Interferon type I gene expression in chronic hepatitis C

Interferon type I gene expression in chronichepatitis C

Sabine Mihm1, Michael Frese2, Volker Meier1, Perdita Wietzke-Braun1, Jens-Gerd Scharf1,Ralf Bartenschlager2 and Giuliano Ramadori1

1Division of Gastroenterology and Endocrinology, Department of Internal Medicine, Georg-August-Universitat, Gottingen, Germany and 2Department of Molecular Virology, Institute of Hygiene, University ofHeidelberg, Heidelberg, Germany

Hepatitis C virus (HCV) frequently causes chronic liver disease. The cause of viral persistence might be aninappropriate type I interferon (IFN) induction. To analyze the host’s IFN response in chronic hepatitis C, wemeasured the transcription level of type I IFN genes as well as type I IFN-regulated genes in livertissue and corresponding blood samples from patients with chronic hepatitis C, nonviral liver diseases, anda suspected but later excluded liver disease. Competitive and real-time RT-PCR assays were used to quantifythe messenger RNA (mRNA) levels of all known IFN-a, IFN-b, and IFN-k genes and those of some IFN-regulatedgenes. We failed to detect any hepatic type I IFN mRNA induction, although liver tissue of chronichepatitis C patients contained high numbers of some type I IFN-inducible effector mRNA molecules. Analysisof peripheral blood samples, however, showed a clear type I IFN induction. Parallel experiments employing HCVreplicon cell lines revealed that replication of HCV RNA is not sufficient to induce any type I IFN nor toinduce directly type I IFN-regulated genes such as MxA. In conclusion, our data provide evidence for theabsence of an induction of type I IFN genes by HCV in the human liver and argue for a further development oftype I IFN-based therapies.Laboratory Investigation (2004) 84, 1148–1159, advance online publication, 21 June 2004; doi:10.1038/labinvest.3700135

Keywords: HCV replicons; IFN-a; IFN-b; IFN-l; MxA

Hepatitis C virus (HCV) infection is a major cause ofchronic liver disease, which in many cases proceedsto fatal liver cirrhosis and hepatocellular carcinoma.Worldwide, an estimated 170 million people arepersistently infected with the virus,1 indicating thatHCV is able to escape innate immune responsemechansims. Currently, hepatitis C patients aretreated with interferon alpha (IFN-a) given eitheralone or in combination with the nucleoside analogribavirin. Owing to improved application schemes,now up to half of the patients benefit from IFN-a-based therapies as evidenced by sustained viruseradication.2

IFNs are divided into type I and type II IFNs. Thetype I genes encode 13 closely related IFN-asubtypes, IFN-b, and the more distantly relatedIFN-k, IFN-o, and IFN-e.3 The product of the only

type II gene, IFN-g, exhibits no sequence homologyto other IFNs but possesses some of their biologicalproperties (see below). Most recently, by genomicscreening, a novel cytokine family has been dis-covered sharing sequence similarities with type IIFNs and the IL-10 family. Accordingly, the mem-bers of this family have been named IFN-l1, IFN-l2,and IFN-l3

4 or IL-29, IL-28A, and IL-28B,5 respec-tively. Like type I IFNs, they are induced by viralinfections and confer antiviral activity.4,5

A hallmark of all IFNs is their ability to enhancethe expression of numerous genes.3 Some of thesegenes encode effector proteins with antiviral activ-ities. An example is MxA, a cytoplasmic GTPasethat inhibits the replication of several RNA viruses.6

Other effector proteins are the double-strandedRNA-dependent protein kinase (PKR) or membersof the family of 20,50oligoadenylate synthetases.3 Incontrast to MxA, their activation requires binding todouble-stranded RNA. While the activation of PKRleads to a translational arrest through phosphoryla-tion of an essential translation initiation factor, theactivation of 20,50oligoadenylate synthetases resultsin the production of small oligoadenylates. These inturn activate the endogenous ribonuclease RNase L,

Received 4 March 2004; revised 28 April 2004; accepted 29 April2004; published online 21 June 2004

Correspondence: Dr S Mihm, PhD, Division of Gastroenterologyand Endocrinology, Department of Internal Medicine, Georg-August-Universitat, Robert-Koch-Stra�e 40, 37075 Gottingen,Germany.E-mail: [email protected]

Laboratory Investigation (2004) 84, 1148–1159& 2004 USCAP, Inc All rights reserved 0023-6837/04 $30.00

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which subsequently cleaves ssRNAs includingmRNAs of viral and host cell origin.

Although both types of IFNs induce the expres-sion of a partially overlapping set of effectorproteins, experiments with genetically targeted micelacking either the type I or type II IFN receptordemonstrate that type I IFNs mediate most of theearly innate immune response against viruses.7 Incontrast, the contribution of IFN-g to virus elimina-tion is believed to be primarily systemic, forexample, by enhancing the expression of proteinsinvolved in antigen processing and presentationsuch as proteasome subunits and major histocom-patibility complex molecules. Furthermore, IFN-gregulates the expression of chemokines thatsupport the adaptive immunity. Another differencebetween the two types of IFNs is their origin. Type IIFNs are produced by many, if not all, virus-infectedcells. However, there is evidence that some celltypes such as hepatocytes are poor IFN producers,8

whereas a certain blood leukocyte population,termed natural IFN-producing cells, is specializedfor the production of high amounts of type IIFNs (reviewed in Colonna et al9). In contrastto type I IFNs, the expression of IFN-g is restrictedto activated T lymphocytes and natural killercells.

Recently, it has been demonstrated that both typesof IFNs efficiently inhibit HCV replication in thehuman hepatoma cell line Huh-7.10–13 Several IFN-induced proteins have been analyzed for theirability to interfere with HCV replication, but effectorproteins that inhibit HCV replication have not yetbeen identified.11,14 A previous approach comparinggene expression in HCV-infected liver tissues withthat in noninfected livers by using the technique ofsuppression subtractive hybridization revealed anenhanced (ie greater than five-fold) expression ofsome IFN-regulated genes in the HCV-infectedlivers.15 Among these are the type I IFN-regulatedgenes MxA, interferon inducible protein 44 (IFI-44),and interferon inducible protein 56 (IFI-56K). Onthe one hand, these data indicate that especiallythose genes that are upregulated in chronic hepatitisC might not be those that are capable to inhibit HCVreplication sufficiently. On the other hand, thesedata indicate ongoing type I IFN-regulated processesand suggest that HCV might induce the expressionof type I IFNs in liver cells.

Our current study addresses the question whetheror not HCV replication triggers the induction ofendogenous type I IFNs. We thus analyzed theexpression of all known IFN-a, IFN-b, and IFN-lgenes in human liver tissue biopsy samples andmatching blood samples from patients with chronichepatitis C. To study the effect of virus replicationon type I IFN and on type I IFN-regulated geneexpression more directly in a homogenous hepato-cyte-derived cell population, a number of Huh-7 celllines containing HCV replicons were employed inparallel.

Materials and methods

Patients

As part of a routine clinical evaluation, liver biopsyprocedures from a total of 60 consecutive out-patients with chronic hepatitis C (n¼ 39; 19 female,20 male; mean age 44.6 years, range 18–67), andnonviral liver diseases (n¼ 12; six female, six male;mean age 45 years, range 27–63) were performed.Additionally, liver biopsy specimens were obtainedfrom patients with a suspected but later excludedliver disease that thus can be referred to as ‘healthyliver controls’ (n¼ 9; four female, five male; meanage 52 years, range 29–67). Chronic HCV infectionwas diagnosed by the detection of HCV-specificantibodies and HCV RNAs in the patient’s serum.Liver disease was confirmed histopathologically asdescribed.16,17 Inflammatory activity was estimatedto be mild in 15 patients, moderate in 19, and severein 4. Histopathological findings and diagnoses forpatients with nonviral liver diseases comprisedprimary biliary cirrhosis (n¼ 2), steatosis (n¼ 4),cryptogenic hypodense liver foci (n¼ 1), hydropicswelling of hepatocytes (n¼ 1), cryptogenic cirrho-sis (n¼ 2), primary sclerosing cholangitis (n¼ 1),and Stauffer’s syndrome (n¼ 1). Diagnoses of pa-tients with the absence of any hepatic histopatholo-gical condition included non-Hodgkin’s lymphoma(n¼ 1), dermatomyositis (n¼ 1), gastric cancer(n¼ 1), and mesenterial cyst (n¼ 1). Additionalsamples were taken to rule out focal nodularhyperplasia (n¼ 1), and other liver diseases inpatients who showed slightly elevated serum trans-aminase activities (n¼ 4). Patients with concomitantnon-C viral infections and those with continuedalcohol or other drug abuse were excluded. In-formed consent was obtained from each patient. Thestudy was approved by the local ethical committeeof the Georg-August-University, Gottingen and con-formed to the ethical guidelines of the 1975Declaration of Helsinki.

Cells and Viruses

The human hepatoma cell line Huh-7 has beenoriginally described by Nakabayashi et al.18 TheHuh-7 cell clones 5-15 and 9-13 (containing thesubgenomic HCV replicons I389/NS3-30 and I377/NS3-30, respectively) and the cell clones 20-1 and 21-5(containing the genomic replicon I389/Core-30/5.1)were previously described.19–22 Maps showing thegenetic organization of the replicons used in thisstudy have already been published.13 The Huh-7 cellclones Neo 3/1 and Neo 3/2 have been generatedafter transfection of naive Huh-7 cells with pcDNA3(Invitrogen, Karlsruhe, Germany). Human 293 cellswere kindly provided by Friedemann Weber (Uni-versity of Freiburg, Germany). All cells were main-tained in Dulbecco’s modified Eagle’s mediumsupplemented with 10% fetal calf serum, 200 U/ml

Endogenous type I interferon in hepatitis CS Mihm et al

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Laboratory Investigation (2004) 84, 1148–1159

of penicillin G, and 200 mg/ml of streptomycin. Forcells that have been transfected with HCV repliconsor pcDNA3, the culture medium was additionallysupplemented with 0.25–1 mg/ml of G418.

Newcastle disease virus (NDV) strain H53 (kindlyprovided by Peter Staeheli, University of Freiburg,Germany) was propagated by inoculating the allan-toic cavity of 10-day-old embryonated chicken eggs.Stock virus contained 8.8� 108 50% tissue cultureinfective doses per ml as determined on CEC32 cells(kindly provided by P Staeheli).

Preparation of Peripheral Blood Mononuclear Cells

Heparinized peripheral blood samples were takenon the same day when liver biopsy procedures wereperformed. Peripheral blood mononuclear cells(PBMCs) obtained from approximately 30 ml ofperipheral blood were isolated by Ficoll densitycentrifugation and dextran sedimentation accordingto Boyum.23 Cell preparations were routinely as-sessed for viability (495%) by trypan blue exclu-sion. In a typical experiment, a PBMC preparationconsisted of more than 98% lymphocytes/mono-cytes and less than 2% granulocytes as determinedby the morphology of cells stained according toPappenheim.

Isolation of Total Cellular RNA

Total cellular RNA was prepared by CsCl density-gradient ultracentrifugation using guanidinium iso-thiocyanate24,25 essentially as described.26 Startingmaterial was homogenized liver tissue, PBMCs, orHuh-7 cells containing HCV replicons. Finally, RNAwas dissolved in distilled water and the RNAconcentration was determined photometrically.

Quantification of Gene Expression by CompetitiveRT-PCR

An amount of 2 mg of total cellular RNA was reversetranscribed as described previously.26 Quantifica-tion of cDNAs corresponding to transcripts ofinterest were performed by using internal cDNAstandards (IS).26 In brief, IS were constructed to becomplementary to and to compete with oligonucleo-tide primers and for amplification of target se-quences. Target cDNAs were amplified in thepresence of 10- and two-fold serial dilutions of theIS. The amount of target transcripts was thencalculated on the basis of the known molecularquantity of the IS and related to the amount of areference mRNA (albumin, b-actin, or glycerinalde-hydephosphate dehydrogenase (GAPDH)), whichhad been quantified in parallel (Figure 1). Primersequences, annealing temperature, the number of

Figure 1 Analysis of transcript expression by quantitative RT-PCR. cDNAs corresponding to 32 ng of total cellular RNA were coamplifiedwith decreasing amounts of an IS in 10-fold dilution series. Concentrations of the IS (amol/reaction) are given above the lanes for IFN-aand b-actin (upper left and lower left panels, respectively). Based on the amount of the IS that yielded a stronger amplification signal thanthe corresponding target cDNA (arrows in the left panels), a second round of reactions was carried out in which the IS was titrated bytwo-fold dilutions. IS concentrations that yielded amplification products of comparable intensities as determined by visualization, orvalues in-between (arrows in the right panels) were taken as a basis for subsequent calculations. In the example shown, the ratio of IFN-a(0.0000935 amol) to b-actin transcripts (0.75 amol) is 0.12� 10�3.


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cycles carried out, the size of the target amplifica-tion product, and the size of the IS amplificationproduct are given in Table 1.

Quantification of Gene Expression by Real-Time PCR

A measure of 2 mg of total cellular RNA was used forreverse transcription as described above except thatrandom hexamers (6 mM) were used for priming.Complementary DNAs (cDNAs) derived from livertissue (corresponding to 32 ng of RNA) or PBMC orHuh-7 cell preparations (corresponding to 8 ng ofRNA) were amplified using the ABI Prism 7000sequence detection system. Quantification of IFN-a2,IFN-a8, IFN-a21, IFN-b, MxA, and GAPDH cDNAswas performed using the 50 nuclease assay-basedAssays on Demand (Applied Biosystems, Darmstadt,Germany) according to the supplier’s instructions.The amount of albumin, IFN-l1, and IFN-l2/l3

cDNAs was determined by using primer and 6-carboxy-fluorescein-amedit (FAM)-labeled MinorGroove Binder (MGB) probes that had been designedby the PrimerExpress software (Applied Biosystems)(albumin: forward 50-AAC ACA AAG ATG ACAACC CAA ACC-30, reverse 50-GCA GTG CAC ATCACA TCA ACC T-30, probe 50-CCC CCG ATT GGT G-30; IFN-l1: forward 50-CAC GCG AGA CCT CAA ATATGT G-30, reverse 50-AGG GTG GGT TGA CGT TCTCA-30, probe 50-CCG ATG GGA ACC TG-30; IFN-l2/l3:forward 50-GCC ACA TAG CCC AGT TCA AGT C-30;reverse 50-GGC ATC TTT GGC CCT CTT AAA-30,probe 50-CTC CAC AGG AGC TGC-30). The amountof HCV RNA in relation to a cellular referencetranscript was determined using a primer pair fromthe 50 UTR and a mixture of two FAM-labeledTAMRA TaqMan probes that recognize most of theviral subtypes with comparable amplification effi-ciency (forward 50-CAG AAA GCG TCT AGC CAT

GG-30, reverse 50-CGC AGA CCA CTA TGG CTC TC-30, probe 1a/3 50-TAG TAY GAG TGT CGT GCA GCCTCC AGG-30, probe 1b 50-TTA GTA TGA GTG TTGTGC AGC CTC CAG G-30).27 Amplification of b-actincDNA was monitored in the presence of SYBR GreenI dye (Applied Biosystems) (forward 50-AAC ACAAAG ATG ACA ACC CAA ACC-30, reverse 50-GCAGTG CAC ATC ACA TCA ACC T-30). Fractionalthreshold cycles (CT) expressing the initial concen-tration of target sequence were determined accord-ing to the supplier’s guidelines. Relative mRNAquantification was calculated by using the arith-metic formula 2�DCT. Note that DCT is the differencebetween the CT of a given target cDNA and anendogenous reference cDNA. Thus, this value yieldsthe amount of the target normalized to an endogen-ous reference. For instance, a DCT of �10 means thatthe ratio of target to reference mRNAs is 2�10 or1:1024.

IFN Promoter Analysis

Cells were seeded into six-well plates and the nextday, monolayers of about 80% confluency werecotransfected with 1mg of plasmid paLuc or p-125Luc (kindly provided by Takashi Fujita, TheTokyo Metropolitan Institute of Medical Science,Tokyo, Japan) and 0.1 mg of pRL-SV40 (Promega,Madison, WI, USA) by using Optimem (Life Tech-nologies, Gaithersburg, MD, USA) and the DAC-30transfection reagent (Eurogentec, Seraing, Belgium).Note that the reporter plasmids paLuc and p-125Luccontain the firefly luciferase gene under control ofan IFN-a and IFN-b promoter enhancer element,respectively,28 whereas pRL-SV40 contains the Re-nilla luciferase gene under control of the constitu-tive simian virus 40 (SV40) promoter. At 12 h aftertransfection, cells were either incubated with NDV

Table 1 Specifications for quantitative, competitive RT-PCR assays

Gene Primer for competitive RT-PCR Tm (1C)a Number of cycles Size of product (bp)

mRNA IS

IFN-a 50-GAAGCTTYCTCCTGYYTGAWGGACAGA-30 68 36 372 50650-GGGGATCCTCTGACAACCTCCCANGCACA-30

IFN-b 50-TTTCAGTGTCAGAAGCTCCT-30 60 41 364 47650-TGGCCTTCAGGTAATGCAGA-30

MxA 50-CTGTGGCCATACTGCCAGGA-30 61 30 482 30250-ACTCCTGACAGTGCCTCCAA-30

PKR 50-CAGGCACGACAAGCATAGAA-30 60 36 430 32050-CTACTCCCTGCTTCTGACGG-30

RNase L 50-GCAGAAATGCCTTGATCCAT-30 60 36 402 26050-AGTCTTCAGCAGGAGGGTGA-30

b-Actin 50-GTGGGGCGCCCCAGGCACCA-30 62 28 538 37050-CTCCTTAATGTCACGCACGAT-30

GAPDH 50-ACCACAGTCCATGCCATCAC-30 59 30 452 26050-TCCACCACCCTGTTGCTGTA-30

Albumin 50-CTTGAATGTGCTGATGACAGG-30 58 28 157 22350-GCAAGTCAGCAGGCATCTCATC-30

aMelting temperature.


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(multiplicity of infection¼ 2) or left untreated. After18 h of further cultivation, cells were lysed andluciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madi-son, WI, USA). Finally, firefly luciferase readingswere divided by the corresponding Renilla lucifer-ase activities to correct for differences in transfec-tion efficiency.

Statistical Analysis

Statistics were calculated by using the PC-STATIS-TIK software package version 4.0 (Hoffmann-Soft-ware, Giessen, Germany). Gaussian distributed datawere analyzed by parametric t-tests for independentsamples or by analysis of variance (ANOVA). P-values of less than 0.05 were considered statisticallysignificant.

Results

Expression of MxA, IFN-a, IFN-b, and IFN-k in theLiver of Hepatitis C Patients

MxA protein expression is a sensitive biologicalmarker for ongoing virus replication and/or thepresence of type I IFNs.29 It has been shownpreviously that liver biopsy samples of hepatitis Cpatients contain elevated MxA mRNA levels alongwith those of other type I IFN-regulated genes.15,30,31

To find out whether the observed expression of typeI IFN-regulated genes in HCV-infected livers mightbe induced by intrahepatically produced IFNs, wetook liver specimens from patients with chronichepatitis C, liver disorders unrelated to viral infec-tions, and a suspected but later excluded liverdisease and analyzed the samples for the expressionof type I IFN genes and that of type I IFN-regulatedgenes by using quantitative RT-PCR techniques (fordetails, see the Materials and methods section). Inline with previous findings, we found that MxA ishighly expressed in the liver of hepatitis C patients.The median amount of MxA mRNA in samples ofhepatitis C patients was 11- and 15-fold higher thanthat in patients with nonviral liver diseases andhealthy livers, respectively (Figure 2a). Next, wequantified the type I IFN expression by usingdegenerated primers that amplified mRNAs of all13 IFN-a subtypes (subsequently referred to as IFN-an) and specific primers that amplified IFN-bmRNAs. The median IFN/albumin mRNA ratio inthe liver of chronic hepatitis C patients was0.2� 10�3 for IFN-an (Figure 2b) and 0.006� 10�3

for IFN-b (Figure 2c), respectively. These levels didnot differ significantly from those in liver specimensderived from patients with liver disorders unrelatedto viral infections or from those in patients withhealthy livers indicating that HCV replication doesnot trigger the production of type I IFNs in the liver.

To exclude that members of the IFN-l familysubstitute for type I IFNs in HCV-infected livers, arandomly chosen subset of the previously usedbiopsy samples was analyzed for the presence ofIFN-l mRNAs. We used primers that either specifi-cally bind to IFN-l1 transcripts or those thatrecognize an evolutionary conserved sequence pre-sent in IFN-l2 and IFN-l3 mRNAs. Figure 2d and eshows that similar amounts of IFN-l mRNAs weredetected in samples of diseased livers irrespective ofwhether HCV had caused the pathological changes.However, a slight but statistically significant highernumber of IFN-l2/l3 mRNAs was found in HCV-infected liver samples compared to healthy controllivers (Figure 2e). Nevertheless, it is unlikely thatthe slight increase in the expression of IFN-l2 orIFN-l3 accounts for the expression of MxA in HCV-infected tissues. First, the livers of patients withnonviral liver diseases and those infected with HCVboth contain elevated levels of IFN-l2/l3 mRNAs,but only the latter have elevated MxA mRNA levelsalso. Second, data from a previous experiment inwhich we used suppression subtractive hybridiza-tion to compare the gene expression in HCV-infectedliver tissue to that in noninfected tissues of acomparable histopathological status, revealed noevidence for an enhanced intrahepatic expressionof any of the IFN-l genes in hepatitis C patients (RPatzwahl and S Mihm, unpublished observation).Taken together, the results suggest that the activa-tion of IFN-regulated genes in hepatitis C patients isnot associated with an intrahepatic production oftype I IFNs.

Expression of MxA, IFN-a, IFN-b, and IFN-k in PBMCsof Hepatitis C Patients

PBMCs have been reported to comprise a specia-lized population of leukocytes that possess theability to produce high amounts of type I IFNs.9

This knowledge prompted us to investigate whetheran infection with HCV activates the type I IFNexpression in peripheral blood cells. For thisanalysis, we chose all available hepatitis C patients’blood samples that were obtained at the time theliver biopsy was taken (n¼ 12). As controls, bloodsamples were taken from healthy volunteers insteadof patients with healthy livers because the lattersuffered from diseases other than hepatitis C and,therefore, might have an abnormal PBMC geneexpression profile. In accordance with a previousreport,32 we found that MxA mRNA was moderatelybut significantly elevated in PBMCs of chronichepatitis C patients (Figure 3a). Furthermore, RNApreparations of PBMC from hepatitis C patientscontained 23-fold higher levels of IFN-an than thosefrom healthy individuals (Figure 3b). This differ-ence was significant irrespective of whether IFN-an

mRNA levels were related to b-actin or GAPDH asreference transcripts. Similar to IFN-an, PBMCs of


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chronic hepatitis C patients also contained elevatedmRNA levels of IFN-b (Figure 3c), IFN-l1 (Figure3d), IFN-l2/l3 (Figure 3e), and the IFN regulatoryfactor 7 (IRF-7) (data not shown). The data demon-strate that chronic HCV infection activates theexpression of type I IFNs and type I IFN-regulatedgenes in blood cells indicating that the patients’capacity to activate type I IFNs is not generallyimpaired.

Expression of MxA, IFN-a, IFN-b, and IFN-k in Huh-7Cells Containing HCV Replicons

To corroborate the supposition that ongoing HCVreplication does not activate type I IFN genes ininfected hepatocytes, we analyzed the effect of HCVRNA replication on the innate immune response of

Huh-7 cells. In contrast to the situation in vivo, inwhich only a proportion of hepatocytes is virusinfected, the Huh-7 cell culture system for HCVreplication consists of a cell population in whichvirtually all cells contain HCV RNAs.19 In a firstexperiment, we addressed the question whetherHCV RNA replication induces the expression type IIFN mRNAs in cell culture. Total RNA from cellscontaining the subgenomic HCV replicons I389/NS3-30 and I377/NS3-30 (cell clones 5-15 and 9-13,respectively), the genomic replicon I389/Core-30/5.1(cell clones 20-1 and 21-5), and from control cellswithout HCV replicons (naive cells and the neomy-cin-resistant cell clones Neo 3/1 and Neo 3/2) wasused to determine the mRNA levels of several type IIFNs by real-time RT-PCR. Figure 4 shows that cellswith and without HCV replicons contained similaramounts of IFN-a2 (Figure 4a), IFN-a8 (Figure 4b),

Figure 2 Intrahepatic expression of MxA, IFN-a, IFN-b, and IFN-l in hepatitis C patients. Liver biopsy specimens taken from patientswith chronic hepatitis C, nonviral liver diseases, and from those with a suspected but later excluded liver disease were quantified withrespect to (a) MxA mRNAs, (b) all subtypes of IFN-a mRNAs (IFN-an), (c) IFN-b mRNAs, (d) IFN-l1 mRNAs, and (e) IFN-l2/l3 mRNAs inrelation to albumin mRNAs by using quantitative RT-PCR assays. Medians are indicated by horizontal bars. Levels of significance areindicated (ns, not significant). Similar results were obtained when data were related to b-actin or GAPDH (data not shown).


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IFN-a21 (Figure 4c), and IFN-b (Figure 4d). Inaddition, we found that the presence of HCVreplicons does not increase the amount of IFN-ltranscripts (data not shown).

For some IFN-regulated genes, evidence has beenfound that their expression is induced by virusreplication without a preceding type I IFN produc-tion.33–36 These findings prompted us to investigatewhether HCV replication in Huh-7 cells leads to theactivation of IFN-regulated genes even if we couldnot detect an increase in the number of type I IFNmRNAs. As shown in Figure 4e, the presence ofHCV replicons did not cause a general increase inthe amount of MxA mRNAs although we observedthat mRNA levels slightly varied between differentcell clones. To exclude the possibility that differentamounts of HCV RNAs account for the clonaldifferences in MxA expression, the number of viral

nucleic acids was determined, too. Under theexperimental conditions chosen, the number of viralnucleic acids was quite comparable in all Huh-7 cellclones (Figure 4f) suggesting that the presence ofHCV RNAs does not enhance MxA gene expression.Furthermore, we quantified mRNA levels of addi-tional IFN-regulated genes including those thatencode IFI-44, IFI-56K, and PKR. In no case didwe detect an enhanced expression in cells with HCVreplicons (data not shown). Thus, we conclude thatHCV replication has no direct effect on the activa-tion of type I IFN-regulated genes.

As an alternative readout for the activation of typeI IFN genes, we determined the type I IFN promoteractivity in the presence or absence of HCV RNAs.Naive Huh-7 cells, several Huh-7 cell clones with orwithout HCV replicons, and 293 cells were transi-ently transfected with luciferase reporter constructs

Figure 3 Expression of MxA, IFN-a, IFN-b, and IFN-l in PBMCs of hepatitis C patients. PBMCs were isolated from blood samples takenfrom patients with hepatitis C at the time of liver biopsy and from healthy volunteers. RNA was prepared and analyzed for the amount of(a) MxA mRNAs, (b) all subtypes of IFN-an mRNAs, (c) IFN-b mRNAs, (d) IFN-l1 mRNAs, and (e) IFN-l2/l3 mRNAs in relation to b-actinas a reference by quantitative RT-PCR assays. Medians are indicated by horizontal bars. Levels of significance are given (ns, notsignificant).


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containing IFN-a or IFN-b promoter sequences. At12 h after transfection, the cells were infected withNDV or left untreated. After an additional incuba-

tion period of 18 h, luciferase activities weredetermined and normalized for transfection efficien-cies (see Materials and methods for details). Figure 5

Figure 4 Expression of type I IFN genes in Huh-7 cells with and without HCV replicons. Naive Huh-7 cells, those that carry a neomycinresistance gene (cell clones Neo 3/1 and Neo 3/2), those that contain subgenomic replicons (cell clones 5-15 and 9-13), and those thatcontain genomic HCV replicons (cell clones 20-1 and 21-5) were seeded into multiple cell culture dishes and cultivated for 72 h. Cellswere harvested, total RNA was prepared and analyzed for the amount of (a) IFN-a2 mRNAs, (b) IFN-a8 mRNAs, (c) IFN-a21 mRNAs, (d)IFN-b mRNAs, (e) MxA mRNAs, and (f) HCV RNA in relation to b-actin mRNAs using quantitative RT-PCR assays in triplicate. Meanvalues and standard errors of the mean are given. The results of one representative experiment out of three are shown.


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shows that untreated Huh-7 cells exhibited muchlower IFN-a and IFN-b baseline promoter activitiesthan 293 cells. Furthermore, IFN promoter activitiesin Huh-7 cells did not increase in response to thetreatment with NDV. In contrast, 293 cells showed a38- and 25-fold enhancement of their IFN-a and IFN-b promoter activities, respectively, in response to theNDV treatment. These findings indicate that Huh-7cells in general are rather poor IFN producers.Interestingly, the presence of HCV replicons had aweak stimulatory effect on the expression of bothtype I IFN promoter constructs. However, this effectwas only observed with untreated cells and not withNDV-infected cells. The biological relevance of theobserved slight increase in the IFN promoteractivities is not clear since we did not detectincreased amounts of type I IFN mRNAs by real-time RT-PCR (Figure 4a–d). Taken together, the dataobtained by the HCV replicon system are in line

with the weak intrahepatic type I IFN expressionobserved in HCV-infected individuals.

Discussion

The present study demonstrates the absence ofdetectable amounts of type I IFN mRNAs in anumber of individual liver samples of chronichepatitis C patients in spite of an enhanced expres-sion of type I IFN-regulated mRNAs (Figure 2). Theexpression of all known type I IFNs was monitoredincluding those of the recently described IFN-lfamily. Our findings are in line with earlierobservations made on selected single tissue samplesor small pools of samples that had been comparedby DNA chip arrays or by related techniques. Forinstance, the analysis of a subtracted library (tran-scriptome of an HCV-infected liver tissue minus thatof noninfected tissues with similiar histopathologi-cal changes) revealed an enhanced expression ofsome IFN-regulated genes but not that of type I IFNgenes themselves.15 Moreover, when HCV-associatedliver cirrhosis was compared to nondiseased livertissue, a limited gene expression analysis using acDNA microarray of 874 genes revealed the induc-tion of the type I IFN-inducible peptide 6–16 in theabsence of IFN-a2.

37 A more comprehensive oligo-nucleotide microarray analysis of more than 9000genes documented a higher expression of four IFN-inducible genes in HCV-associated hepatocellularcarcinoma specimens than in noninfected nontu-morous liver, but not that of type I IFN genes.38 Amicroarray analysis performed by Honda et al,39 incontrast, revealed an increased intrahepatic expres-sion of IFN-a genes in chronic hepatitis C patients.The deviant findings of this analysis might be due tothe fact that Honda et al compared HCV-infectedliver biopsy samples to uninfected tissue samplessurgically obtained from noncancerous liver parts ofliver tumor patients.

Experimentally infected chimpanzees served asan animal model in several studies on the innateimmune response against HCV. Oligonucleotidemicroarrays revealed that liver biopsy samples ofacute and persistently HCV-infected animals con-tain high amounts of type I IFN-inducedmRNAs.40,41 Elevated type I IFN mRNAs, however,were described only in one report so far.42

In our experiments, we employed a number ofHCV replicons to analyze the effect of HCV replica-tion on the IFN gene expression in cell culture. Wefound that the presence of HCV replicons does notenhance the transcription of type I IFN genes andtype I IFN-regulated genes (Figure 4). Furthermore,ongoing HCV replication had only minor effects onthe expression of a luciferase reporter that iscontrolled by a type I IFN promoter (Figure 5).These findings are in line with data of a most recenttranscriptome analysis, in which we used anoligonucleotide microarray that contains probe sets

Figure 5 IFN-a and IFN-b promoter activity in Huh-7 cells withand without HCV replicons. Naive Huh-7 cells, those that carry aneomycin resistance gene (cell clone Neo 3/1), those that containsubgenomic HCV replicons (cell clone 9-13), those that containgenomic HCV replicons (cell clones 20-1 and 21-5), and 293 cellswere cotransfected with two luciferase reporter plasmids. Oneplasmid encodes the firefly luciferase either under control of an(a) IFN-a or (b) IFN-b promoter. The other plasmid encodes theRenilla luciferase under control of the constitutive SV40 promoter(see Materials and methods for details). At 12 h after transfection,cells were incubated with NDV or left untreated. After 18 h, cellswere lyzed and luciferase activities were determined (measure-ments were taken in duplicate). Columns represent the quotient ofthe firefly and Renilla luciferase readings. The figure shows theresult of a single representative experiment.


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for all known human type I IFN genes to analyze thegene expression in Huh-7 cells with and withoutHCV replicons (M Trippler and M Frese, unpub-lished results). Our finding that the incubation ofHuh-7 cells with NDV is not sufficient to inducetype I IFN mRNAs is in complete agreement withearlier findings made by Keskinen et al8 whoanalyzed IFN production in several human hepato-ma lines after stimulation with various agents andviruses. In HepG2 and Huh-7 cells, for example,they found no detectable IFN-a or IFN-b productionin response to the stimuli applied. Thus, the authorssuggested that hepatoma cells may have an intrinsi-cally poor ability to produce type I IFNs, which maycontribute to their inability to efficiently resist viralinfections. Furthermore, our results are in line witha recent observation of Lanford et al43 who failed todetect IFN-b mRNA induction in naive Huh-7 cellsafter stimulation with poly-IC or in the presence ofHCV replicons. The authors speculated that the lackof IFN-b inducibility in Huh-7 cells is the result of adefect in the signaling pathway for dsRNA. How-ever, the view of poor type I IFN inducibility inhepatoma cells has recently been challenged by Foyet al44 and Fredericksen et al45, who both usedSendai virus (SENV) to successfully trigger the IFNsystem in Huh-7 cells as evidenced by an increasedproduction of IFN-b. Thus, it remains an openquestion whether hepatocytes/hepatoma cells aregenerally impaired in sensing ongoing viral replica-tion.

Interestingly, it has recently been noted thatongoing HCV replication interferes with severallevels of the IFN system as do many other viruses.3

For example, Foy et al reported that the HCVprotease NS3/4A inhibits the phosphorylation ofIRF-3, a transcription factor required for the activa-tion of type I IFN genes. These findings arecompatible with our results in the replicon systemand with the situation in the HCV-infected liver asevidenced by the present study.

The observation that some type I-regulated effec-tor genes are activated in chronic hepatitis C mightbe due to secondary inflammatory events. Thisprocess, however, is obviously not able to terminatethe infection because the induction is either nothigh enough for an efficient virus elimination or theexpressed effector proteins do not affect HCVreplication. In fact, the effector protein that is mostfrequently found in HCV infection, MxA, has beenshown not to inhibit HCV RNA replication inhuman hepatoma cells.11

The finding that type I IFN mRNA molecules areeasily detectable in peripheral blood samples butnot in the liver of chronic hepatitis C patients mightbe due to the selective recruitment of peripheralcells. The liver of hepatitis C patients expressesinterferon-inducible protein 10 (IP-10), a chemokinethat attracts monocytes and activated T-helpercells.15,46–48 The main producers of type I IFNs,however, are the so-called natural IFN producing

cells (IPCs) or plasmacytoid dendritic cells (pDCs).9

Despite expression of very high levels of the IP-10receptor CXCR3, natural IFN producing cells do notrespond efficiently to CXCR3 ligands.49 Uponactivation, the CXCR3 appears to be nonfunctional.On the other hand, activated natural IFN-producingcells do have a propensity to migrate to secondarylymphoid organs, rather than to sites of inflamma-tion.50 This behavior might be due to the expressionof the lymph node homing molecule 1-selectin.50

The absence of detectable hepatic type I IFN mRNAmolecules substantiates the rationale for applyingtype I IFNs. Although being not yet as effective asdesired, improvements of application schedules inthe past clearly led to improvements of therapyresponse. Thus, our data argue for further develop-ment of type I IFN-based therapies.

Acknowledgements

We thank all physicians of the Division of Gastro-enterology who were involved in liver biopsyprocedures and patients’ care and control for theirkind cooperation, Friedemann Weber, Peter Staehe-li, and Takashi Fujita for reagents and helpfuldiscussions, Waltraut Kopp and Ulrike Herian forexpert technical assistance, and Kerry Mills forcritical reading of the manuscript. Supporting grantswere obtained from the Deutsche Forschungsge-meinschaft (SFB402/C1 and SFB 402/C6) and theBundesministerium fur Bildung und Forschung(Kompetenznetz Hepatitis, Contract No. 01KI0102).

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Interferon type I gene expression in chronic hepatitis C