Functional IDO Cells Initiate Tolerogenesis in the Absence Of

Kynurenine Pathway Enzymes in Dendritic Cells Initiate Tolerogenesis in the Absence of Functional IDO

This information is current as Maria L. Belladonna, Ursula Grohmann, Paolo Guidetti, of October 2, 2021. Claudia Volpi, Roberta Bianchi, Maria C. Fioretti, Robert Schwarcz, Francesca Fallarino and Paolo Puccetti J Immunol 2006; 177:130-137; ; doi: 10.4049/jimmunol.177.1.130 http://www.jimmunol.org/content/177/1/130 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Kynurenine Pathway Enzymes in Dendritic Cells Initiate Tolerogenesis in the Absence of Functional IDO1

Maria L. Belladonna,* Ursula Grohmann,* Paolo Guidetti,† Claudia Volpi,* Roberta Bianchi,* Maria C. Fioretti,* Robert Schwarcz,† Francesca Fallarino,* and Paolo Puccetti2*

Dendritic cell (DC) tryptophan catabolism has emerged in recent years as a major mechanism of peripheral tolerance. However, there are features of this mechanism, initiated by IDO, that are still unclear, including the role of enzymes that are downstream of IDO in the kynurenine pathway and the role of the associated production of kynurenines. In this study, we provide evidence that 1) murine DCs express all enzymes necessary for synthesis of the downstream product of tryptophan breakdown, quinolinate; 2) IFN-␥ enhances transcriptional expression of all of these enzymes, although posttranslational inactivation of IDO may prevent metabolic steps that are subsequent and consequent to IDO; 3) overcoming the IDO-dependent blockade by provision of a downstream quinolinate precursor activates the pathway and leads to the onset of suppressive properties; and 4) tolerogenic DCs Downloaded from can confer suppressive ability on otherwise immunogenic DCs across a Transwell in an IDO-dependent fashion. Altogether, these data indicate that kynurenine pathway enzymes downstream of IDO can initiate tolerogenesis by DCs independently of tryptophan deprivation. The paracrine production of kynurenines might be one mechanism used by IDO-competent cells to convert DCs lacking functional IDO to a tolerogenic phenotype within an IFN-␥-rich environment. The Journal of Immunology, 2006, 177: 130–137. http://www.jimmunol.org/ he tolerogenic pathway of L-tryptophan catabolism has used mixture of D,L-stereoisomers (15, 16), in which the L-isomer been implicated, with either protective or disease-promot- is expected to be the active form of the inhibitor (13). T ing roles, in a variety of physiopathologic conditions, Two major theories have been proposed to account for tolerance ranging from pregnancy (1) to transplantation (2), and from auto- induction via tryptophan catabolism. One theory posits that tryp- immunity (3) and inflammation (4–6) to infection (7) and neopla- tophan breakdown suppresses T cell proliferation by critically re- sia (8, 9). There is growing evidence that tryptophan catabolism ducing the availability of this indispensable amino acid in local may be involved in the development of depressive-like behaviors tissue microenvironments (17). The other theory assumes that in both humans and mice (10, 11). In the experimental models of downstream metabolites of tryptophan catabolism, collectively immune regulation, a central role for IDO has been established known as kynurenines, act to suppress immune reactivity, proba- by guest on October 2, 2021 either by using the competitive enzyme inhibitor 1-methyl-trypto- bly through direct interactions with effector T lymphocytes and 3 phan (1-MT) under in vivo or in vitro conditions or by using other cell types (10). These are not mutually exclusive possibili- dendritic cells (DCs) ex vivo from IDO-deficient mice (12). How- ties, and each might have a role in the immunobiology of IDO ever, pharmacologic or genetic inhibition of IDO activity may not (16), including unique actions by specific kynurenines such as be completely informative, because IDO could not be the only 3-hydroxyanthranilic acid (3-HAA) (18) and the downstream relevant target of 1-MT (13), and IDO-deficient mice, which are product quinolinic acid (QUIN) (19). Clarification of the relative genetically altered hosts, might have abnormal development or dif- contributions of the different mechanisms would be crucial to an ferentiation of their DC lineage. Interestingly, these animals man- improved understanding of the complex role of tryptophan catab- ifest compensatory or redundant mechanisms for tolerogenesis in olism in health and disease and to the implementation of immu- vivo (14). Also relevant in this regard is the recent and rather notherapies targeting IDO function (13). puzzling finding that the D-isomer of 1-MT is apparently more IDO activity is regulated at both transcriptional and posttrans- effective at inhibiting tryptophan catabolism than the universally lational levels (20), and IFN-␥ represents the principal regulator of Indo transcription (21). We have previously shown that the default functional programs of murine splenic CD8Ϫ and CD8ϩ DCs in *Department of Experimental Medicine, University of Perugia, Perugia, Italy; and †Maryland Psychiatric Research Center, University of Maryland School of Medicine, the presentation in vivo of poorly immunogenic peptides are dis- Baltimore, MD 21228 parate but flexible (22). IFN-␥, however, fails to modify the default Ϫ Received for publication February 22, 2006. Accepted for publication April 12, 2006. immunogenic function of CD8 DCs, although it does potentiate ϩ The costs of publication of this article were defrayed in part by the payment of page the basic tolerogenic program of CD8 DCs in an IDO-dependent charges. This article must therefore be hereby marked advertisement in accordance fashion. The cytokine, in fact, will not confer tryptophan-catabo- with 18 U.S.C. Section 1734 solely to indicate this fact. Ϫ lizing activity on CD8 DCs, although the resultant IDO protein 1 This work was supported by the Italian Association for Cancer Research. expression is comparable in the two DC subsets (23). 2 Address correspondence and reprint requests to Dr. Paolo Puccetti, Department of In this study, we examined the role of kynurenines that are phys- Experimental Medicine, Section of Pharmacology, University of Perugia, Via del Giochetto, Perugia 06126, Italy. E-mail address: [email protected] iologically generated downstream of the initial and rate-limiting 3 Abbreviations used in this paper: 1-MT, 1-methyl-tryptophan; DC, dendritic cell; step mediated by IDO in tryptophan degradation. In addition, in 3-HAA, 3-hydroxyanthranilic acid; QUIN, quinolinic acid; siRNA, small interfering IDO-competent DCs, we compared 1-MT and Indo gene silencing RNA; GC/MS, gas chromatography with electron capture negative ionization mass for modulation of the potent tolerogenic function induced by spectrometry; KYN, L-kynurenine; PI, propidium iodide; 3-HK, 3-hydroxykynure- nine; AA, anthranilic acid; Treg, regulatory T cell. IFN-␥ in this subset. We obtained evidence that Indo silencing in

CD8ϩ DCs had effects comparable to pharmacologic inhibition of Kynurenine and quinolinic acid measurements the enzyme. Perhaps of greater interest, initiating the kynurenine Ϫ These procedures, using gas chromatography with electron capture nega- pathway in CD8 DCs downstream of IDO led to the production tive ionization mass spectrometry (GC/MS), have previously been de- of QUIN and imparted tolerogenic properties to those cells in the scribed in detail (28, 29). Briefly, 10 ␮l of culture supernatant was diluted absence of tryptophan consumption. Thus, similar to CD8ϩ DCs, (1/5, v/v) in water. Fifty microliters of internal standard (100 nM 3,5- their CD8Ϫ counterparts are competent for the portion of the path- pyridinedicarboxylic acid and 200 nM homophenylalanine) were added, and proteins were precipitated with HCl (25 ␮l, 5 N). Fatty compounds way that is subsequent to IDO, and can further metabolize the were removed by extraction with chloroform (100 ␮l). After centrifugation initial kynurenine when provided externally. This suggests that the (10 min, 10,000 ϫ g), 100 ␮l of the acidic supernatant were added to a paracrine production of kynurenines could be one mechanism glass tube containing 50 ␮l of 62.5 mM tetrabutylammonium hydrogen whereby IDO-competent cells affect the activity of DCs in which sulfate. The mixture was lyophilized and resuspended in 25 ␮l of acetone containing 7.5% of diisopropylethylamine and 3% pentafluorobenzyl bro- functional IDO is blocked posttranslationally. mide. The samples were incubated in sealed tubes for 15 min at 60–65°C. Fifty microliters of decane and 750 ␮l of water were then added, the sam- Materials and Methods ples were thoroughly mixed, and the decane phase was removed. One Mice and reagents microliter of the decane phase was injected into a GC/MS system consisting of a Trace GC coupled with a Trace MS quadrupole mass spectrometer Female DBA/2J (H-2d) mice were obtained from Charles River Breeding (ThermoFinnigan). Chromatographic separation was achieved using a Laboratories. Synthetic kynurenines (used in vitro at 10 or 100 ␮M ac- 30-m Rtx-5MS capillary column (0.25-mm internal diameter, 0.25-␮m film cording to the experimental design) and racemic 1-MT (D,L-isomers) were thickness; Restek) with helium as a carrier gas. A split/splitless injection port purchased from Sigma-Aldrich. The kynurenine 3-monooxygenase inhib- (1-␮l injection volume) was used. The temperature program was as follows: itor PNU 156561 (4,5-dichlorobenzoylalanine; synthesized in the Pharma- 155°C for 1.25 min, 40°C/min to 275°C, 10°C/min to 320°C, 1 min at 320°C,

cia and Upjohn Research Laboratories of Nerviano, Italy) has been char- injection port 228°C. The GC/MS uses electron capture negative ion chemical Downloaded from acterized previously (24). The P815AB peptide (amino acid sequence ionization mass spectroscopy with methane as the reagent gas. The ion source LPYLGWLVF) was synthesized and puriﬁed as described previously (22, temperature was 220°C. Selected ion monitoring was performed by recording 25). All in vivo studies were performed in compliance with National and signals of characteristic (M-PFB)Ϫ ions. Perugia University Animal Care and Use Committee guidelines. Skin test assay DC preparation, treatments, and immunization A skin test assay was used for measuring class I-restricted delayed-type

Splenic DCs were purified by magnetic-activated sorting using CD11c Mi- hypersensitivity responses to synthetic peptides as described previously http://www.jimmunol.org/ croBeads and MidiMacs (Miltenyi Biotec), in the presence of EDTA to (22, 30, 31). Results were expressed as the increase in footpad weight of disrupt DC-T cell complexes. Cells were Ͼ99% CD11cϩ, Ͼ99% MHC peptide-injected footpads over that of vehicle-injected counterparts. Values I-Aϩ, Ͼ98% B7-2ϩ, Ͻ0.1% CD3ϩ, and appeared to consist of 90–95% (mean Ϯ SD) are compiled data from independent experiments with at CD8Ϫ, 5–10% CD8ϩ, and 1–5% B220ϩ cells. DC populations were further least six mice per group per experiment. The statistical analysis was per- separated into CD8Ϫ and CD8ϩ fractions by means of CD8␣ MicroBeads formed using Student’s paired t test by comparing the mean weight of (Miltenyi Biotec) (22, 25). The CD8Ϫ fraction was ϳ45% CD4ϩ and typ- experimental footpads with that of control counterparts. ϩ ϩ ically contained Ͻ0.5% contaminating CD8 cells. Less than 1% CD8 ϩ and Ͻ5% CD8Ϫ DCs expressed the B220 marker, respectively (26). DCs Induction of apoptosis by IDO DCs Ϫ ␥ Ϫ were exposed to 100 ng/ml IL-12 (CD8 DCs) or 200 U/ml IFN- (CD8 Conditions for inducing and assaying IDO-dependent apoptosis have been ϩ ␮ ϩ Ϫ or CD8 DCs) for 24 h at 37°C in the presence or absence of 4 M 1-MT described previously (32). Briefly, CD8 or CD8 DCs (2 ϫ 105), treated by guest on October 2, 2021 (27). For immunization, cells were washed between and after incubations overnight with IFN-␥ in the presence or absence of 10 ␮M L-kynurenine ␮ before peptide loading (5 M,2hat37°C), irradiation, and i.v. injection (KYN), were washed and cultured for 48 h with 4 ϫ 105 F76 cells, a ϫ 5 Ϫ ϩ into recipient hosts. A total of 3 10 IL-12-treated CD8 DCs was Th1-type P815AB-specific CD4 T cell clone, in the presence of 5 ␮M ␥ ϩ Ϫ ϩ injected in combination with 5% IFN- -treated CD8 or CD8 DCs. P815AB peptide. At the end of the coculture, the CD11c cells were removed by the use of CD11c MicroBeads (Miltenyi Biotec), and the re- Small interfering RNA (siRNA) synthesis and transfection mainder of the population was surface stained with anti-CD4-PE and Ј FITC-labeled annexin V and propidium iodide (PI; BD Pharmingen). For The siRNA sequences specific for murine Indo (sense, 5 -GGGCUUCU ϩ UCCUCGUCUCUtt-3Ј; antisense, 5Ј-AGAGACGAGGAAGAAGCCCtt- measurement of apoptosis, a gate was set on CD4 T cells, and the per- Ј centage of cells in the very early stages of apoptosis was determined by 3 ) were selected, synthesized, and annealed by the manufacturer (Am- ϩ bion). For transfection, siRNAs (6.7 ␮g) in 30 ␮l of transfection buffer (20 annexin V staining excluding PI cells. mM HEPES, 150 mM NaCl; pH 7.4) were pipetted into a sterile Eppendorf Transwell DC conditioning tube. In a separate polystyrene tube, 6.7 ␮g of 1,2-dioleoyl-3-trimethyl- ammonium-propane were mixed with 30 ␮l of transfection buffer, and then DC conditioning by IDO-competent cells was performed using a 6.5-mm both solutions were mixed gently by pipetting several times. After incu- Transwell system (Corning) with a 0.4-␮m pore polycarbonate membrane bation at room temperature for 20 min, the mixture was added to 1 ml of insert. CD8Ϫ DCs (106) were added to the lower chamber, while CD8ϩ complete medium containing 106 DCs and incubated for 48 h at 37°C, with DCs (106) were added to the upper chamber. Cultures were set up in the or without IFN-␥ during the last 18 h of incubation. Cells were then re- presence of 200 U/ml IFN-␥, and, after 18 h, CD8Ϫ DCs were recovered, covered, washed, and immediately used for in vitro or in vivo experiments. extensively washed, and used for in vivo sensitization. Control treatment consisted of Negative Control siRNA (Ambion). Results PCR analysis Indo silencing and 1-MT have comparable effects on functional RT-PCR analysis was performed (30 cycles, annealing temperature of IDO in vitro and in vivo 60°C) using sense and antisense primers, as follows: Indo sense, 5Ј- GAAGGATCCTTGAAGACCAC-3Ј; Indo antisense, 5Ј-GAAGCTGCG IDO catalyzes the initial and rate-limiting step in tryptophan deg- ATTTCCACCAA-3Ј; Sod2 sense, 5Ј-AGACCTGCCTTACGACTATG- radation to QUIN through the sequential formation of KYN, 3-hy- 3Ј; Sod2 antisense, 5Ј-AGCGGAATAAGGCCTGTTGT-3Ј. droxykynurenine (3-HK) or alternatively anthranilic acid (AA), Real-time PCR were run on a Chromo4 Four-Color Real-Time Detector and 3-HAA (Fig. 1). Thus, pharmacologic blockade of IDO by the (Bio-Rad) using specific primers as follows: Indo sense, 5Ј-GAAGGATC CTTGAAGACCAC-3Ј; Indo antisense, 5Ј-GAAGCTGCGATTTCCAC use of 1-MT results in impaired formation of KYN and the down- CAA-3Ј; Kynu sense, 5Ј-AAATTCCCTTGGCCTTCAAC-3Ј; Kynu anti- stream metabolites including QUIN. siRNA technology is a means sense, 5Ј-GCGTTTGCCTACATCATGG-3Ј; Kmo sense, 5Ј-GCCTTG of posttranscriptional gene silencing that can be used to modulate AAAGCCATTGGTC-3Ј; Kmo antisense, 5Ј-GCACTGTGAGTAC gene expression in murine DCs (25, 27). To comparatively exam- CCCTTCC-3Ј; Haao sense, 5Ј-ACGCCGTGTGAGAGTGAAGT-3Ј; Ј Ј ine the effects of 1-MT and posttranscriptional Indo silencing, Haao antisense, 5 -GTAATACCTCAGCCCATCC-3 . PCR products were ϩ normalized to murine Gapdh using specific primers (sense, 5Ј-GCCTTC splenic DCs were fractionated and the CD8 fraction, treated or CGTGTTCCTACCC-3Ј; antisense, 5Ј-CAGTGGGCCCTCAGATGC-3Ј). not with IFN-␥, was transfected with Indo-specific or control 132 KYNURENINE PATHWAY ENZYMES IN TOLERANCE

DC populations in the spleens of DBA/2 mice consist of CD8Ϫ (ϳ90%) and CD8ϩ (ϳ10%) fractions that mediate the respective immunogenic and tolerogenic presentation of the synthetic tumor/ self nonapeptide P815AB. Upon transfer into recipient hosts, peptide-loaded CD8Ϫ DCs initiate immunity, and CD8ϩ DCs initiate anergy, when Ag-specific skin test reactivity is measured at 2 wk after cell transfer (30, 31). On cotransferring cytokine-conditioned DCs of the two subsets, the IDO-inducing tolerogenic potential of IFN-␥ acting on CD8ϩ DCs enables these cells to prevail over the adjuvant properties of IL-12 acting on CD8Ϫ DCs, resulting in lack of skin test reactivity (22, 34–36). P815AB-pulsed, IL-12- treated CD8Ϫ DCs were injected in combination with 5% CD8ϩ DCs, either untreated or treated with IFN-␥, with or without con- comitant 1-MT or Indo siRNA treatment (Fig. 2D). The results FIGURE 1. Kynurenine pathway of tryptophan catabolism in mamma- showed that IFN-␥ was an absolute requirement for the occurrence lian cells, in which IDO catalyzes the initial and rate-limiting step under of suppressive effects by CD8ϩ DCs cotransferred with IL-12- transcriptional regulation by IFN-␥. Indicated in the figure are the enzyme primed CD8Ϫ DCs. However, the effect was equally ablated by activities, and the corresponding genes, leading to the production of im- treatment of the tolerogenic fraction with 1-MT or Indo siRNA munologically relevant metabolites. before DC cotransfer. This confirms that IDO is a major pharmacologic target of racemic 1-MT in our experimental setting of Downloaded from ϩ ␥ siRNA, resulting in efficient and selective inhibition of Indo tran- CD8 DC conditioning in vitro with IFN- . script expression at 48 h of transfection (Fig. 2A). This was accompanied by inhibition of IDO function as measured by the pro- Detection of transcriptional but not functional expression of Ϫ ␥ duction of KYN by IFN-␥-treated CD8ϩ DCs (Fig. 2B) as well as kynurenine pathway enzymes in CD8 DCs treated with IFN- by their ability to induce apoptosis of a Th1 clone, as documented Using RT-PCR, we previously showed that IFN-␥ activates Indo http://www.jimmunol.org/ previously (32, 33) (Fig. 2C). All of these functional effects in transcription in both CD8ϩ and CD8Ϫ DCs, resulting in compa- vitro were mimicked by the use of 1-MT in place of Indo siRNA. rable expressions of IDO protein in the two subsets (21, 23). In this by guest on October 2, 2021

FIGURE 2. Indo silencing has effects comparable to 1-MT on functions mediated by IDO in vitro and in vivo. A, Kinetic RT-PCR analysis of Indo expression in CD8ϩ DCs treated with Indo-specific siRNA in the presence or absence of IFN-␥. Control cells were treated with negative control (nc) siRNA. Expression of the IFN-␥-inducible Sod2 gene was also assayed as a specificity control. B, Effect of IFN-␥ on tryptophan (100 ␮M) conversion to KYN in CD8ϩ DCs previously subjected to 1-MT treatment or Indo silencing by siRNA for 48 h. KYN levels in 5-h supernatants were measured by GC/MS, p Ͻ 0.005 (IFN-␥ vs medium treatment). C, Effect of Indo ,ء .and the results are mean Ϯ SD of three different experiments, each performed in triplicate silencing on IDO-dependent apoptosis mediated by IFN-␥-treated CD8ϩ DCs. Apoptosis was measured in Ag-specific CD4ϩ T cells cultured with CD8ϩ DCs pre-exposed to IFN-␥ with or without Indo siRNA or 1-MT. Representative dot plots are shown of annexin V staining of gated CD4ϩ PIϪ cells. Numbers within boxes indicate percentages of CD4ϩ apoptotic cells in one experiment representative of three. The fold increase values in apoptosis rate induced by IFN-␥ in the remainder experiments were 2.9 and 2.5, respectively, and these values decreased to Ͻ1.1 upon Indo silencing. D, Indo silencing ablates the tolerogenic potential of CD8ϩ DCs. Combinations of IL-12-treated CD8Ϫ and CD8ϩ DCs subjected to various treatments (indicated) were p Ͻ 0.0001 (experimental ,ء .loaded with P815AB and injected into recipient mice to be assayed at 2 wk for skin test reactivity to the eliciting peptide vs control footpads). Compiled data from three independent experiments. The Journal of Immunology 133 study, we used real-time PCR for quantitative and comparative detection of high levels of QUIN. Collectively, these data indi- assessment of Indo expression in CD8Ϫ and CD8ϩ DCs and ex- cated that DCs are endowed with the whole enzymatic machinery tended the examination to a series of genes coding for enzymes necessary for the production of one of the end products of the downstream of IDO, namely Kmo (coding for kynurenine 3-mono- kynurenine pathway, QUIN. However, these data do not clarify oxygenase), Kynu (kynureninase), and Haao (3-hydroxyanthrani- whether the inability of IFN-␥ to activate the pathway in the CD8Ϫ late 3,4-dioxygenase) (Fig. 3A). The results confirmed enhanced subset of DCs is due solely to an inefficient rate-limiting step (i.e., transcriptional activity of Indo by IFN-␥ in CD8Ϫ DCs, to an ex- the one mediated by IDO) or to a combination of the latter with tent comparable to CD8ϩ counterparts. Similar to Indo albeit to a posttranscriptional regulation of downstream enzymes. lesser extent, the expressions of Kmo, Kynu, and Haao were all Ϫ enhanced by IFN-␥ in both DC subsets. Despite the increase in KYN conversion into QUIN occurs in CD8 DCs, resulting in gene transcription, IFN-␥ induced little or no production of KYN tolerogenic properties and QUIN in CD8Ϫ DCs (Fig. 3B), possibly accounting for the We investigated whether the enzyme response induced by IFN-␥ failure of the recombinant cytokine to promote tolerogenic prop- downstream of IDO in CD8Ϫ DCs is associated with the produc- erties in this subset (21, 23, 35). In contrast, in CD8ϩ DCs, IFN-␥ tion of QUIN when the precursor KYN is added to the culture resulted in early (5 h) production of KYN, followed by later (18 h) medium. We first validated the model by adding KYN to a culture of Indo siRNA-treated CD8ϩ DCs and found that the addition of the external precursor was indeed associated with the production of QUIN when the cells had been exposed to IFN-␥ (Fig. 4A). Similar to the latter cells, CD8Ϫ DCs produced high levels of QUIN if cotreated with IFN-␥ and KYN. The ability of CD8Ϫ DCs Downloaded from to release QUIN in response to IFN-␥ and the externally added precursor prompted us to investigate whether synthesis of QUIN was associated with the acquisition of properties typical of the tolerogenic CD8ϩ DC subset. In the experimental setting illustrated above, we measured apoptosis of CD4ϩ cells cultured with CD8Ϫ DCs either untreated or treated with IFN-␥ and KYN, singly http://www.jimmunol.org/ or in combination (Fig. 4B). The results revealed an ability of the CD8Ϫ DCs to induce apoptosis in ϳ25% of Th1 target cells upon coexposure to IFN-␥ and KYN. We also asked whether the CD8Ϫ DCs conditioned by KYN and IFN-␥ treatment would be capable of suppressive properties in vivo when cotransferred with otherwise immunogenic DCs. In an experimental model system similar to that of Fig. 2D, P815AB-pulsed and IL-12-primed CD8Ϫ DCs were injected in combination with 5% CD8Ϫ DCs cotreated with by guest on October 2, 2021 a mixture of IFN-␥ and KYN, either alone or together with PNU 156561, a selective inhibitor of kynurenine 3-monooxygenase (Fig. 5A). The results showed that the combined effects of IFN-␥ and KYN turned CD8Ϫ DCs from immunogenic to suppressive, thus mimicking the behavior of the CD8ϩ DC subset. However, inhibiting the conversion of KYN into 3-HK, and thus preventing synthesis of QUIN, abolished the onset of suppressive properties in the CD8Ϫ DC fraction. Of interest, the mice sensitized with the DC population containing 5% IFN-␥/KYN-treated CD8Ϫ DCs were not amenable to subsequent immunogenic priming to the peptide when performed 1 mo after the first exposure (Fig. 5B). Again, this effect is similar to that observed with IFN-␥-treated CD8ϩ DCs (35) and indicates the onset of an Ag-specific tolerant state. Taken together, these data suggest that even in the absence of functional IDO, kynurenine pathways enzymes, if activated by IFN-␥ in the presence of an appropriate precursor, catalyze the synthesis of QUIN, and the effect is associated with DC acquisition of tolerogenic properties. FIGURE 3. CD8Ϫ DCs express kynurenine pathway enzymes but produce little or no KYN and QUIN. A, Induction of Indo, Kmo, Kynu, and IDO-competent DCs transfer tolerogenic potential across a Haao transcripts in CD8ϩ and CD8Ϫ DCs by IFN-␥. Sorted CD8ϩ and cell-impermeable membrane Ϫ CD8 DCs were exposed to rIFN-␥ for 4 h. At the end of the culture, The finding that kynurenine pathway enzymes downstream of IDO mRNA levels of genes related to the kynurenine pathway were quantified can initiate tolerogenesis in DCs lacking functional IDO prompted Ϯ by real-time PCR using Gapdh normalization. Data (means SD of three us to investigate whether IDO-competent cells might turn other- experiments using triplicate samples) are presented as normalized tran- wise immunogenic DCs into tolerogenic cells via soluble mole- script expression in the samples relative to normalized expression in the ϩ ␥ Ϫ respective control cultures, i.e., cells unexposed to IFN-␥ but maintained in cules. CD8 DCs were exposed overnight to IFN- , and CD8 ϭ DCs were added to the lower chamber of a Transwell system. The medium alone (fold change 1; dotted line). B, Conversion of tryptophan ϩ (100 ␮M) to KYN and QUIN by CD8ϩ vs CD8Ϫ DCs in response to CD8 cells had been treated with either negative control or Indo- Ϫ exposure to IFN-␥ for 5 or 18 h. Values are means Ϯ SD of three inde- specific siRNAs. The CD8 DCs were then recovered and assayed Undetectable levels. in vivo for their ability to suppress induction of skin test reactivity ,ء .pendent experiments performed in triplicate 134 KYNURENINE PATHWAY ENZYMES IN TOLERANCE

FIGURE 5. The combined effects of IFN-␥ and KYN confer tolerogenic potential in vivo to CD8Ϫ DCs. Combinations of IL-12-treated CD8Ϫ DCs and a minority fraction (5%) of the same cells not treated with IL-12 but exposed to 200 U/ml IFN-␥ and 10 ␮M KYN were loaded with P815AB. A group of IFN-␥/KYN-treated CD8Ϫ DCs was also exposed to 20 ␮M PNU 156561, a selective inhibitor of kynurenine 3-monooxygenase. The

cells were then transferred into recipient hosts. Mice were assayed for skin Downloaded from test reactivity on day 15 after cell transfer (A) or, alternatively, were treated on day 30 with an otherwise effective priming consisting of P815AB- pulsed CD8Ϫ DCs, to be assayed for skin test reactivity on day 45 (B). p Ͻ 0.0001 (experimental vs ,ء .Compiled data from three experiments control footpads). http://www.jimmunol.org/

intermediates. Although all kynurenines are found in high concen- tration in urine, none of them has so far been assigned an important physiological function in peripheral organs. In the brain, the kynurenine pathway yields several intermediates, including the free radical generator 3-HK, the excitotoxic N-methyl-D-aspartate FIGURE 4. Synthesis of QUIN by CD8Ϫ DCs is associated with ability receptor agonist QUIN as well as its antagonist, kynurenic acid. to induce apoptosis. A, Effective conversion of KYN into QUIN by IFN- Astrocytes, neurons, and microglia all express IDO, but only mi- by guest on October 2, 2021 ␥-treated CD8Ϫ DCs. CD8Ϫ DCs and CD8ϩ DCs treated with negative croglial cells are capable of producing substantial quantities of control (nc) or Indo siRNA were compared for ability to catabolize KYN QUIN (39). Although KYN can be produced in the brain to a to QUIN on activating kynurenine pathway enzymes with IFN-␥. Cells were cultured for 18 h in the presence of 200 U/ml IFN-␥ and 100 ␮M KYN before measurement of QUIN concentrations in supernatants. Data p Ͻ ,ء .are the means Ϯ SD of three experiments with triplicate samples 0.01 (IFN-␥ vs medium treatment). B, Induction of potential for apoptosis by IFN-␥ and KYN (10 ␮M) in CD8Ϫ DCs. Apoptosis of Ag-speciﬁc CD4ϩ T cells was measured as in Fig. 2C. Numbers within boxes indicate percentages of CD4ϩ apoptotic cells in one experiment representative of three. The fold increase in apoptosis rate induced by KYN ϩ IFN-␥ in the other two experiments was 4.0 and 6.8, respectively.

to P815AB (Fig. 6). According to the experimental model described above, a minority fraction of conditioned CD8Ϫ DCs was added to an IL-12-treated counterpart, and the mixture was transferred into recipients to be assayed at 2 wk for the development of skin test reactivity. The results showed that otherwise nontolero- FIGURE 6. IDO-competent DCs added across a Transwell to a culture Ϫ genic CD8 DCs became suppressive as a result of coculture in the of nontolerogenic cells induce suppressive properties in the latter via IDO- ϩ Transwell system with CD8 DCs treated with negative control dependent mechanisms. CD8ϩ DCs (106) were added to the upper chamber but not Indo siRNA. This result demonstrates that IDO-competent of a Transwell system, the bottom chambers of which contained an equal Ϫ DCs can confer tolerogenic potential on cells lacking functional number of CD8 DCs. rIFN-␥ was present in the cultures at 200 U/ml, and the CD8ϩ DCs had been transfected with Indo siRNA or negative control IDO by means of tryptophan catabolism and the production of Ϫ soluble diffusible molecules. (nc) siRNA. After 18 h, the recovered CD8 DCs were pulsed with P815AB and added to a majority fraction (95%) of peptide-pulsed, IL-12- treated DCs. The cells were then transferred into recipient mice that were Discussion assayed at 2 wk for P815AB-specific skin test reactivity. The contents of The kynurenine pathway is a major route of L-tryptophan catabo- the lower and upper chambers of the Transwell are indicated in the figure. p Ͻ 0.0001 (experimental vs ,ء .lism, resulting in the production of nicotinamide adenine dinucle- Compiled data from three experiments otide and other neuroactive (37, 38) and immunomodulatory (10) control footpads). The Journal of Immunology 135 moderate degree, the cerebral pathway is driven mainly by blood- synthetic 3-HAA is also taken up by DCs, resulting in the produc- borne KYN, which enters from the circulation using the large neu- tion of QUIN (data not shown). tral amino acid transporter. In the brain, KYN is rapidly taken up DCs have several modes at their disposal to mediate tolerogenic by astrocytes and microglial cells, to be converted into end prod- functions, and metabolic pathways of immune regulation are but ucts that are then released extracellularly (37). one possibility (48). A distinctive feature of tryptophan catabolism In the periphery, the immunosuppressive pathway of tryptophan as an effector mechanism of suppression is that its function is com- catabolism has been shown to contribute to successful pregnancy patible with a directive role of Tregs in orchestrating peripheral (1) and transplantation (2), as well as to effective control of auto- tolerance (30). We have previously shown that the default immu- immunity (3). We have proposed a model of transplantation tol- nogenic or tolerogenic program of Ag presentation by DC subsets erance that might apply to both transplantation and autoimmunity is highly flexible, and that otherwise immunogenic DCs can be (10). In this model, IFN-␥ acts on tolerogenic DCs to activate IDO, rendered tolerogenic by the actions of inhibitory ligands expressed resulting in the onset of specific tolerance through the induction of by T cells (2, 22, 26, 27). Unlike soluble or cell-bound CTLA-4, tryptophan starvation and the production of immunomodulatory IFN-␥ is unable per se to induce functional IDO expression in kynurenines. Extensive work in our as well as others’ laboratories CD8Ϫ DCs (21). Our current data demonstrate that, in the presence has subsequently shown that this model 1) accommodates the ac- of IFN-␥, CD8Ϫ DCs can be rendered tolerogenic by the nearby tivity of regulatory T cells (Tregs) that, expressing surface production of kynurenines that are taken up by the cells and then CTLA-4, engage B7 coreceptor molecules on DCs and initiate the metabolized to downstream products of the pathway. This mech- ␥ production of autocrine IFN- (12, 30); 2) applies to different sub- anism is qualitatively different from that of CTLA-4, which acts on sets of DCs, including plasmacytoid DCs (15, 26); and 3) suggests Ϫ

CD8 DCs to activate IDO, making it functionally responsive to Downloaded from that the IDO mechanism is quite general in nature, in that, for IFN-␥ up-regulation (21). On the one hand, the bulk of these data example, not only DCs but also CD4ϩ T cells (40) and neutrophils demonstrates that tryptophan consumption may not always be an (7) respond to CTLA-4 engagement of their surface B7 molecules absolute requirement for the DCs to exert suppressive properties. with the activation of IDO, depending on the release of autocrine On the other hand, it is possible that, to meet the needs of ﬂexi- IFN-␥. bility and redundancy, the paracrine production of kynurenines is Despite much evidence for such a general role of tryptophan

exploited by IDO-competent DCs to convert DCs that are not com- http://www.jimmunol.org/ catabolism as a mechanism of immunosurveillance (17), there are petent to a tolerogenic phenotype. several mechanistic features of the pathway that await further clar- A model of cross-talk of DCs and T cells in tryptophan catab- ification (19). Among these are the relative contributions of IDO olism within an IFN-␥-rich environment is illustrated in Fig. 7. and the remainder pathway enzymes to immunosuppression (41), The model, far from detracting from the role of tryptophan star- as well as the possible function of specific kynurenines as inducers vation in mediating specific effects of the IDO mechanism (16), or mediators of immunoregulation (18, 33, 42–44). In this study, we have provided evidence that 1) IDO is the major relevant target reinforces the concept that tryptophan deprivation and tryptophan of pharmacologic inhibition of the kynurenine pathway when racemic 1-MT is used as an enzyme inhibitor; 2) DCs express the by guest on October 2, 2021 entire metabolic machinery necessary for synthesis of QUIN; 3) IFN-␥ enhances the transcriptional expression of all these enzymes, which may in fact respond as a coordinately regulated clus- ter of genes (45); yet, posttranslational inactivation of IDO occurs in nontolerogenic CD8Ϫ DCs, and this prevents the action of enzymes consequent to IDO function; and 4) bypassing the IDO- associated blockade by the addition of a downstream QUIN precursor initiates the pathway and leads to the onset of suppressive properties in CD8Ϫ DCs treated with IFN-␥. Human macrophages are endowed with the ability to convert tryptophan to QUIN (46, 47); yet, no information was previously available regarding the functional expression of kynurenine pathway enzymes other than IDO in human or murine DCs. We found that the splenic CD8ϩ subset of murine DCs could be induced by IFN-␥ to release high levels of QUIN. Despite functional lack of Ϫ FIGURE 7. A model of cross-talk of DCs and T cells in tryptophan the pathway regulatory enzyme IDO in CD8 DCs, these cells catabolism. In local tissue microenvironments, the activity of IDO com- ␥ ϩ could nevertheless be induced by IFN- to produce QUIN in the petent DCs (IDO ) driven by IFN-␥ will result in the sustained production presence of KYN, a downstream precursor of the end product. of kynurenines (Kyn). These, in turn, could recruit other cell types to the Activation of the pathway was accompanied by the appearance of regulatory response, including DCs in which the function of IDO, but not tolerogenic properties in vivo. Thus, the current study may be the other kynurenine pathway enzymes, is inhibited posttranslationally first to demonstrate the presence of a functional kynurenine path- (IDOϪ). The combined effects of tryptophan (Trp) starvation (16), caused ϩ way in murine DCs and, interestingly, to specifically associate the by IDO DCs, and the high kynurenine production, resulting from the ϩ Ϫ tolerogenic phenotype with the function of a coordinately regu- actions of IDO and IDO DCs, is expected to induce a great variety of lated set of enzymes, as observed upon treatment with IFN-␥.In effects on target T cells (18, 33, 44) and other cell types (42, 43). Tregs would have a crucial role in establishing an IFN-␥-rich environment, either addition, similar to what was observed with astrocytes and micro- via CTLA-4/B7-dependent interaction with DCs (30) or by direct produc- glial cells (37), our current data demonstrate that KYN is taken up tion of the cytokine (49). Because the combined effects of tryptophan star- by DCs. The kynurenine 3-monooxygenase-dependent conversion vation and tryptophan catabolites include the induction of a regulatory of KYN into QUIN by DCs further demonstrates that specific in- phenotype in naive CD4ϩ T cells (51), an IDO-based positive feedback tracellular metabolism of kynurenines occurs in those cells. Of loop might be locally operative, resulting in Treg generation by Tregs interest in this regard, we have also found that similar to KYN, (dotted line). 136 KYNURENINE PATHWAY ENZYMES IN TOLERANCE catabolites may act singly or in combination to establish a regu- oxide in maintaining peripheral tolerance by CD4ϩCD25ϩ regulatory T cells. latory environment most suitable to the control of local inflamma- J. Immunol. 174: 5181–5186. 19. Moffett, J. R., and M. A. Namboodiri. 2003. Tryptophan and the immune re- tory or systemic T cell-mediated responses. The model also em- sponse. Immunol. Cell. Biol. 81: 247–265. phasizes the likely dual role of tryptophan catabolism both as an 20. Braun, D., R. S. Longman, and M. L. Albert. 2005. A two-step induction of effector system (30, 49) and an inducer of Treg activity (7, 44, 50, indoleamine 2,3 dioxygenase (IDO) activity during dendritic-cell maturation. Blood 106: 2375–2381. 51). Because 1-MT has been shown to act synergistically with 21. Orabona, C., P. Puccetti, C. Vacca, S. Bicciato, A. Luchini, F. Fallarino, cytoreductive chemotherapy in an experimental tumor setting (9), R. Bianchi, E. Velardi, K. Perruccio, A. Velardi, et al. 2006. Toward the identification of a tolerogenic signature in IDO-competent dendritic cells. Blood 107: the identification of proper enzyme targets for pharmacologic in- 2846–2854. hibition of the kynurenine pathway may pave the way to improved 22. Grohmann, U., R. Bianchi, C. Orabona, F. Fallarino, C. Vacca, A. Micheletti, strategies of antitumor chemotherapy (13). More generally, under- M. C. Fioretti, and P. Puccetti. 2003. Functional plasticity of dendritic cell subsets as mediated by CD40 versus B7 activation. J. Immunol. 171: 2581–2587. standing the molecular mechanisms that preside over control of 23. Fallarino, F., C. Vacca, C. Orabona, M. L. Belladonna, R. Bianchi, B. Marshall, IDO expression by DCs may provide new opportunities for de- D. B. Keskin, A. L. Mellor, M. C. Fioretti, U. Grohmann, and P. Puccetti. 2002. ϩ signing ways to induce or abrogate tolerance in physiopathologic Functional expression of indoleamine 2,3-dioxygenase by murine CD8␣ dendritic cells. Int. Immunol. 14: 65–68. conditions (21, 25, 52). 24. Wu, H. Q., P. Guidetti, J. H. Goodman, M. Varasi, G. Ceresoli-Borroni, C. Speciale, H. E. Scharfman, and R. Schwarcz. 2000. Kynurenergic manipula- tions influence excitatory synaptic function and excitotoxic vulnerability in the rat Disclosures hippocampus in vivo. Neuroscience 97: 243–251. The authors have no financial conflict of interest. 25. Orabona, C., M. L. Belladonna, C. Vacca, R. Bianchi, F. Fallarino, C. Volpi, S. Gizzi, M. C. Fioretti, U. Grohmann, and P. Puccetti. 2005. Cutting edge: silencing suppressor of cytokine signaling 3 expression in dendritic cells turns

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