IDO 活性

Toll-Like Receptor-3Ligation-Induced Indoleamine2, 3-Dioxygenase Expression in Human Trophoblasts
Bo Wang,Kaori Koga,Yutaka Osuga,Ingrid Cardenas,Gentaro Izumi,
Masashi Takamura,Tetsuya Hirata,Osamu Yoshino,Yasushi Hirota,
Miyuki Harada,Gil Mor,and Yuji Taketani
Department of Obstetrics and Gynecology(B.W.,K.K.,Y.O.,G.I.,M.T.,T.H.,O.Y.,Y.H.,M.H.,Y.T.), University of Tokyo,Tokyo113-8655,Japan;and Department of Obstetrics and Gynecology and Reproductive Science(I.C.,G.M.),Yale University School of Medicine,New Haven,Connecticut06520
Indoleamine2,3-dioxygenase(IDO)is an enzyme that degrades an essential amino acid,trypto-phan,and plays a role in inhibiting the proliferation of T cells and intracellular pathogens.Inhib-iting IDO in mice leads to fetal rejection,suggesting its significance in establishing pregnancy. Toll-like receptor3(TLR-3)is a key component of the innate immune system that recognizes viral double-stranded RNA and triggers immune reactions by producing type I ing a human trophoblast cell culture system,we studied the effect of TLR-3ligation on IDO expression and function by treating trophoblasts with polyinosinic-polycytidylic acid[poly(I:C)](a synthetic double stranded RNA,which mimics viral RNA).Real-time PCR and Western blot analysis revealed that IDO mRNA and protein expression was significantly induced by poly(I:C).The activity of IDO was also increased by poly(I:C)given that the L-kynurenine concentrations were elevated in con-ditioned media.Conditioned media from poly(I:C)-treated trophoblasts were found to inhibit the proliferation of human T cells significantly.Poly(I:C)was also shown to induce interferon(IFN)-␤mRNA expression in trophoblasts.Recombinant human IFN-␤increased IDO mRNA expression in trophoblasts more rapidly than poly(I:C).Pretreating with neutralizing antibody against IFN-␤significantly suppressed IDO induction by poly(I:C).Collectively we have demonstrated that liga-tion of TLR-3by poly(I:C)induces IDO expression in human first-trimester trophoblasts via an IFN-␤-dependent pathway.These findings suggest that upon viral infection,trophoblasts induce IDO and in turn contribute to antimicrobial activity and maintenance of fetomaternal tolerance. (Endocrinology152:4984–4992,2011)
T he fetomaternal interface is an immunologically unique site that must maintain host defense against possible pathogens and promote tolerance to the allo-geneic fetus(1).Therefore,the immune system at the fetomaternal interface must be precisely controlled to meet the paradoxical task of defending against potential pathogens without compromising fetal survival.In this context,innate immune responses against microorgan-isms at the fetomaternal interface play a significant role (1,2).
Cells of the innate immune system express a series of receptors known as pattern recognition receptors that rec-ognize and bind to sequences known as pathogen-associ-ated molecular patterns,which are uniquely expressed on the surface of microorganisms.Toll-like receptors(TLR) are the major family of pattern recognition receptors.To date,10functional human TLR have been characterized and each TLR responds to a particular ligand(3).For example,TLR-4mediates responses toward Gram-nega-tive bacterial lipopolysaccharide(4),whereas TLR-3me-diates immune responses toward viral double-stranded RNA(dsRNA)(5).Indeed,TLR signals must strike a del-icate balance between clearing infection and causing path-ological effects on the host.
ISSN Print0013-7227ISSN Online1945-7170
Printed in U.S.A.
Copyright©2011by The Endocrine Society
doi:10.1210/en.2011-0278Received March10,2011.Accepted August30,2011. First Published Online September27,2011Abbreviations:BrdU,5-Bromo-2Ј-deoxyuridine;dsRNA,double-stranded RNA;FBS,fetal bovine serum;GAPDH,glyceraldehyde-3-phosphate dehydrogenase;IDO,indoleamine 2,3-dioxygenase;IFN,interferon;IRF3,IFN regulatory factor3;poly(I:C),polyinosinic-poly-cytidylic acid;TLR,Toll-like receptor.
R E P R O D U C T I O N-D E V E L O P M E N T
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Among various types of cells in the fetomaternal inter-face,we focused on trophoblasts and investigated their roles in innate immunity.Studies have demonstrated that trophoblasts can recognize and respond to pathogens through the expression of TLR.For example,TLR-4li-gation promotes cytokine production,which may be in-volved in the pathogenesis of preeclampsia(6),whereas ligation of TLR-2induces apoptosis in first-trimester trophoblasts(7).As a result of TLR-3ligation,tropho-blasts produce cytokines[including interferon(IFN)-␤], chemokines that mount active immune responses(8) and antiviral molecules(9).All these findings show that trophoblasts respond to pathogens through TLR and further affect pregnancy.
Pregnant women may be exposed to a variety of patho-gens including bacteria,fungus,and virus.Among them, viral infections cause highly diversified reactions to preg-nant women,depending on the type of virus and the con-dition of hosts.Some are asymptomatic(10),but preterm labor(11–13)and fetal congenital anomalies of the central nervous system(14)and the cardiovascular system(15, 16)have been reported.Pregnancy can also increase sus-ceptibility and the severity of viral infections such as H1N1(17).This wide spectrum of the clinical conse-quence of viral infection during pregnancy implies various immunological reactions,especially in the placenta.
Indoleamine2,3-dioxygenase(IDO)is the rate-limiting enzyme in the kynurenine pathway of tryptophan catab-olism(18,19).IDO plays a paradoxical role because it has antimicrobial activity in human cells(20)but also has immunosuppressive properties.Of note,IDO is crucial to the fetus in that it maintains maternal tolerance(21).IDO has been shown to be expressed(10)and functioning as an enzyme for tryptophan catabolism(22)in human tropho-blasts.On the other hand,it has been recently reported that TLR ligation induces IDO expression in human cells such as gingival fibroblasts(23)and astrocytes(24).These findings prompted us to hypothesize that,in addition to previously reported molecules that are enhanced by TLR-3ligation,IDO may also be induced by TLR-3stim-ulation in trophoblasts,thereby contributing to viral res-olution and the maintenance of maternal tolerance for fetus upon viral infection during pregnancy(10). Materials and Methods
Reagents
Fetal bovine serum(FBS)was purchased from Life Technol-ogies,Inc.-Invitrogen(Carlsbad,CA),and the same batch of serum was used in all experiments.Polyinosinic-polycytidylic acid[poly(I:C)],a synthetic analog of viral dsRNA,was pur-chased from Invivogen(San Diego,CA).Recombinant human IFN-␤was purchased from PBL Biomedical Laboratories(Pis-cataway,NJ;1ngϭ160U).Rabbit antibody raised against human IFN-␤was purchased from PBL for neutralization ex-periments.Protease inhibitor cocktail was purchased from Roche Diagnostics GmbH(Mannheim,Germany).Antihuman IDO mouse monoclonal antibody was purchased from Oriental Yeast(Nagahama,Japan).Normal mouse IgG1was purchased from Amersham Biosciences(Little Chalfont,UK).Rabbit anti-human vinculin antibody(the loading control)was purchased from Santa Cruz Biotechnology,Inc.(Santa Cruz,CA).Horse-radish peroxidase-conjugated antimouse antibody was pur-chased from Amersham Biosciences and antirabbit IgG from Santa Cruz Biotechnology.The enhanced chemiluminescence Western blotting system was purchased from Amersham Biosci-ences.All other reagents were purchased from Sigma-Aldrich Corp.(St.Louis,MO)unless stated otherwise.
Isolation and culture of trophoblasts from
first-trimester placenta
First-trimester placentas(7–12wk of gestation,nϭ9)were obtained from elective termination of normal pregnancies per-formed after obtaining written informed consent under a study protocol approved by the Institutional Review Board of the Uni-versity of Tokyo.Women’s ages ranged from18to34yr.
Primary trophoblasts from first-trimester placentas were pre-pared as described previously(25).Briefly,tissue specimens were washed with cold PBS to remove excess blood.Placental villi were scraped from fetal membranes,transferred to trypsin-EDTA(Life Technologies,Inc.-Invitrogen)digestion buffer with deoxyribonuclease I(15U/ml)and incubated at37C for20min with shaking.An equal volume of DMEM/F12(Life Technolo-gies,Inc.-Invitrogen)containing10%FBS was added to inacti-vate trypsin and the tissue was filtered through nylon cell strainers with apertures of100␮m.Trophoblasts in the filtrate were cen-trifuged at200g for5min.The resultant cell pellet was resus-pended in5ml of DMEM/F12containing10%FBS and layered onto3ml of Ficoll-Paque PLUS(GE Healthcare,Uppsala,Swe-den)in a15-ml conical tube.The tube was centrifuged at120ϫg for20min.The cellular interface containing trophoblasts was collected and resuspended in red blood cells lysis buffer at room temperature for5min and then centrifuged at200ϫg for5min. After washing with PBS once,the trophoblasts were incubated with anti-CD45antibodies coupled to magnetic beads(Dynal Biotech,Oslo,Norway)for20min at4C to exclude contami-nating leukocytes.Cells bound to the beads were removed by a magnetic collector,and the suspension containing unbound cells was centrifuged at200ϫg for5min.The resulting isolated cells were resuspended in DMEM/F12containing10%FBS,penicil-lin(100U/ml),streptomycin(100␮g/ml),and amphotericin B (0.25␮g/ml)and cultured at37C/5%CO
2
.Purification of the trophoblasts was determined by immunocytochemical staining for CK7(Sigma-Aldrich)and CD45(Covance,Princeton,NJ). Purity of the trophoblasts was greater than95%as determined by positive staining for CK7and negative staining for CD45.
Culture of cell line
The immortalized trophoblast cell line,Swan71,which was derived by telomerase-mediated transformation of7-wk cytotro-
Endocrinology,December2011,152(12):4984–4985
phoblasts(26),was cultured at37C/5%CO
2
in RPMI1640 supplemented with10%FBS and antibiotics.
Treatment of trophoblasts with poly(I:C),IFN-␤, and antibodies
When the trophoblasts reached70–80%confluence in1–2d, media were removed and replaced with fresh media containing 10%FBS.To evaluate the effect of poly(I:C)on mRNA and protein expression of IDO and IFN-␤,primary trophoblasts were treated with poly(I:C)as indicated.In time-course experi-ments,Swan71cells were incubated with either poly(I:C)(10␮g/ml)or IFN-␤(25ng/ml)for the time period indicated(0,2,4, 6,8,12,and24h).To neutralize IFN-␤activity,primary tro-phoblasts were preincubated with rabbit antihuman IFN-␤(20␮g/ml)or nonimmune rabbit IgG for30min and then stimulated by poly(I:C).
Quantitative RT-PCR
Total RNA was isolated from cells using an RNAeasy minikit (QIAGEN,Hilden,Germany)and then treated with ribonu-clease-free recombinant deoxyribonuclease(Roche Diagnostics GmbH).Reverse transcription was performed using ReverTra Ace(Toyobo,Osaka,Japan).One microgram of total RNA was reverse transcribed in a20-␮l total volume.PCR amplification was performed using the LightCycler480System(Roche Ap-plied Science,Mannheim,Germany).IDO primers(sense,5Ј-GGTCATGGAGATGTCCGTAA-3Ј;antisense,5Ј-ACCAAT AGAGAGACCAGGAAGAA-3Ј)were used to amplify a227-bp fragment.IFN-␤primers(sense,5Ј-GCTCTCCTGTTGTGCT TCTCCACTACAGC-3Ј;antisense,5Ј-CTGACTATGGTCCA GGCACAGTGACTGTACTCC-3Ј)were used to amplify a 476-bp fragment.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)primers(Toyobo)(sense,5Ј-ACCACAGTCCATGC CATCAC-3Ј;antisense,5Ј-TCCACCACCCTGTTGCTGTA-3Ј) were used to amplify a452-bp fragment.The conditions for PCR were as follows:IDO,45cycles at95C for10sec,62C for10 sec,and72C for10sec;IFN-␤,45cycles at95C for10sec,72 C for10sec,and72C for20sec;GAPDH,30cycles at95C for10sec,64C for10sec,and72C for18sec.All PCR were followed up with melting curve analysis.The expression level of each mRNA was normalized according to GAPDH levels in each case.
Western blot analysis
Cultured trophoblasts were homogenized in a lysis buffer[10 m M Tris-HCl,50m M NaCl,2m M EDTA,1%Triton X-100(pH 7.0)]with a protease inhibitor cocktail.Protein(30␮g)was separated by10%SDS-PAGE and then transferred onto nitro-cellulose membranes.The blots were blocked in Tris-buffered saline-0.1%Tween20containing5%nonfat milk and then in-cubated with antibodies at4C overnight.The membranes were incubated with primary antibodies;antihuman IDO antibody (1:800)or antihuman vinculin antibody(1:400)as a loading control.The membranes were further incubated with a second-ary antibody,a horseradish peroxidase-conjugated antimouse (1:1000)or antirabbit(1:5000)IgG for1h at room temperature. Signals were developed using an enhanced chemiluminescence Western blotting system.Densitometric analysis was performed using ImageJ(National Institutes of Health,Bethesda,MD).Kynurenine assay
IDO-dependent catabolism of tryptophan produces kynure-nine.Hence,the enzymatic activity of IDO can be evaluated by measuring the level of kynurenine in conditioned media(27). After stimulating Swan71cells with poly(I:C),70␮l of Swan71 conditioned media was mixed with35␮l of30%trichloroacetic acid,vortexed,and centrifuged at8000ϫg for5min.Then75␮l of the supernatant was added to an equal volume of Ehrlich reagent(100mg of p-dimethylbenzaldehyde in5ml of glacial acetic acid)in a96-well plate,and the absorbance was read at an OD of492nm.The concentration of kynurenine was determined by referring to a standard curve of defined kynurenine concen-trations(0–100␮M).
Analysis of T cell proliferation
To evaluate the effect of IDO produced by trophoblasts on T cell proliferation,we cultured T cells with conditioned media from the trophoblast culture and analyzed DNA synthesis in T cells by measuring5-bromo-2Ј-deoxyuridine(BrdU)incorpora-tion.Conditioned media were prepared by culturing Swan71 cells with or without poly(I:C)for24h.T cells were isolated from peripheral blood mononuclear cells from healthy,nonpregnant volunteers by using a CD4-positive T cell isolation kit II(Milte-nyi Biotec,Bergisch Gladbach,Germany)and stimulated with phytohemagglutinin(5␮g/ml).The cells(2ϫ105cells in100␮l media per well)were cultured in96-well flat-bottom plates,con-taining90%(vol/vol)Swan71cells’conditioned media.To con-firm the tryptophan-catabolizing effect of IDO,L-trypotophan (100␮M)was added to the conditioned media.After72h,10␮l of BrdU solution was added to each well and incubated for an additional24h.BrdU uptake was measured using the cell pro-liferation ELISA system based on BrdU incorporation(Amer-sham Biosciences).The immune complexes were detected by the subsequent substrate reaction,and the resultant color was read at450nm in the DigiScan microplate reader(ASYS Hitech GmbH,Eugendorf,Austria).
Statistical analysis
Data analysis was performed using the statistical software package SPSS for Windows(Chicago,IL).All data were checked for their normal distribution by using the Kolmogorov-Smirnov test,and if significant,nonparametric statistical analysis was applied.Parametric variables underwent the Student’s t test.Sta-tistical significance was considered as PϽ0.05.
Results
Poly(I:C)induced IDO mRNA and protein expression in the first-trimester trophoblasts RT-PCR results revealed that first-trimester tropho-blasts did not express IDO mRNA constitutively.In contrast,IDO mRNA expression was induced by12h treatment of poly(I:C)(10␮g/ml)(Fig.1A).IFN-␥,which is known to induce IDO in many cell types,was used as a positive control(28,29).The induction was dose depen-dent(Fig.1B).We further analyzed protein expression of IDO by Western blot analysis.Similar to mRNA results,
4986Wang et al.TLR-3-Induced Trophoblast IDO via IFN-␤Endocrinology,December2011,152(12):4984–4992
first-trimester trophoblasts do not express IDO protein constitutively,but expression was induced by poly(I:C)in a dose-dependent manner (Fig.1,C1).Induction of IDO by poly(I:C)was also demonstrated in the Swan 71cells (Fig.1,C2).
Poly(I:C)induced IDO activity in the first-trimester trophoblasts
Given that poly(I:C)induced IDO mRNA and protein expression in trophoblasts,we next assessed the effect of
poly(I:C)on the enzymatic activity of IDO in trophoblasts by measuring kynurenine concentrations in the con-ditioned media.As illustrated in Fig.2,in comparison with conditioned media from untreated trophoblasts in which kynurenine levels did not change,the concentration of kynurenine in condi-tioned media from poly(I:C)-treated trophoblasts increased over time.The difference in kynurenine levels,be-tween poly(I:C)-treated cells and un-treated cells,peaked at 24h but was significant at 8h (P Ͻ0.01)and re-mained significant for up to 36h.This result indicated that poly(I:C)induces IDO enzymatic activity in trophoblasts and catabolizes tryptophan in the con-ditioned media.
Conditioned media from poly(I:C)-treated trophoblasts suppressed T cell DNA synthesis
The effect of poly(I:C)treatment of trophoblasts on T cell response was then evaluated (Fig.3).In comparison with T cells cultured in control condi-tioned media,DNA synthesis in T cells cultured in conditioned media taken from poly(I:C)-treated Swan 71cells was significantly reduced.We con-firmed that the residual poly(I:C)in the conditioned me-dium had no effect on T cells (data not shown).The DNA synthesis of T cells was significantly,although not com-pletely,restored when L -tryptophan was added to the con-ditioned media from poly(I:C)-treated Swan 71cells.This restoring effect of L -tryptophan indicates that the catab-olism and depletion of tryptophan by IDO is responsible for the reduction of T cell DNA synthesis,although it is possible that other factors may also contribute to this reduction.
Poly(I:C)induced IFN-␤and IFN-␤induced IDO in the first-trimester primary trophoblasts
The mechanism by which TLR-3ligation induces IDO in trophoblasts was then investigated by focusing on the possible involvement of IFN-␤.IFN-␤mRNA was not constitutively expressed in primary trophoblasts but was induced by poly(I:C)(Fig.4A).This result is consistent with findings observed in HTR-8/SVneo cells,a tropho-blast cell line (9).IFN-␤induction of IDO was then in-vestigated.As shown in Fig.4,B and C,recombinant
Untreated
1 10Poly (I:C) µg/ml
IDO Vinculin
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FIG.1.IDO induction by poly(I:C)in human first-trimester trophoblasts.A,Primary first-trimester trophoblasts were cultured in the absence or presence of poly(I:C)(10␮g/ml)or IFN-␥(25ng/ml,positive control)for 12h.IDO mRNA expressions were evaluated by RT-PCR followed by electrophoresis.IDO mRNA expression was not detected in untreated
trophoblasts but was induced by poly(I:C).B,Quantitative RT-PCR analysis indicated that
poly(I:C)(1or 10␮g/ml,12h)significantly increased the IDO mRNA level in a dose-dependent manner in primary trophoblasts (n ϭ4).Values are the mean ϮSE .*,P Ͻ0.05(vs.control);**,P Ͻ.01(vs.control).C1,Primary trophoblasts were cultured in the absence or presence of 1or 10␮g/ml of poly(I:C)for 24h.IDO protein expression was evaluated by Western blot analysis.Vinculin,a housekeeping protein,was used as a loading control.IDO protein
expression was not detected in untreated trophoblasts but was induced by poly(I:C)in a dose-dependent manner.One of the representative results is shown.C2,Untreated Swan 71
trophoblast cells do not express IDO protein,whereas 10␮g/ml of poly(I:C)(24h)induces IDO protein expression.
Culture duration, hs
K y n u r e n i n e (µM )FIG.2.IDO activity in the conditioned media of Swan 71trophoblasts.Swan 71cells were cultured with or without 10␮g/ml of poly(I:C).Conditioned media were collected at the indicated time points (0,4,8,12,24,and 36h)and concentrations of kynurenine were determined.Data represent mean ϮSD of quadruplicate determinations.*,P Ͻ0.001vs.untreated at the same indicated time points.
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IFN-␤(25ng/ml)induced IDO mRNA (Fig.4B)and pro-tein (Fig.4C)in primary trophoblasts.Aggregated densi-tometry data showed a significant induction of IDO mRNA and protein by IFN-␤(n ϭ5:P Ͻ0.01,n ϭ6:P Ͻ0.01,respectively).
The induction of IDO by poly(I:C)was preceded by that of IFN-␤
The time-dependent nature of IFN-␤and IDO induc-tion by poly(I:C)was then investigated in Swan 71cells.The induction of IDO mRNA was detected at 4h after poly(I:C)treatment and peaked at 8h (Fig.5A).However,IFN-␤mRNA levels peaked at 2h after poly(I:C)treat-ment,which preceded the induction of IDO (Fig.5B).In-vestigation of the time dependence of IDO induction by IFN-␤revealed the earliest sign of induction at 2h and peaked at 4h (Fig.5C).
Blocking IFN-␤canceled the IDO induction by poly(I:C)
Given the possible involvement of IFN-␤in IDO in-duction by poly(I:C),anti-IFN-␤antibody was added to the culture system.Primary trophoblasts were pretreated with neutralizing antibody against human IFN-␤(20␮g/
ml)or control IgG for 30min followed by treatment with poly(I:C)(10␮g/ml)for 8h.Quantitative RT-PCR was per-formed to detect changes in IDO mRNA levels.Neutralizing antibody against IFN-␤inhibited the induction of IDO by poly(I:C)in trophoblasts (Fig.6)thereby confirming that IFN-␤mediates the poly(I:C)stimulated induction of IDO.
Discussion
In the present study,we have demonstrated for the first time that IDO,which is involved in both antiviral activity and fetomaternal tolerance,is induced by the TLR-3li-gand poly(I:C)in human trophoblasts.This study has demonstrated that poly(I:C)induces the expression of IDO mRNA and protein in trophoblasts.Furthermore,our findings confirm that poly(I:C)induces IDO enzy-matic activity and depletes tryptophan and consequently reduces DNA synthesis in T cells.In terms of elucidating the signaling pathway,IFN-␤mediates the induction of IDO by poly(I:C)because blocking IFN-␤specifically re-duced this poly(I:C)effect.
It has been previously demonstrated by our group that ligation of TLR-3by the viral ligand,poly(I:C),causes
T c e l l s B r d U i n c o r p o r a t i o n (o p t i c a l d e n s i t y 450 n m )
0.4
0.81.2N o P o l y (I :C ) C M
P o l y (I :C ) C M
N o P o l y (I :C ) C M +T r y p t o p h a n
P o l y (I :C ) C M +T r y p t o p h a n
*
*
FIG.3.Suppression of T cell DNA synthesis by conditioned media from poly(I:C)-treated Swan 71trophoblasts.Conditioned media were prepared by culturing Swan 71cells with or without 10␮g/ml of poly(I:C)for 24h.T cells were cultured for 72h in conditioned media with 5␮g/ml of phytohemagglutinin in the presence or absence of 100␮M of L -tryptophan.DNA synthesis of T cells was determined by BrdU incorporation.Conditioned media from poly(I:C)-treated Swan 71cells suppressed the DNA synthesis of T cells.This suppression was restored when L -tryptophan was added to the culture.One of the
representative data is shown (mean ϮSEM of hexaplicate experiments).*,P Ͻ0.0001.
GAPDH
IFN-β
IDO
GAPDH
B
A
C
P o l y (I :C )
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Vinculin
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d
FIG.4.Poly(I:C)induced IFN-␤and IFN-␤induced IDO expression in primary first-trimester trophoblasts.A,Primary trophoblasts were cultured with or without 10␮g/ml of poly(I:C)for 12h.IFN-␤mRNA expressions were evaluated by analysis of RT-PCR followed by electrophoresis.IFN-␤was not constitutively expressed in first-trimester trophoblasts,but poly(I:C)induced IFN-␤expression.B,Primary trophoblasts were cultured with or without 25ng/ml of recombinant human IFN-␤for 12h.IDO mRNA expression was detected and analyzed by using RT-PCR.GAPDH was used as a loading control.C,Primary trophoblasts were cultured with or without 25ng/ml of recombinant human IFN-␤for 24h.IDO protein expression was detected by using Western blot analysis.Vinculin was used as loading control.Both IDO mRNA and protein expression were induced by IFN-␤.
4988Wang et al.TLR-3-Induced Trophoblast IDO via IFN-␤Endocrinology,December 2011,152(12):4984–4992
trophoblasts to secrete chemokines such as regulated upon activation,normal T cell expressed,and secreted and an-tiviral factors such as IFN-␤,secretory leukocyte protease inhibitor,2Ј,5Ј-oligoadenylate synthetase,myxovirus-resistance A,and apolipoprotein B mRNA-editing en-zyme-catalytic polypeptide-like 3G (9).These chemokines and antiviral factors are thought to modulate further im-mune responses and in turn contribute to the pathogenesis of pregnancy complications.As a result of the current study,IDO,which is a strong immune modulating factor,can now be added to the list of factors produced by tro-phoblasts upon TLR-3ligation.
Previous studies have demonstrated that IDO has an-tiviral effects in various types of human cells.Bodaghi et al.(30)demonstrated that IDO controls human cytomeg-
alovirus replication in human retinal pigment epithelial cells.Similar IDO effects were reported in human astro-cytes for herpes simplex virus (31)and in epithelial,en-dothelial,and astroglial cells for measles virus (32).The mechanism by which IDO inhibits the replication of virus is the degradation of tryptophan,which is essential for virus replication (30,31).In the case of a viral infection such as cytomegalovirus,the placenta and hence tropho-blasts are targets for virus.In the current study,we found that TLR-3ligation,which mimics viral infection,induces IDO expression and promotes tryptophan catabolism in first-trimester primary trophoblasts.This result further confirms our previous findings and other studies that sug-gest that trophoblasts or the placenta act as an active bar-rier to resolve infection and protect the fetus (1,33–35).On the other hand,IDO in the fetomaternal interface is also known to play an important role in maintaining tol-erance for the establishment of a successful pregnancy.Munn et al.(21)proposed that the expression of IDO in the placenta is crucial to prevent immunological rejection of the fetus.They administered L -methyl-tryptophan,a pharmacological inhibitor of IDO,to pregnant mice and demonstrated rapid T cell-induced rejection of allogenic fetuses (21).In human pregnancy,IDO expression and activity have been demonstrated in the human placenta (36,37),and reduced IDO expression in the human pla-centa and/or decidua has been linked to pathological preg-nancy such as preeclampsia (38,39),retarded intrauterine development (37),and miscarriage (40).In terms of the localization of IDO within the fetomaternal interface,the expression by human first-trimester trophoblasts is con-troversial (36,37,41).Some studies did not detect IDO in placental villi (37,41),whereas Kudo et al.(36)
demon-
010********
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20406080100
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FIG.5.Time-course studies of IDO induction by poly(I:C)(A),IFN-␤induction by poly(I:C)(B),and IDO induction by IFN-␤(C).Trophoblast Swan 71cells were stimulated with 10␮g/ml of poly(I:C)or 25ng/ml of IFN-␤for the indicated time periods (0,2,4,6,8,12,and 24h).Quantitative RT-PCR was performed to detect changes in mRNA
expression:the fold increases of IFN-␤mRNA are indicated.Values are the mean ϮSEM of triplicate cultures.Note that IDO induction by poly(I:C)was preceded by IFN-␤induction by poly(I:C)(A vs.B)and IFN-␤
induced IDO more rapidly than poly(I:C)(C vs.A).
Poly (I:C)Control IgG Anti-IFN-β IgG
20406080I D O m R N A
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FIG.6.Neutralizing antibody against IFN-␤inhibited IDO induction by poly(I:C)in primary trophoblasts.Primary trophoblasts were pretreated with neutralizing antibody against human IFN-␤(anti-IFN-␤IgG)or nonimmune rabbit IgG (control IgG)for 30min followed by treatment with 10␮g/ml of poly(I:C)for 8h.The level of IDO mRNA was evaluated by the analysis of quantitative RT-PCR.Values are the mean ϮSE of triplicate cultures.In comparison with controls,
neutralizing antibody against IFN-␤significantly inhibited poly(I:C)-induced IDO mRNA expression.*,P Ͻ0.001.
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