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Characterization of type I interferon pathway during hepatic differentiation of human pluripotent stem cells and hepatitis C virus infection

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long-term survival of engrafting cells in the body, not only do the cells have to execute liver-specific function but also withstand the physical strains and invading pathogens. The cellular innate immune system orchestrated by the interferon (IFN) pathway provides the first line of defense against pathogens. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN-induced genes during hepatic differentiation of pluripotent stem cells. To address this objective, we derived endodermal cells (day 5 post-differentiation), hepatoblast (day 15) and hepatocyte-like cells (day 21) from human embryonic stem cells (hESCs). Day 5, 15 and 21 cells were stimulated with IFN-alpha and subjected to IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that the IFN-alpha treatment activated STAT-JAK pathway in differentiating cells. Transcriptome analysis indicated stage specific expression of classical and non-classical IFN-stimulated genes (ISGs). Subsequent validation confirmed the expression of novel ISGs including RASGRP3, CLMP and TRANK1 by differentiated hepatic cells upon IFN treatment. Hepatitis C virus replication in hESC-derived hepatic cells induced the expression of ISGs - LAMP3, ETV7, RASGRP3, and TRANK1. The hESC-derived hepatic cells contain intact innate system and can recognize invading pathogens. Besides assessing the tissue-specific functions for cell therapy applications, it may also be important to test the innate immune function of engrafting cells to ensure adequate defense against infections and improve graft survival. (C) 2015 The Authors. Published by Elsevier B.V.

Cloning and characterization of interferon stimulated genes Viperin and ISG15, and their promoters from snakehead Channa argus

By suppression subtractive hybridization, rapid amplification of cDNA ends and gene walking methods, interferon stimulated genes (ISGs), Viperin and ISG15, and their promoters have been cloned and characterized from snakehead Channa argus. The Viperin cDNA was found to be 1474 nt and contain an open reading frame (ORF) of 1059 nt that translates into a putative peptide of 352 amino acid (aa). The putative peptide of Viperin shows high identity to that in teleosts and mammals except for the N-terminal 70 aa. The ISG15 cDNA was found to be 758 nt and contain an ORF of 468 nt that translates into a putative peptide of 155 aa. The putative peptide of ISG15 is composed of two tandem repeats of ubiquitin-like (UBL) domains, and a canonical conjugation motif (LRGG) at C-terminal. Viperin and ISG15 promoter regions were characterized by the presence of interferon stimulating response elements (ISRE) and gamma-IFN activation sites (GAS). ISRE is a feature of IFN-induced gene promoter and partially overlaps interferon regulatory factor (IRF) 1 and IRF2 recognition sites. GAS is responsible for the gamma-IFN mediated transcription. One conserved site for NF-kappa B was found in the promoter region of Viperin. This is the first report of conservative binding motif for NF-kappa B in accordance with the consensus sequence (GGGRN-NYYCC) among teleost ISG promoters. Moreover, there were also TATA, CAAT and Sp1 transcription factor sites in Viperin and ISG15 promoters. In 5' untranslated region (UTR), snakehead ISG15 gene contains a single intron, which differs from Viperin gene. The transcripts of Vipeirn and ISG15 mRNA were mainly expressed in head kidney, posterior kidney, spleen and gill. The expression levels in liver were found to increase obviously in response to induction by IFN-inducer poly I : C.

The innate immune response to grass carp hemorrhagic virus (GCHV) in cultured Carassius auratus blastulae (CAB) cells

Virus infection of mammalian cells activates an innate antiviral immune response characterized by production of interferon (IFN) and the subsequent transcriptional upregulation of IFN-stimulated genes (ISGs) by the JAK-STAT signaling pathway. Here, we report that a fish cell line, crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells, can produce IFN activity and then form an antiviral state after infection with UV-inactivated grass carp hemorrhagic virus (GCHV), a double-stranded (ds) RNA virus. From UV-inactivated GCHV-infected CAB cells, 15 pivotal genes were cloned and sequenced, and all of them were shown to be involved in IFN antiviral innate immune response. These IFN system genes include the dsRNA signal sensing factor TLR3, IFN, IFN signal transduction factor STAT1, IFN regulatory factor IRF7, putative IFN antiviral effectors Mx1, Mx2, PKR-like, Viperin, IFI56, and other IFN stimulated genes (ISGs) IFI58, ISG15-1, ISG15-2, USP18, Gig1 and Gig2. The identified fish IFN system genes were highly induced by active GCHV, UV-inactivated GCHV, CAB IFN or poly(I).poly(C), and showed similar expression patterns to mammals. The data indicate that an IFN antiviral innate immune response similar to that in mammals exists in the UV-inactivated GCHV-infected CAB cells, and the IFN response contributes to the formation of an antiviral state probably through JAK-STAT signaling pathway. This study provides strong evidence for existence of IFN antiviral innate immune response in fish, and will assist in elucidating the origin and evolution of vertebrate IFN system. (c) 2006 Elsevier Ltd. All rights reserved.

Identification of two novel interferon-stimulated genes from cultured CAB cells induced by UV-inactivated grass carp hemorrhage virus

Interferon (IFN) exerts its antiviral effect by inducing the expression of a number of IFN-stimulated genes (ISGs) to establish a host antiviral state. Earlier studies identified some important fish IFN system genes from IFN-induced CAB cells (crucian carp Carassius auratus L. embryonic blastulae cells) after treatment with UV-inactivated GCHV (grass carp hemorrhage virus). Herein, the cloning of 2 novel IFN-stimulated genes, termed Gig1 and Gig2, is described for the same cell system. The complete cDNA sequences of Gig1 and Gig2 contain 1244 bp encoding for a 194-amino-acid protein and 693 bp for a 158-amino-acid protein, respectively. A search of public databases revealed that these are 2 novel IFN-stimulated genes, since neither significant homologous genes nor conserved motifs were identified. Active GCHV, UV-inactivated GCHV and CAB IFN-containing supernatant (ICS) induced transcription of these genes and distinct kinetics were observed. An analysis of differences in expression between the 2 genes and the IFN signal factors CaSTAT1 and CaIRF7 indicated that GCHV infection activated different signal pathways for their up-regulation. Upon virus infection, the transcription of Gig1 but not of Gig2 is strongly suppressed by cycloheximide (CHX). In contrast, following treatment with CAB IFN-containing supernatant, CHX does not inhibit either gene transcription. The results suggest that GCHV infection can induce expression of both Gig1 and Gig2 via newly synthesized CAB IFN, most probably through the JAK-STAT signal pathway, and can also directly activate Gig2 transcription without ongoing protein synthesis.

Identification and expression analysis of two IFN-inducible genes in crucian carp (Carassius auratus L.)

Interferon (IFN) exerts its antiviral effects mainly through activation of a subset of IFN-stimulated genes (ISG), but relatively few of fish ISGs have been isolated and characterized so far. Here, we report two fish ISGs, termed CaIF158 and CaIF156, cloned from a subtractive cDNA library constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. Database search revealed that both ISGs had a high-level homology with all members of a well conserved gene family with multiple tetratricopeptide repeat (TPR) motifs, including human IF160, IF158, IF156, IFI54 and their homologues in some other mammalian species. The transcripts of CaIF158 and CaIF156 were undetectable in CAB cells but could be induced by active GCHV, UV-inactivated GCHV or CAB IFN. Analysis of expression difference between them and IFN signal factors, CaSTAT1 and CaIRF7, indicated that their transcriptions were mediated possibly through JAK-STAT signal pathway, which was further supported by the induction analysis in UV-inactivated GCHV infected, IFN-treated and untreated cells in the presence or absence of cycloheximide (CHX), a potent inhibitor of protein synthesis. In addition, a pufferfish (Fugu rubrides) DNA sequence representing putative FrIFI56 was also revealed when CalF158 and CalF156 were used to search the pufferfish genome database. Phylogenetic analysis showed that these fish ISGs form a unique clad independent of mammalian homologues, reflecting a distant evolutionary relationship from mammals. These studies identified the first teleost IFI56 and IFI58 orthologues. (C) 2003 Elsevier B.V All rights reserved.

An unbiased genetic screen reveals the polygenic nature of the influenza virus anti-interferon response

UNLABELLED: Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, interferon (IFN)-dependent upregulation of IFN-stimulated genes (ISGs), and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterized populations and selected mutants of wild-type viruses selected by passage through non-IFN-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that nonsynonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterization induced IFN and ISGs and were unable to counteract the effects of exogenous IFN, yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs but nonetheless replicated to high titers. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in non-IFN-responsive cells allows the virus to relax from these constraints and find a new genome consensus within its sequence space.

IMPORTANCE: In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses, and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes to interferon evasion have not been well characterized. Here, we examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titers had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine-tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response.

IFN stimulated gene expression in the liver is a better predictor of treatment response in chronic hepatitis c than the IL28b (IFN lambda 3) genotype

Background: Therapy of chronic hepatitis C (CHC) with pegIFNa/ribavirin achieves sustained virologic response (SVR) in ~55%. Pre-activation of the endogenous interferon system in the liver is associated non-response (NR). Recently, genome-wide association studies described associations of allelic variants near the IL28B (IFNλ3) gene with treatment response and with spontaneous clearance of the virus. We investigated if the IL28B genotype determines the constitutive expression of IFN stimulated genes (ISGs) in the liver of patients with CHC. Methods: We genotyped 93 patients with CHC for 3 IL28B single nucleotide polymorphisms (SNPs, rs12979860, rs8099917, rs12980275), extracted RNA from their liver biopsies and quantified the expression of IL28B and of 8 previously identified classifier genes which discriminate between SVR and NR (IFI44L, RSAD2, ISG15, IFI22, LAMP3, OAS3, LGALS3BP and HTATIP2). Decision tree ensembles in the form of a random forest classifier were used to calculate the relative predictive power of these different variables in a multivariate analysis. Results: The minor IL28B allele (bad risk for treatment response) was significantly associated with increased expression of ISGs, and, unexpectedly, with decreased expression of IL28B. Stratification of the patients into SVR and NR revealed that ISG expression was conditionally independent from the IL28B genotype, i.e. there was an increased expression of ISGs in NR compared to SVR irrespective of the IL28B genotype. The random forest feature score (RFFS) identified IFI27 (RFFS = 2.93), RSAD2 (1.88) and HTATIP2 (1.50) expression and the HCV genotype (1.62) as the strongest predictors of treatment response. ROC curves of the IL28B SNPs showed an AUC of 0.66 with an error rate (ERR) of 0.38. A classifier with the 3 best classifying genes showed an excellent test performance with an AUC of 0.94 and ERR of 0.15. The addition of IL28B genotype information did not improve the predictive power of the 3-gene classifier. Conclusions: IL28B genotype and hepatic ISG expression are conditionally independent predictors of treatment response in CHC. There is no direct link between altered IFNλ3 expression and pre-activation of the endogenous system in the liver. Hepatic ISG expression is by far the better predictor for treatment response than IL28B genotype.

Mécanismes de subversion de l'immunité innée par le virus de l'Hépatite C (VHC)

L'hépatite C pose un problème de santé publique majeur, dans la mesure où le risque de développer une infection chronique est relativement élevé (40 à 60%) et où la résistance au traitement de choix - l’interféron alpha pégylé et la ribavirine - touche près de la moitié des patients. Cette persistence virale repose avant tout sur de puissantes stratégies d’évasion du système immunitaire inné de l’hôte par le virus. Dans ce projet, nous nous sommes intéressés à la caractérisation de la réponse antivirale dans des hépatocytes primaires humains normaux et chroniquement infectés avec le VHC, un domaine encore largement inconnu dû à la difficulté d’obtenir ce type de matériel primaire. Nous avons étudié la fonctionnalité de deux voies majeures de détection des pathogènes viraux suite à l’exposition d’hépatocytes primaires humains à de l’ARNdb intracellulaire, via le récepteur et adaptateur RIG-I/MDA5-CARDIF, et extracellulaire via TLR3-TRIF, mimant ainsi les étapes précoces de la détection d’un virus par la cellule hôte. Nous avons établi par RT-PCR quantitatif et analyse transcriptomique par microarray, que ces deux voies de stimulation sont fonctionnelles dans des hépatocytes primaires normaux et que leur activation entraîne à la fois l’expression de gènes antiviraux communs (ISG56, ISG15, CXCL10, …) mais aussi spécifiques avec les gènes IL28A, IL28B et IL29 qui sont une signature de l’activation de la voie de détection de l’ARNdb intracellulaire. La protéine virale NS3/4A joue un rôle majeur à la fois dans le clivage de la polyprotéine virale initiale, mais aussi en interférant avec les cascades de signalisation engagées suite à la détection par la cellule hôte de l’ARN du VHC. Plus particulièrement, nous avons démontré que l’expression ectopique de NS3/4A dans des hépatocytes primaires humains normaux entraîne une diminution significative de l’induction des gènes antiviraux dûe au clivage de CARDIF au cours de l’activation de la voie de signalisation médiée par RIG-I. Nous avons également démontré que l’expression de la NS3/4A entraîne des modifications de l’expression de gènes-clé impliqués dans la régulation de l’apoptose et du programme de mort cellulaire, en particulier lorsque la voie TLR3 est induite. L’ensemble de ces effets sont abolis en présence de BILN2061, inhibiteur spécifique de NS3/4A. Malgré les stratégies de subversion de l’immunité innée par le VHC, nous avons démontré l’induction significative de plusieurs ISGs et chemokines dans des hepatocytes primaires provenant de patients chroniquement infectés avec le VHC, sans toutefois détecter d’interférons de type I, III ou certains gènes antiviraux précoces comme CCL5. Ces observations, concomitantes avec une diminution de l’expression de CARDIF et une correlation inverse entre les niveaux d’ARNm des ISGs et l’ARN viral révèlent une réponse antivirale partielle dûe à des mécanismes interférents sous-jacents. Cette réponse antivirale détectable mais inefficace est à mettre en lien avec l’échec du traitement classique PEG-IFN-ribavirine chez la moitié des patients traités, mais aussi en lien avec l’inflammation chronique et les dommages hépatiques qui mènent ultimement au développement d’une fibrose puis d’une cirrhose chez une grande proportion de patients chroniquement infectés.

Caractérisation de la voie d'activation des interférons de type I

Codirecteur de recherche: Dr Sylvain Meloche

Atuação do interferon-tau no reconhecimento materno da gestação

Maternal recognition of pregnancy is a process by which the conceptus signals its presence to the mother in order to prolong the life of the corpus luteum (CL) thus maintaining the pregnancy. This process occurs between days 15 and 19 after fertilization and is the most important biological challenge for obtaining satisfactory reproductive indices in bovine. Interferon-tau (IFN-τ) glycoprotein -secreted in the uterus by the conceptus- has a paracrine action inhibiting the expression of estrogen receptors (ESR1) and oxytocin (OXTR) in the endometrium, thus preventing the release of luteolytic pulses of prostaglandin F2 alpha (PGF2α), hormone responsible for the onset of luteolysis. IFN-τ also increases the expression of several interferon-stimulated genes (ISGs) in the uterus, CL, and blood cells. Direct endocrine action of IFN-τ on extrauterine tissues stimulates ISGs expression, which in the corpus luteum seems to be involved with luteal resistance to luteolytic action of PGF2α. This review discusses recent findings on the luteolysis mechanism in the bovine and endocrine and paracrine mechanisms such as IFN-τ during the maternal recognition of pregnancy.

Anti-HIV state but not apoptosis depends on IFN signature in CD4+ T cells

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4(+) T cell target population, we examined gene expression in CD4(+) T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-alpha-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-alphaR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-alpha. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4(+) T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4(+) T cells, which induces an anti-HIV state but not apoptosis.

Interferon response of the cystic fibrosis bronchial epithelium to major and minor group rhinovirus infection

Rhinoviruses (RVs) are associated with exacerbations of cystic fibrosis (CF), asthma and COPD. There is growing evidence suggesting the involvement of the interferon (IFN) pathway in RV-associated morbidity in asthma and COPD. The mechanisms of RV-triggered exacerbations in CF are poorly understood. In a pilot study, we assessed the antiviral response of CF and healthy bronchial epithelial cells (BECs) to RV infection, we measured the levels of IFNs, pattern recognition receptors (PRRs) and IFN-stimulated genes (ISGs) upon infection with major and minor group RVs and poly(IC) stimulation. Major group RV infection of CF BECs resulted in a trend towards a diminished IFN response at the level of IFNs, PRRs and ISGs in comparison to healthy BECs. Contrary to major group RV, the IFN pathway induction upon minor group RV infection was significantly increased at the level of IFNs and PRRs in CF BECs compared to healthy BECs.

Distinct Molecular Events during Secretory Granule Biogenesis Revealed by Sensitivities to Brefeldin A

The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their temporal order and interrelationships, we have developed a pulse–chase protocol that follows the synthesis and packaging of a sulfated hormone, pro-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated into POMC predominantly on N-linked endoglycosidase H-resistant oligosaccharides. Subcellular fractionation and pharmacological studies confirm that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sulfation, POMC undergoes a number of molecular events before final storage in dense-core granules. The first step involves the transfer of POMC from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20°C block prevents its proteolytic conversion to mature adrenocorticotropic hormone. Proteolytic cleavage products were found in vesicular fractions corresponding to ISGs, suggesting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little unregulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage event within the maturing granule. The second step of granule biogenesis thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregulated release to one that favors intracellular storage. By using BFA, we show that the two processes occurring in ISGs may be uncoupled: although the unregulated secretion from ISGs is impaired by BFA, proteolytic processing of POMC within this organelle proceeds unaffected. The finding that BFA impairs constitutive secretion from both the TGN and ISGs also suggests that these secretory processes may be related in mechanism. Finally, our data indicate that the unusually high levels of unregulated secretion often associated with endocrine tumors may result, at least in part, from inefficient storage of secretory products at the level of ISGs.

Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays

The pleiotropic activities of interferons (IFNs) are mediated primarily through the transcriptional regulation of many downstream effector genes. The mRNA profiles from IFN-α, -β, or -γ treatments of the human fibrosarcoma cell line, HT1080, were determined by using oligonucleotide arrays with probe sets corresponding to more than 6,800 human genes. Among these were transcripts for known IFN-stimulated genes (ISGs), the expression of which were consistent with previous studies in which the particular ISG was characterized as responsive to either Type I (α, β) or Type II (γ) IFNs, or both. Importantly, many novel IFN-stimulated genes were identified that were diverse in their known biological functions. For instance, several novel ISGs were identified that are implicated in apoptosis (including RAP46/Bag-1, phospholipid scramblase, and hypoxia inducible factor-1α). Furthermore, several IFN-repressed genes also were identified. These results demonstrate the usefulness of oligonucleotide arrays in monitoring mammalian gene expression on a broad and unprecedented scale. In particular, these findings provide insights into the basic mechanisms of IFN actions and ultimately may contribute to better therapeutic uses for IFNs.