Microbes and Infection 12 (2010) 587e597 www.elsevier.

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Original article

5-Lipoxygenase is a key determinant of acute myocardial inammation and mortality during Trypanosoma cruzi infection
Wander R. Pavanelli a,1, Fredy R.S. Gutierrez a,1, Flavia S. Mariano a, Cibele M. Prado b, a c Beatriz Rossetti Ferreira , Mauro Martins Teixeira , Claudio Canetti d, Marcos A. Rossi b, Fernando Q. Cunha e, Joao S. Silva a,*
a

o o o Department of Biochemistry and Immunology, Ribeira Preto School of Medicine, University of Sa Paulo, Av. Bandeirantes, 3900 - 14049-900 Ribeira Preto, SP, Brazil b o o Department of Pathology, Ribeira Preto School of Medicine, University of Sa Paulo, Brazil c Department of Biochemistry and Immunology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil d Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Brazil e o o Department of Pharmacology, Ribeira Preto School of Medicine, University of Sa Paulo, Brazil Received 28 November 2009; accepted 30 March 2010 Available online 8 April 2010

Abstract This study provides evidence supporting the idea that although inammatory cells migration to the cardiac tissue is necessary to control the growth of Trypanosoma cruzi, the excessive inux of such cells during acute myocarditis may be deleterious to the host. Production of lipid mediators of inammation like leukotrienes (LTs) along with cytokines and chemokines largely inuences the severity of inammatory injury in response to tissue parasitism. T. cruzi infection in mice decient in 5-lipoxygenase (5-LO), the enzyme responsible for the synthesis of LTs and other lipid inammatory mediators, resulted in transiently increased parasitemia, and improved survival rate compared with WT mice. Myocardia from 5-LO/ mice exhibited reduced inammation, collagen deposition, and migration of CD4, CD8, and IFN-g-producer cells compared with WT littermates. Moreover, decreased amounts of TNF-a, IFN-g, and nitric oxide synthase were found in the hearts of 5-LO/ mice. Interestingly, despite of early higher parasitic load, 5-LO/ mice survived, and controlled T. cruzi infection. These results show that efcient parasite clearance is possible in a context of moderate inammatory response, as occurred in 5-LO/ mice, in which reduced myocarditis protects the animals during T. cruzi infection. 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Trypanosoma cruzi; Myocarditis; 5-Lipoxygenase; Leukotrienes; Inammation

1. Introduction Chagas heart disease (CHD) is among the most severe clinical manifestations of the infection with the hemoagellate protozoan Trypanosoma cruzi and constitutes a major problem to public health in South and Central America. This systemic infection is characterized by an acute phase when circulating
* Corresponding author. Tel.: 55 16 3602 3242; fax: 55 16 3633 6840. E-mail address: jsdsilva@fmrp.usp.br (J.S. Silva). 1 These authors contributed equally to this work and should be considered as co-rst. 1286-4579/$ - see front matter 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2010.03.016

parasites and inconstant inammatory reaction can be found at the site of parasite inoculation. Also, intense inammation can undergo at the myocardium during this stage of disease, due to parasite tropism to cardiomyocytes. Thereafter, the myocardial aggression is usually controlled leading to the indeterminate phase of the disease. However, around 30% of patients eventually develop chronic chagasic cardiomyopathy (CCC), which consist of severe, uncontrolled myocarditis with intense brosis and is clinically manifested as complex arrhythmias, ventricular aneurism, heart failure, thromboembolism, and sudden death, mainly due to structural disturbances and intense brosis of cardiac tissue as well as microvascular changes [1,2].

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Although the precise factors governing disease progression remain poorly understood, the cascade of inammatory events that occurs at the cardiac tissue during the acute phase of the infection certainly contributes to the onset and severity of CCC [3]. Indeed, some studies suggest that cardiac inltrating leukocytes may contribute to myocardial dysfunction during T. cruzi infection by releasing or inducing the release of harmful arachidonic acid metabolites such as LTs, which leads to altered cardiac physiology [4]. LTs constitute a class of potent proinammatory mediators that participate directly and indirectly in pathophysiology of cardiovascular diseases [5,6]. Their biosynthesis derives from the oxidation of arachidonic acid by 5-lipoxygenase (5-LO) within granulocytes, macrophages and mast cells at early stages of inammation. The migration of leukocytes to the myocardium is closely regulated by the production of chemoattractants and expression of their receptors in tissue-invading cells [3,7]. In fact, the chemokines CCL3, CCL4, and CCL5 are produced by macrophages and cardiomyocytes soon after the infection, and modulate the leukocytes migration, phagocytosis and destruction of parasites by macrophages in a nitric oxide(NO)dependent manner [8,9]. The heart-inltrating inammatory cells produce several cytokines including INF-g and TNF-a, that are essential for the control of tissue parasitism [7]. These data pointed out an important role for cardiac tissue inammation in host protective response [10,11]. The intensity of acute cellular inammation in response to T. cruzi is also dependent on a series of signals triggered by factors produced by injured tissue. Such signals comprise arachidonic acid derivatives, including LTB4 and cysteinyl LTs (LTC4, LTD4 and LTE4) [12e15]. 5-LO is the enzyme responsible for synthesis of LTs, and is primarily expressed in granulocytes, macrophages, dendritic cells and mast cells [16,17]. LTB4 is synthesized by resident and recruited leukocytes during infections with bacteria, fungi, viruses, and protozoa [18], and activates macrophages to kill the intracellular forms of T. cruzi [19]. In addition, LTB4 controls the migration and effector functions of leukocytes in sites of inammation [20]. Finally, LTs are also able to modify the production of cytokines by T cells and monocytes [21,22]. In accordance, the blockade of LTB4 receptor in mice infected with T. cruzi results in increased parasitemia, suggesting that it may contribute to host protective response [23]. Therefore, we investigate the participation of 5-LO and its derivative LTB4 in the pathogenesis of myocarditis during acute phase of experimental infection with T. cruzi. Our results show that despite of increased peak of parasitemia, mice decient of 5-LO are able to arrest the parasite burden and exhibit increased resistance to T. cruzi infection, since they show increased survival associated to reduced cardiac damage and scarce tissue parasitism. 2. Methods 2.1. Animals Female wild type (WT) and 5-LO decient (5-LO/) mice of the 129 strain, 6e8 weeks old, were maintained under

standard conditions in the animal houses at our institutions. All procedures had prior approval from the local animal ethics committee. 2.2. Parasites and experimental infection The Y strain of T. cruzi was grown in the rhesus monkey kidney epithelial cells (LLC-MK2 Line). WT and 5-LO/ mice were infected by an intraperitoneal (i.p.) injection of 100 blood trypomastigotes from a strain-matched mouse. The parasitemia was evaluated in alternated days, starting on 5 days post-infection (p.i.) in a sample of 5 mL of blood obtained from the tail vein. Survival rates were determined in independent groups of 20 animals until 60 days p.i. 2.3. Histological analysis After 10, 15 and 20 days p.i. with T. cruzi, hearts were removed from mice, xed in neutral buffered 10% formaldehyde in PBS, and 5-mm slices prepared and stained either with hematoxylin and eosin(H&E) or with picrosirius red. Inammation was scored by counting the total numbers of inammatory cells in 40 microscope elds (x 200) per tissue section of ve mice from each group and the quantications were performed in three noncontiguous sections in a blinded manner by two researchers as previously described [24]. A method for the quantitative examination of the heart tissue was carried out in picrosirius red stained slides at medium power light microscopic elds (x 250): a 100-point ocular integration eyepiece II (Carl Zeiss) was used to estimate the volume fraction (%) of brosis in picrosirius-red-stained sections. Twenty elds of heart tissue were analyzed for each mouse, as described [25]. 2.4. Measurement of cytokines and LTB4 Concentrations of cytokines in tissue homogenates from heart, liver, and spleens were measured by ELISA. The IFN-g (OpTEIA, BD Bioscience, San Diego-CA), TNF-a, IL-4, and IL-10 (Duoset R&D Systems, Minneapolis-MN), were assayed following the manufacturer instructions. The reaction was reveled with peroxidase-conjugated streptavidin (Vector Laboratories, Burlingame-CA) followed by the substrate containing TMB (Promega, Madison-WI) as a chromogen. Optical densities (O.D.) of samples were then read at 450 nm and the concentrations of cytokines were calculated by extrapolating the O.D. obtained from a standard curve of each recombinant cytokine. The levels of LTB4 in heart homogenates were determined by enzyme immunoassay using a commercial kit (BiotrakTM, Amersham Pharmacia Biotech, UK) according to the manufacturers instructions. 2.5. NO quantication Blood was obtained from the retro-orbital plexus of mice at 10, 15, and 20 days p.i. Nitrate was reduced to nitrite with nitrate

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reductase and the nitrite concentration determined by the Griess method. In this assay, 0.1 mL of reductase-treated serum was incubated for 10 min with 0.1 ml of Griess reagent in a microplate, and then the O.D. was read at 550 nm [26]. The NO2 concentration was calculated from the O.D. values of a standard curve of NaNO2(1e200 mmol/L) as previously described [27]. 2.6. Immunohistochemistry Hearts of mice on days 10, 15 and 20 p.i. with T. cruzi were removed, embedded in optimal cutting temperature medium (Tissue-Tek; Sakura Torrance-CA), and stored in liquid N2. Immunohistochemistry was performed in different frozen tissue sections with each of the following antibodies: rabbit IgG anti-mouse NOs2, rat anti-mouse CD8, rat anti-mouse CD4, goat anti-mouse IFN-g, or rabbit polyclonal serum antiT. cruzi, all diluted in phosphate buffered saline containing 3% (w/v) of nonfat dry milk. The staining was performed avidineperoxidase complex and 3, 30 -diaminobenzidine (DAB, Vector Laboratories, Burlingame, Canada), and counterstaining with Mayers hematoxylin. A semi-quantitative method similar to that for picrosirius red was performed in T. cruzi immunostained tissues. In the case of CD4, CD8, and IFN-g, the number of stained cells in a xed area of 50 squared micrometers was counted. 2.7. Statistic analysis Results are expressed as means SEM of three independent experiments. Students t-test was used to analyze the statistical signicance of the observed differences. The KaplaneMeier method was used to compare survival times of the studied groups. Values of P 0.05(*)or P 0.01(**) were considered signicant. 3. Results 3.1. Production of LTB4 in cardiac tissue of mice in the acute phase of infection with T. cruzi First we asked if LTB4 is produced in the cardiac tissue during the acute phase of T. cruzi infection. The levels of LTB4 were measured in heart homogenates from mice belonging to each group at different time points after infection or in not infected control. The results showed that in WT mice the production of LTB4 is gradually increased along the infection, peaking in day 15 p.i., and starts to diminish on day 20 p.i. In addition, the levels of LTB4 in hearts from infected 5-LO/ mice were above baseline from control not infected mice, clearly indicating that LTB4 is present in the heart during the infection with T. cruzi and that its production is dependent of 5-LO (Fig. 1A). 3.2. Survival and parasitemia of WT and 5-LO/ mice infected with T. cruzi To address the relevance of 5-LO activity in the resistance to the infection, parasitemia and survival analyses of WT and

5-LO/ mice infected with T. cruzi were evaluated. The deciency of 5-LO enzyme resulted in transient, enhanced and slightly anticipated peak of parasitemia compared with the control group (9th and 11th days p.i., respectively; Fig. 1B). However, both groups of animals achieved the complete

Fig. 1. Production of LTB4 in infected T. cruzi mice and the involvement of 5LO in control of parasites proliferation. The levels of LTB4 in the heart homogenate from WT and 5-LO/ mice infected or not with T. cruzi (A),. The dashed line represents the levels of LTB4 in heart from control (not infected) mice. The parasitemia (B) and mortally (C) of infected WT or 5LO/ are showed. The data showed represent the mean SEM of the results obtained using ve mice and are representative of three independent experiments. *P < 0.05 compared with infected mice.

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control of parasitemia after 17 p.i. days. Despite the control of parasitemia in both groups, the survival of 5-LO/ mice was signicantly higher. All WT mice succumbed to infection by day 30 p.i., while at the same time a stable survival rate of 65% was observed (Fig. 1C). 3.3. Evaluation of heart pathology in WT and 5-LO/ mice infected with T. cruzi Since LTB4 is produced during the infection and 5-LO/ mice are more resistant to the infection, we next examined if the intensity of myocarditis was associated with this proinammatory molecule. At day 10 p.i., the myocardium of WT and 5-LO/ mice showed a similar mild myocarditis, characterized by a discrete interstitial inammatory inltrate composed mainly of lymphomononuclear cells. At day 15 p.i., WT mice presented a severe myocarditis associated with an inammatory inltrate mainly composed of lymphomonuclear cells. In contrast, the myocardium of 5-LO/ mice showed a moderate myocarditis also accompanied by an inammatory inltrate of lymphomononuclear cells. At day 20 p.i., the myocardium from WT mice presented a strikingly severe myocarditis, associated with an inammatory inltrate composed of lymphomononuclear cells. In contrast, the myocardium from 5-LO/ mice showed a moderate myocarditis (Fig. 2A). In fact, the analysis of inammatory score of cardiac tissues from both mice groups showed a signicant decreased leukocytic inltration in the 5LO/ infected mice, compared with WT mice (Fig. 2B). These results suggest that 5-LO plays a role in the pathogenesis of acute myocarditis during T. cruzi infection. 3.4. Detection of T. cruzi antigens and collagen deposition in cardiac tissues of WT and 5-LO/ infected mice We next evaluated if the reduced inammation observed in 5-LO/ mice effects parasite clearance of the myocardium. Immunohistochemistry for detection of parasite antigens demonstrated a dual effect of 5-LO deciency on tissue parasitism. First, compared with WT mice, 5-LO/ mice displayed increased amounts of T. cruzi antigens (approximately two folds) at day 15 p.i. In contrast, at the day 20 p.i., the levels of parasite antigens of 5-LO/ mice decreased signicantly compared with WT mice (Fig. 2C). These data suggest that although 5-LO/ mice have a reduced inammatory response, they control the parasite load in the cardiac tissue. The collagen deposition on cardiac tissue was evaluated by picrosirius red stained sections of heart on days 10, 15 and 20 p.i. Data obtained by conventional or polarization microscopy revealed mild interstitial brosis in both WT and 5-LO/ mice on days 10 and 15 p.i.. The brosis was manifested by a discrete increase in the amount of perimysial and endomysial collagen as compared with control hearts from noninfected controls. At day 20 p.i., the WT mice exhibited a marked diffuse interstitial myocardial brosis characterized by thicker perymisial collagen bers extending into the endomysium in

comparison with the more discrete brosis in the myocardium from 5-LO/ mice (Fig. 3A). In fact, the quantication of myocardial brosis in heart sections of both groups of mice revealed that in general, the brosis was more noticeably on day 20 p.i. than in previous periods. Of note, the volume fraction of brosis was nearly two times higher in the myocardium from WT than that from 5-LO/ mice on 20 day p.i. (Fig. 3B). Therefore, these results support the hypothesis that the absence of LTs abrogated the inammatory response and brosis in cardiac tissue of T. cruzi infected mice. 3.5. Characterization of the inammatory inltrate in myocardia of WT and 5-LO/ T. cruzi-infected mice The phenotype of lymphocytes that migrated to the cardiac tissue of 5-LO/ mice was determined by immunohistochemistry. The results demonstrated a progressive increase of CD4 and CD8 cells in the heart of WT mice along the analyzed time points. In 5-LO/ mice, despite to accumulate leukocytes into the cardiac tissue, the amount was signicantly lower compared with that of infected WT mice in all time points studied (Fig. 4A and B). Also, the number of IFN-g positive cells increased gradually along the time in WT mice, but not in 5LO/ mice (Fig. 4C). Together, these results conrm a reduction in the number of leukocytes in the inammatory lesions of heart of 5-LO/ mice infected with T. cruzi. 3.6. Production of cytokines and NO in WT and 5-LO/ mice infected with T. cruzi To further study the inammatory response in the heart, liver and spleen of WT and 5-LO/ mice, we evaluated the production of TNF-a, IFN-g, IL-10, and IL-4 in tissues from infected WT and 5-LO/ mice. The results showed that the production of TNF-a, IFN-g, and IL-4 in the heart and liver were higher on days 15 and 20 p.i. in WT compared with 5LO/-infected mice. In addition, higher TNF-a and IL-4 concentration (but not IFN-g) was also detected in the spleens of WT mice. Conversely, the IL-10 production was signicantly lower in liver of infected-WT mice compared with 5LO/ mice on days 15 and 20 p.i. (Fig. 5). In accordance, the immunostaining for nitric oxide synthase (iNOS) was lower in the heart tissue of 5-LO/ than that of WT infected mice (Fig. 6A). This data correlated with the increased levels of nitrite and nitrate found in the sera of WT mice (Fig. 6B). This reduced production of proinammatory cytokines and NO, along with increased levels of the anti-inammatory cytokine IL-10, explain the reduced cardiac inammatory response in 5-LO/ mice infected with T. cruzi. 4. Discussion Lipid mediators such as LTs and prostaglandins participate in the recruitment of leukocytes to the inamed tissue [28,29]. These mediators are produced from arachidonic acid by either the cyclooxygenase pathway (leading to prostaglandins and thromboxanes) or the 5-LO pathway to form LTs. We have

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Fig. 2. Histological evaluation and T. cruzi antigens in heart of T. cruzi-infected mice. WT and 5-LO/ mice were infected with T. cruzi and the heart collected, xed and examined under photomicroscope. Representative photomicrographs of hearts from WT and 5-LO/ mice on days 15 and 20 p.i. with T. cruzi are showed (A). The inammatory score (B) and the percentage of T. cruzi antigens per area in myocardium (C) are also showed. The data represent the mean SEM of the results obtained using ve mice and are representative of three independent experiments. *P < 0.05 compared with infected WT mice. Scale bars 50 mm.

previously showed that the treatment of infected mice with the COX-2 inhibitors sodium salicylate or meloxicam dramatically reduced the blood parasites load and delayed the death curve of the infected animals. Moreover, this treatment restored the production of IL-2 to control levels and lymphocyte proliferation. These data pointed out a role for PGE2 in mediating the immunosuppression occurring in experimental T. cruzi infection [30]. Here we attempted to evaluate the affect of the deciency of another metabolic pathway for arachidonic acid: the 5-LO enzyme. The myocardial inammatory response observed during T. cruzi infection is essential for eliminating the parasite but

also accounts for the extensive tissue damage and subsequent morbidity [3,31]. Our results suggest that exacerbated myocarditis is detrimental to the host during the acute phase of the infection. LTs are essential in the killing of T. cruzi by in vitro infected macrophages [19]. Moreover, the treatment of T. cruzi-infected mice with a BLT1 receptor antagonist was accompanied by increased parasitemia, but not lethality [23]. In accordance, our current data revealed an increased LTB4 production during the acute phase of T. cruzi infection. 5-LO/ -infected mice presented reduce leukocyte migration to the myocardium and also exhibited a higher parasitic load than WT mice. Despite these facts, 5-LO/-infected mice were

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Fig. 3. Cardiac brosis of WT and 5-LO/ mice on the acute phase of T. cruzi infection. WT and 5-LO/ mice were infected with T. cruzi, euthanized, and the hearts processed for detection of collagen. The photomicrographs of cardiac tissues of mice stained with picrosirius red at day 20 p.i. (A) and the quantication of collagen deposition (B) are showed. The data represent the mean SEM of the results obtained using ve mice and are representative of three independent experiments. *P < 0.05 compared with infected WT mice. Scale bars 150 mm.

able to control parasite burden and survived to the challenge. Of note, similar results were obtained in mice treated with MK886, an inhibitor of LTs biosynthesis (See Supplementary Fig. 1). The inammatory response in the heart is characterized by the inltration of macrophages, CD4, and CD8 lymphocytes that produces reactive species of nitrogen and oxygen, as well as cytokines (mainly IFN-g, TNF-a, and IL-10). In spite of being necessary for parasite killing within phagocytes, nitric

oxide is a complex mediator which excessive production, as commonly observed during T. cruzi infection, has been showed to cause apoptosis of inammatory cells, autonomic neuronal cells and cardiomyocytes [27,32,33], resulting in immune suppression, extensive tissue destruction [27,34] and may even facilitate the parasite survival. The high levels of NO observed in WT mice on 20 day p.i could impact the efciency of immune response, allowing the proliferation and persistence of parasite in the tissue. Therefore, lower NO

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Fig. 4. CD4, CD8, and INF-g cells in cardiac tissue of WT and 5-LO/ mice infected with T. cruzi. CD4 (A), CD8 (B) and INF-g cells (C) were quantied in frozen sections of cardiac tissue processed for immunohistochemistry. The data represent the mean SEM of the results obtained using ve mice and are representative of three independent experiments. *P < 0.05 compared with infected WT mice.

production as that observed 5-LO/-infected mice could be enough to control parasite replication avoiding extensive myocardial damage. Infected 5-LO/ mice exhibited reduced levels of leukocytes and cytokines in the myocardium and also showed reduced levels of myocardial brosis at a late stage of acute phase of the infection (day 20 p.i.), which is a clear consequence of the decreased tissue destruction. Thus, in addition to the production of cytokines and chemokines [3] observed during the very early stages of cardiac inammation, the lipid mediators derived from 5-LO metabolism, especially LTB4, participate in the induction of such inammatory damage into parasitized cardiac tissues during the infection with T. cruzi. Indeed, it was recently demonstrated that 5-LO products are responsible for oxidative stress in erythrocytes during this infection [35].

The biologically active products of arachidonic acid oxidation by 5-LO play a role in the pathogenesis of various inammatory disorders like asthma [36], cystic brosis [37] psoriasis [38], rheumatoid arthritis [39], and inammatory bowel disease [40]. Among these mediators, LTs are known to participate actively in the control of infections by protozoa, as demonstrated in several studies [41e43]. Despite that LTB4 and LTC4 have been shown to participate in parasite uptake and killing by phagocytes and cardiac myoblast in vitro [19,44], the role of these molecules in host resistance and induction of myocarditis during T. cruzi infection remains unknown. LTB4 promote the recruitment of inammatory cells including activated CD4 and CD8 T lymphocytes [29], and contribute to increase the uptake and intracellular destruction of T. cruzi, as well as the production of IFN-g and NO by phagocytes [23,44]. Unlike LTB4, the cys-LTs(LTC4) do not induce leukocyte migration into inamed tissue, but contribute to inammation via increased vascular permeability and subsequent edema [45]. Moreover, they have also been described as detrimental factors to heart contractility [4]. Thus, the absence of 5-LO seems to prevent the harmful effects of these mediators on heart contractile function. In addition to LTs, lipoxins (Lx) are also derived from arachidonic acid metabolism and have an antagonistic role, inhibiting inammatory reactions [46]. The role of Lx in determining the myocardial inammation was not studied, although our results show that the deciency in 5-LO do not appears to overwhelms signicantly the production of Lx, as the predominant effect observed in 5-LO/ mice is a reduced cardiac inammation. However, specic inhibition of Lx should help to clarify separately the role of these antiinammatory lipid mediators in the myocarditis induced by T. cruzi. The increased amounts of parasites transiently observed in the blood and in the cardiac tissue of 5-LO/ mice (on days 9 and 15 p.i., respectively) are probably due to the reduced levels of NO in serum, as well as diminished migration of CD4 and CD8 T cells to the myocardium, in addition to reduced production of IFN-g. All these factors are essential for controlling T. cruzi replication. A report using the 5-LO inhibitor nordihydroguaiaretic acid (NDGA) showed similar results in terms of parasitemia and tissue parasitism [35]. Indeed, mice lacking CD4 or CD8 cells, as well as IFN-g producing cells, are incapable to control the infection [7,47,48]. Nevertheless, 5-LO/ mice were lastly able to control the parasite burden in the circulation and in the cardiac tissue (on days 13 and 20 p.i., respectively). These data are in accordance with previously published reports that inhibition of LTB4 (one product of 5-LO) was accompanied by higher levels of parasitemia, but not lethality in mice infected with T. cruzi [23]. Our results clearly show that 5-LO/ mice present reduced index of brosis associated to low levels of IL-4 and TNFa compared with WT mice. These cytokines are released by inammatory cells and promote brosis [25,49]. IL-4 promotes progression of brosis by stimulating the synthesis of collagen [50]. In addition to TNF-a and IL-4, TGF-b is

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Fig. 5. Production of IFN-g, TNF-a, IL-10, and IL-4 in the heart, liver, and spleen of WT and 5-LO / mice infected with T. cruzi. The tissues of WT and 5-LO/ mice were homogenized in PBS in presence of proteases inhibitor and the concentrations of IFN-g, TNF-a, IL-10, and IL-4 evaluated by ELISA. The data showed represent the mean SEM of the results obtained using ve mice and are representative of three independent experiments. *P < 0.05 compared with infected WT mice.

a well known pro-brotic cytokine which plays a prominent role in the structural alterations caused by T. cruzi at later stages of infection [51]. Further investigations focused on the chronic heart pathology should demonstrate if absence of 5LO can lead to reduced matrix remodeling and perhaps reduced chronic morbidity. The absence of 5-LO results in decreased production of proinammatory cytokines (TNF-a and INF-g), but did not signicantly enhance the levels of the anti-inammatory cytokine IL-10 in the heart. Of note, the effect of 5-LO deciency in the production of cytokines is the same in cardiac and hepatic tissue, whereas in the spleens from 5-LO/ mice only TNF-a is signicantly reduced. Thus, added to the central role of LTs in recruiting inammatory cells, these lipid

mediators are also important modulators of the cytokine environment produced during T. cruzi infection. In conclusion, our ndings suggest that during experimental infection with T. cruzi, the products of 5-LO activity are required but not essential for the control of systemic and cardiac parasite burden by immune cells. More important, this study provides evidence that apart from being the intensity of the myocardial inammation determinant for control the growth of the pathogen, it can also be associated with acute mortality. Consequently, it is possible that therapeutic approaches aiming to reduce the intensity of myocarditis, in combination with anti-parasitic therapy, could be useful to control T. cruzi-induced myocardial damage and the resulting morbidity. Although further studies are required on these issues, this report constitutes an incentive to

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Fig. 6. Activation of nitric oxide synthase in cardiac tissues and levels of nitric oxide in the sera of mice during infection with T. cruzi. The inducible nitric oxide synthase (iNOS) was revealed in frozen sections of cardiac tissue processed for immunohistochemistry (A) and concentration of nitrite/nitrate in sera from WT and 5-LO/ mice were determined by the Griess method (B). The data showed represent the mean SEM of the results obtained using ve mice and are representative of three independent experiments. *P < 0.05 compared with infected WT mice. Scale bars 50 mm.

focus on the selective inhibition of 5-LO in the development of immunomodulatory therapies during this infection. Acknowledgements ` Supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, The Millennium Institute for Vaccine Development and Technology, and Conselho Nacional de Desen volvimento Cientco e Tecnologico. Appendix. Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.micinf.2010.03.016.

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