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Combination therapy with benznidazole and doxycycline shows no additive effect to monotherapy with benznidazole in mice infected with the VL-10 strain of the Trypanosoma cruzi
IJCA-27861; No of Pages 6 International Journal of Cardiology xxx (xxxx) xxx
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Combination therapy with benznidazole and doxycycline shows no additive effect to monotherapy with benznidazole in mice infected with the VL-10 strain of the Trypanosoma cruzi Ana Cláudia Alvarenga Carneiro a, G.P. Costa a, Cyntia Silva Ferreira b, Isalira Peroba Rezende Ramos e, Mariáurea Matias Sarandy d, Ana Luísa Junqueira Leite a, A.P.J. Menezes a, B.M. Silva b, K.O.P.C. Nogueira c, A.C.C. Carvalho e, Reggiani Vilela Gonçalves d, André Talvani a,⁎ a
Laboratório de Imunobiologia da Inflamação, Departamento Ciências Biológicas, Universidade Federal de Ouro Preto, Minas Gerais, Brazil Laboratório de Biologia e Tecnologia de Micro-organismos, Departamento Ciências Biológicas, Universidade Federal de Ouro Preto, Minas Gerais, Brazil c Laboratório de Biomateriais e Patologia Experimental, Departamento Ciências Biológicas, Universidade Federal de Ouro Preto, Minas Gerais, Brazil d Laboratório de Patologia Experimental, Departamento de Biologia Animal, Universidade Federal de Viçosa Minas Gerais, Brazil e Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil b
a r t i c l e
i n f o
Article history: Received 25 February 2019 Received in revised form 20 June 2019 Accepted 15 July 2019 Available online xxxx Keywords: Trypanosoma cruzi VL-10 strain Inflammation Benznidazole Doxycycline Echocardiography
a b s t r a c t Background: Chagas heart disease is the most important clinical manifestation of Trypanosoma cruzi infection. Pharmacological therapies have been proposed aiming to reduce inflammatory response and cardiac damage in infected hosts. In this study, we investigated the use of doxycycline (Dox), in a sub-antimicrobial dose, in monotherapy and in combination with benznidazole (Bz) during the acute phase of infection with the VL-10 strain of T. cruzi, evaluating the therapeutic effect during the acute and chronic phases of the infection. Methods and results: C57BL/6 mice were treated for 20 days with Dox (30 mg/kg), Bz (100 mg/kg), or both drugs in combination starting 9 days after infection. Parasitemia was measured during the acute phase and the animals were monitored for 12 months, after which echocardiography analysis was performed. Blood samples were obtained from euthanized mice for CCL2, CCL5, IL-10 analysis, and cardiac fragments were collected for histopathological evaluation. Dox treatment did not ameliorate parasitological/inflammatory parameters but reduced the cardiac collagen neoformation (CN) in 35%. In contrast, Bz administration reduced parasitemia, plasma levels of CCL2 and CCL5, and cardiac infiltration during acute infection, and reduced the level of IL-10 and CN (95%) at 12 months. Dox was unable to improve ejection fraction, while Bz treatment ameliorated the ejection fraction. No additive effect was observed in combination therapy. Conclusion: Dox monotherapy is not effective in the acute or chronic phases of experimental cardiomyopathy induced by the VL-10 strain of T. cruzi. Furthermore, combination therapy with Dox does not potentiate the effects of Bz monotherapy. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma cruzi infection represents a relevant social problem that still occurs in many countries, especially in Latin America. Cardiac involvement is present in many cases of Chagas disease and can be considered as the more severe clinical form of T. cruzi infection [1,2]. This cardiac disease results in a remodeling of the collagen matrix and subsequent fibrosis, leading to myocarditis, increased tissue rigidity, diastolic
⁎ Corresponding author at: Laboratory of Immunobiology of Inflammation, DECBI/ICEB, Campus Morro do Cruzeiro, Universidade Federal de Ouro Preto, 35400-000 Ouro Preto, Minas Gerais, Brazil. E-mail address: [email protected] (A. Talvani).
and systolic dysfunction, functional loss and, in some cases, severe dilated cardiomyopathy associated with ventricular arrhythmias [3,4]. Benznidazole (Bz) is the most commonly used drug against T. cruzi in Brazil and is used to reduce or eliminate the parasites that trigger these cardiac inflammatory processes [5–8]. Unfortunately, Bz has low efficacy and leads to undesired adverse reactions. For this reason, new anti-T. cruzi compounds have been tested in the last decade, as well as anti-inflammatory drugs that could be useful in preserving cardiac morpho-functional structure. These drugs include angiotensin converting enzyme (ACE) inhibitors, beta-blockers, statins, and, more recently, Doxycycline (Dox) [9–12]. Doxycycline is a broad-spectrum bacteriostatic agent invented and clinically developed in the early 1960s by Pfizer Inc., NY, USA [13]. Besides its original antibacterial role, Dox has emerged as a co-adjuvant therapy in non-bacterial illnesses such as
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Please cite this article as: A.C.A. Carneiro, G.P. Costa, C.S. Ferreira, et al., Combination therapy with benznidazole and doxycycline shows no additive effect to monotherapy with b..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.07.047
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malaria and tick-borne rickettsial diseases [14]. Dox has also stood out as a potent inhibitor of metalloproteinases and suppressor of the hydrolases (e.g., phospholipase A2 and alpha-amylase) when administered in low doses (b50 mg/kg), which can reflect, in part, its capacity to scavenge reactive oxygen species and suppress inflammatory cytokines involved in distinct inflammatory disorders [15,16]. Due to the inefficiency of current treatments against T. cruzi, new pharmacological strategies directed towards the modulation of systemic and/or tissue-specific inflammatory response have been pursued, with the aim of minimizing tissue damage caused by this protozoan. The purpose of this study was to identify whether Dox and Bz therapies could positively modify immune and cardiac parameters during acute (30 days) and long-term (12 months) infection in C57BL/6 mice infected with the VL-10 strain of T. cruzi. 2. Methods 2.1. Animals C57BL/6 male mice, aged 6–8 weeks, were bred and maintained at the Center of Animal Science (CCA) at the Federal University of Ouro Preto (UFOP), Brazil. The procedures adopted are in accordance with the ethical principles of animal experimentation preestablished by the National Council for Control of Animal Experimentation (CONCEA). This research was previously approved by the Ethics Committee on Animal Research of UFOP-CEUA (protocol n° 2016/63).
2.6. Echocardiography Transthoracic echocardiographic examination was performed on mice after 12 months of infection, under anesthesia with continuous oxygen flow, maintaining similar heart rates between the groups to avoid the influence of heart rates on cardiac contractility indexes. Each anesthetized animal was placed in a dorsal decubitus position on a surgical table for transducer positioning in the left hemithorax of the animal. VisualSonics Vevo 770 equipment was used with a 30 MHz transducer. The images were acquired at a frequency of 14 MHz for resolution optimization. At time of visualization of the left ventricle (transverse and longitudinal sections), Simpson analysis was used in B mode (bi-dimensional) to obtain measurements of the following variables: ejection fraction (EF%); fractional area change (FAC%); end-diastolic volume (EDV); and end-systolic volume (ESV). Additionally, analysis was also performed in M-mode (one-dimensional) to obtain measurements of the following variables: left ventricular mass (mg); and left ventricular volume (μL) at the papillary muscle levels. 2.7. Statistical analysis The data obtained in this study are expressed as the mean ± standard deviation. The GraphPad Prism v.5 (GraphPad Software, San Diego, CA, USA) program was used and data were analyzed using the Kolmogorov-Smirnov test to confirm normality patterns. Depending on the nature of the data, one-way analysis of variance (ANOVA) with post-test Bonferroni correction, and Kruskal-Wallis test with post-test Tukey-Kramer and Dunn's tests were used for multiple comparisons. For all analyses, a significance level of p ≤ 0.05 was adopted.
3. Results and discussion 3.1. Parasitemia curve
2.2. Parasites and infection The male C57BL/6 mice (60 days of birth) were infected intraperitoneally with 5 × 103 blood trypomastigote forms of T. cruzi strain VL-10, which had been stored in liquid nitrogen and maintained in vivo by successive passages in Swiss mice. Parasitemia was determined daily by optic microscopy analysis of 5 μL samples, after tail bleeding [17].
2.3. Treatments and euthanasia Treatment with doxycycline (Dox; 30 mg/kg) and benznidazole (Bz; 100 mg/kg) was initiated 9 days after infection by T. cruzi. After the establishment of T. cruzi infection, the drugs were administered for 20 consecutive days orally (gavage) at a volume of 0.2 mL diluted in fetal bovine serum (PBS) + carboxymethylcellulose (CMC) 0.5%. Animals (n = 6 for the acute phase and n = 13 for the chronic phase), for each group evaluated were sorted into groups: (i) uninfected animals that received PBS + CMC; (ii) infected, untreated mice that received PBS + CMC; (iii) infected mice treated with Dox; (iv) infected mice treated with Bz; and (v) infected mice treated with a combination of Dox + Bz. Animals in the acute and chronic phases were euthanized after 30 days and 12 months of infection, respectively, according to euthanasia practice guidelines regulated by the CONCEA. Blood and cardiac tissues were collected for immunoenzymatic assays and histopathological analysis.
2.4. Immunoassays Immunoenzymatic assays were employed to evaluate the concentrations of the chemokines CCL5 and CCL2, and the regulatory cytokine IL-10. Plasma samples were isolated, and each assay performed according to the manufacturer's protocol (PeproTech Inc., Rocky Hill, NJ, USA). At the end of the reaction, 100 μL/well of the chromogen substrate ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), Sigma-Aldrich Inc., St. Louis, MO, USA) was added for 60 min followed by dark incubation at room temperature. Finally, samples were read in a spectrophotometer (Emax Molecular Devices, Sunnyvale, CA, USA) using wavelengths of 405 nm and 630 nm.
2.5. Histopathological analysis Heart fragments were collected and immediately fixed in 10% buffered formaldehyde. The next day, the fixative was replaced with 70% ethanol. The tissues were then processed and embedded in histological resin (HistoResin, Leica Basic Resin©). Then, the blocks were submitted to microtomy with 3-μm-thick sections and stained with hematoxylin and eosin (H&E) for quantification of the inflammatory infiltration. All histopathological analysis was digitized using the microscope Leica DM5000B (Leica Application Suite, UK, version 2.4.0 R1) with a coupled micro-camera and processed through the Leica QWin V3 image analyzer program. The inflammatory process was evaluated by quantifying all cellular nuclei randomly selected within a total area of 35,493.7 μm2 – equivalent to 25 fields of analyzed myocardium (40× magnification). Inflammation was quantified by counting the number of cardiac cellular nuclei in infected heart tissue compared with the number of cardiac cellular nuclei observed in the tissue of non-infected mice.
Dox treatment did not inhibit replication of the VL-10 strain of T. cruzi, with no difference observed between the untreated and Doxtreated infected animals. On the other hand, Bz reduced the peak and profile of the parasitemia curve, maintaining activity both in monotherapy and in combination with Dox, as shown in Fig. 1. In this way, as the parasite load is closely related to the inflammatory response, any drug capable of reducing parasitemia (such as Bz) is likely to partially reduce the systemic and/or local inflammatory processes [3]. There are many factors that influence the parasite level in the bloodstream during the acute phase, such as the genetic background of the parasite strain and the host, as well as other external factors such as temperature and host stress [18,19]. However, a higher immune response occurs during the initial stage of infection in humans and experimental models. Controlling the immune elements, by reduction of parasites or by direct pharmacological interference, could minimize the deleterious action of immune cells or their inflammatory mediators. However, it is as yet uncertain as to whether this early intervention would result in notable changes in chronic cardiac pathology. 3.2. Evaluation of inflammatory markers Several studies have already shown that the expression and production of inflammatory mediators both increase during the acute and chronic phases of animal model and human T. cruzi infection [9,12,20]. Furthermore, part of this immune profile observed in experimental infections was dependent on the genetics of the T. cruzi strain [21]. In the present study, we observed a reduction in CCL2 and CCL5 levels in mice treated with Bz; this profile was maintained when Bz was used in combination with Dox. However, Dox did not reduce these plasma chemokines in the acute phase of infection with the VL-10 strain of T. cruzi (Fig. 2A and C). Another study, using different genetic variations of the parasite, different strategies and treatment times, but the same sub-therapeutic dose than our study (30 mg/kg), demonstrated that Dox leads to a strong decrease in the clonal expansion of T cells, resulting in a reduction in levels of the inflammatory cytokines and chemokines, and in a lower expression of matrix metalloproteinases [22]. In this sense, it is worth emphasizing that Dox was previously used not only for its primary bacteriostatic action, but for its inhibition actions on MMPs at sub-therapeutic doses [13,16]. Some common adverse effects caused by the class of tetracyclines and their semi-
Please cite this article as: A.C.A. Carneiro, G.P. Costa, C.S. Ferreira, et al., Combination therapy with benznidazole and doxycycline shows no additive effect to monotherapy with b..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.07.047
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Fig. 1. Parasitemia curve. The blood parasites were daily evaluated in C57BL/6 mice infected with 5000 trypomastigote forms of the VL-10 strain of T. cruzi. These animals were grouped according to the treatment with Benznidazole (Bz), Doxycycline (Dox) or a combination of both drugs. The data at each point of the curve are representative of the mean of the parasitemia/day for each group.
synthetic forms are gastrointestinal disorders, including anorexia, vomiting, diarrhea, mouth ulcerations and irritation of the perianal region [14] but no one was observed in the animals under sub-therapeutic dose of Dox. On the other hand, after 12 months of infection (when physiological and pathological disturbances become more evident), we expected to observe partial stabilization of systemic and local inflammatory processes. In contrast with observations during the acute phase, plasma CCL2 levels did not change in any of the chronic infection groups (Fig. 2B). Additionally, the only change observed in CCL5 levels was an increase in plasma concentration in mice treated with Dox + Bz compared to uninfected animals (Fig. 2D). However, even with the potential immunoregulation of Bz during the acute phase, Bz decreased the profile of IL-10 in the acute and chronic phases of infection (Fig. 2E and F). IL-10 has a protective role against fatal acute myocarditis in a murine model of T. cruzi infection, and in chagasic individuals. A relevant factor in the immunoregulatory activity of this cytokine against T. cruzi infection is the evidence that IL-10 mRNA is already detected 15 days after infection, suggesting its participation in the initial events of the disease [23]. However, the VL-10 strain of T. cruzi, used in our current study, induces a smaller cardiac inflammatory response when compared to other virulent strains in C57BL/6 mice model, or when used in other mammalian models (e.g., Swiss mice and dogs) [9,24]. These parasite-host interactions influence the inflammatory and regulatory patterns of the mediators.
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Fig. 2. Plasma CCL-2, CCL5, and IL-10 levels in Trypanosoma cruzi-infected mice under different therapeutic strategies. Concentrations of CCL2 (A, B), CCL5 (C, D) and IL-10 (E, F) in plasma from C57BL/6 mice infected with T. cruzi VL-10 strain are presented according the proposed therapy with Benznidazole (Bz), Doxycycline (Dox) or a combination of both drugs during the acute and chronic phases of the infection. Data are represented as mean ± standard deviation, where equal symbols (*, &, #, α, δ) mean statistical difference (p ≤ 0.05) between groups (One-Way ANOVA test, post-test of Tukey).
3.3. Histopathological analysis In this section, cardiac tissue from uninfected animals is represented in Fig. 3A and G. During the acute phase, all infected, untreated animals presented both diffuse and localized cardiac inflammatory processes, as shown in Fig. 3B. Mice treated with Bz (Fig. 3C) and with Dox + Bz (Fig. 3E) showed no evidence of cardiac inflammatory response, in contrast with mice treated with Dox (Fig. 3D), however this inflammatory process was similar to that observed in the infected untreated mice, as evidenced by the quantitative analysis shown in Fig. 3F. This matches expectations as Dox does not regulate production of inflammatory chemokines. During the chronic phase of infection, no inflammatory infiltrate was observed in the infected, untreated, or in the groups that received treatment (Fig. 3L). In the chronic phase of experimental T. cruzi infection, we observed 55% of the cardiac collagen neoformation in parasite-infected animals, 35% in Dox-treated-animals, an absence in those mice under Bz therapy and 30% in those animals receiving both Dox + Bz (data not shown). Besides immune modulatory action, another important role proposed for the use of sub-antimicrobial doses of doxycycline in treating cardiac diseases is its inhibitory action on the matrix metalloproteinases (MMPs), mainly MMP-2 and -9, preventing their non-physiological remodeling of the collagen matrix [22,25,26].
Please cite this article as: A.C.A. Carneiro, G.P. Costa, C.S. Ferreira, et al., Combination therapy with benznidazole and doxycycline shows no additive effect to monotherapy with b..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.07.047
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3.4. Echocardiography Since only Bz affected inflammatory mediators and cardiac infiltration in the VL-10 T. cruzi-infected mice, we expected preservation of cardiac function in Bz-treated mice, but not in those treated with only Dox. Indeed, infection with the parasite reduced the ejection fraction (EF%) in all animals, but in those treated with Bz, we observed a well-preserved EF% (Fig. 4A). Our results in this experimental model corroborate studies that propose the EF as an important variable in the prognostic determination of chagasic individuals, since decrease in EF% is strongly associated with worsening of diastolic function, and with increases in the dimensions of the atrium and left ventricle [27]. There was no significant difference in the fractional cardiac area (FCA) between the infected, treated and untreated groups. However, the untreated infected group presented a reduced FCA when compared to the control group, as shown in Fig. 4B. In humans, fractional variation of the two-dimensional area is one of the most commonly-used measures for the quantitative estimation of left ventricular function, as this measure has been identified as an important factor in the prognosis of heart failure arising from myocardial infarction [28]. Another important parameter evaluated was the left ventricle (LV) mass. We observed an increase of this mass in the untreated infected mice when compared to the uninfected animals, but Dox monotherapy reduced this parameter (Fig. 4C), possibly through indirect action on the MMPs. Echocardiographic studies suggested that left ventricular mass may be a relevant variable in the prognosis of patients with heart failure [29]. Increased LV mass is an important risk factor for the development of ejection fraction decline and, consequently, the onset of heart failure.
Finally, we observed a decrease in diastolic volume in groups treated with Bz (monotherapy or in combination), in comparison with the untreated infected group (Fig. 4D). This decrease in end-diastolic volume may be the result of a decrease in compliance and a partial decrease in ventricular diastolic relaxation, and, consequently, in the ventricular ejection [30]. Studies have shown that diastolic dysfunction parameters were the determinants of cardiac functional capacity in mammalians, regardless of systolic function [31]. Since the end-systolic volume of the group treated with Bz and the group treated with Dox + Bz decreased compared to the infected group, as shown in Fig. 4 E, more blood was pumped into the animal body. In humans, left ventricular systolic function, as measured by the ejection fraction, remains a strong prognostic marker regardless of the clinical status of each individual [28–31]. In summary, our results show that Bz and its combination with sub-therapeutic dose of Dox reduced plasma levels of CCL2 and CCL5, and inflammatory infiltrate in cardiac tissue in the acute phase of infection. During the chronic phase, treatment with Bz maintained reduced plasma levels of IL-10 in infected animals, but alone or in combination with Dox resulted in improvement of echocardiographic parameters, such as ejection fraction and end-diastolic and end-systolic volumes.
4. Conclusions Sub-therapeutic dose of Dox, in monotherapy, during the acute phase of T. cruzi infection (VL-10 strain) was not effective to ameliorate the murine cardiomyopathy in both, acute and chronic
Fig. 3. Photomicrographs of histological sections of the heart. C57BL/6 mice were infected with T. cruzi VL-10 strain and treated or not with Doxycycline (Dox) and/or Benznidazole (Bz) in the acute phase of the infection. (A, G) normal cardiac histological appearance in uninfected mice; (B) moderate and focal inflammatory infiltrate in infected and untreated mice in the acute phase; (C, I) absence of inflammatory infiltrate in infected mice treated with Bz in the acute (C) and chronic (I) phases; (D) discrete and focal inflammatory process in infected mice treated with the Dox in the acute phase; (H) absence of inflammatory process in untreated infected mice in the chronic phase; (J) absence of inflammatory process in mice treated with Dox in the chronic phase; (E, K) absence of inflammatory process in mice treated with the combination of Bz + Dox in the acute (E) and chronic (K) phases; (F, L) Analysis of inflammatory infiltrate in cardiac muscle tissue in infected mice treated with Bz, Dox and combination in the acute (F) and chronic (L) phases. The data are represented as mean ± standard deviation (n = 6), where equal symbols (*, &, #, α, δ) indicate statistical difference (p ≤ 0.05) between groups (One-Way ANOVA, post-test of Tukey). HematoxylinEosin. Magnification 40×. Bar = 50 μm2.
Please cite this article as: A.C.A. Carneiro, G.P. Costa, C.S. Ferreira, et al., Combination therapy with benznidazole and doxycycline shows no additive effect to monotherapy with b..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.07.047
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Fig. 4. Echocardiogram in mice infected with the VL-10 strain of Trypanosoma cruzi. Mice were treated with Doxycycline (Dox) and/or Benznidazole (Bz) in the acute phase and the echocardiography analyzed in the chronic phase of infection. Graphical representation of the results of the comparative analyzes of the ejection fraction (A); fractional area variation (FAC%) (B); mass of the left ventricle (C); end-diastolic volume (D); and end-systolic volume (E). The data are representative of the mean ± standard deviation, where (*, #, &) (p ≤ 0.05) indicates statistical difference between groups (One-Way ANOVA test, Tukey post-test).
phases. Furthermore, combination therapy with Bz does not potentiate the effects of Bz monotherapy in chagasic cardiomyopathy.
Financial support Universidade Federal de Ouro Preto (UFOP), Centro Nacional de Biologia Estrutural e Bioimagem (CENABIO/UFRJ). Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors are grateful to the CAPES and FAPEMIG for the support of the graduate scholarship and AT and ACCC acknowledge CNPq and FAPERJ (ACCC) for a research productivity fellowship.
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Combination therapy with benznidazole and doxycycline shows no additive effect to monotherapy with benznidazole in mice infected with the VL-10 strain of the Trypanosoma cruzi Ana Cláudia Alvarenga Carneiro a, G.P. Costa a, Cyntia Silva Ferreira b, Isalira Peroba Rezende Ramos e, Mariáurea Matias Sarandy d, Ana Luísa Junqueira Leite a, A.P.J. Menezes a, B.M. Silva b, K.O.P.C. Nogueira c, A.C.C. Carvalho e, Reggiani Vilela Gonçalves d, André Talvani a,⁎ a
Laboratório de Imunobiologia da Inflamação, Departamento Ciências Biológicas, Universidade Federal de Ouro Preto, Minas Gerais, Brazil Laboratório de Biologia e Tecnologia de Micro-organismos, Departamento Ciências Biológicas, Universidade Federal de Ouro Preto, Minas Gerais, Brazil c Laboratório de Biomateriais e Patologia Experimental, Departamento Ciências Biológicas, Universidade Federal de Ouro Preto, Minas Gerais, Brazil d Laboratório de Patologia Experimental, Departamento de Biologia Animal, Universidade Federal de Viçosa Minas Gerais, Brazil e Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil b
a r t i c l e
i n f o
Article history: Received 25 February 2019 Received in revised form 20 June 2019 Accepted 15 July 2019 Available online xxxx Keywords: Trypanosoma cruzi VL-10 strain Inflammation Benznidazole Doxycycline Echocardiography
a b s t r a c t Background: Chagas heart disease is the most important clinical manifestation of Trypanosoma cruzi infection. Pharmacological therapies have been proposed aiming to reduce inflammatory response and cardiac damage in infected hosts. In this study, we investigated the use of doxycycline (Dox), in a sub-antimicrobial dose, in monotherapy and in combination with benznidazole (Bz) during the acute phase of infection with the VL-10 strain of T. cruzi, evaluating the therapeutic effect during the acute and chronic phases of the infection. Methods and results: C57BL/6 mice were treated for 20 days with Dox (30 mg/kg), Bz (100 mg/kg), or both drugs in combination starting 9 days after infection. Parasitemia was measured during the acute phase and the animals were monitored for 12 months, after which echocardiography analysis was performed. Blood samples were obtained from euthanized mice for CCL2, CCL5, IL-10 analysis, and cardiac fragments were collected for histopathological evaluation. Dox treatment did not ameliorate parasitological/inflammatory parameters but reduced the cardiac collagen neoformation (CN) in 35%. In contrast, Bz administration reduced parasitemia, plasma levels of CCL2 and CCL5, and cardiac infiltration during acute infection, and reduced the level of IL-10 and CN (95%) at 12 months. Dox was unable to improve ejection fraction, while Bz treatment ameliorated the ejection fraction. No additive effect was observed in combination therapy. Conclusion: Dox monotherapy is not effective in the acute or chronic phases of experimental cardiomyopathy induced by the VL-10 strain of T. cruzi. Furthermore, combination therapy with Dox does not potentiate the effects of Bz monotherapy. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma cruzi infection represents a relevant social problem that still occurs in many countries, especially in Latin America. Cardiac involvement is present in many cases of Chagas disease and can be considered as the more severe clinical form of T. cruzi infection [1,2]. This cardiac disease results in a remodeling of the collagen matrix and subsequent fibrosis, leading to myocarditis, increased tissue rigidity, diastolic
⁎ Corresponding author at: Laboratory of Immunobiology of Inflammation, DECBI/ICEB, Campus Morro do Cruzeiro, Universidade Federal de Ouro Preto, 35400-000 Ouro Preto, Minas Gerais, Brazil. E-mail address: [email protected] (A. Talvani).
and systolic dysfunction, functional loss and, in some cases, severe dilated cardiomyopathy associated with ventricular arrhythmias [3,4]. Benznidazole (Bz) is the most commonly used drug against T. cruzi in Brazil and is used to reduce or eliminate the parasites that trigger these cardiac inflammatory processes [5–8]. Unfortunately, Bz has low efficacy and leads to undesired adverse reactions. For this reason, new anti-T. cruzi compounds have been tested in the last decade, as well as anti-inflammatory drugs that could be useful in preserving cardiac morpho-functional structure. These drugs include angiotensin converting enzyme (ACE) inhibitors, beta-blockers, statins, and, more recently, Doxycycline (Dox) [9–12]. Doxycycline is a broad-spectrum bacteriostatic agent invented and clinically developed in the early 1960s by Pfizer Inc., NY, USA [13]. Besides its original antibacterial role, Dox has emerged as a co-adjuvant therapy in non-bacterial illnesses such as
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malaria and tick-borne rickettsial diseases [14]. Dox has also stood out as a potent inhibitor of metalloproteinases and suppressor of the hydrolases (e.g., phospholipase A2 and alpha-amylase) when administered in low doses (b50 mg/kg), which can reflect, in part, its capacity to scavenge reactive oxygen species and suppress inflammatory cytokines involved in distinct inflammatory disorders [15,16]. Due to the inefficiency of current treatments against T. cruzi, new pharmacological strategies directed towards the modulation of systemic and/or tissue-specific inflammatory response have been pursued, with the aim of minimizing tissue damage caused by this protozoan. The purpose of this study was to identify whether Dox and Bz therapies could positively modify immune and cardiac parameters during acute (30 days) and long-term (12 months) infection in C57BL/6 mice infected with the VL-10 strain of T. cruzi. 2. Methods 2.1. Animals C57BL/6 male mice, aged 6–8 weeks, were bred and maintained at the Center of Animal Science (CCA) at the Federal University of Ouro Preto (UFOP), Brazil. The procedures adopted are in accordance with the ethical principles of animal experimentation preestablished by the National Council for Control of Animal Experimentation (CONCEA). This research was previously approved by the Ethics Committee on Animal Research of UFOP-CEUA (protocol n° 2016/63).
2.6. Echocardiography Transthoracic echocardiographic examination was performed on mice after 12 months of infection, under anesthesia with continuous oxygen flow, maintaining similar heart rates between the groups to avoid the influence of heart rates on cardiac contractility indexes. Each anesthetized animal was placed in a dorsal decubitus position on a surgical table for transducer positioning in the left hemithorax of the animal. VisualSonics Vevo 770 equipment was used with a 30 MHz transducer. The images were acquired at a frequency of 14 MHz for resolution optimization. At time of visualization of the left ventricle (transverse and longitudinal sections), Simpson analysis was used in B mode (bi-dimensional) to obtain measurements of the following variables: ejection fraction (EF%); fractional area change (FAC%); end-diastolic volume (EDV); and end-systolic volume (ESV). Additionally, analysis was also performed in M-mode (one-dimensional) to obtain measurements of the following variables: left ventricular mass (mg); and left ventricular volume (μL) at the papillary muscle levels. 2.7. Statistical analysis The data obtained in this study are expressed as the mean ± standard deviation. The GraphPad Prism v.5 (GraphPad Software, San Diego, CA, USA) program was used and data were analyzed using the Kolmogorov-Smirnov test to confirm normality patterns. Depending on the nature of the data, one-way analysis of variance (ANOVA) with post-test Bonferroni correction, and Kruskal-Wallis test with post-test Tukey-Kramer and Dunn's tests were used for multiple comparisons. For all analyses, a significance level of p ≤ 0.05 was adopted.
3. Results and discussion 3.1. Parasitemia curve
2.2. Parasites and infection The male C57BL/6 mice (60 days of birth) were infected intraperitoneally with 5 × 103 blood trypomastigote forms of T. cruzi strain VL-10, which had been stored in liquid nitrogen and maintained in vivo by successive passages in Swiss mice. Parasitemia was determined daily by optic microscopy analysis of 5 μL samples, after tail bleeding [17].
2.3. Treatments and euthanasia Treatment with doxycycline (Dox; 30 mg/kg) and benznidazole (Bz; 100 mg/kg) was initiated 9 days after infection by T. cruzi. After the establishment of T. cruzi infection, the drugs were administered for 20 consecutive days orally (gavage) at a volume of 0.2 mL diluted in fetal bovine serum (PBS) + carboxymethylcellulose (CMC) 0.5%. Animals (n = 6 for the acute phase and n = 13 for the chronic phase), for each group evaluated were sorted into groups: (i) uninfected animals that received PBS + CMC; (ii) infected, untreated mice that received PBS + CMC; (iii) infected mice treated with Dox; (iv) infected mice treated with Bz; and (v) infected mice treated with a combination of Dox + Bz. Animals in the acute and chronic phases were euthanized after 30 days and 12 months of infection, respectively, according to euthanasia practice guidelines regulated by the CONCEA. Blood and cardiac tissues were collected for immunoenzymatic assays and histopathological analysis.
2.4. Immunoassays Immunoenzymatic assays were employed to evaluate the concentrations of the chemokines CCL5 and CCL2, and the regulatory cytokine IL-10. Plasma samples were isolated, and each assay performed according to the manufacturer's protocol (PeproTech Inc., Rocky Hill, NJ, USA). At the end of the reaction, 100 μL/well of the chromogen substrate ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), Sigma-Aldrich Inc., St. Louis, MO, USA) was added for 60 min followed by dark incubation at room temperature. Finally, samples were read in a spectrophotometer (Emax Molecular Devices, Sunnyvale, CA, USA) using wavelengths of 405 nm and 630 nm.
2.5. Histopathological analysis Heart fragments were collected and immediately fixed in 10% buffered formaldehyde. The next day, the fixative was replaced with 70% ethanol. The tissues were then processed and embedded in histological resin (HistoResin, Leica Basic Resin©). Then, the blocks were submitted to microtomy with 3-μm-thick sections and stained with hematoxylin and eosin (H&E) for quantification of the inflammatory infiltration. All histopathological analysis was digitized using the microscope Leica DM5000B (Leica Application Suite, UK, version 2.4.0 R1) with a coupled micro-camera and processed through the Leica QWin V3 image analyzer program. The inflammatory process was evaluated by quantifying all cellular nuclei randomly selected within a total area of 35,493.7 μm2 – equivalent to 25 fields of analyzed myocardium (40× magnification). Inflammation was quantified by counting the number of cardiac cellular nuclei in infected heart tissue compared with the number of cardiac cellular nuclei observed in the tissue of non-infected mice.
Dox treatment did not inhibit replication of the VL-10 strain of T. cruzi, with no difference observed between the untreated and Doxtreated infected animals. On the other hand, Bz reduced the peak and profile of the parasitemia curve, maintaining activity both in monotherapy and in combination with Dox, as shown in Fig. 1. In this way, as the parasite load is closely related to the inflammatory response, any drug capable of reducing parasitemia (such as Bz) is likely to partially reduce the systemic and/or local inflammatory processes [3]. There are many factors that influence the parasite level in the bloodstream during the acute phase, such as the genetic background of the parasite strain and the host, as well as other external factors such as temperature and host stress [18,19]. However, a higher immune response occurs during the initial stage of infection in humans and experimental models. Controlling the immune elements, by reduction of parasites or by direct pharmacological interference, could minimize the deleterious action of immune cells or their inflammatory mediators. However, it is as yet uncertain as to whether this early intervention would result in notable changes in chronic cardiac pathology. 3.2. Evaluation of inflammatory markers Several studies have already shown that the expression and production of inflammatory mediators both increase during the acute and chronic phases of animal model and human T. cruzi infection [9,12,20]. Furthermore, part of this immune profile observed in experimental infections was dependent on the genetics of the T. cruzi strain [21]. In the present study, we observed a reduction in CCL2 and CCL5 levels in mice treated with Bz; this profile was maintained when Bz was used in combination with Dox. However, Dox did not reduce these plasma chemokines in the acute phase of infection with the VL-10 strain of T. cruzi (Fig. 2A and C). Another study, using different genetic variations of the parasite, different strategies and treatment times, but the same sub-therapeutic dose than our study (30 mg/kg), demonstrated that Dox leads to a strong decrease in the clonal expansion of T cells, resulting in a reduction in levels of the inflammatory cytokines and chemokines, and in a lower expression of matrix metalloproteinases [22]. In this sense, it is worth emphasizing that Dox was previously used not only for its primary bacteriostatic action, but for its inhibition actions on MMPs at sub-therapeutic doses [13,16]. Some common adverse effects caused by the class of tetracyclines and their semi-
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Fig. 1. Parasitemia curve. The blood parasites were daily evaluated in C57BL/6 mice infected with 5000 trypomastigote forms of the VL-10 strain of T. cruzi. These animals were grouped according to the treatment with Benznidazole (Bz), Doxycycline (Dox) or a combination of both drugs. The data at each point of the curve are representative of the mean of the parasitemia/day for each group.
synthetic forms are gastrointestinal disorders, including anorexia, vomiting, diarrhea, mouth ulcerations and irritation of the perianal region [14] but no one was observed in the animals under sub-therapeutic dose of Dox. On the other hand, after 12 months of infection (when physiological and pathological disturbances become more evident), we expected to observe partial stabilization of systemic and local inflammatory processes. In contrast with observations during the acute phase, plasma CCL2 levels did not change in any of the chronic infection groups (Fig. 2B). Additionally, the only change observed in CCL5 levels was an increase in plasma concentration in mice treated with Dox + Bz compared to uninfected animals (Fig. 2D). However, even with the potential immunoregulation of Bz during the acute phase, Bz decreased the profile of IL-10 in the acute and chronic phases of infection (Fig. 2E and F). IL-10 has a protective role against fatal acute myocarditis in a murine model of T. cruzi infection, and in chagasic individuals. A relevant factor in the immunoregulatory activity of this cytokine against T. cruzi infection is the evidence that IL-10 mRNA is already detected 15 days after infection, suggesting its participation in the initial events of the disease [23]. However, the VL-10 strain of T. cruzi, used in our current study, induces a smaller cardiac inflammatory response when compared to other virulent strains in C57BL/6 mice model, or when used in other mammalian models (e.g., Swiss mice and dogs) [9,24]. These parasite-host interactions influence the inflammatory and regulatory patterns of the mediators.
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Fig. 2. Plasma CCL-2, CCL5, and IL-10 levels in Trypanosoma cruzi-infected mice under different therapeutic strategies. Concentrations of CCL2 (A, B), CCL5 (C, D) and IL-10 (E, F) in plasma from C57BL/6 mice infected with T. cruzi VL-10 strain are presented according the proposed therapy with Benznidazole (Bz), Doxycycline (Dox) or a combination of both drugs during the acute and chronic phases of the infection. Data are represented as mean ± standard deviation, where equal symbols (*, &, #, α, δ) mean statistical difference (p ≤ 0.05) between groups (One-Way ANOVA test, post-test of Tukey).
3.3. Histopathological analysis In this section, cardiac tissue from uninfected animals is represented in Fig. 3A and G. During the acute phase, all infected, untreated animals presented both diffuse and localized cardiac inflammatory processes, as shown in Fig. 3B. Mice treated with Bz (Fig. 3C) and with Dox + Bz (Fig. 3E) showed no evidence of cardiac inflammatory response, in contrast with mice treated with Dox (Fig. 3D), however this inflammatory process was similar to that observed in the infected untreated mice, as evidenced by the quantitative analysis shown in Fig. 3F. This matches expectations as Dox does not regulate production of inflammatory chemokines. During the chronic phase of infection, no inflammatory infiltrate was observed in the infected, untreated, or in the groups that received treatment (Fig. 3L). In the chronic phase of experimental T. cruzi infection, we observed 55% of the cardiac collagen neoformation in parasite-infected animals, 35% in Dox-treated-animals, an absence in those mice under Bz therapy and 30% in those animals receiving both Dox + Bz (data not shown). Besides immune modulatory action, another important role proposed for the use of sub-antimicrobial doses of doxycycline in treating cardiac diseases is its inhibitory action on the matrix metalloproteinases (MMPs), mainly MMP-2 and -9, preventing their non-physiological remodeling of the collagen matrix [22,25,26].
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3.4. Echocardiography Since only Bz affected inflammatory mediators and cardiac infiltration in the VL-10 T. cruzi-infected mice, we expected preservation of cardiac function in Bz-treated mice, but not in those treated with only Dox. Indeed, infection with the parasite reduced the ejection fraction (EF%) in all animals, but in those treated with Bz, we observed a well-preserved EF% (Fig. 4A). Our results in this experimental model corroborate studies that propose the EF as an important variable in the prognostic determination of chagasic individuals, since decrease in EF% is strongly associated with worsening of diastolic function, and with increases in the dimensions of the atrium and left ventricle [27]. There was no significant difference in the fractional cardiac area (FCA) between the infected, treated and untreated groups. However, the untreated infected group presented a reduced FCA when compared to the control group, as shown in Fig. 4B. In humans, fractional variation of the two-dimensional area is one of the most commonly-used measures for the quantitative estimation of left ventricular function, as this measure has been identified as an important factor in the prognosis of heart failure arising from myocardial infarction [28]. Another important parameter evaluated was the left ventricle (LV) mass. We observed an increase of this mass in the untreated infected mice when compared to the uninfected animals, but Dox monotherapy reduced this parameter (Fig. 4C), possibly through indirect action on the MMPs. Echocardiographic studies suggested that left ventricular mass may be a relevant variable in the prognosis of patients with heart failure [29]. Increased LV mass is an important risk factor for the development of ejection fraction decline and, consequently, the onset of heart failure.
Finally, we observed a decrease in diastolic volume in groups treated with Bz (monotherapy or in combination), in comparison with the untreated infected group (Fig. 4D). This decrease in end-diastolic volume may be the result of a decrease in compliance and a partial decrease in ventricular diastolic relaxation, and, consequently, in the ventricular ejection [30]. Studies have shown that diastolic dysfunction parameters were the determinants of cardiac functional capacity in mammalians, regardless of systolic function [31]. Since the end-systolic volume of the group treated with Bz and the group treated with Dox + Bz decreased compared to the infected group, as shown in Fig. 4 E, more blood was pumped into the animal body. In humans, left ventricular systolic function, as measured by the ejection fraction, remains a strong prognostic marker regardless of the clinical status of each individual [28–31]. In summary, our results show that Bz and its combination with sub-therapeutic dose of Dox reduced plasma levels of CCL2 and CCL5, and inflammatory infiltrate in cardiac tissue in the acute phase of infection. During the chronic phase, treatment with Bz maintained reduced plasma levels of IL-10 in infected animals, but alone or in combination with Dox resulted in improvement of echocardiographic parameters, such as ejection fraction and end-diastolic and end-systolic volumes.
4. Conclusions Sub-therapeutic dose of Dox, in monotherapy, during the acute phase of T. cruzi infection (VL-10 strain) was not effective to ameliorate the murine cardiomyopathy in both, acute and chronic
Fig. 3. Photomicrographs of histological sections of the heart. C57BL/6 mice were infected with T. cruzi VL-10 strain and treated or not with Doxycycline (Dox) and/or Benznidazole (Bz) in the acute phase of the infection. (A, G) normal cardiac histological appearance in uninfected mice; (B) moderate and focal inflammatory infiltrate in infected and untreated mice in the acute phase; (C, I) absence of inflammatory infiltrate in infected mice treated with Bz in the acute (C) and chronic (I) phases; (D) discrete and focal inflammatory process in infected mice treated with the Dox in the acute phase; (H) absence of inflammatory process in untreated infected mice in the chronic phase; (J) absence of inflammatory process in mice treated with Dox in the chronic phase; (E, K) absence of inflammatory process in mice treated with the combination of Bz + Dox in the acute (E) and chronic (K) phases; (F, L) Analysis of inflammatory infiltrate in cardiac muscle tissue in infected mice treated with Bz, Dox and combination in the acute (F) and chronic (L) phases. The data are represented as mean ± standard deviation (n = 6), where equal symbols (*, &, #, α, δ) indicate statistical difference (p ≤ 0.05) between groups (One-Way ANOVA, post-test of Tukey). HematoxylinEosin. Magnification 40×. Bar = 50 μm2.
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Fig. 4. Echocardiogram in mice infected with the VL-10 strain of Trypanosoma cruzi. Mice were treated with Doxycycline (Dox) and/or Benznidazole (Bz) in the acute phase and the echocardiography analyzed in the chronic phase of infection. Graphical representation of the results of the comparative analyzes of the ejection fraction (A); fractional area variation (FAC%) (B); mass of the left ventricle (C); end-diastolic volume (D); and end-systolic volume (E). The data are representative of the mean ± standard deviation, where (*, #, &) (p ≤ 0.05) indicates statistical difference between groups (One-Way ANOVA test, Tukey post-test).
phases. Furthermore, combination therapy with Bz does not potentiate the effects of Bz monotherapy in chagasic cardiomyopathy.
Financial support Universidade Federal de Ouro Preto (UFOP), Centro Nacional de Biologia Estrutural e Bioimagem (CENABIO/UFRJ). Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors are grateful to the CAPES and FAPEMIG for the support of the graduate scholarship and AT and ACCC acknowledge CNPq and FAPERJ (ACCC) for a research productivity fellowship.
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Please cite this article as: A.C.A. Carneiro, G.P. Costa, C.S. Ferreira, et al., Combination therapy with benznidazole and doxycycline shows no additive effect to monotherapy with b..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.07.047