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The Journal of Immunology, 2006, 177: 6271-6280.
Copyright © 2006 by The American Association of Immunologists, Inc.

Trypanosoma cruzi-Mediated IFN-{gamma}-Inducible Nitric Oxide Output in Macrophages Is Regulated by iNOS mRNA Stability1

Marc Bergeron* and Martin Olivier2,*,{dagger}

* Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Quebec, Pavillon Centre Hospitalier de l’Université Laval, and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Sainte-Foy, Quebec, Canada G1V 4G2; and {dagger} Research Institute of the McGill University Health Centre, Centre for the Study of Host Resistance, Departments of Medicine, Microbiology, and Immunology, McGill University, Montreal, Quebec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although the effects of activated macrophages (M{phi}) on the intracellular parasite Trypanosoma cruzi are well documented, little is known about how host-M{phi} functions are affected by this pathogen before activation. This study is aimed at assessing the capacity of T. cruzi infection to modulate J77.4 murine M{phi} NO generation following IFN-{gamma} stimulation, and identifying mechanisms regulating this modulation. Results show that parasite infection potentiates M{phi} to produce inducible NO synthase (iNOS) mRNA and protein as well as NO following IFN-{gamma} stimulation above IFN-{gamma} alone controls. This potentiation occurs through the concomitant activation of NF-{kappa}B, ERK1/ERK2 MAPK, and stress-activated protein kinase signaling pathways. Activation of the JAK/STAT pathway by IFN-{gamma} then leads to STAT1{alpha} translocation and the transcription of a stable iNOS mRNA species. A decreased rate of iNOS mRNA degradation results in elevated levels of iNOS protein and NO production. Maximal iNOS expression is likely achieved through NF-{kappa}B activation by T. cruzi, whereas iNOS mRNA stability results from ERK1/ERK2 MAPK and stress-activated protein kinase activation by the infection. Taken together, our data show that T. cruzi-infected M{phi} NO generation is controlled at both pre- and posttranscriptional levels and relies on signaling pathway cross-talk. This is the first report of a parasite pathogen capable of heightening host mRNA stability.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Trypanosoma cruzi is the parasite responsible for Chagas’ disease, a disease that often leads to the progressive destruction of hollow organ and nervous tissues. The World Health Organization estimates that almost a quarter of Latin America’s population is permanently at risk, with over 15 million people infected with the parasite (<http://www.who.int/tdr/diseases/chagas/default.htm>). T. cruzi is usually transmitted by cone-nosed bugs, but because the parasite can also be transmitted through blood transfusion (1) and congenitally (2), increased travel and immigration threatens to widen the risk of contracting Chagas’ disease outside of endemic areas. No vaccine is currently available, and the inherent resistance of some strains to chemotherapeutic agents reduces the effectiveness of existing treatments (3).

Macrophages (M{phi})3 are good producers of NO in response to IFN-{gamma} (4). NO released by IFN-{gamma}-activated M{phi} is involved in host resistance to several pathogens including parasitic protozoa and helminths (reviewed in Ref. 5). Although several reports have linked M{phi}-derived NO with host protection and parasite clearance during T. cruzi infection (6, 7, 8), few, if any, have examined IFN-{gamma}-related responses in nonactivated M{phi} infected with this parasite. In this context, it seems that some pathogens, including the related parasite Leishmania, down-modulate mouse M{phi} NO generation (9). Understanding how T. cruzi affects NO production in M{phi} is important because these cells are the first to be infected during the onset of murine Chagas’ disease (10), are infected before IFN-{gamma} detection in serum (8), and play a key part in host defense against this parasite (7, 8, 11, 12). Furthermore, NO has been shown to lead to apoptosis (13, 14, 15, 16) and the characteristic suppression of T cell proliferation during acute T. cruzi infections (17, 18, 19). T. cruzi-induced M{phi} apoptosis has been suggested to play a role in the spread of the infection through the release of viable parasites (20). However, NO-induced apoptosis has also been suggested as a means of both limiting host tissue damage during the acute phase of the disease and promoting the chronic phase (14, 15). Due to the host of effects that NO exerts, it would therefore seem that NO generation by M{phi} during the early stages of the infection may be of significant importance to the outcome of Chagas’ disease.

At the cellular level, numerous signaling pathways have been shown to be involved in the production of NO in response to IFN-{gamma}. Of these, the JAK/STAT and NF-{kappa}B signaling pathways seem most important (21, 22, 23, 24). Although the signaling cascades leading to the release of NO by activated mouse M{phi} are relatively well established, the intracellular signaling events leading to NO production in T. cruzi-infected M{phi} remain unknown. Knowledge of these events is essential to understanding the manner in which T. cruzi modulates NO production in M{phi}.

In this study, we were interested in verifying the effect of T. cruzi infection on NO generation in response to IFN-{gamma} in mouse M{phi} and identifying the mechanisms through which this NO production is achieved. Our results show that T. cruzi infection potentiates mouse M{phi} to produce iNOS (inducible NO synthase) mRNA and iNOS protein in response to IFN-{gamma} at levels higher than IFN-{gamma} controls alone, thereby increasing NO release. Our data also suggest that NO generation is achieved through a web of signaling pathways that interact at the pretranscriptional level, leading to STAT1{alpha}, AP-1, and NF-{kappa}B activation along with increased iNOS mRNA posttranscriptional stability. This is the first report that shows increased parasite-induced host cell iNOS mRNA stability and that this stability relies on the ERK1/ERK2 MAPK and the stress-activated protein kinase (SAPK)/JNK signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Recombinant murine IFN-{gamma} (105 U/ml) was acquired from Invitrogen Life Technologies. JAK2 signal transduction inhibitor AG-490 (N-benzyl-3,4-dihydroxybenzylidenecyanoacetamide) and NF-{kappa}B translocation inhibitor CAPE (caffeic acid phenethyl ester) were purchased from BIOMOL Research Laboratories, whereas ERK1/ERK2, MEK1/2, and p38 signal transduction inhibitors (apigenin (4',5,7-trihydroxyflavone), PD98059 (2'-amino-3'methoxyflavone), and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), respectively) were obtained from Calbiochem-Novabiochem. SAPK/JNK SP600125 was purchased from Tocris. Actinomycin D was purchased from Sigma-Aldrich. TRIzol was acquired from Invitrogen Life Technologies. Finally, [{alpha}-32P]dCTP and [{gamma}-32P]dATP (3000 Ci/mmol) isotopes were obtained from NEN Life Science Products.

Abs and DNA probes

Rabbit anti-iNOS Ab was purchased from Cedarlane. Rabbit anti-actin mAb was obtained from Sigma-Aldrich. Rabbit anti-phospho-JAK2 Ab was purchased from Upstate Biotechnology. Anti-phospho-ser727-STAT1{alpha} and anti-phospho-tyr701-STAT1{alpha} Abs were provided by Dr. D. Frank (Harvard Medical School, Boston, MA). Anti-STAT1{alpha}, anti-c-Jun, anti-c-Fos, anti-JunB, and anti-JunD Abs were purchased from Santa Cruz Biotechnology. Rabbit anti-phospho-MEK1/2, anti-phospho-ERK1/ERK2, anti-phospho-SEK1/MKK4 MKK4 (stress-activated protein kinase/Erk kinase or Jun kinase kinase (JNKK)), anti-phospho-p38, anti-phospho-SAPK/JNK, anti-phospho-c-Jun, and corresponding non-phospho Abs were acquired from New England Biolabs. Finally, anti-rabbit and anti-mouse HRP-conjugated goat Abs were obtained from Affini-Pure (Jackson ImmunoResearch Laboratories). The iNOS and GAPDH cDNA probes were provided by Dr. D. Radzioch (McGill University, Montreal, Quebec, Canada).

Transcription factor target sequences

The iNOS/{gamma}-IFN-activated site (GAS)-containing oligonucleotide (GAS/iNOS) was synthesized in our laboratory using the sequence published by Gao et al. (23). AP-1 and NF-{kappa}B consensus oligonucleotides, along with the Sp1 oligonucleotide, were purchased from Santa Cruz Biotechnology.

Cell lines and parasite cultures

J77.4 murine M{phi} and National Institutes of Health 3T3 fibroblasts were obtained from the American Type Culture Collection. They were maintained in DMEM (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Invitrogen Life Technologies). Cells were grown at 37°C in a humidified incubator with 5% CO2. For murine bone marrow-derived M{phi} (MBMDM), bone marrow from BALB/c mice was extracted from the femurs and tibias of the animals, and the cells were separated with the use of a buffer coat (Wisent). Cells were allowed to adhere overnight, and the resulting supernatant was transferred into 24-well plates and differentiated into M{phi} using recombinant M{phi}-CSF (PeproTech). Cells were further cultured in this way for 7 days before experiments in DMEM supplemented with 10% heat-inactivated FBS. Cells were grown at 37°C in a humidified incubator with 5% CO2. The strain of T. cruzi used in this study was a gift from Dr. A. Gustavo Guevara (Hospital of Los Andes, Quito, State of Pichicha, Ecuador). It was isolated in Pedreo Carbo, Guayas, Ecuador, from Triatoma dimidiata, was characterized as belonging to zymodeme Z1, and is now known as the Ecua0 strain. Trypomastigotes were collected from National Institutes of Health 3T3 fibroblast cell monolayer supernatants 5 days after infection.

Nitrite measurements

M{phi} were seeded in 24-well tissue culture plates at 2.5 x 105 cells per well and incubated in the presence or absence of T. cruzi trypomastigotes. Parasite:M{phi} ratios of 5:1, 10:1, and 20:1 were used for dose-response assays, and experiments lasted 24 h. For time course assays, the same parasite:M{phi} ratios were used, and the time points examined were 3, 4, 6, 12, and 24 h. For experiments where infected M{phi} were stimulated with IFN-{gamma} (100 U/ml), infections (same parasite:M{phi} ratios) were maintained anywhere from 3 to 24 h, and the wells were washed with PBS to remove any remaining free parasites before IFN-{gamma} stimulation. There were no significant differences between experiments where preinfections lasted 3, 4, 6, 12, or 24 h or the parasite:M{phi} ratio used; therefore, subsequent experiments used 4-h preinfections and a 5:1 parasite:M{phi} ratio. Uninfected M{phi} served as negative controls, whereas positive controls were 24-h IFN-{gamma}-stimulated M{phi}, because this allowed a detectable amount of nitrite to be produced while avoiding M{phi} overcrowding. At the end of each time point, supernatants were collected and nitrite was measured using the Griess reaction (25) with NaNO2 as the standard, as previously described in Ref. 26 .

Second messenger phosphorylation experiments

M{phi} were plated in 6-well tissue culture plates (1–2 x 106cells/well) and incubated with or without T. cruzi trypomastigotes for up to 4 h. In experiments where infected M{phi} were stimulated with IFN-{gamma} (100 U/ml), the wells were washed with PBS to remove any remaining free parasites before IFN-{gamma} stimulation. Uninfected M{phi} were used as negative controls, whereas positive controls were M{phi} stimulated with IFN-{gamma}. Total M{phi} protein was then used for Western blot analyses. Results show peak messenger phosphorylation (5–30 min poststimulation).

Western blotting

Total M{phi} protein obtained from nitrite measurement and second messenger phosphorylation experiments were subjected to Western blot analysis. Western blots were adapted from methods outlined previously (27). Briefly, M{phi} were collected and disrupted in cold lysis buffer (20 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 10% (v/v) glycerol, 1% (v/v) Igepal CA-640, 10 µM NaF, 1 mM sodium orthovanadate, 100 µg/ml PMSF, 25 µg/ml aprotinin, and 25 µg/ml leupeptin), and total M{phi} protein (20–30 µg/lane) submitted to SDS-PAGE (10%), according to methods adapted from Refs. 28 to30 . Protein concentrations were determined using BCA Protein Assay Reagent (Pierce). The separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore). Membranes were blocked and incubated in the presence of various primary and HRP-conjugated secondary Ab. Results were visualized using the NEN chemiluminescence detection system. For experiments using signal transduction inhibitors (JAK2 (AG-490), MEK (PD98059), ERK1/ERK2 (apigenin), p38 (SB203580), SAPK/JNK (SP600125), and NF-{kappa}B (CAPE)), inhibitors were added 30 min to 1 h before M{phi} infection with T. cruzi. Final concentrations are as indicated in the figures. Proteins obtained from nitrite measurement experiments were blotted against the anti-iNOS Ab, whereas those obtained for second messenger phosphorylation analyses were blotted against anti-phospho-JAK2, anti-phospho-ser727-STAT1{alpha}, anti-phospho-tyr701-STAT1{alpha}, anti-phospho-MEK1/2, anti-phospho-ERK1/ERK2, anti-phospho-SEK1/MKK4, anti-phospho-p38, anti-phospho-SAPK/JNK, and anti-phospho-c-Jun Abs. The anti-actin, anti-STAT1{alpha}, and nonphosphorylated second messenger Abs were used to evaluate protein loading.

mRNA extraction and Northern blotting

M{phi} were seeded in 25-cm2 flasks (5 x 106 M{phi} per flask upon sampling) and infected or not with T. cruzi trypomastigotes. Parasite:M{phi} ratios of 5:1, 10:1, and 20:1 were used for dose-response assays, and experiments lasted up to 24 h. For time course assays, a 5:1 parasite:M{phi} ratio was used, and the time points examined were 2, 4, 8, 12, and 24 h. For experiments where infected M{phi} were stimulated with IFN-{gamma} (100 U/ml), infections lasted 24 h, and the cells were washed with PBS to remove any remaining free parasites before IFN-{gamma} stimulation. Uninfected M{phi} served as negative controls, whereas IFN-{gamma}-stimulated M{phi} were used as positive controls. Total M{phi} mRNA was extracted using TRIzol reagent, and Northern blotting was done according to the method outlined in Ref. 31 . Briefly, mRNA samples (10–20 µg/lane) were separated in a 1.2% agarose/formaldehyde gel. The mRNA was subsequently transferred onto a Nytran SuPerCharge nylon transfer membrane (Schleicher & Schuell) before being prehybridized overnight at 42°C. iNOS or GAPDH 32P random prime-labeled cDNA probes (106 cpm/ml prehybridization solution) were then added to the prehybridization solution and allowed to hybridize overnight. Membranes were washed 3 x 15 min in 2 x SSC/0.1% SDS and 3 x 15 min in 0.2 x SSC/0.1% SDS at 42°C. Results were visualized by autoradiography. The housekeeping gene GAPDH was probed to verify that wells were equally loaded. iNOS mRNA stability was determined using actinomycin D. Briefly, M{phi} were infected or not for 24 h and then stimulated with IFN-{gamma} for another 24 h. Actinomycin D (5 µg/ml) was then added, and M{phi} were sampled at 2, 4, and 8 h. The levels of remaining iNOS mRNA were quantified by Northern blot, followed by densitometry using an Alpha Imager 2000 digital imaging and analysis system (Alpha Innotech).

EMSAs

M{phi} were plated in 25-cm2 flasks (1–2 x 106 M{phi} per flask by the end of the experiment) and incubated with trypomastigotes (5:1 parasites per M{phi}) for 30-min to 4-h periods. For postinfection IFN-{gamma} stimulation, flasks were washed with PBS to remove any remaining free parasites before IFN-{gamma} (100 U/ml) stimulation. Time points were taken between 30 min and 4 h postinfection or poststimulation. After each time point, M{phi} were washed and scraped into cold PBS. Nuclear proteins were extracted using modifications of the method outlined in Ref. 32 . Briefly, M{phi} were sedimented for 5 min at 1500 rpm and resuspended in 1 ml of cold PBS, transferred to Eppendorf tubes, and centrifuged at 13,000 rpm for 25 s. Cells were resuspended and incubated on ice for 15 min in a hypotonic solution (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF) to cause cell swelling. Twenty-five microliters of 10% Igepal CA-640 was then added, and M{phi} were vortexed and centrifuged for 30 s at 13,000 rpm. Pellets were resuspended and incubated on a shaking platform 15 min at 4°C in lysis buffer containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF. Lysates were then spun 5 min at 14,000 rpm, and supernatants containing nuclear proteins were transferred to new Eppendorf tubes. Nuclear proteins from lysates (6 µg/lane) were subsequently allowed to hybridize with 32P-labeled target oligonucleotide sequences (GAS/iNOS, AP-1, NF-{kappa}B) and analyzed by EMSA using a 4% polyacrylamide gel (33, 34). Results were visualized by autoradiography. The sequences of the oligonucleotides used were 5'-CTTTTCCCCTAACAC-3' for STAT1{alpha} (GAS/iNOS), 5'-CGCTTGATGACTCAGCCGGAA-3' for AP-1, and 5'-AGTTGAGGGGACTTTCCCAGGC-3' for NF-{kappa}B. Results show peak nuclear translocation that occurred within the first 2 h of infection.

Statistical analyses

Means were compared using a one-way ANOVA, followed by Fisher’s protected least significant differences test. Values of p < 0.05 were deemed statistically significant. Calculations were done using the StatView statistical software (SAS Institute).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NO modulation by T. cruzi infection

In the sequence of events leading to the establishment of the parasite inside the host, M{phi} are infected before any IFN-{gamma} secretion by NK cells and T lymphocytes (8, 10), the main IFN-{gamma} producers during murine Chagas’ disease, or NO production by M{phi} (35). In fact, during the course of the infection, there is a 5-day lag between IFN-{gamma} secretion and NO generation (8), at which time parasite-infected M{phi} are actually exposed to IFN-{gamma}. It has been shown that in this context, Leishmania parasites inhibit J77.4 mouse M{phi} NO production upon IFN-{gamma} stimulation (9). Given the fact that M{phi}-derived NO plays a key role in parasite clearance, the key question we wanted to address in this study was whether or not T. cruzi infection modified M{phi} NO output in response to IFN-{gamma}, because this might affect disease progression. M{phi} were thus infected with the parasite and subsequently stimulated with IFN-{gamma}. Our results show that T. cruzi-infected M{phi} treated with IFN-{gamma} release significantly more NO than IFN-{gamma}-stimulated M{phi} do both in J77.4 cells (Fig. 1A) and primary MBMDM (Fig. 1B) (fold increases over all experiments ranged from 1.57 to 3.94 with a mean of 2.37 and a SD of 0.87). The same trend holds true for iNOS protein (Fig. 1A) and mRNA expression levels (Fig. 1E). This last result prompted us to conclude that NO generation and iNOS expression might be regulated at the pretranscriptional level. It is well established that T. cruzi infection by itself does not induce NO production. To verify whether it was also the case in our system, M{phi} were infected with the parasite alone as a control. As expected, the infection of M{phi} with T. cruzi does not elicit NO generation or iNOS protein synthesis, whether it be in a dose- (Fig. 1, A and C) or time- (Fig. 1D) dependent manner. Taken together, these results suggest that, although T. cruzi infection alone does not induce NO production, it does potentiate mouse M{phi} in their ability to generate NO upon IFN-{gamma} stimulation. The next question we therefore wanted to address was what could account for such iNOS expression and NO outputs. We set out to verify whether or not cell signaling alterations could be involved, given that Leishmania-induced M{phi} dysfunctions have been linked to impaired Ca2+ and protein kinase C signaling, as well as to Src homology protein-1 protein tyrosine phosphatase-mediated JAK2 signaling alterations (36, 37, 38, 39, 40).


Figure 1
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  		FIGURE 1. The effects of T. cruzi infection on IFN-{gamma}-mediated murine M{phi} NO production. A, iNOS protein expression and NO production of T. cruzi-infected M{phi} stimulated with IFN-{gamma} (100 U/ml), as determined by Western immunoblotting and nitrite measurement using the Griess reaction, respectively. Identical letters over bars indicate that corresponding data are not statistically different (p < 0.05), as determined by one-way ANOVA followed by Fisher’s protected least significant differences test. B, NO production of T. cruzi-infected MBMDM stimulated with IFN-{gamma} (100 U/ml), as determined by nitrite measurement using the Griess reaction. Identical letters over bars indicate that corresponding data are not statistically different (p < 0.05), as determined by one-way ANOVA followed by Fisher’s protected least significant differences test. C, Northern blot analysis of iNOS mRNA expression in parasite-infected M{phi}. D, iNOS mRNA transcription kinetics over time in T. cruzi-infected M{phi}, as determined by Northern blot analysis. E, Northern blot analysis of iNOS mRNA expression kinetics over time in T. cruzi-infected M{phi} stimulated with IFN-{gamma}. All of the results reported in this figure are representative of at least three independent experiments.

 
Signaling pathways involved in T. cruzi-induced NO production

M{phi} NO generation has been shown to proceed mainly through the JAK2/STAT1{alpha} signaling pathway (24). To verify the involvement of this and other pathways in our system, M{phi} were preincubated with established pharmacological inhibitors before infection and IFN-{gamma} stimulation. Initially, J77.4 M{phi} were preincubated with AG-490 (10–75 µM), a specific inhibitor of JAK2, before being infected with the parasite and further stimulated with IFN-{gamma}. As shown in Fig. 2A, AG-490 curbs the IFN-{gamma}-induced NO production of T. cruzi-infected M{phi} in a dose-dependent manner. Given the fact that STAT1{alpha} can also be phosphorylated by ERK1/ERK2 MAPK (24, 41), the implications of abrogating this pathway were examined. M{phi} were therefore incubated with PD98059 (1–40 µM), a specific inhibitor of MEK1/2, or apigenin (10–75 µM), a specific inhibitor of ERK1/ERK2, before being infected with T. cruzi and further stimulated with IFN-{gamma}. The use of PD98059 or apigenin significantly curtails in a dose-dependent manner iNOS and NO generation in T. cruzi-infected M{phi} treated with IFN-{gamma} (Fig. 2, B and C, respectively). Although these results do not allow us to discriminate between parasite and IFN-{gamma} effects, they do, however suggest a role for both the JAK2/STAT1{alpha} and ERK1/ERK2 MAPK signaling pathways in T. cruzi-induced M{phi} NO production. To further establish the significance of these two signal transduction pathways, we next looked at the phosphorylation levels of different key second messengers involved in these pathways.


Figure 2
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  		FIGURE 2. The effects of JAK2 (AG-490) and MAPK (PD98059 and apigenin) inhibitors on IFN-{gamma}-mediated NO generation of T. cruzi-infected mouse M{phi}. Cells were preincubated with increasing doses of the appropriate inhibitor before infection and IFN-{gamma} (100 U/ml) stimulation. iNOS protein expression and NO generation were determined as previously described in Fig. 1A. A, Effect of AG-490 (10–75 µM) on NO production of T. cruzi-infected M{phi} treated with IFN-{gamma}. B, Effect of PD98059 (1–40 µM) on NO generation of T. cruzi-infected M{phi} stimulated with IFN-{gamma}. C, Effect of apigenin (10–75 µM) on NO production of T. cruzi-infected M{phi} treated with IFN-{gamma}. Identical letters over bars indicate that corresponding data are not statistically different (p < 0.05). Results are representative of three independent experiments.

 
The JAK2/STAT1{alpha} and ERK1/ERK2 MAPK signaling pathways cannot account for the observed T. cruzi-mediated NO production

The infection of murine M{phi} with T. cruzi leads to the phosphorylation of STAT1{alpha} on its serine 727 residue (Fig. 3A), of MEK1/2 and of ERK1/ERK2 (Fig. 3B), but not of JAK2 or the tyrosyl residue of STAT1{alpha} (Fig. 3A). However, stimulation of T. cruzi-infected M{phi} with IFN-{gamma} results in the additional phosphorylation of JAK2 and the tyrosyl residue of STAT1{alpha} (Fig. 3A). Interestingly enough, no additive or synergistic effect between T. cruzi and IFN-{gamma} can be seen (Fig. 3, A and B). Therefore, it would seem that, although the JAK2/STAT1{alpha} and ERK1/ERK2 MAPK signaling pathways are necessary for NO generation, they are not directly responsible for T. cruzi-mediated NO production. It would also seem that IFN-{gamma}-induced JAK2 phosphorylation acts as the trigger without which no NO secretion can occur. In light of these results, we hypothesized that incomplete STAT1{alpha} phosphorylation by the parasite would not lead to nuclear translocation of that transcription factor without IFN-{gamma} triggering. As predicted, no STAT1{alpha} translocation takes place in T. cruzi-infected M{phi} (Fig. 3C); upon IFN-{gamma} stimulation, however, STAT1{alpha} does translocate to the nucleus (Fig. 3C). Again, no additive or synergistic effect was observed. Taken together, these results suggest that the signaling events leading to iNOS expression in T. cruzi-infected M{phi} proceed essentially as they do in M{phi} stimulated with IFN-{gamma}, although they differ completely in output levels. Our initial results suggesting that NO production of T. cruzi-infected M{phi} is controlled at the pretranscriptional level and the activation of the ERK1/ERK2 MAPK pathway (Fig. 3B), prompted us to verify whether the parasite activated other signaling pathways. Indeed, it has been suggested that the ERK1/ERK2 MAPK and p38 pathways might be involved in iNOS mRNA stability (42). Furthermore, it has recently been shown that c-Jun inhibition destabilized iNOS mRNA in LPS-activated M{phi} (43). We therefore decided to examine these possibilities by looking at the SAPK pathways.


Figure 3
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  		FIGURE 3. The effects of T. cruzi infection on murine M{phi} STAT1{alpha} activation. Cells were infected with the parasite and some were further stimulated with 100 U/ml IFN-{gamma}. A, Phosphorylation status of the JAK2/STAT1{alpha} signaling pathway messengers, as determined by Western immunoblot analyses. B, Western blot analyses of the phosphorylation status of representative second messengers of the ERK1/ERK2 MAPK signaling pathway. Results shown here depict peak phosphorylation levels of respective anti-phospho-Abs. C, EMSA showing the nuclear translocation of STAT1{alpha} in T. cruzi-infected M{phi} treated or not with 100 U/ml IFN-{gamma}. Binding specificity was assessed by the addition of a 100-fold molar excess of unlabeled STAT1{alpha} (specific) or Sp1 (nonspecific) oligonucleotide to protein lysates obtained from IFN-{gamma}-stimulated M{phi}. Results are representative of three independent experiments.

 
T. cruzi induces the phosphorylation of SAPK involved in parasite-mediated NO generation

IFN-{gamma}-induced NO production in T. cruzi-infected M{phi} is curbed in a dose-dependent manner when using SB203580, a specific inhibitor of p38 (Fig. 4A), or SP600125, a specific inhibitor of SAPK/JNK (Fig. 4B). Furthermore, T. cruzi infection of mouse M{phi} results in the phosphorylation of the SAPK SEK1/MKK4, p38, SAPK/JNK, and c-Jun (Fig. 4C). The infection of M{phi} also leads to the nuclear translocation of AP-1 (Fig. 4D), a transcription factor acting downstream of the p38 and SAPK/JNK SAPK pathways. Supershift assays show that the AP-1 complexes that result from T. cruzi infection are potentially made up of the following proteins: c-Jun and JunB mainly, although c-Fos and JunD also seem to be involved (Fig. 4D). Once again, no additive or synergistic effect occurs between the parasite and IFN-{gamma} (Fig. 4, C and D), suggesting that these pathways are not directly involved in T. cruzi-mediated generation. However, those results point toward iNOS mRNA stability as a possible mechanism that could account for NO production.


Figure 4
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  		FIGURE 4. The effects of T. cruzi infection on the SAPK pathways of mouse M{phi}. A and B, M{phi} were preincubated with increasing doses of SB203580 (0.1–10 µM) (A) or SP600125 (5–30 µM) (B) before being infected and subsequently treated with IFN-{gamma} (100 U/ml). iNOS protein expression and NO generation were determined by Western immunoblotting and nitrite measurement, as described previously. Identical letters over bars indicate that corresponding data are not statistically different (p < 0.05). C, Western blot analyses of the phosphorylation status of representative second messengers of the SAPK signaling pathways. Results depict peak phosphorylation levels of respective anti-phospho-Abs. D, EMSA showing the nuclear translocation of AP-1 in T. cruzi-infected M{phi} treated or not with 100 U/ml IFN-{gamma}. Binding specificity was assessed by the addition of a 100-fold molar excess of unlabeled AP-1 (specific) or Sp1 (nonspecific) oligonucleotide to protein lysates obtained from IFN-{gamma}-stimulated M{phi}. Supershift assays were conducted by incubating the nuclear proteins extracted from parasite-infected M{phi} with specific Abs against c-Jun, c-Fos, JunB, and JunD for 1 h at 4°C, before reaction with [32P]-labeled AP-1 oligonucleotide. Results are representative of three independent experiments.

 
NF-{kappa}B nuclear translocation occurs during the infection of mouse M{phi} with T. cruzi

Although the contribution of NF-{kappa}B has been shown to be minimal in NO production of IFN-{gamma}-stimulated M{phi} (24), it has been shown to be required for maximal iNOS expression (44). We therefore decided to examine whether or not NF-{kappa}B could account in any way for the observed NO generation in T. cruzi-infected M{phi}. Our results show that the treatment of M{phi} with CAPE (0.1–10 µM), an NF-{kappa}B inhibitor, reduces NO output of T. cruzi-infected M{phi} stimulated with IFN-{gamma} in a dose-dependent manner (Fig. 5A). Our data also show that the infection of M{phi} with T. cruzi does lead to NF-{kappa}B nuclear translocation (Fig. 5B). However, IFN-{gamma} stimulation of T. cruzi-infected M{phi} does not lead to an increase in NF-{kappa}B translocation (Fig. 5B). It would therefore seem that, although NF-{kappa}B does not explain NO generation above control levels by itself, it might be required for maximal NO production of IFN-{gamma}-treated, T. cruzi-infected M{phi}.


Figure 5
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  		FIGURE 5. The effects of T. cruzi infection on mouse M{phi} NF-{kappa}B activation. A, M{phi} were preincubated with increasing doses of CAPE (0.1–10 µM) before being infected and subsequently treated with IFN-{gamma} (100 U/ml). iNOS protein expression and NO generation were assessed as described previously. Identical letters over bars indicate that corresponding data are not statistically different (p < 0.05). B, EMSA showing the nuclear translocation of NF-{kappa}B in T. cruzi-infected M{phi} treated or not with 100 U/ml IFN-{gamma}. Binding specificity was assessed by the addition of a 100-fold molar excess of unlabeled NF-{kappa}B (specific) or Sp1 (nonspecific) oligonucleotide to protein lysates extracted from IFN-{gamma}-stimulated M{phi}. Results are representative of three independent experiments.

 
T. cruzi infection of mouse M{phi} leads to increased iNOS mRNA stability

To assess the effect of T. cruzi infection on the stability of iNOS mRNA, control and parasite-infected M{phi} were stimulated with IFN-{gamma} (100 U/ml). Actinomycin D (5 µg/ml) was then added, and the amounts of iNOS mRNA remaining at different time points were determined. As shown in Fig. 6A, iNOS mRNA from IFN-{gamma}-treated M{phi} is degraded at a constant rate. In contrast, T. cruzi-infected M{phi} iNOS mRNA levels remain stable for 4 h before they too start to drop. Therefore, it would seem that iNOS mRNA stability could by and large explain the quantities of NO that are generated upon IFN-{gamma} stimulation of T. cruzi-infected M{phi}. Furthermore, using PD98059 (40 µM), SB203580 (10 µM), and SP600125 (30 µM), we were able to demonstrate that parasite-mediated iNOS mRNA stability was achieved via the ERK1/ERK2 and SAPK/JNK signaling pathways (Fig. 6B).


Figure 6
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  		FIGURE 6. The effect of T. cruzi infection on murine M{phi} iNOS mRNA stability. A, T. cruzi induces iNOS mRNA stability of J77.4 mouse M{phi}. B, iNOS mRNA stability is achieved through ERK1/ERK2 MAPK and SAPK/JNK signaling. iNOS mRNA stability was determined using Northern blot analyses of actinomycin D-treated (5 µg/ml) IFN-{gamma}-stimulated (100 U/ml) control ({square}) or parasite-infected ({blacksquare}) M{phi}. Densitometric quantification of iNOS mRNA decay was normalized to GAPDH. Results are representative of three independent experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is well established that T. cruzi infection elicits NO generation in IFN-{gamma}-activated M{phi} (6, 45, 46, 47, 48) and that these cells are recognized as efficient NO producers upon IFN-{gamma} stimulation (4). The effect of T. cruzi infection on M{phi} NO production in response to IFN-{gamma}, however, has not been studied in detail. This is an important issue given that M{phi} are the initial host cells of the parasite (10), that the colonization of T. cruzi within the host occurs before IFN-{gamma} activation of M{phi} (8), and that these cells play a major role in parasite clearance. In this study, we show that T. cruzi infection potentiates murine M{phi} to accumulate more iNOS mRNA, iNOS protein, and NO following IFN-{gamma} stimulation.

Modulation of M{phi} NO generation by T. cruzi infection differs from that caused by other intracellular protozoan parasites, and this modulation is probably in tune with the pathogenesis of Chagas’ disease. It is of biological interest to note that T. cruzi infection leads to an up-regulation of several signaling pathways and NO release, whereas Leishmania, a closely related kinetoplastid species, results in the opposite. In this case, lack of NO production can be attributed to an increase in Src homology protein-1 protein tyrosine phosphatase activity that leads to altered JAK2 signaling (38, 39, 40). NO serves as a major means of parasite control and, as such, may keep T. cruzi in check during the first weeks of the infection, thereby avoiding the death of the host. Past that stage of the disease, however, NO is inconsequential as far as parasite population control is concerned (8), given that the humoral response has taken over.

IFN-{gamma}-induced NO production in murine M{phi} relies heavily on the translocation of STAT1{alpha} to the nucleus (21, 22, 23, 24). Our data are reminiscent of those results. The infection of murine M{phi} with T. cruzi led to the phosphorylation of STAT1{alpha} on its ser727 residue, of MEK1/2 and ERK1/ERK2, but not of JAK2 or the tyr701 residue of STAT1{alpha}. T. cruzi infection has been shown to induce phosphorylation in murine M{phi} (49), especially the phosphorylation of ERK1/ERK2 MAPK (50, 51), SEK1/MKK4, SAPK/JNK, and p38 (51). Stimulation of T. cruzi-infected M{phi} with IFN-{gamma} resulted in the additional phosphorylation of JAK2 and the tyrosyl residue of STAT1{alpha}, thereby leading to NO generation. The upstream second messengers and cell surface receptors responsible for T. cruzi MAPK activation remain to be identified, although a TLR, TLR-2, has recently been suggested as one such possible cell surface receptor that could be activated by parasite-derived glycosylphosphatidylinositol anchors (51). Activation of the NF-{kappa}B signaling pathway has been recognized to lead to NO production in response to IFN-{gamma} (21, 22). However, its contribution seems marginal at best, as suggested by recent results obtained in our laboratory (24). Nonetheless, we decided to verify whether or not this transcription factor played any part in T. cruzi-mediated NO generation following IFN-{gamma} stimulation, because this parasite has been shown to elicit TNF-{alpha} synthesis and autocrine stimulation during M{phi} infection (52), and because NF-{kappa}B activation following T. cruzi-derived glycosylphosphatidylinositol anchor stimulation has been documented (51). Pretreatment of M{phi} with the NF-{kappa}B translocation inhibitor CAPE before parasite infection and IFN-{gamma} stimulation only partially curtailed NO production (as compared with JAK2, MAPK, and SAPK inhibitors), despite full parasite NF-{kappa}B nuclear translocation induction. Indeed, no further translocation occurred upon additional IFN-{gamma} treatment. What these findings demonstrate is that NF-{kappa}B activation alone by the infection is not sufficient for NO release. STAT1{alpha} remains the key transcription factor that needs to be activated in order for NO production to occur. However, this does not exclude the possibility that NF-{kappa}B might be needed to achieve maximal iNOS expression and that, in conjunction with iNOS mRNA stability, accounts for IFN-{gamma}-induced NO generation of T. cruzi-infected M{phi}.

It is interesting to note that, even though use of JAK2, MEK 1/2, ERK1/ERK2, and NF-{kappa}B inhibitors did not allow us to discriminate between the effects of parasite and IFN-{gamma}, higher concentrations of ERK1/ERK2 and NF-{kappa}B inhibitors were needed to achieve equivalent NO down-modulation as compared with IFN-{gamma} M{phi} activation values previously published (24). This points to effects of the parasite on ERK1/ERK2 MAPK and NF-{kappa}B that might act differently than IFN-{gamma}, especially when considering the mRNA stability results that we obtained. Although this model accounts for the manner in which NO is produced, it does not necessarily explain the increased quantities of NO generated by parasite-infected M{phi} submitted to IFN-{gamma} treatment. One highly plausible explanation we found lies in the fact that the parasite infection heightens iNOS mRNA stability through the activation of the ERK1/ERK2 MAPK and SAPK/JNK pathways. iNOS mRNA accumulation in the cell may therefore account for the increase of iNOS protein and NO. Modulation of iNOS mRNA as a mechanism of regulation of NO production has previously been reported in different systems, including mouse M{phi} (53, 54, 55). Modulation of host mRNA stability by viral and bacterial pathogens has been extensively shown (56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71). There is also one report of host mRNA stability modulation by a parasite given that Entamoeba histolitica infection of M{phi} has been shown to lead to the destabilization of TNF-{alpha} and c-Fos mRNAs (72). However, this is the first report that demonstrates that a parasitic infection can lead to increased host mRNA stability. Our results show that iNOS mRNA stability was sustained for over 4 h before dropping in parasite-infected M{phi}, whereas mRNA degradation seemed to be linear in IFN-{gamma}-stimulated M{phi}. As previously mentioned, Bhat et al. (42) have suggested that iNOS mRNA might be stabilized through the activation of the ERK1/ERK2 MAPK and p38 pathways, and that c-Jun is responsible for iNOS mRNA stability in LPS-activated J77.4 M{phi} (43). Our results show that iNOS mRNA is stabilized through the action of the ERK1/ERK2 MAPK and SAPK/JNK pathways, but not by the p38 one as suggested by Bhat et al. (42). p38, however, does in fact act in some other manner as shown by Fig. 4A. We are presently investigating the role of p38 in this context. Among the many cis and trans factors that are thought to affect mRNA stability, one likely candidate is the AU-rich elements (AREs) that are present in many short-lived mRNAs that code for cytokines, proto-oncogenes, and inducible growth factors (73). iNOS mRNA contains several of these AREs (74). One possible explanation for the observed iNOS mRNA stability in our system might come from MAPK- and SAPK-induced factors that could either act as AU-binding protein repressors or deny AU-binding protein access to AREs. Whatever the mechanism, however, having elevated amounts of iNOS mRNA in the cell, even for a few hours, would result in higher iNOS protein synthesis and NO generation, given that several ribosomes will attach to a single mRNA copy and that a single protein will catalyze several reactions.

In summary, we suggest that NO release in T. cruzi-infected M{phi} proceeds using signaling pathway cross-talk between the JAK/STAT, MAPK, SAPK/JNK, and NF-{kappa}B pathways. First, the infection leads to the activation of NF-{kappa}B, the partial activation of STAT1{alpha}, and the signaling pathways involved in iNOS mRNA stability (ERK1/ERK2 and SAPK/JNK MAPK). The next step consists in the complete activation of STAT1{alpha} upon IFN-{gamma} stimulation of JAK2, thereby allowing STAT1{alpha} to translocate to the nucleus and the transcription of a stable species of iNOS mRNA. Finally, maximal iNOS expression coupled with a slower degradation rate of this mRNA species allows its accumulation within the M{phi}, which leads to elevated iNOS protein synthesis and the liberation of massive amounts of NO.


   Acknowledgments
 
We acknowledge Dr. D. Frank (Harvard Medical School, Boston, MA) for providing the anti-phospho-ser727-STAT1{alpha} and anti-phospho-tyr701-STAT1{alpha} Ab, and Dr. D. Radzioch (McGill University, Montreal, Quebec, Canada) for providing the iNOS and GAPDH cDNA probes. We also thank Drs. J. Drummelsmith and A. Bergeron for careful editing of the manuscript as well as Dr. Sachiko Sato and team for technical help.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Canadian Institutes of Health Research (to M.O.). M.O. is a member of the Canadian Institutes of Health Research Group in Host-Pathogen Interactions. M.O. is the recipient of a Canadian Institutes of Health Research Investigator Award and is a Burroughs Wellcome Fund Awardee in Molecular Parasitology. M.B. is the recipient of a Laval University/Canadian Institutes of Health Research Doctoral Award. Back

2 Address correspondence and reprint requests to Dr. Martin Olivier, Research Institute of the McGill University Health Centre, Centre for the Study of Host Resistance, Departments of Medicine, Microbiology, and Immunology, McGill University, Montreal, Quebec, Canada H3A 2B4. E-mail address: martin.olivier{at}mcgill.ca Back

3 Abbreviations used in this paper: M{phi}, macrophage; iNOS, inducible NO synthase; SAPK, stress-activated protein kinase; CAPE, caffeic acid phenethyl ester; MBMDM, murine bone marrow-derived M{phi}; ARE, AU-rich element. Back

Received for publication August 5, 2005. Accepted for publication August 10, 2006.


   References
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 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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