Indoleamine 2,3-dioxigenase (IDO) is critical for ... - The FASEB Journal

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Indoleamine 2,3-dioxigenase (IDO) is critical for host resistance against Trypanosoma cruzi Carolina Paola Knubel,* Fernando Fabiań Martínez,* Ricardo E. Fretes,† Cintia Díaz Lujan,† Martín Gustavo Theumer,* Laura Cervi,* and Claudia Cristina Motrań*,1 *Centro de Investigaciones en Bioquímica Clínica e Inmunología–Consejo Nacional de Investigaciones Científicas y Tecnicas (CIBICI-CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, and †Instituto de Biología Celular, Facultad de Medicina, Universidad Nacional de Co´rdoba, Co´rdoba, Argentina Indoleamine 2,3-dioxigenase (IDO) is an inflammatory cytokine-inducible rate-limiting enzyme of the tryptophan (Trp) catabolism, which is involved in the inhibition of intracellular pathogen replication as well as in immunomodulation. Here we demonstrated the effect of IDO-dependent Trp catabolism on Trypanosoma cruzi resistance to acute infection. Infection with T. cruzi resulted in the systemic activation of IDO. The blocking of IDO activity in vivo impaired resistance to the infection and exacerbated the parasite load and infectionassociated pathology. In addition, IDO activity was critical to controlling the parasite’s replication in macrophages (Mos), despite the high production of nitric oxide produced by IDO-blocked T. cruziinfected Mos. Analysis of the mechanisms by which IDO controls the parasite replication revealed that T. cruzi amastigotes were sensitive to L-kynurenine downstream metabolites 3-hydroxykynurenine (3HK) and 3-hydroxyanthranilic acid, while 3-HK also affected the trypomastigote stage. Finally, 3-HK treatment of mice acutely infected with T. cruzi was able to control the parasite and to improve the survival of lethally infected mice. During infection, IDO played a critical role in host defense against T. cruzi; therefore, the intervention of IDO pathway could be useful as a novel antitrypanosomatid therapeutic strategy.—Knubel, C. P., Martínez, F. F., Fretes, R. E., Díaz Lujan, C., Theumer, M. G., Cervi, L., Motrań, C. C. Indoleamine 2,3-dioxigenase (IDO) is critical for host resistance against Trypanosoma cruzi. FASEB J. 24, 2689 –2701 (2010). www.fasebj.org ABSTRACT

Key Words: Infection 䡠 parasites 䡠 immunomodulation 䡠 kynurenines

TRYPANOSOMA CRUZI, the etiological agent of Chagas disease, is a protozoan parasite that affects ⬎20 million people in Latin America. The infection is initiated by the entry of metacyclic trypomastigotes (tps) into the mammalian host and the subsequent invasion by these parasites of a wide variety of host cell types. Within host 0892-6638/10/0024-2689 © FASEB

cells, T. cruzi converts into an amastigote stage that replicates in the host cell cytoplasm (1). As immune control of the infection is established and the infection progresses into the chronic phase, the parasites are restricted predominantly to muscle tissues. The onset of human pathology may be extremely diverse and depends on the parasite biology as well as its relationship with the host. During the very early stages of infection, this parasite is found within macrophages (Mos), where its replication can be either inhibited or favored, leading to dissemination to other sites within the body (2). Experimental evidence indicates that control of T. cruzi parasitism during the early phase of infection is critically dependent on effective Mo activation, as well as on the up-regulation of the inducible nitric oxide synthase enzyme (iNOS) and on subsequent nitric oxide (NO) production; in mice, NO is considered to be the principal effector molecule killing the intracellular amastigotes (3, 4). Nevertheless, the presence of high levels of NO and proinflammatory cytokines may at the same time be detrimental, given their participation in tissue injury (5). Therefore, in order to limit the potential damage produced by an excessive inflammatory reaction, the immune system has evolved a series of immunoregulatory mechanisms, such as down-regulatory cytokines, regulatory T-cell induction, and indoleamine 2,3-dioxigenase (IDO) up-regulation. IDO is an intracellular enzyme that is constitutively expressed in several human and mouse cells. Being present in innate immune cells, such as Mos and dendritic cells (DCs), IDO catalyzes the initial ratelimiting step of tryptophan (Trp) catabolism, thus leading to the production of immunoregulatory catabolites (collectively known as kynurenines) (6). 1 Correspondence: Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Co´rdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, Co´rdoba, 5000, Argentina. E-mail: [email protected] doi: 10.1096/fj.09-150920

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The IDO gene promoter contains multiple sequence elements that confer responsiveness to proinflammatory mediators, thereby demonstrating the strong correlation between inflammation and induced IDO expression (7). Early studies documented the ability of IDO to inhibit the proliferation of facultative intracellular pathogens in vitro through the consumption of the essential amino acid Trp, thus showing that IDO forms part of the innate host defense against infections (8, 9). In addition, Trp starvation and the accumulation of kynurenines inhibit T-cell proliferation, promote T-cell death or anergy, and exert differential effects on helper T-cell responses (10 –13). Moreover, IDO-competent DCs have a role in generating the acquired regulatory T (Treg) cells that drive peripheral tolerance (14). Although IDO might have a direct effect in vitro on the replication of some kinds of pathogens, the specific contribution of IDO to host defense against different infections in vivo is not predictable because of the complex role of IDO in immunoregulation and the unique host-parasite relationship. Thus, the induction of immunosuppression and the generation of Treg cells during infection are particularly relevant, given that one undesirable consequence of inducing IDO expression by the innate system might be the suppression of the specific T-cell response before pathogen clearance. On the other hand, the restriction of pathogen growth and the prompt activation of immunoregulatory mechanisms (key to control pathogenic inflammation) are one highly desirable possible effect of IDO activation. We evaluated the role of IDO in the control of in vivo infection with T. cruzi. This study clearly demonstrates that IDO expression is up-regulated during T. cruzi infection in mice and that IDO activity is critical to control T. cruzi replication during the acute phase of this infection. In addition, T. cruzi amastigotes were sensitive to l-kynurenine downstream metabolites 3-HK and 3-HAA, while 3-HK affected also the tp stage. Finally, 3-HK treatment of mice acutely infected with T. cruzi was able to control the parasite replication and to improve the survival of lethally infected mice.

MATERIALS AND METHODS

Female BALB/c mice, 6 – 8 wk old, obtained from Comisioń Nacional de Energía Ato´mica (CNEA; Buenos Aires, Argentina), were i.p. infected with 500 tps from T. cruzi, as described previously (15). The studies were approved by the Institutional Review Board and Ethical Committee of the Faculty of Chemical Sciences, National University of Cordoba (Res 459/09). The Tulahuen strain of T. cruzi was used, which was maintained by weekly i.p. inoculations in mice. Drugs We purchased 1-methyl-d-tryptophan (1-MT), l-tryptophan (Trp), l-kynurenine (Kyn), 3-hydroxy-dl-kynurenine (3-HK), Vol. 24

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Western blotting for IDO Spleen cells or tissue cell extracts (100 ␮g) treated with RIPA buffer were analyzed by 12% SDS-PAGE and transferred to a nitrocellulose membrane. IDO protein was revealed by immunoblotting using polyclonal rabbit anti-IDO antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by horseradish peroxide-coupled secondary antibody (SigmaAldrich) and visualized using a chemiluminescence substrate (GE Healthcare, Piscataway, NJ, USA). Anti-p38 (Santa Cruz Biotechnology) was used as loading control. HPLC analysis of L-kynurenine The l-kynurenine concentration was measured by HPLC. Mouse sera were kept at ⫺20°C until measurement. Eighty microliters of serum was diluted with 80 ␮l of potassium phosphate buffer (0.05 M, pH 6.0) containing the internal calibrator 3-nitro-l-tyrosine (100 ␮M). Protein was precipitated with 25 ␮l of trichloroacetic acid (2 M). Then the tubes with the precipitate were immediately vortex-mixed and centrifuged for 10 min at 13,000 g. One hundred microliters of the supernatant was analyzed by HPLC as described previously (16) using a Hewlett Packard HP1100 series system (Hewlett Packard, Palo Alto, CA, USA). Assay of IDO activity Fresh spleen cells, tissues, or cultured Mos were washed 3 times before being suspended in ice-cold PBS and disrupted by sonication for 30 s in an ice bath at a power of 100 W. The homogenate was centrifuged at 800 g for 10 min at 4°C, and the supernatant was then centrifuged at 15,000 g for 15 min at 4°C. The resultant supernatant was used for the colorimetric assay of IDO activity, as described by Kudo and Boyd (17). In vivo IDO inhibition 1-MT was dissolved in 50 mM sodium carbonate buffer (pH 10) before use. Mice were given 1-MT in the drinking water (1 mg/ml), beginning at the day of infection and continuing until death, while the controls received only water. Monitoring of acute infection

Mice and parasites

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3-hydroxyanthranilic acid (3-HAA), quinolinic acid (QA) and 3-nitro-l-tyrosine (⬃99% pure) from Sigma-Aldrich (St. Louis, MO, USA). Potassium phosphates and acetonitrile for the HPLC elution buffer (Merck, Darmstadt, Germany) were of HPLC grade. Aminoguanidine (AG) was obtained from Sigma-Aldrich.

Blood was collected at different times postinfection (pi). Erythrocytes were lysed in a 0.87% ammonium chloride buffer, and viable tps were counted in a Neubauer chamber. Mice were weighed every other day following infection to monitor the systemic repercussion of the acute disease. Histological studies The heart and the skeletal muscle from the quadriceps were removed at different times pi, fixed in buffered 10% formalin (pH 7.0), and embedded in paraffin. Organs were cut with a microtome to obtain 5-␮m slices, collecting 1 section every 100 ␮m of the tissue until 300 ␮m of each organ was sectioned, thus obtaining 3 of each organ belonging to 3 levels, which were then stained with hema-

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toxilyn/eosin (H/E). At least 25 fields from each section were checked for parasites and histopathology under an ⫻40 objective of a photonic microscope (Axioskop; Carl Zeiss Vision, Oberkochen, Germany) in a blind study. Twelve random digital images per organ were taken at the 3 levels (4/level) with a Hitachi color camera (Hitachi, Tokyo, Japan) being employed to measure the number of amastigotes, the area and number of nests of the amastigotes, and the number of inflammatory infiltrates. Measurements were performed by the quantitative image analysis system Axiovision 3.0.6 (Carl Zeiss).

3-HK treatment

Bone marrow-derived Mos

Statistical analysis

Bone marrow-derived Mos were generated by using L-cell conditioned medium as described previously (18).

Survival data were analyzed using the Kaplan-Meier test, and group comparisons by the Student’s t test, depending on the normality of the data. The statistical significance level was chosen as P ⬍ 0.05.

In vitro trypanocidal activity assay Mos (1⫻106) were cultured on 12-mm round glass coverslips in a 24-well plate with 1-MT, IFN-␥, IFN-␥ plus LPS, Trp, Kyn, 3-HK, 3-HAA, QA, or medium alone for 24 h, before being infected with 2 ⫻ 106 T. cruzi tps per well. To avoid Mo reinvasion of tps, noninternalized parasites were removed by 2 washes with RPMI medium 24 h later. The growth of parasites in Mos was evaluated by counting the intracellular amastigotes by immunofluorescence assays 48 h later, as described previously (19). In some experiments, Mos were cultured with 1-MT or medium alone for 24 h before being infected by 4 h. After washing to avoid reinvasion and the effect of the drugs on extracellular parasites, the cultures were treated with 20 ␮M of 3-HK or 3-HAA for 20 h and washed. Then, the growth of parasites in Mos was evaluated by counting the intracellular amastigotes, as described above. The trypanocidal effect of the different Trp metabolites was also tested on bloodstream tps according to a standard WHO protocol with minor modifications (20). NO assay The production of NO was measured indirectly by assaying nitrites in the culture supernatant using the Griess reaction ,as described previously (21). Video microscopy and quantitative motility assays Tps were monitored by video microscopy using a Zeiss Axiovert 200 microscope equipped with an ⫻60 objective (Supplemental Videos 1 and 2). Images were captured every second for a total of 50 s using a Zeiss Axiocam digital camera, and a trace of individual cells was monitored using WCIF ImageJ software (Wright Cell Imaging Facility, Toronto, ON, Canada). Tp ultrastructural study The pellet of the treated parasites was fixed in Karnovsky solution (5% gluteraldehyde and 4% formaldehyde) for 2 h at room temperature, then washed and postfixed in 1% osmium tetroxide. Afterward, parasite cells were dehydrated in acetone and embedded in Araldite (Electron Microscopy Sciences, Hatfield, PA, USA). The ultrathin sections were contrasted with uranyl acetate and lead citrate. Observations were made using a LEO 906E transmission electron microscope (Carl Zeiss). IDO IN HOST DEFENSE AGAINST T. CRUZI

Groups of 6 mice (6 – 8 wk old) maintained under standard conditions were infected with 1000 bloodstream T. cruzi tps by the i.p. route. Five days after infection, mice were daily treated with 3-HK (1 mg/kg/d) for 5 consecutive days [days postinfection (dpi) 5–10] by the i.p. route. We resuspended 3-HK in 0.1 M PBS, with this vehicle also being employed as a negative control. The levels of parasitemia were monitored every 2 d as described above, and the number of deaths was recorded daily.

RESULTS Experimental T. cruzi infection induces up-regulation of IDO expression and functional activity To assess IDO expression in T. cruzi infection, we infected BALB/c mice with 500 tps of T. cruzi. The tp dose was selected in view of the fact that almost all 500-tp-infected mice were able to develop the acute infection and progress to the chronic phase. At different times pi, IDO protein was detected by Western blot in spleen, skeletal muscle, and heart. A 42-kDa band corresponding to IDO was up-regulated in infected mice but not in uninfected ones (Fig. 1A), with this up-regulation starting as early as at dpi 3 (not shown). In addition, IDO activity was determined in spleen, skeletal muscle, and heart, and l-kynurenine production was quantified in sera at different times pi. In all tested tissues, IDO activity was higher in infected than in uninfected mice (Fig. 1B) and was undetectable in T. cruzi tp lysates (not shown). In the same way, the concentrations of Kyn in sera were also higher in infected than in uninfected mice (Fig. 1C). IDO blockade impairs resistance to T. cruzi infection To evaluate the role of IDO in T. cruzi infection, IDO activity was inhibited in infected mice using 1-MT, an IDO pharmacological inhibitor (22). Prior to the evaluation of the course of infection, we confirmed that, at least up to 14 dpi, IDO activity was inhibited in 1-MTtreated mice (Supplemental Fig. 1A, B). When the mice were monitored for survival, body weight, parasitemia, and histopathology, the results demonstrated that the treatment with 1-MT significantly impaired resistance to infection. As shown in Fig. 2A, B, 1-MT treatment significantly decreased survival in infected mice. For a 30-d treatment period, analyzing 3 independent experiments, 73% of the control mice survived compared to only 39% of the 1-MT-treated mice (Fig. 2B). Related to this, mice treated with 1-MT exhibited more parasitemia than control animals from dpi 10 to the peak of 2691

Figure 1. T. cruzi experimental infection induces up-regulation of IDO expression and functional activity. A) Western blot analysis for IDO detection in whole spleen, skeletal muscle, and heart lysates from uninfected (NI) or 15 d infected (I) mice. Data show one I or NI mouse representative of ⱖ10 mice of each strain assayed at different times pi. B) IDO activity determined in extracts of spleen, skeletal muscle, and heart. Bars represent average ⫾ sd value of activity (U/mg protein) obtained in tissue extracts from 3 mice/time point. C) Concentration of l-kynurenine in sera by HPLC. Bars represent average ⫾ sd serum l-kynurenine concentration (␮M) from 3 mice/time point. Experiments shown are representative of 3 independent tests. *P ⬍ 0.05, **P ⬍ 0.001 vs. NI.

parasitemia, which occurred on dpi 18 for both groups (Fig. 2C). In line with this findings, body weight on dpi 26 onwards of 1-MT-treated mice was significantly below that of control mice (Fig. 2D). The increased susceptibility to infection observed in 1-MT-treated mice was associated with an extensive parasite growth and the presence of numerous inflammatory foci in heart and skeletal muscle. An histological analysis of hearts from control mice on dpi 14 revealed typical histopathological alterations of acute chagasic myocarditis, with a few nests of T. cruzi amastigotes and scarce lymphomononuclear inflammatory infiltrates (Fig. 3Aa, B). In contrast, hearts from 1-MTtreated mice showed an important myocarditis in subendocardia, with the myocardia and epicardia showing significant between-group differences in the number and size of parasite nests (Fig. 3Ab, c; B). An histological analysis of skeletal muscle from control mice revealed

the presence of inflammatory infiltrates and a few nests of T. cruzi amastigotes (Fig. 3Ad, B). For 1-MT-treated mice, there were significant between-group differences in the number of lymphomononuclear inflammatory foci, amastigote nests, and amastigotes per nest (Fig. 3Ae, f; B). In addition, we observed that treatment with 1-MT did not adversely affect survival or normal histology in uninfected mice (not shown). IDO is critical for the control of T. cruzi amastigote growth in Mos The fact that IDO activity was necessary to control early parasite replication and improve mouse survival might have been the result of 2 not mutually exclusive mechanisms: the intracellular Trp starvation and/or the downstream metabolites produced by IDO activity in-

Figure 2. IDO blockade impairs resistance to T. cruzi infection. Survival, parasitemia, and body weight were determined at different times pi in 500-tp infected mice, treated (1-MT) or not (control) with 1-MT. A) Survival rate of 1-MT- or control-infected mice. B) Mice treated or not with 1-MT scored for survival at 30 dpi. Figure incorporates data from all 3 independent experiments; P value based on Kaplan-Meier test. C) Quantification of parasitemia (tp/ml blood). Results are means ⫾ sd of 6 – 8 mice/group. D) Body weight (g) at different times pi. *P ⬍ 0.05 vs. control. Experiments shown in A, C, D are representative of 3 independent tests; 6 – 8 mice/group.

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Figure 3. IDO blockade is associated with extensive parasite growth and the presence of lymphomononuclear inflammatory infiltrates in target organs. Heart and skeletal muscle sections obtained on dpi 15 from 500-tp-infected mice, treated (1-MT) or not (control) with 1-MT, were stained with H/E. A) Representative histological sections of heart (a–c) and skeletal muscle (d–f) from control (a, d) or 1-MT-treated mice (b, c, e, f). Thin arrow indicates focal mononuclear cell infiltrates. Thick arrow indicates nests of amastigotes (a, b, d, e: ⫻100; c, f: ⫻400). B) Quantitation of foci of cellular inflammatory infiltrates, number of nests of amastigotes, number of amastigotes in the nests, and size of nests at 3 levels of heart and skeletal muscle from control (n⫽3) and 1-MT-treated (n⫽4) groups, employing Axio-Vision 3.0.6 software. *P ⬍ 0.05 vs. control.

hibiting parasite replication or promoting parasite death, and/or the IDO activity developing, with precise timing, an adequate immune response able to control the infection (23) and immunopathology (13, 24, 25). Here we have focused our study on the first of these mechanisms by hypothesizing that IDO activity could be crucial to inhibit parasite replication or promote parasite death. To study the effect of IDO activity on the regulation of parasite growth in vitro, bone marrow-derived Mos were cultured in the absence or presence of different 1-MT concentrations for 24 h before being infected with T. cruzi tps. After 24 h, the noninternalized parasites were removed by washing, and the infected cells were cultured for another 48 h. Then the intracellular IDO IN HOST DEFENSE AGAINST T. CRUZI

amastigotes were counted by immunofluorescence assay, with the results shown in Fig. 4A, B. IDO blockade was shown to result in a strong stimulatory effect on intracellular parasite growth that was dependent on the 1-MT dose. Because the proinflammatory mediators involved in iNOS up-regulation and NO production are also able to induce IDO activity, we investigated the specific contribution of inducible IDO and iNOS in the control of T. cruzi replication in Mos. To carry this out, Mos were cultured in medium alone or medium containing IFN-␥ plus LPS (to induce iNOS and IDO activity up-regulation) for 24 h before being infected. The results presented in Fig. 4D, E show that the infection of Mos with T. cruzi-induced iNOS and IDO activity. In addi2693

Figure 4. IDO is critical for control of T. cruzi infection in vitro. Mos were incubated for 24 h with medium or activated with IFN-␥ (10 ng/ml) plus LPS (10 ␮g/ml) in the presence or absence of 1-MT (100 ␮M) and/or AG (100 ␮M). Cells were infected with T. cruzi tps, and intracellular parasites were counted by immunofluorescence assay. Bars represent relative units, calculated by dividing number of intracellular parasites (n/200 cells) in differentially treated cultures by number of intracellular parasites (n/200 cells) in medium-treated cultures. Nitrite concentration and IDO activity were assayed in 24 h pi supernatant or cell extracts, respectively. A) At 72 h pi, Mos were observed at ⫻1000. Representative field for each group is shown. B) Intracellular parasite numbers counted in Mos that were previously treated with different 1-MT concentrations (solid bars) or medium (open bar). Values are averages ⫾ sd from 3 different independent experiments. *P ⬍ 0.05 vs. medium. C) Representative field for each cell group (⫻1000). D) iNOS activity. *P ⬍ 0.05 vs. medium; **P ⬍ 0.01 vs. infected Mos without IFN-␥ ⫹ LPS; #P ⬍ 0.05 (continued on next page) 2694

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tion, activation with IFN-␥ plus LPS previous to Mo infection resulted in iNOS (Fig. 4D) and IDO (Fig. 4E) activity up-regulation and a significant inhibitory effect of intracellular amastigote growth (Fig. 4C, F). However, when under the same culture conditions, IDO was blocked using 1-MT, the inhibitory effect of intracellular amastigote growth induced by IFN-␥ plus LPS was reversed (Fig. 4C, F) despite significantly higher iNOS activity observed in 1-MT-treated Mos (Fig. 4D). Next, we studied whether iNOS and IDO had synergistic effects on the control of T. cruzi replication. Mos were treated with an iNOS inhibitor in the presence or absence of 1-MT, and the participation of each enzyme (or both) on intracellular parasite replication was studied. The parasite replication in IFN-␥ plus LPS-activated Mos was significantly lower than that observed in nonactivated Mos (Fig. 4G). When activated Mos were treated with an iNOS inhibitor, despite a significant increase in the parasite replication occurring compared with cultures in the absence of AG, the levels of parasite replication were notably lower than those observed under IDO-blocking conditions (Fig. 4G). In addition, the parasite replication when both enzymes were inhibited together was similar to that observed under IDO-blocking conditions (Fig. 4H). Furthermore, the blocking of IDO activity resulted in an increase of iNOS activity as described previously (26) (Fig. 4H), while treatment with AG was unable to significantly modify the IFN-␥ plus LPS- induced IDO activity (Fig. 4I). Taken together, these results demonstrated that IDO activity is critical to control in vitro T. cruzi replication in Mos. Related to this, although iNOS activity is also important to partially control parasite replication, it is effective only when IDO is active. IDO promotes inhibition of intracellular T. cruzi replication through the L-kynurenine catabolites To address the mechanisms by which IDO blockade affect parasite replication, we investigated whether Trp supplementation could prevent IDO-mediated inhibition of parasite replication. When Mos were incubated with different Trp concentrations and subsequently infected, the parasite replication increased in direct proportion to the Trp added (Fig. 5A, B). However, the number of intracellular parasites was significantly lower than those observed in 1-MT-treated cultures, suggesting that Trp depletion is not the only mechanism responsible for IDO-mediated inhibition of parasite replication. In agreement, when Mos were treated with 1-MT plus different Trp concentrations (a culture condition that avoided possible effects of kynurenines on parasite growth), the number of

amastigotes was comparable to those observed in cultures treated only with 1-MT (Fig. 5B). Next, we considered whether kynurenines could affect parasite replication. Kyn, 3-HK, 3-HAA, or QA was added to Mos 24 h prior to infection, and the intracellular parasite replication was analyzed as described above. The addition of Kyn at various concentrations ranging from 50 to 200 ␮M did not affect parasite growth compared with cultures treated with medium alone (Fig. 5A, C). In addition, supplementation with Kyn (even at concentrations 20 times greater than those in the serum of infected mice; Fig. 5C) did not affect parasite growth in cultures treated with 1-MT. In contrast, 20 ␮M of 3-HK, 3-HAA, or QA all were able to reduce the number of intracellular parasites in 1-MT-treated cultures (Fig. 5D). Furthermore, 3-HK treatment (but not 3-HAA or QA treatment) was able to reduce the number of intracellular parasites in IDO active cells. QA but not Kyn, 3-HK, or 3-HAA resulted in Mo toxicity when analyzed by Annexin-V and 7 AAD (Supplemental Fig. 2), and, consequently QA was not considered for further studies. To study whether these compounds could be affecting the penetrative capacity of extracellular parasites, Mos were cultured with 1-MT or medium alone for 24 h before being infected by 4 h. After washing to avoid reinvasion and the effect of the compounds on extracellular parasites, the cultures were treated with 20 ␮M of 3-HK or 3-HAA for 20 h, washed, and the intracellular parasites were quantified. As shown in Fig. 5E, the treatment of T. cruzi-infected Mos with 3-HK or 3-HAA reduced the number of intracellular parasites in cultures treated with 1-MT. In addition, neither 3-HK nor 3-HAA was able to reduce the number of intracellular parasites in IDO active cells. Taken together, these results demonstrate that 3-HK and 3-HAA promoted the inhibition of intracellular T. cruzi replication. The fact that Trp-derived metabolites are necessary to promote optimal human DC activation on TLR stimulation (27) prompted us to investigate if the control of intracellular T. cruzi replication exerted by 3-HK or 3-HAA was mediated by Mo activation. Mos were treated with 3-HK or 3-HAA in the presence or absence of 1-MT before being infected, and then the Mo activation status (release of inflammatory cytokines and the expression of activation markers) was studied. As shown in Supplemental Fig. 3A, IDO inhibition with 1-MT in T. cruzi-infected cells led to a significant reduced production of IL-12p70 and TNF but did not modify IL-6 or IFN-␥ production. In addition, the assayed compounds did not modify the T. cruzi-induced IL-12 or TNF production (Supplemental Fig. 3A). The

vs. culture without 1-MT. E) IDO activity. *P ⬍ 0.05 vs. medium alone. **P ⬍ 0.01 vs. infected Mos without IFN-␥ ⫹ LPS. F) Intracellular parasite numbers. *P ⬍ 0.05 vs. medium; **P ⬍ 0.05 vs. culture without 1-MT. G) Intracellular parasite numbers. *P ⬍ 0.05 vs. medium; **P ⬍ 0.05 vs. culture without 1-MT. H) iNOS activity. *P ⬍ 0.05 vs. medium; **P ⬍ 0.05 vs. culture without 1-MT. I) IDO activity. *P ⬍ 0.01 vs. culture without 1-MT, **P ⬍ 0.05 vs. medium. Bars in D–I represent averages ⫾ sd from 2 (F–H)or 3 (D, E, I) independent experiments. IDO IN HOST DEFENSE AGAINST T. CRUZI

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Figure 5. IDO promotes inhibition of intracellular T. cruzi replication through production of l-kynurenine catabolites. Mos were treated with medium alone or indicated drugs (Trp: 50 –200 ␮M; Kyn: 50 –200 ␮M; 3-HK, 3-HAA, and QA: 20 ␮M) for 24 h, before being infected with T. cruzi tps. At 24 h pi, cells were washed and incubated for another 48 h; cells were then fixed, and intracellular parasites were counted by immunofluorescence assay. Bars represent relative units, calculated as in Fig. 4. A) At 72 h pi, Mos were observed at ⫻1000. Representative field for each group is shown. B, C) Intracellular parasite numbers after treatment with Trp (B) or Kyn (C). *P ⬍ 0.05, **P ⬍ 0.01 vs. medium. D) Intracellular parasite numbers. *P ⬍ 0.05 vs. medium; **P ⬍ 0.05 vs. 1-MT alone. E) Mos were cultured with 1-MT or medium alone for 24 h before being infected by 4 h. After washing, cultures were treated with 20 ␮M 3-HK or 3-HAA for 20 h. Cells were washed and incubated for another 48 h; cells were then fixed, and intracellular parasites were counted by immunofluorescence assay. Relative intracellular parasite numbers are shown. *P ⬍ 0.05 vs. 1-MT alone. Values are averages ⫾ sd from 3 independent experiments.

supplementation of 1-MT-treated cultures with 3-HK but not 3-HAA restored IL-12 production, whereas both L-kynurenine metabolites totally restored TNF production. Taken together, only TNF production was associated with the ability of the Mos to control the parasite replication. In terms of the expression of activation markers, the addition of 3-HK or 3-HAA to infected Mos treated with 1-MT marginally modified MHC class II or CD86 expression, although under these culture conditions there were substantial differences in T. cruzi replication (Fig. 5D). These results suggested that 3-HK and 3-HAA exert the control of T. cruzi replication in Mos by a mechanism that may involve the induction of TNF secretion or by direct effect on the amastigote stage. 2696

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3-HK has a direct effect on T. cruzi bloodstream tps The fact that Mo cultures treated with 3-HK, before but not after being infected, showed a reduced number of intracellular parasites in IDO active cells (Figs. 5D, E) suggested that 3-HK could be affecting the penetrative capacity of extracellular parasites. Then, we explored the effect of the Trp downstream metabolites on bloodstream tps. We cultured tps in the presence of different concentrations of Kyn, 3-HK, 3-HAA, or gentamicin as a negative control for 24 h, and the remaining live parasites were counted in a Neubauer chamber. At 24 h posttreatment, neither of the compounds used showed in vitro trypanocydal activity against T. cruzi tps (not shown). However, 20 ␮M of 3-HK (but not Kyn, 3-HAA,


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or QA) induced morphological changes on T. cruzi tps, which could be observed under the optical microscope. Also, 3-HK-exposed tps suffered changes such as swelling, vacuolization, and decreased motility (not shown and Supplemental Videos 1 and 2). The motility of untreated and 3-HK-treated tps was monitored by video microscopy, and traces of typical cell movements are illustrated in Fig. 6A. Untreated trypanosomes were highly motile cells, with a mean velocity of ⬃17.2 ␮m/s second, whereas 3-HK-treated parasites had a distinct pattern of displacement that was up to 16 times slower

than control cells (Fig. 6A and Supplemental Videos 1 and 2). Electron microscopy analysis of untreated tps revealed all the typical ultrastructural characteristics of the trypanosomatids, with the flagellar pocket, the flagellum, and a normal mitochondrion next to the cell periphery being seen (Fig. 6Ba, b). Tps treated with 3-HK presented numerous ribosome in the cytoplasm (Fig. 6B; R) and large mitochondrion (Fig. 6B; M), with dilated cristae together with the kinetoplast (Fig. 6B; K) within the mitochondrion showing dispersed arrange-

Figure 6. 3-HK has a direct effect on bloodstream T. cruzi tps. A) Motility traces of control or 3-HK (20 ␮M)-treated tps. Trypanosomes were monitored by video microscopy using a Zeiss Axiovert 200 microscope equipped with an ⫻60 objective (Supplemental Videos 1 and 2). Images were captured every second up to a total of 50 s using a Zeiss Axiocam digital camera. Positions of individual cells are plotted at 1-s intervals; traces were obtained using WCIF ImageJ software. Representative traces are shown. Average velocity of control or 3-HK-treated cells was calculated using WCIF ImageJ (control, n⫽6; 3-HK, n⫽8). B) Ultrastructural examination of the tps treated with 3-HK (20 ␮M) or medium for 24 h. a, b) General view of untreated tps showing normal structures; ⫻27,800. c–f) Tps treated with 3-HK. M, mitochondrion; MS, subpeculliar microtubules; N, nucleus; F, flagellum; K, kinetoplast; R, ribosomes. Scale bars ⫽ 0.35 ␮M (a); 0.21 ␮M (b); 0.46 ␮M (c); 0.76 ␮M (d); 0.38 ␮M (e); 0.21 ␮M (f ). IDO IN HOST DEFENSE AGAINST T. CRUZI

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ment of its kDNA content (Fig. 6Bc, d). Treated parasites also exhibited changes in the nucleus (Fig. 6B; N) presenting a different disposition of its chromatin and large nucleoli, together with a poorly defined nuclear membrane (Fig. 6Be). Moreover, microtubules of the flagellum and subpelicular microtubules were compatible with normal structure (Fig. 6Bf). These results demonstrate that 3-HK control T. cruzi replication by a direct effect on amastigote and bloodstream tps. Treatment with 3-HK improves the resistance to T. cruzi infection Next, we investigated whether supplying exogenous 3-HK to mice would result in 3-HK-dependent effects detectable over the course of T. cruzi infection. Five days after T. cruzi infection with a lethal dose of 1000 tps (28), the mice were treated daily with 3-HK or PBS (1 mg/kg/d⫽4.4 ␮mol/kg/d) for 5 consecutive days (dpi 5–10) by the i.p. route. Mice infected with T. cruzi and injected with PBS rendered high levels of parasitemia, causing death between dpi 16 and 21 (Fig. 7A). By contrast, although 3-HK was administered for only 5 d, 65% of 3-HK-treated mice survived to acute infection and displayed lower parasitemia. Thus, at the peak of parasitemia of nontreated mice (d 16), 3-HK treated mice presented a significant reduction in circulating parasites (9.8⫾0.6⫻106 vs. 4.2⫾1⫻10 6 parasites/ml). In addition, Giemsastained smears of blood of mice treated with 3-HK showed tps presenting swelling and vacuolization as observed during tp treatment in vitro, while the slender (highly infective) forms of tps were present only in blood from nontreated mice (Fig. 7B). In one experiment representative of 2, the median survival time of mice treated with placebo did not exceed

40 d, with 100% lethality, while 60% of mice treated with 3-HK survived infection (Fig. 7C). For a 30-d treatment period, with 2 independent experiments, 64.7% of the 3-HK-treated mice survived, whereas only 21.4% of nontreated mice survived (Fig. 7D).

DISCUSSION It is well known that IDO activity in Mos forms part of the innate host defense against infection. Because Mos are the most important cells for T. cruzi replication, they are a major cell population involved in the control of the infection in vivo, in particular for reticulotropic strains such as the Tulahuen used in the present study (29). Thus, during the early phase of infection, the Mos could act as host cells for the parasite and as effector cells in the early IDO-dependent antiparasite response. However, it is commonly assumed that the main arm of Mos to fight T. cruzi infection is its capacity to release NO (3, 30 –32), although Cummings and Tarleton (33) have demonstrated that mice deficient in iNOS or NOS2 exhibit resistance to infection comparable to that of wild-type mice. In the present study, we demonstrated that T. cruzi infection of Mos induced significant IDO and iNOS activity. The inhibition of IDO activity suppressed the capacity of Mos to control parasite replication, although the iNOS activity was unmodified by 1-MT treatment. Similar results were observed when the iNOS and IDO activities were induced by the infection plus IFN-␥/LPS treatment, with activated Mos treated with 1-MT being defective in the clearance of T. cruzi even though the inhibition of IDO activity upregulated iNOS activity as described previously (26). As was expected, the intracellular growth of T. cruzi was increased in activated Mos treated with AG, but surpris-

Figure 7. Treatment with 3-HK improves the resistance to T. cruzi infection. BALB/c mice infected with 1000 bloodstream T. cruzi tps were treated daily with 3-HK (1 mg/kg/d) from dpi 5 to 10 by i.p. route. A) Parasitemia. Results are means ⫾ sd of 6 – 8 animals/group and are representative of 2 independent experiments. *P ⬍ 0.05. B) Giemsa-stained smears of blood from 3-HK-treated mice or controls. Inset: slender form of tps (⫻1500). C) Survival rate. Data are representative of 1 of 2 independent experiments. D) Mice treated with 3-HK or controls were scored for survival at 30 dpi. Figure incorporates data from 2 independent experiments; P value based on Kaplan-Meier test.

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ingly, the parasite replication under iNOS blocking conditions was markedly smaller than that observed under IDO-blocking conditions. In addition, INOS and IDO inhibition did not show any synergistic effects. Taken together, these results highlight the essential role played by IDO in controlling T. cruzi replication in Mos. One interesting point is how the T. cruzi parasite stimulates IDO activation. TLR2 and TLR9 recognition by T. cruzi-derived components is involved in the innate iNOS induction in T. cruzi-infected Mos (34, 35), whereas neither the identity of the receptor that induces IDO expression in Mos exposed to T. cruzi nor the parasite ligands that trigger this receptor has been identified yet. However, elegant work by Koga et al. (36) demonstrated that Mos and DCs from MyD88⫺/⫺ TRIF⫺/⫺ mice, in which TLR-dependent activation of innate immunity is eliminated, are defective in the clearance of T. cruzi and also show impaired induction of IFN-␤ during T. cruzi infection. These authors suggest that MyD88-dependent resistance to T. cruzi infection is correlated with the induction of inflammatory cytokines such as IL-12 and TNF (inducers of IDO), while TRIF-dependent resistance depends on production of IFN-␤ and subsequently on IFN-␤ inducible genes. Because they found that induction of GTPase IFN-inducible p47 (IRG47), implicated in the immune innate response to protozoan parasites (37–39), is impaired in T. cruzi-infected cells from MyD88⫺/⫺, TRIF⫺/⫺, MyD88⫺/⫺, and IFNAR1⫺/⫺ mice, in addition to the fact that the knockdown of IRG47 in MyD88⫺/⫺ Mos increased intracellular parasite replication, they proposed IRG47 to be the IFN-inducible molecule responsible for Mo resistance. However, because the evidence presented in favor of IRG47 is to a certain extent indirect, the defective clearance of T. cruzi in double-knockout Mos could be the result of deficiency occurring in several type I IFN-inducible antimicrobial molecules, with IDO being one of the most important. In agreement, IDO, LRG-47 (another member of the p47 GTPase family), and iNOS are all STAT1regulated antimicrobial effector enzymes (40). One mechanistic hypothesis to explain the correlation between IDO activity and control of parasite growth assumes that reduced access to free Trp inhibits parasite replication (the Trp-depletion hypothesis). An alternative hypothesis is that the Trp downstream metabolites produced by IDO activity inhibit parasite replication or promote parasite death. Chlamydia pneumoniae, Toxoplasma gondii, certain bacteria, and viruses, which are either intracellular or else live in close association with a host cell, depend on exogenous Trp and are sensitive to Trp depletion by IDO activity (8, 41– 43). In addition, Vincendeau et al. (44) have reported that the inhibition of IDO in Trypanosoma musculi (extracellular trypanosoma)-infected mice increased the number of circulating parasites. But, in another study, it was demonstrated that the intracellular replication of T. cruzi in human fibroblasts was not IDO IN HOST DEFENSE AGAINST T. CRUZI

depend on Trp concentration or controlled by IFN-␥and/or TNF-induced IDO activity (45). In our experimental model, the slight but dose-dependent increase in parasite replication observed after exogenous Trp supplementation suggests that T. cruzi replication was promoted by Trp. However, the fact that the levels of parasite replication observed in Trp-supplemented cultures were significantly lower than those observed in 1-MT-treated cultures could imply that the growth restriction of IDO against T. cruzi was the result of another mechanism as well as Trp starvation. The Trp downstream catabolites 3-HAA and QA have been shown to be involved in selective apoptosis in vitro of murine thymocytes and Th1 cells (13). In addition, QA has protective effects in mice with candidiasis, by a mechanism that may depend on activation of the local host Mos (46), whereas combined treatment with l-kynurenine and IFN-␥ protected mice with chronic granulomatous disease from invasive pulmonary aspergillosis by the generation of l-kynurenine downstream metabolites (47). In the present study, we observed that the supplementation of 1-MT-treated Mos with 3-HK or 3-HAA before or after being infected restored the Mo trypanocidal activity and their capacity to produce TNF, suggesting that each one of these catabolites on their own has a direct or a TNF-mediated effect on the amastigote stage of T. cruzi. On the other hand, the fact that the treatment of Mos with 3-HK, before but not after being infected, helped with parasite clearance in IDO active cells suggested that 3-HK could be affecting the penetrative capacity of extracellular parasites. In accord, we demonstrated that 3-HK was toxic against the bloodstream tp stage of the parasite inducing morphological and motility changes without changes in the viability. In addition, 3-HKexposed tps suffered ultrastructural changes, showing kinetoplasts with structural alterations, many ribosomes in the cytoplasm, large mitochondrion, and a poorly defined nuclear membrane that could explain the motility changes. Furthermore, our in vivo results showed that the therapeutically administration of 3-HK decreased the parasitemia of acute infected mice and improved their survival, suggesting that the pharmacologic intervention of IDO pathway could be used as a novel antitrypanosomatid therapeutic strategy. However, because the majority of Chagas patients reside in resource-limited settings, it is desirable that the drug be given orally. Therefore, further experiments are in process in our laboratory to analyze optimal doses, the therapeutic effect of 3-HK administration by oral route, and the toxicity of this compound. The fact that IDO is essential for DC activation and chemotactic responsiveness to chemokines (23) suggests that IDO activity plays an important role in the generation of the specific T cell response. Therefore, our results demonstrating that IDO inhibition induced an increase of bloodstream and tissue parasites could also be, in 1-MT-treated mice, associated with an impaired immune response (23). However, the prolifera2699

tive response and cytokine profiles studied at 14 dpi in splenocytes and lymph node cells stimulated with a total parasite homogenate as well as the levels of IgG antibodies against the parasite did not reveal significant differences between infected mice treated with 1-MT and controls (data not shown). Another important consequence of IDO activation is to induce T-cell apoptosis, anergy, and Treg cells, which results in the control of an excessive inflammatory response but could also allow parasite replication (14). The fact that infected mice treated with 1-MT presented significantly more inflammatory foci than control mice in target tissues may be associated with the loss of inflammation control by IDO inhibition. However, in experiments not reported here, we found that at 14 dpi the levels of proinflammatory mediators (NO, IL-1␤, TNF, IL-6, and IL-12) in the target tissues and sera and the absolute number of CD4⫹ CD25⫹ Foxp3⫹ cells in spleen did not show any differences between infected mice treated with 1-MT and controls (data not shown). Thus, at 14 dpi, the exacerbated inflammatory infiltrate observed in infected 1-MTtreated mice might have been associated with the parasite load in the tissues rather than with the loss of inflammation control. Experiments are currently being carried out in our laboratory to evaluate if IDO inhibition during the acute phase of the infection is able to modify the specific and autoreactive immune response as well as the outcome of chronic Chagas disease. C.P.K. thanks Agencia Nacional de Promocioń Científica y Tećnica, and F.F.M. thanks the Consejo Nacional de Investigaciones Científicas y Tećnicas (CONICET) of Argentina for the fellowships granted. C.C.M., L.C., and M.G.T. are members of the Scientific Career of CONICET of Argentina. The authors are grateful to Cecilia Sampedro and Carlos Mas [Centro de Investigaciones en Química Biolo´gica de Co´rdoba (CIQUIBIC)-CONICET] for assistance with video microscopy. The authors thank Eva Acosta Rodriguez for critical reading of the manuscript and native speaker Dr. Paul Hobson, who revised the manuscript. This work was supported by grants from CONICET of Argentina, Agencia Nacional de Promocioń Científica y Tećnica (PICT 25511), Ministerio de Ciencia y Tecnología de la Provincia de Co´rdoba, and Secretaría de Ciencia y Tećnica–Universidad Nacional de Co´rdoba (grants to C.M.). The funding bodies had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no competing interests.

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