Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 112(8): 551-560, August 2017 551 Bone marrow cell migration to the heart in a chimeric mouse model of acute chagasic disease Camila Iansen Irion, Bruno Diaz Paredes, Guilherme Visconde Brasil, Sandro Torrentes da Cunha, Luis Felipe Paula, Alysson Roncally Carvalho, Antonio Carlos Campos de Carvalho, Adriana Bastos Carvalho, Regina Coeli dos Santos Goldenberg/+ Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil BACKGROUND Chagas disease is a public health problem caused by infection with the protozoan Trypanosoma cruzi. There is currently no effective therapy for Chagas disease. Although there is some evidence for the beneficial effect of bone marrow-derived cells in chagasic disease, the mechanisms underlying their effects in the heart are unknown. Reports have suggested that bone marrow cells are recruited to the chagasic heart; however, studies using chimeric mouse models of chagasic cardiomyopathy are rare. OBJECTIVES The aim of this study was to investigate the migration of bone marrow cells to the heart after T. cruzi infection in a model of chagasic disease in chimeric mice. METHODS To obtain chimerical mice, wild-type (WT) C57BL6 mice were exposed to full body irradiation (7 Gy), causing bone marrow ablation. Then, bone marrow cells from green fluorescent protein (GFP)-transgenic mice were infused into the mice. Graft effectiveness was confirmed by flow cytometry. Experimental mice were divided into four groups: (i) infected chimeric (iChim) mice; (ii) infected WT (iWT) mice, both of which received 3 × 104 trypomastigotes of the Brazil strain; (iii) non-infected chimeric (Chim) mice; and (iv) non-infected WT mice. FINDINGS At one-month post-infection, iChim and iWT mice showed first degree atrioventricular block with decreased heart rate and treadmill exercise parameters compared to those in the non-infected groups. MAIN CONCLUSIONS iChim mice showed an increase in parasitaemia, myocarditis, and the presence of amastigote nests in the heart tissue compared to iWT mice. Flow cytometry analysis did not detect haematopoietic progenitor cells in the hearts of infected mice. Furthermore, GFP+ cardiomyocytes were not detected in the tissues of chimeric mice. Key words: Chagas disease - chimeric mice - heart - bone marrow Chagas disease (CD), caused by the protozoan Try- CD4+ and CD8+ T cells are essential for parasite control panosoma cruzi, is an important health problem in Cen- and host survival (Tarleton et al. 1996). In the chronic tral and South America (Rassi Jr et al. 2010). Reports phase, parasites are rarely found in the blood. However, from World Health Organization (WHO 2016) estimate small and progressive inflammatory foci continue to de- that 6-7 million people are infected worldwide. Although velop for years, culminating in the destruction of cardio- a decrease in incidence has been observed in endemic myocytes, formation of fibrosis, and the development of countries, CD is increasing in other places, such as the dilated cardiomyopathy (Gutierrez et al. 2009) in approx- United States of America, Canada, Japan, Australia, and imately 30% of infected individuals, which can progress Europe due to migratory movement (Rassi Jr et al. 2010). to congestive heart failure (Soares et al. 2004). At this Heart impairment is the most severe and frequent mani- stage, heart transplantation is often the only therapeutic festation of this disease. During the acute phase, infec- option. However, cell therapy has emerged as a promis- tion with T. cruzi leads to high parasitaemia and tissue ing alternative treatment. Recent advances have shown parasitism, which can result in an exacerbated immune the beneficial effects of progenitor cells from bone mar- response in the heart (Gutierrez et al. 2009), including in- row (BM) in different tissues (Epperly et al. 2003, Abedi filtration of CD4+ and CD8+ T lymphocytes and macro- et al. 2007), including the heart (Orlic et al. 2001, Yoshio- phages, with a predominance of CD8+ T cells (Machado ka et al. 2005, Goldenberg et al. 2008, Jasmin et al. 2012, et al. 2005). Depletion of lymphocytes in murine models Souza et al. 2014). In a mouse model of chronic chagasic results in high susceptibility to T. cruzi, showing that cardiomyopathy, mononuclear cells from BM decreased inflammatory infiltrates and fibrosis (Soares et al. 2004) and prevented dilatation of the right ventricle (Golden- berg et al. 2008). In an acute model, mesenchymal stem cells from BM also promoted a reduction of right ven- tricular dilatation (Jasmin et al. 2012). Although there is strong evidence for a beneficial ef- doi: 10.1590/0074-02760160526 fect of BM-derived cells in chagasic and other cardio- Financial support: CNPq, CAPES, FAPERJ, INCT, Ministério da Saúde + Corresponding author: [email protected] myopathies, the mechanisms underlying the effects of Received 7 December 2016 BM cells in the heart are still unknown. Reports have Accepted 14 March 2017 shown that BM cells are recruited to repair the heart in online | memorias.ioc.fiocruz.br 552 Bone marrow cells migrate to the heart • Camila Iansen Irion et al. a model of myocardial infarction (Orlic et al. 2001, Fa- blood (PB) samples (20 µL) collected from the tail vein zel et al. 2006). Transplantation of BM expressing green were incubated with 200 µL of erythrocyte-lysing solu- fluorescent protein (GFP) into irradiated mice has been tion (BD FACS Lysing Solution, BD Biosciences) for 15 used to screen and study BM cell participation in tissue min at room temperature, washed with PBS, centrifuged repair in different organs, including the heart (Epperly at 300 × g for 3 min, and then suspended in PBS (300 et al. 2003, Abedi et al. 2007, Fujita et al. 2007, Souza et μL) for flow cytometry analysis (BD FACSAria IIu). al. 2014). Chimeric mice have been used in myocardial Chimeric mice with greater than 70% engraftment of infarction models to show the beneficial effects of BM GFP-positive cells in PB were included in the study. CD117+ cells (Fazel et al. 2006) and granulocyte colony- Preparation of antigens (lysed parasites) - To avoid stimulating factor (G-CSF) (Fujita et al. 2007) on cardiac high mortality of irradiated mice after T. cruzi infection, tissue. However, there are few studies of CD using chi- the mice were injected with lysed parasites before in- meric mouse models (Soares & dos Santos 2009, Souza et fection. To obtain lysed parasites, a monkey fibroblast al. 2014). In this study, we investigated BM cell migration cell line (LLC-MK2) was infected with T. cruzi trypo- to the heart after T. cruzi infection in an experimental mastigotes (Brazil strain; at a 1:1 parasite:cell ratio). The model of acute CD in chimeric mice and compared para- cells were maintained in DMEM supplemented with 2% sitaemia, myocarditis, parasite nests, and cardiac func- foetal bovine serum (FBS) at 37ºC with 5% CO . From tion in infected wild-type (WT) and chimeric mice. 2 day five to eight, parasites were harvested from the MATERIALS AND METHODS culture supernatant, centrifuged (1200 × g for 15 min), resuspended in 1 mL of PBS, and counted in a Neubau- Animals - Male and female C57Bl/6 mice and transgenic 6 green fluorescent protein-expressing mice [C57Bl/6-Tg(- er chamber. A suspension containing 10 parasites/mL CAG-EGFP) C14-Y01-FM131O (GFP mice)] were donated in PBS was lysed by three freeze-thaw cycles in a wa- by Dr Masaru Okabe (Okabe et al. 1997) and housed in ter bath at 37ºC and liquid nitrogen for 5 min each. A our animal facility at the Carlos Chagas Filho Biophys- complete loss of viability was verified by the absence of ics Institute (UFRJ, Rio de Janeiro, Brazil). The facility parasite bodies via light microscopy. The lysed parasite is temperature controlled (23ºC), with a 12-12h light-dark suspension (200 µL) was injected by the intraperitoneal cycle and free access to water and standard chow. The in- (IP) route 22 days before T. cruzi inoculation. vestigation was carried out in accordance with the Guide T. cruzi infection and screening - Infected groups of for the Care and Use of Laboratory Animals [DHHS pub- mice (males and females, aged 4-5 months) were inoc- lication no. (NIH) 85-23, revised in 1996, Office of Sci- ulated by IP injection with 3 × 104 bloodstream trypo- ence and Health Reports, Bethesda, MD 20892, USA] and mastigotes of the Brazil strain. Five days post infection all animal protocols were approved by the Ethics Commit- (dpi), parasitaemia was assessed by counting the number tee on the Use of Animals in Scientific Experimentation of trypomastigotes in PB diluted in 0.85% ammonium (Health Science Centre of the Federal University of Rio de chloride buffer in a Neubauer chamber, three times a Janeiro), under protocol number 163/13. week. Data are expressed as the number of trypomastig- Generation of chimeric mice - To generate chimeric otes per millilitre of blood. Mortality was assessed daily. mice, 34 C57Bl/6 mice (weighing approximately 25 g) Experimental groups - Animals were divided into were exposed to whole body radiation (7 Gy) in a linear four groups: (i) the iChim group (n = 17) and (ii) the accelerator (VarianClinac 2100 CD) used for radiother- iWT group (n = 17), which were composed of chimeric apy, resulting in BM ablation as previously described and WT C57Bl/6 mice, respectively, that received T. cru- (Paredes et al. 2012). BM cells were harvested from zi lysate (ip) at 52 days-post irradiation (dpir) and were the femurs and tibiae of GFP transgenic mice at two infected with T. cruzi 22 days after lysate injection (74 months of age by centrifugation (1500 × g for 3 min). dpir), and age-matched non-infected controls in the (iii) To isolate mononuclear cells, Bone Marrow Cells BMCs Chim group (n = 13) and (iv) WT group (n = 14).