Type I Interferon Regulates the Expression of Long Non-Coding RNAs

Interferons (IFNs) are key players in the antiviral response. IFN sensing by the cell activates transcription of IFN-stimulated genes (ISGs) able to induce an antiviral state by affecting viral replication and release. IFN also induces the expression of ISGs that function as negative regulators to limit the strength and duration of IFN response. The ISGs identified so far belong to coding genes. However, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long noncoding RNAs (lncRNAs). To address whether IFN can also regulate the expression of lncRNAs, we analyzed the transcriptome of HuH7 cells treated or not with IFNα2 by expression arrays. Analysis of the arrays showed increased levels of several well-characterized coding genes that respond to IFN both at early or late times. Furthermore, we identified several IFN-stimulated or -downregulated lncRNAs (ISRs and IDRs). Further validation showed that ISR2, 8 and 12 expression mimics that of their neighboring genes GBP1, IRF1 and IL6, respectively, all related to the IFN response. These genes are induced in response to different doses of IFNα2 in different cell lines at early (ISR2 or 8) or later (ISR12) time points. IFNβ also induced the expression of these lncRNAs. ISR2 and 8 were also induced by an influenza virus unable to block the IFN response but not by other wild-type lytic viruses tested. Surprisingly, both ISR2 and 8 were significantly upregulated in cultured cells and livers from patients infected with HCV. Increased levels of ISR2 were also detected in patients chronically infected with HIV. This is relevant as genome-wide guilt-by-association studies predict that ISR2, 8 and 12 may function in viral processes, in the IFN pathway and the antiviral response. Therefore, we propose that these lncRNAs could be induced by IFN to function as positive or negative regulators of the antiviral response

Long Non-coding RNAs in Hepatitis C Virus-Infected Cells

Hepatitis C virus (HCV) often leads to a chronic infection in the liver that may progress to steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Several viral and cellular factors are required for a productive infection and for the development of liver disease. Some of these are long non-coding RNAs (lncRNAs) deregulated in infected cells. After HCV infection, the sequence and the structure of the viral RNA genome are sensed to activate interferon (IFN) synthesis and signaling pathways. These antiviral pathways regulate transcription of several cellular lncRNAs. Some of these are also deregulated in response to viral replication. Certain viral proteins and/or viral replication can activate transcription factors such as MYC, SP1, NRF2, or HIF1α that modulate the expression of additional cellular lncRNAs. Interestingly, several lncRNAs deregulated in HCV-infected cells described so far play proviral or antiviral functions by acting as positive or negative regulators of the IFN system, while others help in the development of liver cirrhosis and HCC. The study of the structure and mechanism of action of these lncRNAs may aid in the development of novel strategies to treat infectious and immune pathologies and liver diseases such as cirrhosis and HCC

Identification and analysis of long non-coding RNAs related to viral infection and antiviral response

In the last years, it has been stablished that the cell transcribes for many long non-coding RNAs (lncRNAs). It has been demonstrated that lncRNAs are deregulated in response to changes in cell status and that some lncRNAs act as key regulators of cell proliferation, development or cell homeostasis. However, the function of most lncRNAs is still unknown, and little is known about lncRNAs related to infection and immune response. The aim of this work was to identify and analyze lncRNAs involved in the antiviral cellular response. In the laboratory, they had performed transcriptome studies of control cells, cells infected with Hepatitis C virus (HCV) and/or cells treated with IFN-alpha by microarray and RNA sequencing. They found that IFN-alpha-treatment and/or HCV infection alters the levels of several lncRNAs. This PhD thesis describes the analysis of some of these lncRNAs. Some of the IFN stimulated RNAs (ISRs) ISR2, ISR8 and ISR12, as well as, lncISG15 and lncBST2/BISPR are located in the genome close to well-known IFN stimulated genes (ISGs): GBP cluster, IRF1, IL6, ISG15 and BST2 respectively. This suggests that these lncRNAs might function by regulating the expression of their closer gene. Indeed, BISPR induces the expression of BST2 coding gene. In addition, ISR8 seems to be a key enhancer for certain ISGs and pro-inflammatory genes. Indeed, ISR8 region presents enhancer marks, and disruption of this locus leads to stable cell lines that do not express ISR8 and are unable to induce several ISGs in response to IFN-alpha and/or NF-kB, although they have functional IFN and NF-kB pathways. Surprisingly, the phenotype observed in ISR8-disrupted cells is not rescued by exogenous expression of ISR8 or IRF1 or by decreasing chromatin repressors such as histone deacetylases or DNA methyltransferases to activate gene transcription. Finally, HCV infection induces the expression of many oncogenic lncRNAs. This suggests that the increased rate of liver tumors observed in HCV-infected patients could, in part, result from increased expression of oncogenic lncRNAs. EGOT is a lncRNA induced by the cellular antiviral response and by infection with HCV in patients and cultured cells. EGOT is induced by NF-kB after RIG-I and PKR sensors activation by HCV genome. EGOT inhibition by gapmers leads to higher expression of several ISGs and decreased HCV titer, RNA and protein levels. Therefore, EGOT may function as a negative regulator of the antiviral pathway

Identification and analysis of long non-coding RNAs related to viral infection and antiviral response

In the last years, it has been stablished that the cell transcribes for many long non-coding RNAs (lncRNAs). It has been demonstrated that lncRNAs are deregulated in response to changes in cell status and that some lncRNAs act as key regulators of cell proliferation, development or cell homeostasis. However, the function of most lncRNAs is still unknown, and little is known about lncRNAs related to infection and immune response. The aim of this work was to identify and analyze lncRNAs involved in the antiviral cellular response. In the laboratory, they had performed transcriptome studies of control cells, cells infected with Hepatitis C virus (HCV) and/or cells treated with IFN-alpha by microarray and RNA sequencing. They found that IFN-alpha-treatment and/or HCV infection alters the levels of several lncRNAs. This PhD thesis describes the analysis of some of these lncRNAs. Some of the IFN stimulated RNAs (ISRs) ISR2, ISR8 and ISR12, as well as, lncISG15 and lncBST2/BISPR are located in the genome close to well-known IFN stimulated genes (ISGs): GBP cluster, IRF1, IL6, ISG15 and BST2 respectively. This suggests that these lncRNAs might function by regulating the expression of their closer gene. Indeed, BISPR induces the expression of BST2 coding gene. In addition, ISR8 seems to be a key enhancer for certain ISGs and pro-inflammatory genes. Indeed, ISR8 region presents enhancer marks, and disruption of this locus leads to stable cell lines that do not express ISR8 and are unable to induce several ISGs in response to IFN-alpha and/or NF-kB, although they have functional IFN and NF-kB pathways. Surprisingly, the phenotype observed in ISR8-disrupted cells is not rescued by exogenous expression of ISR8 or IRF1 or by decreasing chromatin repressors such as histone deacetylases or DNA methyltransferases to activate gene transcription. Finally, HCV infection induces the expression of many oncogenic lncRNAs. This suggests that the increased rate of liver tumors observed in HCV-infected patients could, in part, result from increased expression of oncogenic lncRNAs. EGOT is a lncRNA induced by the cellular antiviral response and by infection with HCV in patients and cultured cells. EGOT is induced by NF-kB after RIG-I and PKR sensors activation by HCV genome. EGOT inhibition by gapmers leads to higher expression of several ISGs and decreased HCV titer, RNA and protein levels. Therefore, EGOT may function as a negative regulator of the antiviral pathway

Comprehensive analysis and insights gained from long-term experience of the Spanish DILI Registry

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER); Agencia Española del Medicamento; Consejería de Salud de Andalucía.Background & Aims: Prospective drug-induced liver injury (DILI) registries are important sources of information on idiosyncratic DILI. We aimed to present a comprehensive analysis of 843 patients with DILI enrolled into the Spanish DILI Registry over a 20-year time period. Methods: Cases were identified, diagnosed and followed prospectively. Clinical features, drug information and outcome data were collected. Results: A total of 843 patients, with a mean age of 54 years (48% females), were enrolled up to 2018. Hepatocellular injury was associated with younger age (adjusted odds ratio [aOR] per year 0.983; 95% CI 0.974-0.991) and lower platelet count (aOR per unit 0.996; 95% CI 0.994-0.998). Anti-infectives were the most common causative drug class (40%). Liver-related mortality was more frequent in patients with hepatocellular damage aged ≥65 years (p = 0.0083) and in patients with underlying liver disease (p = 0.0221). Independent predictors of liver-related death/transplantation included nR-based hepatocellular injury, female sex, higher onset aspartate aminotransferase (AST) and bilirubin values. nR-based hepatocellular injury was not associated with 6-month overall mortality, for which comorbidity burden played a more important role. The prognostic capacity of Hy's law varied between causative agents. Empirical therapy (corticosteroids, ursodeoxycholic acid and MARS) was prescribed to 20% of patients. Drug-induced autoimmune hepatitis patients (26 cases) were mainly females (62%) with hepatocellular damage (92%), who more frequently received immunosuppressive therapy (58%). Conclusions: AST elevation at onset is a strong predictor of poor outcome and should be routinely assessed in DILI evaluation. Mortality is higher in older patients with hepatocellular damage and patients with underlying hepatic conditions. The Spanish DILI Registry is a valuable tool in the identification of causative drugs, clinical signatures and prognostic risk factors in DILI and can aid physicians in DILI characterisation and management. Lay summary: Clinical information on drug-induced liver injury (DILI) collected from enrolled patients in the Spanish DILI Registry can guide physicians in the decision-making process. We have found that older patients with hepatocellular type liver injury and patients with additional liver conditions are at a higher risk of mortality. The type of liver injury, patient sex and analytical values of aspartate aminotransferase and total bilirubin can also help predict clinical outcomes