USP18-Based Negative Feedback Control Is Induced by Type I and Type III Interferons and Specifically Inactivates Interferon α Response

Type I interferons (IFN) are cytokines that are rapidly secreted upon microbial infections and regulate all aspects of the immune response. In humans 15 type I IFN subtypes exist, of which IFN α2 and IFN β are used in the clinic for treatment of different pathologies. IFN α2 and IFN β are non redundant in their expression and in their potency to exert specific bioactivities. The more recently identified type III IFNs (3 IFN λ or IL-28/IL-29) bind an unrelated cell-type restricted receptor. Downstream of these two receptor complexes is a shared Jak/Stat pathway. Several mechanisms that contribute to the shut down of the IFN-induced signaling have been described at the molecular level. In particular, it has long been known that type I IFN induces the establishment of a desensitized state. In this work we asked how the IFN-induced desensitization integrates into the network built by the multiple type I IFN subtypes and type III IFNs. We show that priming of cells with either type I IFN or type III IFN interferes with the cell's ability to further respond to all IFN α subtypes. Importantly, primed cells are differentially desensitized in that they retain sensitivity to IFN β. We show that USP18 is necessary and sufficient to induce differential desensitization, by impairing the formation of functional binding sites for IFN α2. Our data highlight a new type of differential between IFNs α and IFN β and underline a cross-talk between type I and type III IFN. This cross-talk could shed light on the reported genetic variation in the IFN λ loci, which has been associated with persistence of hepatitis C virus and patient's response to IFN α2 therapy

Cellular models for the screening and development of anti-hepatitis C virus agents.

International audienceInvestigations on the biology of hepatitis C virus (HCV) have been hampered by the lack of small animal models. Efforts have therefore been directed to designing practical and robust cellular models of human origin able to support HCV replication and production in a reproducible, reliable and consistent manner. Many different models based on different forms of virions and hepatoma or other cell types have been described including virus-like particles, pseudotyped particles, subgenomic and full length replicons, virion productive replicons, immortalised hepatocytes, fetal and adult primary human hepatocytes. This review focuses on these different cellular models, their advantages and disadvantages at the biological and experimental levels, and their respective use for evaluating the effect of antiviral molecules on different steps of HCV biology including virus entry, replication, particles generation and excretion, as well as on the modulation by the virus of the host cell response to infection

Expression and DNA-binding activity of C/EBPα and C/EBPβ in human liver and differentiated primary hepatocytes

International audienceBackground/Aims: Limited information is available on the expression and role of C/EBP factors in human liver and hepatocytes. We investigated the expression and DNA-binding activity of C/EBPα and C/EBPβ in human liver needle biopsies, surgical lobectomies and differentiated cultured hepatocytes derived from lobectomies.Methods: RNA and protein extracts were analyzed by RNAse protection, immunoblot and gel shift assays.Results: C/EBP mRNAs, isoforms and DNA-binding activities were low/undetectable in lobectomies. In contrast, several C/EBPα (47, 45, 35 and 33 kDa) and C/EBPβ isoforms (47, 43, 40, 35 and 21 kDa) were observed in needle biopsies. In cultured hepatocytes, the C/EBP expression pattern dramatically changed with time. C/EBPα mRNA and the 45 kDa isoform increased in parallel, reaching a maximum after 3–4 weeks coincident with weak DNA-binding activity. C/EBPβ mRNA and isoform expression increased rapidly reaching a plateau within 1–2 weeks; all C/EBPβ isoforms were phosphorylated. C/EBPβ exhibited greater DNA-binding activity than C/EBPα, and this activity paralleled C/EBPβ isoform expression.Conclusions: C/EBP isoforms exhibit markedly different expression patterns in lobectomies, needle biopsies and cultured hepatocytes. Stress stimuli during and/or after surgery for lobectomy resections may account for this difference. The pattern of C/EBP isoform expression in long-term highly differentiated cultured hepatocytes is close to that observed in needle biopsies

Ketoconazole and miconazole are antagonists of the human glucocorticoid receptor: consequences on the expression and function of the constitutive androstane receptor and the pregnane X receptor.

International audienceThe constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) play a major part in the control of drug metabolism and transport. We have previously shown that PXR and CAR expression is controlled by the glucocorticoid receptor (GR) and proposed the existence of a signal transmission cascade GR-(PXR/CAR)-drug metabolizing and transporter systems. In the current study, we investigated the effect of ketoconazole and other azole-derived drugs, miconazole and fluconazole, on the transcriptional activity of the human GR (hGR) in HeLa and HepG2 cells, and in primary human hepatocytes. The data show that ketoconazole inhibits GR transcriptional activity and competes with dexamethasone for hGR binding. In primary human hepatocytes, ketoconazole inhibits the expression of 1) GR-responsive genes tyrosine aminotransferase and both PXR and CAR; 2) CAR and PXR target genes, including cytochromes P450 (P450) CYP2B6, CYP2C9, and CYP3A4; UDP-glucuronosyltransferase 1A1, glutathione S-transferases A1 and A2; and transporter proteins (phase III) solute carrier family 21 form A6 and multidrug resistance protein 2. In parallel experiments, ketoconazole affected neither the expression of GR, the expression of glyceraldehyde-3-phosphate dehydrogenase, nor the inducible expression of CYP1A1 and 1A2. Miconazole behaved like ketoconazole, whereas fluconazole had no effect. We conclude that, in addition to their well known inhibitory effect on P450 enzyme activities, ketoconazole and miconazole are antagonists of hGR. These results provide a novel molecular mechanism by which these compounds may exert adverse and toxic effects on drug metabolism and other functions in human