures around the bile ducts. Biliary epithelial cells (cholangiocytes) are the target and potential disease drivers in PSC, yet little is known if cholangiocytes from PSC patients differ from non-PSC controls. To characterize cholangiocytes at early rather than end-stage disease, cholangiocyte organoids (COs) were derived from diseased bile ducts of PSC patients and compared to organoids generated from disease controls. METHODS: Cholangiocytes were obtained during endoscopic retrograde cholangiopancreatography (ERCP) brushing of diseased bile duct areas and expanded as organoids using previously established culture methods. Stable CO lines were analyzed for cell type identity, basic cholangiocyte function, and transcriptomic signature. RESULTS: We demonstrate that cholangiocytes, derived from the damaged area within the bile ducts of PSC patients, can be expanded in culture without displaying functional or genetic disease-related features. We further show that COs from patients who later were diagnosed with dysplasia exhibit higher expression of the cancer-associated genes PGC, FXYD2, MIR4435-2HG, and HES1. CONCLUSIONS: Our results demonstrate that PSC organoids are largely similar to control organoids after culture and highlight the significance of COs as a tool for regenerative medicine approaches as well as their potential for discovering new potential biomarkers for diagnosing cholangiocarcinoma.
RESUMEN
BACKGROUND AND AIMS: Studies have demonstrated that reducing farnesoid X receptor activity with ursodeoxycholic acid (UDCA) downregulates angiotensin-converting enzyme in human lung, intestinal and cholangiocytes organoids in vitro, in human lungs and livers perfused ex situ, reducing internalization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell. This offers a potential novel target against coronavirus disease 2019 (COVID-19). The objective of our study was to compare the association between UDCA exposure and SARS-CoV-2 infection, as well as varying severities of COVID-19, in a large national cohort of participants with cirrhosis. METHODS: In this retrospective cohort study among participants with cirrhosis in the Veterans Outcomes and Costs Associated with Liver cohort, we compared participants with exposure to UDCA, with a propensity score (PS) matched group of participants without UDCA exposure, matched for clinical characteristics, and vaccination status. The outcomes included SARS-CoV-2 infection, symptomatic, at least moderate, severe, or critical COVID-19, and COVID-19-related death. RESULTS: We compared 1607 participants with cirrhosis who were on UDCA, with 1607 PS-matched controls. On multivariable logistic regression, UDCA exposure was associated with reduced odds of developing SARS-CoV-2 infection (adjusted odds ratio [aOR] 0.54, 95% confidence interval [CI] 0.41-0.71, p < 0.0001). Among patients who developed COVID-19, UDCA use was associated with reduced disease severity, including symptomatic COVID-19 (aOR 0.54, 95% CI 0.39-0.73, p < 0.0001), at least moderate COVID-19 (aOR 0.51, 95% CI 0.32-0.81, p = 0.005), and severe or critical COVID-19 (aOR 0.48, 95% CI 0.25-0.94, p = 0.03). CONCLUSIONS: In participants with cirrhosis, UDCA exposure was associated with both a decrease in SARS-CoV-2 infection, and reduction in symptomatic, at least moderate, and severe/critical COVID-19.
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COVID-19 , Cirrosis Hepática Biliar , Humanos , Ácido Ursodesoxicólico/uso terapéutico , COVID-19/complicaciones , Estudios Retrospectivos , SARS-CoV-2 , Cirrosis Hepática/complicaciones , Cirrosis Hepática/tratamiento farmacológicoRESUMEN
Our insight into the pathogenesis of cholestatic liver disease remains limited, partly owing to challenges in capturing the multitude of factors that contribute to disease pathogenesis in vitro. Tissue engineering could address this challenge by combining cells, materials and fabrication strategies into dynamic modelling platforms, recapitulating the multifaceted aetiology of cholangiopathies. Herein, we review the advantages and limitations of platforms for bioengineering the biliary tree, looking at how these can be applied to model biliary disorders, as well as exploring future directions for the field.
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Sistema Biliar/patología , Colestasis/terapia , Ingeniería de Tejidos/métodos , Animales , Colestasis/patología , Células Epiteliales/patología , HumanosRESUMEN
AIMS: Membrane-bound angiotensin-converting enzyme (ACE)2 is the main cellular access point for SARS-CoV-2, but its expression and the effect of ACE inhibition have not been assessed quantitatively in patients with heart failure. The aim of this study was to characterize membrane-bound ACE2 expression in the myocardium and myocardial vasculature in patients undergoing heart transplantation and to assess the effect of pharmacological ACE inhibition. METHODS AND RESULTS: Left ventricular (LV) tissue was obtained from 36 explanted human hearts from patients undergoing heart transplantation. Immunohistochemical staining with antibodies directed against ACE2 co-registered with cardiac troponin T (cTnT) and α-smooth muscle cell actin (SMA) was performed across the entire cohort. ACE2 receptor expression was quantitatively assessed throughout the myocardium and vasculature. ACE2 was consistently expressed throughout the LV myocardium (28.3% ± 22.2% of cardiomyocytes). ACE2 expression was also detected in small calibre blood vessels (range, 2-9 µm), albeit at quantitatively much lower levels (5% ± 9% of blood vessels). There was no significant difference in ACE2 expression between patients receiving ACE inhibitors prior to transplantation and ACE inhibitor-negative controls (P > 0.05). ACE2 expression did not differ significantly between the different diagnostic groups as the underlying reason for heart transplantation (ANOVA > 0.05). N-terminal pro-brain natriuretic peptide (NT-proBNP) (R2 = 0.37, P = 0.0006) and pulmonary capillary wedge pressure (PCWP) (R2 = 0.13, P = 0.043) assessed by right heart catheterization were significantly correlated with greater ACE2 expression in cardiomyocytes. CONCLUSIONS: These data provide a comprehensive characterization of membrane-bound cardiac ACE2 expression in patients with heart failure with no demonstrable effect exerted by ACE inhibitors.
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Enzima Convertidora de Angiotensina 2/metabolismo , Trasplante de Corazón , Inhibidores de la Enzima Convertidora de Angiotensina , HumanosRESUMEN
Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.
Asunto(s)
Enfermedades de los Conductos Biliares/terapia , Conductos Biliares Intrahepáticos/fisiología , Conductos Biliares/citología , Tratamiento Basado en Trasplante de Células y Tejidos , Células Epiteliales/citología , Organoides/trasplante , Animales , Bilis , Conductos Biliares/fisiología , Conductos Biliares Intrahepáticos/citología , Conducto Colédoco/citología , Células Epiteliales/fisiología , Vesícula Biliar/citología , Regulación de la Expresión Génica , Humanos , Hígado/fisiología , Trasplante de Hígado , Trasplante de Células Madre Mesenquimatosas , Ratones , Organoides/fisiología , RNA-Seq , Obtención de Tejidos y Órganos , TranscriptomaRESUMEN
Most of the research behind new medical advances is carried out using either animal models or cancer cells, which both have their disadvantage in particular with regard to medical applications such as personalized medicine and novel therapeutic approaches. However, recent advances in stem cell biology have enabled long-term culturing of organotypic intestinal or hepatic tissues derived from tissue resident or pluripotent stem cells. These 3D structures, denoted as organoids, represent a substantial advance in structural and functional complexity over traditional in vitro cell culture models that are often non-physiological and transformed. They can recapitulate the in vivo architecture, functionality and genetic signature of the corresponding tissue. The opportunity to model epithelial cell biology, epithelial turnover, barrier dynamics, immune-epithelial communication and host-microbe interaction more efficiently than previous culture systems, greatly enhance the translational potential of organotypic hepato-gastrointestinal culture systems. Thus there is increasing interest in using such cultured cells as a source for tissue engineering, regenerative medicine and personalized medicine. This review will highlight some of the established and also some exciting novel perspectives on organoids in the fields of gastroenterology and hepatology.
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Enfermedad , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Organoides/citología , Organoides/fisiología , Animales , Gastroenterología/tendencias , Humanos , Especificidad de Órganos , Medicina de Precisión/métodos , Medicina Regenerativa/métodos , Investigación Biomédica TraslacionalRESUMEN
Cholangiocytes play a crucial role in the pathophysiology of cholestasis. However, research on human cholangiocytes has been restricted by challenges in long-term propagation and large-scale expansion of primary biliary epithelium. The advent of organoid technology has overcome this limitation allowing long-term culture of a variety of epithelia from multiple organs. Here, we describe two methods for growing human cholangiocytes in organoid format. The first applies to the generation of intrahepatic bile ducts using human induced pluripotent stem cells using a protocol of differentiation that recapitulates physiological bile duct development. The second method allows the propagation of primary biliary epithelium from the extrahepatic ducts or gallbladder. Both protocols result in large numbers of cholangiocyte organoids expressing biliary markers and maintaining key cholangiocyte functions.
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Colestasis/metabolismo , Colestasis/patología , Organoides/patología , Conductos Biliares/metabolismo , Conductos Biliares/patología , Vesícula Biliar/metabolismo , Vesícula Biliar/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/metabolismo , Hígado/patología , Organoides/metabolismoRESUMEN
Pediatric liver transplantation is often required as a consequence of biliary disorders because of the lack of alternative treatments for repairing or replacing damaged bile ducts. To address the lack of availability of pediatric livers suitable for transplantation, we developed a protocol for generating bioengineered biliary tissue suitable for biliary reconstruction. Our platform allows the derivation of cholangiocyte organoids (COs) expressing key biliary markers and retaining functions of primary extra- or intrahepatic duct cholangiocytes within 2 weeks of isolation. COs are subsequently seeded on polyglycolic acid (PGA) scaffolds or densified collagen constructs for 4 weeks to generate bioengineered tissue retaining biliary characteristics. Expertise in organoid culture and tissue engineering is desirable for optimal results. COs correspond to mature functional cholangiocytes, differentiating our method from alternative organoid systems currently available that propagate adult stem cells. Consequently, COs provide a unique platform for studies in biliary physiology and pathophysiology, and the resulting bioengineered tissue has broad applications for regenerative medicine and cholangiopathies.