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Highly Efficient Stable Expression of Indoleamine 2,3 Dioxygenase Gene in Primary Fibroblasts Moeen Rezakhanlou, Alireza; Habibi, Darya; Lai, Amy; B. Jalili, Reza; Ong, Christopher J; Ghahary, Aziz Mar 27, 2010

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Highly Efficient Stable Expression of Indoleamine2,3 Dioxygenase Gene in Primary FibroblastsAlireza Moeen Rezakhanlou & Darya Habibi & Amy Lai &Reza B. Jalili & Christopher J. Ong & Aziz GhaharyReceived: 3 September 2009 /Accepted: 20 February 2010 /Published online: 27 March 2010# The Author(s) 2010. This article is published with open access at Springerlink.comAbstract Indoleamine 2,3 dioxygenase (IDO) is a potentimmunomodulatory enzyme that has recently attractedsignificant attention for its potential application as aninducer of immunotolerance in transplantation. We havepreviously demonstrated that a collagen matrix populatedwith IDO-expressing fibroblasts can be applied successfullyin suppressing islet allogeneic immune response. Meanwhile,a critical aspect of such immunological intervention relieslargely on effective long-term expression of the IDO gene.Moreover, gene manipulation of primary cells is known to bechallenging due to unsatisfactory expression of the exogenousgene. In this study, a lentiviral gene delivery system has beenemployed to transduce primary fibroblasts. We used poly-brene to efficiently deliver the IDO gene into primaryfibroblasts and showed a significant increase (about tenfold)in the rate of gene transfection. In addition, by the use offluorescence-activated cell sorting, a 95% pure populationof IDO-expressing fibroblasts was successfully obtained.The efficiency of the IDO expression and the activity ofthe enzyme have been confirmed by Western blotting,fluorescence-activated cell sorting analysis, and Kynurenineassay, respectively. The findings of this study revealedsimple and effective strategies through which an efficientand stable expression of IDO can be achieved for primarycells which, in turn, significantly improves its potential asa tool for achieving immunotolerance in different types oftransplantation.Keywords Lentiviral vector . Indoleamine 2 .3 dioxygenase . Primary fibroblast . Transplantation .Immunogenicity1 IntroductionIndoleamine 2,3-dioxygenase (IDO) is a monomeric, heme-containing enzyme that catalyzes the rate limiting step ofconversion of tryptophan to kynurenine [1]. Recently,tryptophan catabolism has been implicated in immunolog-ical tolerance. One theory proposes that degradation oftryptophan suppresses T cell proliferation by reducing theavailability of this essential amino acid in local tissueenvironments, thereby sensitizing T cells to apoptosis [2].Another theory suggests that the major tryptophan metab-olite, kynurenine, suppresses immune reactivity throughdirect interaction with effector T lymphocytes [3]. Theimmunomodulatory effects of tryptophan deficiency andexcess kynurenine caused by IDO are of particular interestin the field of transplantation [4].Alireza Moeen Rezakhanlou and Darya Habibi contributed equally tothis work.A. Moeen Rezakhanlou :D. Habibi :A. Lai : R. B. Jalili :A. GhaharyBurn and Wound Healing Research Group,Department of Surgery, University of British Columbia,Vancouver, BC, CanadaD. Habibi : C. J. OngThe Prostate Centre at Vancouver General Hospital,Department of Surgery, University of British Columbia,Vancouver, BC, CanadaR. B. JaliliThe Endocrinology and Metabolism Research Centre,University of Tehran/Medical Sciences,Tehran, IranA. Ghahary (*)Burn and Wound Healing Research Lab,Rm 350, Jack Bell Research Centre, 2660 Oak Street,Vancouver, BC, Canada V6H 3Z6e-mail: aghahary@interchange.ubc.caBiol Proced Online (2010) 12:107–112DOI 10.1007/s12575-010-9028-6A critical aspect of an immunological intervention usingIDO is the requirement to achieve effective expression ofthis gene. However, genetic manipulation by non-viraltransfection approaches has been challenging due to theissues of low transfection efficiency, loss of cell viability,and difficulty in obtaining stable transfection [5, 6].Previously, we have shown that by using dermal fibroblaststransduced with an IDO-expressing adenoviral vector, IDOfunctions as a local immunosuppressive factor [7], and localexpression of IDO suppresses islet allogeneic immuneresponse in mouse islet transplantation [8]. Furthermore,we have used the local immunosuppressive effect of IDO inthe development of a non-rejectable skin substitute [9].These findings demonstrate that IDO has considerablepotential for immunoregulation and induction of immuno-tolerance in transplantation.Nevertheless, the immunogenicity and transient geneexpression of adenoviral vectors may hinder its clinical usein transplantation. Thus, in order to sustain the efficacy ofIDO activity in the local environment of transplanted organ,it is essential to prolong the expression of functional IDOprotein. In fact, it was shown that lentiviral vectors canmaintain efficient long-term target gene expression in vivofor more than 4 years [10]. It is has been reported thatpolybrene can markedly enhance the retrovirus transductionefficiency [11]. In this report, we constructed an IDO-expressing lentiviral vector and showed that treatment ofcells with polybrene-enhanced IDO transduction efficiencyalmost ten times. To enrich IDO-expressing cells and therebyoptimize transplantation immunotolerance, we selectivelyisolated the IDO-positive cells by fluorescence-activated cellsorting (FACS) and obtained a greater than 95% purepopulation of IDO-expressing cells.2 Materials and Methods2.1 Plasmid ConstructionsA lentiviral construct for expressing the IDO gene wasgenerated using the pLC-E vector [12] modified from thelentiviral backbone FUGW [13]. For visualization of thelentiviral-mediated IDO expression, a sequence encodingthe red fluorescent mCherry protein under the control of theUbC promoter was incorporated into the vector. The humanIDO gene (NM_002164; a generous gift from Dr. JM Carlinof Miami University) was generated by PCR using a full-length cDNA encoding the gene as template and the forwardprimer (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGCACACGCTATGGAAAACTCCTGG-3′)and reverse primer (5′-GGG GACCACTTTGTACAAGAAA G C T G G G T C C TA A C C T T C C T T C A A A A GGGATTTCTC-3′). The amplified PCR product was firstinserted into an entry vector (pDON201) and then Gateway(Invitrogen) cloned into a lentiviral pLC-E expression vector.The IDO gene is expressed under the control of EF1-αpromoter, and the mCherry red fluorescent gene, a reportergene, is expressed under the control of a ubiquitin promoter.The plasmid was amplified in competent DH10-B bacteriaand purified using the Qiagen Plasmid DNA Maxi-prep kit(Qiagen). Sequence of the IDO/pLC-E construct was con-firmed by DNA sequencing analysis.2.2 Cell CultureSkin samples were collected from 6- to 8-week-old maleC57BL/6 (B6) mice according to the guidelines of theAnimal Policy and Welfare Committee of the University ofBritish Columbia. The samples were then washed in steriledulbecco’s modified eagle medium (DMEM) (InvitrogenLife Technologies, Carlsbad, CA) supplemented with antibi-otic–antimycotic preparation [100 U/ml penicillin, 100 mg/mlstreptomycin, 0.25 mg/ml amphotericin B (Invitrogen)].Cultures of fibroblasts were established as previously described[14] and grown in DMEM supplemented with 10% fetalbovine serum (FBS; Invitrogen Life Technologies, Carlsbad,CA). Confluent cells were released by trypsinization,reseeded onto 75 cm2 cell culture flasks (BD Biosciences,MA), and incubated in a humidified incubator at 37°Csupplied with 5% CO2. Fibroblasts at passages three to fivewere used in all experiments.2.2.1 Lentiviral Vector Production and Cell TransductionReplication-defective lentiviral vectors were generated aspreviously described [15, 16] by transient transfection of293T human kidney cells with the IDO/pLC-E vector, R8.9(packaging plasmid), and VSV-G (envelope plasmid).Some 293T cells were seeded the night before transfectionin DMEM medium supplemented with 10% fetal bovineserum (Invitrogen Life Technologies, Carlsbad, CA). Thecells were replaced with DMEM+10% FBS and trans-fected at 60–70% confluency using ProFectin (MammalianTransfection System Calcium Phosphate; Promega). For each100-mm2 round plate, 10 µg IDO/pLC-E vector, 7.5 µg R8.9plasmid, and 2.5 µg VSV-G plasmid were used. The culturemedium was replaced with a fresh medium containing DMEMand 5% FBS 12–16 h after transfection. Supernatant washarvested 30 h after the medium change. The vector stockswere concentrated by centrifugation at 126,000g for 90 minusing Beckman Ultracentrifuge and stored at −80°C until use.For transduction, 293T cells or mouse fibroblastswere seeded on flat-bottom 6-well cell culture plates(Corning Incorporated, Corning, NY, USA) and incubatedwith the same titer of IDO-lentiviral vector for 24 h in theabsence or presence of 10 ug/ml polybrene. Cell superna-108 Rezakhanlou et al.tant containing lentiviral vectors were removed from theculture 30 h later.2.3 SDS-PAGE and Western BlottingFor detection of the IDO protein expression, transducedcells were harvested 24 h post-transduction and washedtwice with PBS. Cells were then lysed in lysis buffer(50 mM Tris-HCl, pH 7.4; 10 mM EDTA; 5 mM EGTA;0.5% NP40; 1% Triton X-100; and protease inhibitor cocktail(Sigma). Equal amounts of total protein from each individual293T cell culture were separated by 10% sodium dodecylsulfate - polyacrylamide gel electrophoresis (SDS-PAGE).Proteins were then transferred to a PVDFmembrane (MilliporeCorp., Bedford, MA) and immunoblotted with a polyclonalanti-human IDO antibody (Washington Biotechnology Inc.,Baltimore, MD) at final dilution of 1:5,000. Horseradishperoxidase-conjugated goat anti-rabbit IgG was used as thesecondary antibody for the enhanced chemiluminescencedetection system (Amersham Biosciences, UK). Blots werethen stripped and reprobed for β-actin as a control for proteinloading.2.4 Fluorescence MicroscopyThe lentiviral-mediated IDO expression in transduced 293Tcells, and mouse fibroblasts were examined by fluorescencemicroscopy with a Zeiss Axiovert 200 M microscope. Imagesfrom identical areas of cultured cells were recorded using bothfluorescence and bright-field microscopy. Images werecaptured using Northern Eclipse image analysis software.2.5 Kynurenine AssayThe biological activity of IDO was evaluated by measuringthe level of tryptophan degradation product, L-kynurenine,present in the conditioned medium of transduced cells. Theamount of L-kynurenine was measured by a previouslyestablished method [17]. Proteins in the conditioned mediumwere precipitated by trichloroacetic acid. After centrifuga-tion, 0.5 ml of supernatant was incubated with an equalvolume of Ehrlich's reagent at room temperature for 10 min.The reaction mixture was measured spectrophotometricallyat 490 nm. The concentration of kynurenine in the condi-tioned medium was calculated according to a standard curveof defined kynurenine concentration (0–20 mg/ml).2.6 Flow CytometryTo determine the transduction efficiency of the IDO-lentiviral vector, and in order to sort mCherry-positivecells, non-transduced cultured fibroblasts, fibroblasts trans-duced with IDO-lentiviral vector in the absence or presenceof polybrene were trypsinized, collected, and centrifuged.The cell pellet was washed twice with PBS and resuspendedat 106 cells/mL for flow cytometry and at 107 cells/mL forsorting in PBS±2% FBS. For flow cytometry, BD LSRIIwith 630 LP and 670/30 detectors were used. For sorting,BD FACSAria with a blue laser detector was used. In each set,live cells were gated using forward scatter channel vs sidescatter channel followed by gating on the IDO-expressingmCherry-positive cells. The number of mCherry-positivecells for fibroblasts transduced with IDO-lentiviral vector inthe presence of polybrene after sorting was also deter-mined by flow cytometry analysis. The average puritywas greater than 95%.3 Results and Discussion3.1 Characterization of Lentiviral-Mediated IDOExpressionSchematic diagram of the IDO–mCherry lentiviral vector isshown in Fig. 1a. IDO-expressing lentiviral vector prepa-rations were generated. To examine the efficacy of thegenerated lentiviral vector, the vector was first collectedfrom the transfected 293T cells and used to transduce afresh culture of 293T cells. The red fluorescent mCherryprotein acts as a reporter for the promoter activity of theIDO gene, verifying that the IDO gene is present andexpressed in the transduced cells (Fig. 1b). As shown inFig. 1bd, majority of transduced cells express red fluores-cent mCherry protein as a reporter gene for IDO expressioncompared with that of control cells (Fig. 1bb). Figure 1bpanels c and a show the images of the same cells in bright-field. Furthermore, to examine the protein expression of theexogenously introduced IDO gene, lysates of the trans-duced cells were subjected to SDS-PAGE and immuno-blotted with an anti-IDO polyclonal antibody (Fig. 1c). Asseen in Fig. 1c, the IDO expression was positive in bothviral preparations and the transduction was successful.Non-transduced cell lysate was used as a negative controland recombinant IDO protein was used as a positivecontrol. In order to test whether the overexpressed IDOprotein was functional, kynurenine assay was performed tomeasure the concentration of the major tryptophan degra-dation product (L-kynurenine) in the conditioned media. Incomparison with non-transduced cells, the concentration ofL-kynurenine in the conditioned media of transduced cellsincreased nearly threefold (Fig. 1d) and the result wassignificantly different from that of non-transduced cells(P<0.02, n=3). The results show that the IDO-expressinglentiviral vector generated by transient transfection of 293Tcells was functional and suitable for transduction of primaryfibroblasts.Efficient Stable Expression of IDO 1093.2 Transduction of Primary Mouse Fibroblastswith IDO-Expressing Lentiviral VectorDermal fibroblasts were transduced with the IDO-expressing lentiviral vector. To analyze lentiviral-mediatedIDO expression, transduced cells were visualized byfluorescence microscopy 24 h post-transduction (Fig. 2a).The Fig. 2a upper and lower panels are bright-field imagesand fluorescence images, respectively. Fluorescence imagesare images of the same groups of cells captured by thebright-field microscope, and the fluorescence is due toexpression of the mCherry gene. Figure 2a panels a and eas well as b and f represent non-transduced fibroblasts andIDO-lentiviral vector-transduced fibroblasts, respectively.To facilitate delivery of the IDO-lentiviral vector, polybrenewas added to cells at the time of transduction. The cellswere similarly examined by fluorescence microscopy 48 hpost-transduction (Fig. 2a, panels c and g). Transducedfibroblasts in the presence of polybrene were gated andsorted by FACS and examined by fluorescence microscopy(Fig. 2a, panels d and h). As the fluorescent images show,the level of mCherry-positive cells increased in thepresence of polybrene and after sorting. The expression ofFig. 1 Construction of alentiviral-based vector for deliv-ering the IDO gene. a Schematicdiagram of the IDO–mCherrylentiviral construct. b Fluores-cence microscopy analysis ofIDO-expressing cells. Panels aand b as well as c and d representbright-field and fluorescentimages of the IDO–mCherry(red) expression in non-transduced and transduced 293Tcells, respectively. c Lentiviralvector-mediated IDO proteinexpression in transduced 293Tcells. The arrow on the upperband shows the IDO protein. TheIDO-lentiviral vector lanes rep-resent two separate viral prepa-rations. The blot was reprobedwith a β-actin antibody as aloading control. d Kynurenineassay. Functional IDO activitywas evaluated by measuring thecontent of kynurenine in theconditioned media of non-transduced and IDO-lentiviralvector-transduced cells. The datashown are the mean and standarddeviation of kynurenine inconditioned media of three sepa-rate experimentsFig. 2 Transduction of mouse fibroblasts with IDO-expressinglentiviral vector. a Fluorescence microscopy analysis of IDO-lentiviralvector-transduced fibroblasts. Cultured mouse fibroblasts were incubatedwith IDO-lentiviral vector either in the presence or absence of polybrene.Cells that were transduced in the presence of polybrene were then sortedby FACS. Panels a and e, b and f, c and g, as well as d and h representbright-field and fluorescence images of non-transduced fibroblasts,IDO-lentiviral vector-transduced fibroblasts, fibroblasts transduced withIDO-expressing lentiviral vector in the presence of polybrene, andfibroblasts transduced with IDO-expressing lentiviral vector in thepresence of polybrene after sorting, respectively. b These panels showthe Western blot analysis (a) as well as kynurenine levels (b) in differentindicated cells. The data shown are the mean and standard deviation ofkynurenine measurements obtained from three different experiments. cFlow cytometry analysis of the IDO-expressing cell population.Fibroblasts were left either non-transduced or transduced with IDO-expressing lentiviral vector in the absence or presence of polybrene andflow cytometry was then conducted by gating the cells based on theirmCherry fluorescence. The selected cell population was prepared bycell sorting and the number of mCherry-positive cells was greaterthan 95%. Panel a shows non-transduced fibroblasts, b transducedIDO-expressing fibroblasts in the absence of polybrene, c transducedIDO-expressing fibroblasts in the presence of polybrene, and d FACS-sorted IDO-expressing fibroblasts (in the presence of polybrene). Panele shows the percentage of mCherry-positive fluorescent cells in the totalcell population studied as detected by flow cytometry. The data shownare the mean and standard deviation of mCherry-positive cells obtainedfrom three separate experiments„110 Rezakhanlou et al.IDO protein in these cells was determined by Western blotanalysis (Fig. 2b, panel a). IDO protein level increased inthe presence of polybrene and after sorting. The function-ality of the overexpressed IDO protein was assessed bykynurenine assay (Fig. 2b, panel b). The kynureninecontent in the conditioned media of mouse fibroblaststransduced in the presence of polybrene increased approxi-mately twofold when compared with that of control with nopolybrene treatment (P<0.01, n=3). This finding revealedthat polybrene significantly enhanced the transductionefficiency in primary mouse fibroblasts as well as the IDOprotein and its enzymatic activity.Efficient Stable Expression of IDO 1113.2.1 Analysis of Improvement of Lentiviral VectorTransduction by Flow CytometryTo maximize the chances for successful transplantation andreduce the risk of immunorejection, it would be advanta-geous to have a highly pure population of IDO-expressingcells. By FACS, we sorted the IDO–mCherry fluorescentprotein-expressing cells (Fig. 2c). Panel a of Fig. 2c showsthat there is less than 0.02% of autofluorescent cells in thenegative-cell population. As shown in panels b and c ofFig. 2c, treatment of IDO-lentiviral vector-transduced cellsby polybrene increased the number of mCherry-positivecells by tenfold. These cells were then gated and sorted byFACS. Figure 2c, panel d, shows that more than 95% of thecell population after sorting were expressing IDO, and thiswas confirmed by flow cytometry, fluorescence microscopy,and kynurenine assay. The transduction efficiency was shownas percentage ofmCherry-positive fluorescent cells in the totalcell population studied (Fig. 2c, panel e). Transduction ofmouse fibroblasts in the presence of polybrene showed asignificant increase in the number of IDO-expressing cells(P≤0.01, n=3). As a result, we successfully obtained a cellpopulation that contains more than 95% IDO-expressingprimary fibroblasts (Fig. 2c, panels d–e).As efficient gene delivery is a prerequisite for successfulgene therapy, we believe that polybrene significantlyincreases lentiviral-mediated delivery of the IDO gene intotarget cells and that should make this enzyme a promisingand highly attractive strategy for improving the outcome ofcell and possibly organ transplantation.Acknowledgments We would like to thank Dr. Dieter Fink and Ms.Lisa Xu for their support. This work was supported by CanadianInstitutes of Health Research Grant.Competing interest statement The authors declare no competinginterests.Open Access This article is distributed under the terms of theCreative Commons Attribution Noncommercial License which per-mits any noncommercial use, distribution, and reproduction in anymedium, provided the original author(s) and source are credited.References1. 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