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*NITRIC OXIDE
The Journal of Immunology, 2006, 177: 3193-3200.
Copyright © 2006 by The American Association of Immunologists, Inc.

Type I IFNs Stimulate Nitric Oxide Production and Resistance to Trypanosoma cruzi Infection1

Vlaudia M. A. Costa2,*, Karen C. L. Torres{dagger}, Ronaldo Z. Mendonça{ddagger}, Ion Gresser§, Kenneth J. Gollob{dagger} and Ises A. Abrahamsohn3,*

* Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil; {dagger} Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; {ddagger} Instituto Butantan, São Paulo, Brazil; and § Centre de Recherches Biomedicales des Cordeliers, Université Pierre et Marie Curie, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The participation of type I IFNs (IFN-I) in NO production and resistance to Trypanosoma cruzi infection was investigated. Adherent cells obtained from the peritoneal cavity of mice infected by the i.p. route produced NO and IFN-I. Synthesis of NO by these cells was partially inhibited by treatment with anti-IFN-{alpha}beta or anti-TNF-{alpha} Abs. Compared with susceptible BALB/c mice, peritoneal cells from parasite-infected resistant C57BL/6 mice produced more NO (2-fold), IFN-I (10-fold), and TNF-{alpha} (3.5-fold). Later in the infection, IFN-I levels measured in spleen cell (SC) cultures from 8-day infected mice were greater in C57BL/6 than in infected BALB/c mice, and treatment of the cultures with anti-IFN-{alpha}beta Ab reduced NO production. IFN-{gamma} or IL-10 production by SCs was not different between the two mouse strains; IL-4 was not detectable. Treatment of C57BL/6 mice with IFN-I reduced parasitemia levels in the acute phase of infection. Mice deprived of the IFN-{alpha}betaR gene developed 3-fold higher parasitemia levels in the acute phase in comparison with control 129Sv mice. Production of NO by peritoneal macrophages and SCs was reduced in mice that lacked signaling by IFN-{alpha}beta, whereas parasitism of macrophages was heavier than in control wild-type mice. We conclude that IFN-I costimulate NO synthesis early in T. cruzi infection, which contributes to a better control of the parasitemia in resistant mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The protozoan hemoflagellate Trypanosoma cruzi is the etiologic agent of Chagas disease in man and infects a variety of mammalian species. T. cruzi trypomastigotes can infect many different mammalian cell types in which they differentiate and multiply as amastigote forms. Macrophages can act as host cells for the parasite and also as effector cells in the antiparasite immune responses. Several authors have shown that T cell-mediated effector functions are essential for the control of the parasite load and host survival (1, 2, 3, 4).

Control of T. cruzi parasitism during the first weeks of infection is considered to be critically dependent on effective macrophage activation by cytokines. Macrophages activated by IFN-{gamma} and/or TNF-{alpha} synthesize NO, which, in the mouse, is considered the major effector molecule of intracellular amastigotes’ killing (5, 6, 7, 8). Protection conferred by T cells against a T. cruzi challenge infection correlates with higher serum nitrate levels (9) and, conversely, mice treated with inhibitors of NO synthesis (5, 7, 9, 10) or deprived of inducible NO synthase (iNOS)4 genes (11, 12, 13) develop very intense parasitism, underscoring the importance of NO in controlling the parasite load. NO is especially important for the control of parasitism in the early acute phase, but not in the late acute or in the chronic phase of T. cruzi infection (9, 10).

Type I IFNs (IFN-I) were discovered on the basis of their antiviral activity and comprise a conserved family of pleiotropic cytokines that are produced by a variety of cells, including fibroblasts, macrophages, and dendritic cells (14, 15). They are critical to the host antiviral defense and are important to the innate and adaptive immunity to several microorganisms because they activate macrophages and NK cells and costimulate T cell proliferation and differentiation (16, 17, 18).

In the mouse, IFN-I consist of IFN-{alpha} and IFN-beta, and they synergize with LPS to stimulate NO production by mouse macrophages (19, 20, 21). In the mouse model of Leishmania major infection, rIFN-{alpha} plus LPS can activate NO production and destruction of parasites in macrophages (22) and IFN-{alpha}beta stimulates NOS2 activity and controls parasite spreading in the early period after infection (23). Furthermore, treatment of BALB/c mice with low doses of IFN-beta protects from progressive cutaneous leishmaniasis, an effect strictly dependent on iNOS expression and augmented production of IFN-{gamma} and IL-12 (24).

Mice infected with T. cruzi develop antiviral activity in their sera, and treatment of mice with an IFN inducer (25) or with purified IFN-{alpha}beta preparations increases resistance to the infection (26, 27). Exposure of murine cells to live parasites leads to enhanced surface class I MHC expression that is IFN-I dependent and may render parasitized cells more susceptible to T CD8+-mediated cytotoxicity (28).

However, the ability of IFN-I to stimulate iNOS by macrophages could be most important in the innate immune response to T. cruzi infection. The quick limitation of parasitism by innate mechanisms may be especially important for host resistance to T. cruzi because control of parasite proliferation in the first days after infection determines the number of parasites released to the blood (parasitemia) at later time points (29).

Mice lacking the subunit IFNAR1 of IFN-I receptors are completely unresponsive to IFN-{alpha} and IFN-beta (30). These mice provided the opportunity to study in vivo the effect of complete absence of IFN-I signaling on T. cruzi infection.

This work reports on the effect of IFN-I on murine T. cruzi infection and on the mechanisms that control early T. cruzi parasitism. We show that IFN-I exert a significant control of acute-phase parasitemia levels in mice. An important share of the initial NO production by macrophages from the inoculation site, and of splenic origin, was stimulated by IFN-I. Moreover, mice deprived of IFN-I receptor chain IFNAR1 developed increased parasitemia in comparison with the wild-type (WT) controls and also had decreased NO production and iNOS mRNA expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Parasites and infection of mice

C57BL/6 and BALB/c mice used in this study were females, 5–6 wk old, bred, and maintained in specific pathogen-free conditions at the animal facilities of Department of Immunology, Instituto de Ciências Biomédicas, Universidade de São Paulo. All animal procedures were performed in accordance with the Institute’s Ethics Committee on Animal Experimentation, which follows the Brazilian Code for the Use of Laboratory Animals. Mice of the strains 129Sv IFN-{alpha}betaR+/+ and 129Sv IFN-{alpha}betaR0/0 were females, 6–7 wk old, donated by L. Reis from the Ludwig Institute (São Paulo, Brazil). T. cruzi (Y strain) parasites were maintained by weekly i.p. inoculation of BALB/c mice. The mice were killed, and the trypomastigote-rich plasma containing 5000 blood forms was injected i.p. into C57BL/6 and BALB/c mice, and 10,000 blood forms were injected i.p. into 129Sv mice deprived of type I receptor gene, IFN-{alpha}betaR0/0 mice designated IFN-{alpha}betaR knockout (KO) mice, and 129Sv IFN-{alpha}betaR+/+ mice designated WT (30). Parasitemia counts were performed by counting the parasites in 5 µl of citrated blood obtained from the lateral tail veins. Mortality was evaluated by daily inspection of the cages. In a series of experiments conceived to test the effect of IFN-{alpha}beta on the course of infection, groups of C57BL/6 and BALB/c mice were injected s.c. at the base of the tail with purified IFN-{alpha}beta (31) (2000 IU/animal) and subsequently infected with 5000 Y strain blood trypomastigotes by the same route.

Preparation of spleen cell (SC) and peritoneal cell (PC) suspensions

SC suspensions were prepared from mice infected 5, 8, and 11 days previously or from normal mice, depleted of erythrocytes by hypotonic lysis with distilled water, and resuspended in RPMI 1640 complete medium containing 5% FCS, 10 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml) (Sigma-Aldrich). PC suspensions were prepared from mice that had been infected i.p. 6–48 h before and from normal uninfected mice. The cells were harvested from the peritoneal cavity by injecting cold 0.01 M PBS, centrifuged at 500 x g for 5 min at 4°C, and resuspended in RPMI 1640 complete medium to a final concentration of 4 x 106 cells/ml. The PC suspension was dispensed into 24-well flat-bottom plates and incubated at 37°C, 5% CO2, for 16 h, to allow the cells to adhere to the plastic surface, followed by vigorous washing with serum-free medium to remove nonadherent cells. For each experiment, the spleens or PC from three mice were pooled.

In vitro infection of macrophages with T. cruzi

PC were dispensed (2 x 105) into 8-well glass chamber slides (Nunc) incubated 37°C, 5% CO2 for 6 h, to allow the cells to adhere the surface. The cultures were washed to remove nonadherent cells and infected with T. cruzi at 10:1 parasite:cell ratio for additional 12 h. The cultures were washed to remove extracellular parasites, and half the slides were stained with HEMA 3 stain set (Biochemical Sciences). The remaining cultures were incubated for 60 h and then stained with HEMA 3. In a series of preliminary experiments, we compared the number of adherent cells in PC cultures obtained from C57BL/6 or BALB/c mice 24 h after T. cruzi infection. For these, the cells were dispensed into 4-well chamber slides. After a 16-h adherence step, the slides were vigorously washed with PBS and stained, and the cells were counted using a light microscope. The results are reported as the means and SD of the adherent cells counted in twenty x40-microscope fields in three independent experiments.

Cell cultures

SC or PC were cultured in 24-well flat-bottom plates at, respectively, 5 x 106 and 4 x 106 cells/well. Supernatants from the cultures were harvested after 6, 24, 48, and 72 h. Anti-cytokine mAbs were added at the beginning of the culture period: anti-TNF-{alpha} (XT-22, 20 µg/ml), anti-IFN-{gamma} (XMG 1.2, 20 µg/ml), and anti-IL-12 (C17.8, 100 µg/ml). These and the isotype control mAbs were obtained in our laboratory from hybridoma supernatants and isolated by protein G affinity chromatography. The neutralizing anti-IFN-{alpha}beta Ab was a sheep anti-mouse IFN-{alpha}beta antiserum titrated by I. Gresser (Centre de Recherches Biomedicales des Cordeliers, Université Pierre et Marie Curie, Paris, France) for its potency to neutralize the inhibitory activity of IFN-I in a viral neutralizing assay; it was used at a dilution that neutralized 2000 U of IFN-I/ml (31). Isotype control rat anti-beta-galactosidase GL113 (IgG1) and GL117 (IgG2a) mAbs or sheep serum anti-mouse L cells (G-025-501-568 National Institute of Allergy and Infectious Diseases repository, National Institutes of Health) were used as controls for the anti-cytokine Ab treatments.

Measurement of nitrite production and detection of cytokines

Nitrite (NO2–) accumulation in 72-h supernatants of cultured cells was used as an indicator of NO production and was determined by the Griess reaction with sodium nitrite as a standard, as previously described (detection limit: 1.56 µM) (32). Fifty microliters of supernatant were incubated for 10 min, in the dark, at room temperature, with 50 µl of a freshly mixed solution of N-[1-naphthyl]-ethylenediamine (1 mg/ml), sulfanilamide (10 mg/ml), and 5% (v/v) phosphoric acid in distilled water. The absorbance was measured at 540 nm. Production of TNF-{alpha} was measured in supernatants of PC obtained 6 h after infection with trypomastigotes using a two-site sandwich ELISA. This method was standardized in our laboratory using biotinylated anti-TNF-{alpha} (G281-2626, 4 µg/ml; BD Pharmingen) and XT-3 (5 µg/ml). The lower limit detectable TNF-{alpha} in our assay was 0.31 ng/ml. Measurement of IFN-{gamma}, IL-10, and IL-4 in culture supernatants was done by ELISA, as described before (33).

Viral inhibition assay

To quantify IFN-I production in cell culture supernatant samples, dilutions of a standard preparation of IFN-{alpha}beta (Gu02-901-51; National Institutes of Health) were added to L-929 murine fibroblast grown in 96-well tissue culture plates. Duplicate serial dilutions of the supernatants to be tested were added to other culture wells. The cultures were challenged with 30 tissue culture infective doses of encephalomyocarditis virus (34). One IFN unit is defined as the quantity that inhibits the cytopathic effect of encephalomyocarditis virus by 90%. Quantification of IFN-I was done in the presence of anti-IFN-{gamma} mAb XMG 1.2 at 10 µg/ml to block the antiviral effect of IFN-{gamma} possibly present in the supernatants. Specificity control was done by adding anti-IFN-{alpha}beta antiserum at 100 U/ml to triplicate wells in each assay run. Because the antiviral activity was blocked by this procedure and IFN-I in the mouse consists of IFN-{alpha}beta, the measured antiviral activity in the samples is designated interchangeably as IFN-{alpha}beta or IFN-I.

RT-PCR assays

SC suspensions were prepared from mice infected 5 and 8 days previously or from normal mice, as described, and aliquots of 1 x 107 cells were centrifuged and resuspended in TRIzol (Invitrogen Life Technologies). RNA extraction and cDNA synthesis were performed, as previously described (35). PCR were performed by using specific primer pairs, flanking each gene of interest: hypoxanthine-guanine phosphoribosyltransferase (HPRT) (352 bp), sense 5'-GTTGGATACAGGCCAGACTTTGTTG, antisense 5'-GAGGGTAGGCTGGCCTATGGCT; IL-10 (324 bp), sense 5'-CCAGTTTTACCTGGTAGAAGTGATG, antisense 5'-TGTCTAGGTCCTGGAGTCCAGCAGACTCAA; IFN-{gamma} (267 bp), sense 5'-CATTGAAAGCCTAGAAAGTCTG, antisense 5'-CTCATGAATGCATCCTTTTTCG; iNOS (306 bp), sense 5'-TGGGAATGGAGACTGTCCCAG, antisense 5'-GGGATCTGAATGTGATGTTTG.

Primer and sample mixture were incubated in the presence of 10 mM dNTPs and TaqDNA polymerase (Invitrogen Life Technologies). All of the reagents were then mixed in the reaction buffer containing 100 mM Tris-HCl, 500 mM KCl, 1% Triton X-100, and 15 mM MgCl2. Amplification was conducted in a thermal cycler (Techne Genius), conducted for 35 cycles using the following reaction conditions: 94°C melting for 40 s, 59°C annealing for 20 s, and 72°C extension for 1 min. The samples were normalized based on their HPRT content, and the amounts were measured on Image System Nucleovision (Nucleotech Gel Expert program) and expressed as arbitrary units relative to HPRT.

Data analysis

Results are expressed as the arithmetic mean accompanied by the SD. Means of control and experimental groups were compared by Student’s t test and Tukey test. Differences were considered to be significant when p < 0.05. The GraphPad INSTAT statistical analysis program was used to perform the analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Higher resistance of C57BL/6 mice to T. cruzi infection correlates with higher production of NO by peritoneal cavity cells

Following i.p. infection with strain Y blood forms, BALB/c mice developed 5-fold higher parasite counts than similar infected C57BL/6 (Fig. 1A). Moreover, while both mouse strains controlled parasitemia levels, parasite counts remained higher in BALB/c mice, and they were all dead by the 16th day of infection, whereas all C57BL/6 mice survived the infection (data not shown).


Figure 1
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  		FIGURE 1. NO production by cells obtained from the inoculation site is higher in T. cruzi-resistant C57BL/6 than in susceptible BALB/c mice. Mice were infected by the i.p. route with 5000 T. cruzi strain Y blood forms. A, Parasitemia levels of BALB/c and C57BL/6 mice. Means ± SD of eight mice. C57BL/6 vs BALB/c; *, p < 0.05. B, Adherent PC from infected mice (obtained 6 and 24 h after infection) or from normal mice were incubated for 72 h before removing the supernatants for nitrite determination. C, The SC from infected mice were cultured for 72 h before harvest of supernatants. Means ± SD of duplicate experiments. Representative of three independent experiments. C57BL/6 vs BALB/c; *, p < 0.05.

 
As NO is an important effector molecule in the destruction of T. cruzi by activated macrophages, we sought to compare its production in both strains of mice in two distinct compartments: peritoneal cavity cells and spleen. In a series of preliminary experiments, we compared the number of adherent cells in cultures from the peritoneal cavity of C57BL/6 or BALB/c mice. The number of adherent cells was, for BALB/c, 116 ± 10 cells/field, and for C57BL/6, 126 ± 7 cells/field. The morphology of the cells in the preparation was also similar between the two strains of mice and 95% of the cells were macrophages by morphological criteria. As shown in Fig. 1B, NO production by cells from the peritoneal cavity obtained after infection was ~2-fold higher in C57BL/6 mice than in BALB/c. Basal unstimulated nitrite production was not significantly different between the two strains.

In contrast, there were no significant differences in the amount of nitrite measured in SC cultures from BALB/c and C57BL/6 mice on days 8 and 12 of infection (Fig. 1C). On day 5 of infection, consistently higher levels of nitrite were found in SC cultures from infected BALB/c mice, coincident with the observation of extracellular live parasites in the SC cultures from these mice, whereas parasites were not detected and nitrite levels in SC cultures from C57BL/6 mice were not different from uninfected control cultures.

PC from infected C57BL/6 mice produce higher levels of IFN-I and TNF-{alpha} that induces NO production

As shown in Fig. 2A, PC collected from C57BL/6 mice 6 and 24 h after i.p. infection produced much higher levels of IFN-I than cells obtained from infected BALB/c mice. However, for both strains, IFN-I production by PC collected 48 h after infection had fallen to the levels found in uninfected PC cultures (<2 U/ml). We could not detect IL-12 or IL-10 in the supernatants from these cultures. We also tested PC from both mouse strains as to their ability to synthesize TNF-{alpha} after in vitro infection with tissue culture trypomastigotes. After in vitro infection with the parasites at a ratio 5:1, PC obtained from C57BL/6 mice produced much higher levels of TNF-{alpha} than BALB/c PC; addition of neutralizing anti-IFN-{alpha}beta Abs did not modify TNF-{alpha} secretion levels (Fig. 2B). Neither IL-12, nor IL-10, IL-4, or IFN-{gamma} were detected in supernatants obtained up to 72 h of culture, and the addition of anti-IL-10 did not increase IFN-I production levels (data not shown).


Figure 2
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  		FIGURE 2. IFN-{alpha}beta, TNF-{alpha}, and NO production by T. cruzi-infected peritoneal adherent cells. A, The PC were obtained 6 and 24 h after i.p. infection, and adherent cells were cultured for 24 h before harvesting the supernatants for IFN-{alpha}beta determination by biological assay. B, TNF-{alpha} was measured by ELISA in supernatants of peritoneal adherent cell cultures that had been infected with culture trypomastigotes for 6 h and cultivated for further 6 h. C, Adherent PC were treated with anti-TNF-{alpha} mAb XT-22, anti-IFN-{alpha}beta antiserum, or control antiserum (control-AS; National Institute of Allergy and Infectious Diseases repository G-025-501-568) and infected for 6 h with T. cruzi culture trypomastigotes. Supernatants for nitrite determination were obtained after 72 h of culture in the presence of the Abs. Means ± SD of three experiments run in duplicate. C57BL/6 vs BALB/c: *, p < 0.05; anticytokine treatments vs control-AS: **, p < 0.05.

 
To test whether IFN-{alpha}beta or TNF-{alpha} produced by the infected macrophage cultures was indeed stimulating NO synthesis in the same cultures, peritoneal adherent cell cultures were treated with neutralizing Abs to either cytokine and infected with the parasites, and the production of nitrite was measured. Treatment with anti-IFN-{alpha}beta or anti-TNF-{alpha} led to significant reduction of NO production in culture (Fig. 2C), indicating that IFN-{alpha}beta and TNF-{alpha} produced by macrophages infected by T. cruzi were stimulatory of NO synthesis.

SC from C57BL/6 mice transiently produced more IFN-{alpha}beta than BALB/c mice, which stimulates NO production

Production of IFN-I by SC cultures from 8-day infected C57BL/6 mice was significantly greater compared with BALB/c mice (Fig. 3A). Yet, the levels of NO produced by SC from infected C57BL/6 and BALB/c mice were not significantly different (cf Fig. 1C). However, NO production by SC of both strains was, on day 8 of infection, being stimulated by IFN-{alpha}beta and also by IFN-{gamma} and TNF-{alpha}, because treatment of the cultures with the corresponding neutralizing anti-cytokine Abs markedly reduced nitrite levels (Fig. 3B). On day 12 of infection, treatment of BALB/c SC cultures with anti-IFN-{alpha}beta, anti-IFN-{gamma}, or anti-TNF-{alpha} did not modify NO production, whereas only anti-IFN-{gamma} mAb was effective in C57BL/6 SC cultures and reduced NO levels by 40% (data not shown). Semiquantitative evaluation of IFN-{alpha} and IFN-beta mRNA expression by SC at the time points 5, 8, and 12 days of infection showed elevated message levels in comparison with uninfected mice, but no difference in expression between the two strains (data not shown).


Figure 3
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  		FIGURE 3. IFN-{alpha}beta and NO production by SC from T. cruzi-infected mice. A, Production of IFN-{alpha}beta by SC cultures from T. cruzi-infected mice. IFN-{alpha}beta was measured by biological assay in supernatants of SC cultures on the 1st, 5th, 8th, and 12th day after infection with 5000 T. cruzi blood forms. A total of 5 x 106 cells was cultured for 24 h. C57BL/6 vs BALB/c; *, p < 0.05. B, Inhibition of nitrite production by SC cultures treated with anti-IFN-{alpha}beta antiserum, anti-TNF-{alpha} mAb, anti-IFN-{gamma} mAb, or control-AS (National Institute of Allergy and Infectious Diseases repository G-025-501-568). Cultures were obtained from mice on the 8th day after infection with 5000 T. cruzi blood forms. Means ± SD of triplicates. Treated vs untreated cultures; *, p < 0.05. Representative of three independent experiments.

 
Production of IFN-{gamma} and IL-10 in the splenic compartment from infected mice

Production of IFN-{gamma} was detected earlier (5 days) in cultures of SC from infected BALB/c mice, but by day 8 of infection IFN-{gamma} levels were higher (22 ± 3 ng/ml) in SC cultures from C57BL/6 than from BALB/c mice (15 ± 1 ng/ml). However, on day 12 of infection, while IFN-{gamma} production remained at these levels in BALB/c mice, it was greatly reduced in C57BL/6 (3 ± 0.5 ng/ml). Regarding IL-10 and IL-4 production, these cytokines were below detection levels in SC cultures from days 5 and 8 of infection for both strains of mice. On day 12, mean IL-10 levels were 12 ± 3 and 15 ± 2 U/ml for BALB/c and C57BL/6 mice, respectively, and IL-4 was below detection levels (78 pg/ml) for both strains.

Treatment of C57BL/6 mice with IFN-I reduces parasitemia levels

To examine the putative protective activity of IFN-{alpha}beta in the infection, C57BL/6 and BALB/c mice were injected with 2000 U of IFN-{alpha}beta by the s.c. route and immediately challenged at the same site with T. cruzi blood trypomastigotes. Levels of parasitemia in IFN-{alpha}beta-treated C57BL/6 mice were found to be significantly reduced as compared with nontreated controls (Fig. 4A). There was no mortality in either group. In contrast, treatment of BALB/c mice with the same or higher (up to 8000 U) dosage of IFN-{alpha}beta did not modify parasitemia levels and also did not reduce or postponed mortality (data not shown).


Figure 4
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  		FIGURE 4. Effect of treatment with IFN-{alpha}beta and of the disruption of the IFN-{alpha}betaR gene on T. cruzi blood parasitemia levels. A, C57BL/6 mice were treated with IFN-{alpha}beta and infected by the s.c. route with 5,000 T. cruzi blood forms. Means of three animals ± SD. Treated vs untreated; *, p < 0.05. Representative of two experiments. B, Mice deprived of the IFN-{alpha}betaR gene (IFN-{alpha}betaR KO) and their WT counterparts (129Sv) were infected i.p. with 10,000 T. cruzi strain Y blood forms. Means ± SD of eight mice. The 129Sv IFN-{alpha}betaR KO vs 129Sv; *, p < 0.05.

 
The 129Sv IFN-{alpha}betaR KO mice infected with T. cruzi develop higher parasitemia than control WT 129Sv mice

Parasitemia on the eighth day of infection was significantly higher in IFN-{alpha}betaR KO mice than in the WT controls (Fig. 4B). However, IFN-{alpha}betaR KO mice, in contrast with BALB/c mice whose parasitemia levels were of similar magnitude, did not die of the infection. Their survival rate was 100%, which did not differ from the WT 129Sv control mice. The 129Sv mice were relatively resistant to infection with T. cruzi Y strain, and 10,000 blood forms inocula by the i.p. route led to parasitemia levels of the order of 1–2 x 106 parasites/ml, comparable to those seen in C57BL/6 mice inoculated with 5,000 parasites (cf Fig. 1A). All 129Sv mice survived after infection.

Macrophages from IFN-{alpha}betaR KO mice infected with T. cruzi present higher parasite loads than macrophages from infected control WT mice

Macrophages from the peritoneal cavity of IFN-{alpha}betaR KO and 129Sv WT mice infected with T. cruzi were compared as to their ability to become infected and sustain intracellular growth of the parasites. As shown in Fig. 5, by 12 h of infection 46% IFN-{alpha}betaR KO macrophages were parasitized, while in cultures from WT controls only 28% of the cells were parasitized. After 72 h of culture, 56% IFN-{alpha}betaR KO and 37% WT macrophages were infected. In addition, 15% IFN-{alpha}betaR KO macrophages harbored >10 parasites in comparison with only 6% WT macrophages. These results indicate that signaling through IFN-{alpha}beta favors the control of macrophage parasitism by T. cruzi.


Figure 5
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  		FIGURE 5. Peritoneal macrophages from 129Sv IFN-{alpha}betaR KO mice become more infected than macrophages from 129Sv WT mice. Adherent PC were infected with T. cruzi at 10:1 parasite:cell ratio; after 12 (A) or 72 h (B), the chamber slides were processed, and the cells and parasites were counted. The results are the means ± SD of cells counted in 20 x40 microscope fields in three independent experiments. The 129Sv IFN-{alpha}betaR KO vs 129Sv; *, p < 0.05.

 
IFN-{alpha}betaR KO mice have lower NO production by PC and SC than 129Sv WT mice

The comparison of NO production levels between IFN-{alpha}betaR KO and WT mice showed that the former had markedly reduced nitrite production by peritoneal macrophages in comparison with similar cultures from WT mice. In fact, nitrite production by IFN-{alpha}betaR KO macrophages was 4.9 µM ± 0.4, which is only ~10% of the levels found in cultures from WT 129Sv macrophages (55.3 µM ± 2.2). To verify whether the differences in NO production seen in peritoneal macrophage cultures extended to other compartments, we compared nitrite production in cultures of SC from IFN-{alpha}betaR KO and WT mice on days 5 and 8 after infection with T. cruzi. As shown in Fig. 6, nitrite levels produced by SC from infected mice were ~40–50% lower in IFN-{alpha}betaR KO than in 129Sv WT mice. In addition, iNOS mRNA levels expressed by SC obtained from 8-day infected IFN-{alpha}beta KO mice were 3-fold lower in comparison with infected WT mice, whereas no difference in mRNA expression levels for IL-10 and IFN-{gamma} was found between these two groups of mice (data not shown).


Figure 6
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  		FIGURE 6. T. cruzi-infected IFN-{alpha}betaR KO mice produce less NO than 129Sv WT mice. SC cultures from 5-day (A) or 8-day (B) infected mice were treated with anti-IL-12, anti-IFN-{gamma}, or anti-IFN-{alpha}beta, and nitrite levels were measured in supernatants harvested after 72 h. Means ± SD of triplicates. Representative of three independent experiments. Untreated 129Sv IFN-{alpha}betaR KO vs 129Sv: **, p < 0.05; treated vs untreated: *, p < 0.05.

 
That IFN-{alpha}beta was indeed stimulatory of NO production by SC was confirmed by the treatment of WT SC cultures with neutralizing anti-IFN-{alpha}beta antiserum, which reduced nitrite production by 50% on days 5 and 8 of infection (Fig. 6). On the 12th day after infection, the effect of the anti-IFN-{alpha}beta Ab treatment on nitrite levels was no longer observed (data not shown). Addition of anti-IFN-{alpha}beta antiserum to SC cultures from IFN-{alpha}betaR KO mice, as expected, did not modify nitrite levels and confirmed that the antiserum had no specific inhibitory activity on nitrite production. Treatments with anti-IL-12 or anti-IFN-{gamma} mAbs also markedly reduced nitrite production by SC cultures from WT and IFN-{alpha}betaR KO mice. Together, the results show that IFN-{alpha}beta, IFN-{gamma}, and IL-12 costimulate NO production in T. cruzi-infected mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
NO is considered to be the major effector molecule of trypanocidal activity in the mouse. Although it has been shown that IFN-{gamma} and/or TNF-{alpha} stimulate NO production by T. cruzi-infected macrophages (5, 8, 36), the possible stimulatory activity of IFN-I in this process had not hitherto been investigated. We show in the present study that, in addition to TNF-{alpha}, IFN-I were produced by peritoneal cavity cells and by SC obtained from mice infected with T. cruzi (8 days of infection). Treatment of the cell cultures with anti-IFN-{alpha}beta Abs decreased NO levels, indicating that IFN-I are indeed costimulating NO production by infected macrophages from the peritoneal cavity and spleen. Previous work from our laboratory had identified that a significant component of NO production in the first week of infection is independent of IL-12 and of TNF-{alpha} or IFN-{gamma} neutralization by mAb treatment (37). Furthermore, IFN-{gamma}-independent pathways of iNOS induction are present in T. cruzi-infected IFN-{gamma}-receptor-deprived mice (38). In the mouse model of L. major infection, the parasite induces IFN-{alpha}beta and this cytokine plus the parasite act in turn, activating iNOS synthesis by macrophages, thus stimulating innate resistance to this parasite (23). Moreover, treatment of L. major-infected BALB/c mice with IFN-beta up-regulated iNOS expression and protected from progressive infection (24).

Increased levels of antiviral activity in the serum of T. cruzi-infected mice were first reported in 1981 (27). In addition, serum IFN-{alpha}beta levels are higher in infected C57BL/6 and C3H mice in comparison with the extremely susceptible BXH-2 strain or with irradiated mice (29). However, the mechanisms underlying the possible participation of IFN-{alpha}beta in resistance to the infection were at that time unsolved. Our results show that IFN-I production by peritoneal cavity cells and by SC from resistant T. cruzi-infected C57BL/6 mice was higher in comparison with susceptible BALB/c mice. In comparison with BALB/c mice, T. cruzi-infected C57BL/6 mice produced larger amounts of IFN-I-stimulated NO by PC that correlated with better control of acute-phase parasitemia. It was shown by Trischmann (29) that control of T. cruzi proliferation already occurs in the first cycles of intracellular replication, indicating the importance of innate immunity in parasitism control. Even mice completely devoid of functional T or B cells (RAG–/–) can exert some control of parasitism by mechanisms that include NO production and depend on activation by IL-12, IFN-{gamma}, and TNF-{alpha} (36). Both the control of parasitism and host survival are critically dependent on these cytokines because they are active as mediators of the innate, as well as of the acquired immunity, and because IL-12 and IFN-{gamma} are essential for mounting a protective Th1 response. We present evidence that IFN-I also contribute to the early control of T. cruzi parasitism. IFN-{alpha}betaR KO mice, which have a phenotype of IFN-I deficiency (30), developed higher parasitemia levels, but survived at the same rates as their WT counterparts. Thus, in terms of host survival and ultimate resistance to the infection, IFN-I has a secondary role compared with IL-12 and IFN-{gamma}. However, it should be remembered that both these cytokines are essential for Th1 development, whereas, in mice, IFN-I does not stimulate Th1 differentiation (39). In contrast, IFN-I can be synthesized by a variety of epithelial or connective tissue cells in addition to macrophages or lymphocytes, and its synthesis occurs very early after infection. T. cruzi infection induces human fibroblasts to secrete IFN-beta, and a set of IFN-stimulated genes is among the most highly induced transcripts, which are indicative of the quick response to IFN-I (40). We quantified IFN-I in cultures of adherent cells obtained by washing the inoculation site (peritoneal cavity) 6 and 24 h after infection and in SC cultures obtained at later times after infection. In the former cultures, macrophages were the dominant (95%) cell type and probably account for most IFN-I produced, although the contribution of a minor population of fibroblasts cannot be discarded. In SC cultures, dendritic cells and/or lymphocyte populations, in addition to macrophages, could be triggered by T. cruzi to secrete IFN-I. In this regard, T. cruzi surface-derived molecules interact with TLR2 (41) or TLR4 (42) and trigger the synthesis of several proinflammatory cytokines: whether IFN-I synthesis is also stimulated is an interesting possibility that demands further investigation.

The treatment with IFN-{alpha}beta (2000 U) reduced parasitemia levels of C57BL/6 mice during the acute phase by ~50%, but had no effect in BALB/c mice. It should be pointed out that, in our experiments, higher IFN-{alpha}beta doses given to BALB/c mice were not protective and also failed to induce additional protection in C57BL/6 mice. The strict low-dose dependence of IFN-I effect on T. cruzi parasitemia in C57BL/6 mice parallels the observations made using IFN-beta-treated Leishmania-infected mice: only low IFN-beta doses were protective and augmented in vivo iNOS expression (24).

Regardless, the susceptibility of BALB/c to T. cruzi infection could not be overcome by supplying IFN-I at the time of inoculation. Likewise, treatment of C57BL/6 mice (but not of susceptible A/J mice) with the IFN-I inducer Tilerone reduced mortality (25). It is likely that the diminished NO production by BALB/c peritoneal macrophages, in comparison with C57BL/6 mice, results from lesser stimulation due to lower TNF-{alpha} levels (cf Fig. 3B), in addition to lower IFN-I production. In BALB/c mice, the reported higher production of TGF-beta1 and higher arginase activity by macrophages, generating ornithine instead of NO from L-arginine, could also contribute to the low NO production in the early phase of infection (43, 44). Yet, later in the course of infection, NO levels produced by BALB/c SC cultures were high and equivalent to C57BL/6 mice, possibly because activation by IFN-{gamma} compensates for the lower TNF-{alpha} and IFN-I production by BALB/c. An example of cytokine compensatory mechanism is seen in TNFR1–/– mice that are more susceptible to T. cruzi, but ultimately produce similar NO levels as their WT counterparts (45). The difference in susceptibility of BALB/c and C57BL/6 mice to T. cruzi infection could not be correlated with variations of IFN-{gamma} or IL-10 production. Yet, IFN-I levels were significantly higher in the resistant C57BL/6 strain at the early phase of infection and contributed to higher NO production and to better initial parasitism control. Importantly, the determinants of mouse susceptibility to T. cruzi infection are not clearly understood and different genes regulate parasitemia levels and survival (46). In this regard, increased morbidity (but not the parasitic load) of murine T. cruzi infection is associated with higher levels of TNF-{alpha} and lower IL-10 production (36, 47, 48) or with lower ratios of soluble TNF-{alpha} receptors/secreted TNF-{alpha} (49).

Mice deprived of IFN-{alpha}betaR gene developed 3-fold higher parasitemia levels in the acute phase of T. cruzi infection in comparison with their WT counterparts, but without an increase of the mortality rate. In comparison, the lack of IFN-{gamma}, IL-12, or TNFR results in dramatically increased parasitemia and higher mortality (12, 45). However, the absence of signaling via IFN-I makes IFN-{alpha}betaR KO mice less capable of controlling the parasite load early in infection because their macrophages are less efficient at destroying the parasites because of reduced NO production.

The stimulatory effect of endogenously produced IFN-I on NO synthesis in T. cruzi-infected C57BL/6 or 129Sv mice was not dependent on enhancement of IFN-{gamma} production, as evidenced by the finding that neutralization of IFN-I in SC cultures did not affect IFN-{gamma} production levels (data not shown). In addition, IFN-{alpha}betaR KO and 129 Sv mice had similar levels of IFN-{gamma} production in SC cultures. However, IFN-I contribution to improved control of parasitism is most likely not restricted to stimulation of NO synthesis. For example, IFN-I stimulates NK- or T-CD8-mediated cytotoxicity in other infections (24, 50, 51).

Based on our data, we propose that IFN-{alpha}beta stimulate NO production by macrophages as part of the innate immune response to T. cruzi during the initial phase of infection, and thus help control the initial parasite load and parasitemia levels.


   Acknowledgments
 
We thank Dr. Luiz Fernando L. Reis from the Ludwig Institute for the generous gift of the IFN-{alpha}betaR KO and WT 129Sv control mice and for valuable suggestions; Dr. Christian Bogdan for reviewing the manuscript; and Ademir Veras da Silva for the technical assistance.


   Disclosures
Top
 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 investigation was supported by a grant from Fundação de Amparo à Pesquisa do Estado de Sao Paulo and by Conselho Nacional de Pesquisas supplementary fellowships (to I.A.A.). V.M.A.C. was a recipient of Comissao de Aperfeicoamento de Pessoal de Nivel Superior/Programa Institucional de Capacitação Docente e Técnica Ph.D. fellowship, and the experimental work reported herein is part of her doctorate thesis. Back

2 Current address: Departamento de Medicina Tropical, Universidade Federal de Pernambuco, 50670-901, Recife, PE, Brazil. Back

3 Address correspondence and reprint requests to Dr. Ises A. Abrahamsohn, Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Avenida Professor Lineu Prestes 1730, 05508-900, São Paulo, SP, Brazil. E-mail address: iabraham{at}usp.br Back

4 Abbreviations used in this paper: iNOS, inducible NO synthase; IFN-I, type I IFN; HPRT, hypoxanthine-guanine phosphoribosyltransferase; KO, knockout; SC, spleen cell; PC, peritoneal cell; WT, wild type. Back

Received for publication November 4, 2005. Accepted for publication June 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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