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1 1186 Exp Physiol (2012) pp Themed Research Paper Autonomic nervous system modulation affects the inflammatory immune response in mice with acute Chagas disease Marcus Paulo Ribeiro Machado 1,AletheiaMoraesRocha 1,LucasFelipedeOliveira 1, Marília Beatriz de Cuba 1,IgordeOliveiraLoss 1, Lucio Roberto Castellano 2, Marcus Vinicius Silva 1, Juliana Reis Machado 1, Gabriel Antonio Nogueira Nascentes 1, Luciano Henrique Paiva 1, Wilson Savino 3, Virmondes Rodrigues Junior 1, Patricia Chakur Brum 4, Vania Ferreira Prado 5, Marco Antonio Maximo Prado 5, Eliane Lages Silva 1, Nicola Montano 6, Luis Eduardo Ramirez 1 and Valdo Jose Dias da Silva 1 Experimental Physiology 1 Biological Sciences Institute, Triangulo Mineiro Federal University, Uberaba, MG, Brazil 2 Paraiba Federal University, Joao Pessoa, PB, Brazil 3 Oswaldo Cruz Institute, Rio de Janeiro, RJ, Brazil 4 School of Physical Education and Sport, University of Sao Paulo, SP, Brazil 5 Molecular Brain Research Group, Robarts Research Institute, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada 6 Department of Clinical Sciences, University of Milan, Internal Medicine II, L. Sacco Hospital, Milano, Italy The aim of the present study was to evaluate the effects of changes to the autonomic nervous system in mice during the acute phase of Chagas disease, which is an infection caused by the parasite Trypanosoma cruzi. The following types of mice were inoculated with T. cruzi (CHG): wild-type (WT) and vesicular acetylcholine transporter knockdown (KDVAChT) C57BL/6j mice; wild-type non-treated (NT) FVB mice; FVB mice treated with pyridostigmine bromide (PYR) or salbutamol (SALB); and β 2 -adrenergic receptor knockout (KOβ2) FVB mice. During infection and at days after infection (acute phase), the survival curves, parasitaemia, electrocardiograms, heart rate variability, autonomic tonus and histopathology of the animals were evaluated. Negative control groups were matched for age, genetic background and treatment. The KDVAChT-CHG mice exhibited a significant shift in the electrocardiographic, autonomic and histopathological profiles towards a greater inflammatory immune response that was associated with a reduction in blood and tissue parasitism. In contrast, the CHG-PYR mice manifested reduced myocardial inflammation and lower blood and tissue parasitism. Similar results were observed in CHG-SALB animals. Unexpectedly, the KOβ2-CHG mice exhibited less myocardial inflammation and higher blood and tissue parasitism, which were associated with reduced mortality. These findings could have been due to the increase in vagal tone observed in the KOβ2 mice, which rendered them more similar to the CHG-PYR animals. In conclusion, our results indicate a marked immunomodulatory role for the parasympathetic and sympathetic autonomic nervous systems, which inhibit both the inflammatory immune response and parasite clearance during the acute phase of experimental Chagas heart disease in mice. (Received 31 March 2012; accepted after revision 15 June 2012; first published online 18 June 2012) Corresponding author V. J. Dias da Silva: Biological Sciences Institute Federal University of Triangulo Mineiro, Praca Manoel Terra, 330, Centro, Uberaba, MG, Brazil. valdo@mednet.com.br M.P.R.M. and A.M.R. contributed equally to this work. DOI: /expphysiol

2 Exp Physiol (2012) pp Autonomic neuroimmunomodulation during acute Chagas disease 1187 Chagas disease, also known as American trypanosomiasis, was first described by Carlos Chagas in It is caused by the flagellate protozoan Trypanosoma cruzi, and the endemic parasitic infectious disease is highly prevalent throughout Latin America, affecting approximately 12 million people and causing approximately 50,000 deaths annually (Prata, 2001; Coura & Viñas, 2010). Although the pathogenesis of Chagas disease is very complex and not completely understood, particularly with regard to chagasic cardiomyopathy, it is widely accepted that the balance between parasite invasiveness and the host immune response plays a major role in the development and evolution of the acute and chronic manifestations of the disease. In particular, the T helper 1 (Th1) T helper 2 (Th2) response profile ratio, which affects the resistance susceptibility balance to T. cruzi infection, is the critical immune response determinant, as suggested by reports demonstrating that a balanced response is necessary to control T. cruzi infection (Hunter et al. 1997). Others have reported that imbalanced, excessive production of Th1 pro-inflammatory cytokines is a critical protective in vivo mechanism against Chagas disease (Teixeira et al. 2011). The shift towards the Th1 immune response profile resultsinimprovedcontrolofthebloodandtissueparasite levels, although this type of response can also induce an excessive inflammatory response capable of destroying functional cardiomyocytes, inducing tissue necrosis and apoptosis through the massive release of chemokines and consequently promoting the chemotaxis of inflammatory cells to the infected tissue (Teixeira et al. 2011). In addition to cardiomyocytes, intracardiac autonomic neurons may also be affected during infection, which can result in ganglionitis and neuritis during acute and chronic infection and a marked reduction in ganglionic density and neural depopulation (Ramirez et al. 1993; Prata, 2001; Marin-Neto et al. 2007). As a consequence, significant cardiac autonomic dysfunction affecting mainly the parasympathetic branch of the autonomic nervous system has been described in experimental models (Ramirez et al. 1993; Dias da Silva et al. 2003) and patients with Chagas disease (Prata, 2001; Marin-Neto et al. 2007; Pérez et al. 2011) and may be involved in initiating lifethreatening cardiac arrhythmias and sudden death (Prata, 2001; Marin-Neto et al. 2007). Although these data support a direct interaction between the immune and nervous systems for mediating both cardiac and autonomic disturbances, any potential influence of autonomic nervous system dysfunction on the immune response profile observed in Chagas disease cannot be ruled out. Considering recent evidence that both the sympathetic and the parasympathetic branches of the autonomic nervous system can exert significant modulatory effects on the immune response, mainly by inhibiting the Th1 response profile (Sanders, 1998; Elenkov & Chrousos, 1999; Borovikova et al. 2000; Tracey, 2009), a new interpretation of the positive correlation between inflammatory infiltration in the heart and autonomic dysfunction has been proposed. This new interpretation states that cardiac sympathetic and specifically parasympathetic autonomic denervation/dysfunction, which leads to reduced effects on the Th1 response in the heart, could contribute to the increased magnitude of the inflammatory response and possibly to enhanced parasite elimination (Benarroch, 2009). Thus, instead of inflammation leading to the destruction of autonomic fibres and neurons, it is possible that cardiac autonomic denervation/dysfunction contributes to increased inflammation. However, to our knowledge, this hypothesis has not yet been tested in the context of Chagas disease. Moreover, no studies in the literature have performed deliberate modifications of the autonomic nervous system to evaluate the effect on the immune response to T. cruzi. In the present study, we used mice with reduced expression (knockdown) of the vesicular acetylcholine transporter gene (KDVAChT mice) and β 2 -adrenergic receptor knockout mice (KOβ2 mice) to investigate the effects of the loss of autonomic function on the immune response to T. cruzi and inflammation in the heart tissue. In parallel, an additional set of wild-type mice were continuously treated with pyridostigmine bromide (PYR), an anticholinesterase agent, or salbutamol (SALB), a β 2 - adrenergic agonist, to determine the effects of enhanced autonomic function on the immune response during the acute phase of infection. Based on the results presented herein, we can conclude that the autonomic nervous system plays a significant role in modulating the immune response to T. cruzi and has an enormous impact on the morbidity and mortality of chagasic mice. Methods Ethical information All experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no , revised 1996). All procedures were also submitted to and approved by the Commission for Ethics in the Use of Animals in Research of the University of Sao Paulo, Minas Gerais Federal University and Triangulo Mineiro Federal University. Experimental animals Experiments were performed with the following types of mice from various sources: wild-type FVB mice were

3 1188 M. P. R. Machado and others Exp Physiol (2012) pp obtained from the animal facility of the Department of Physiology of Triangulo Mineiro Federal University, Uberaba, MG, Brazil; β 2 -adrenergic receptor gene knockout FVB mice (KOβ2 mice; Chruscinski et al. 1999) were obtained from the School of Physical Education and Sport, University of Sao Paulo, SP, Brazil; and C57BL/6j mice heterozygous for knockdown of the vesicular acetylcholine transporter gene (KDVAChT mice; Prado et al. 2006) and wild-type C57BL/6j mice were obtained from the Department of Pharmacology of Minas Gerais Federal University, Belo Horizonte, MG, Brazil. All animals were male, weighed g and were maintained in the animal facility of the Department of Physiology at the Triangulo Mineiro Federal University on a rodent diet and were given water ad libitum until the beginning of the experimental protocols. The genetically modified animals (KOβ2 mice and KDVAChT mice) were individually genotyped at their original facilities immediately after weaning using specific PCR techniques (Chruscinski et al. 1999; Prado et al. 2006). All the experiments were carried out according to the guidelines laid down by the animal welfare committee of Triangulo Mineiro Federal University. Parasite inoculation To induce experimental Chagas disease, the strains of mice were intraperitoneally inoculated with 1000 or 15,000 trypomastigote forms of the Y or Romildo strains of T. cruzi. An additional set of animals matched for sex, weight and genetic background received intraperitoneal injections of the vehicle, and these were designated as the control non-infected group. After inoculation, all of the animals were observed at least twice daily to monitor their general state and assess mortality for the construction of survival curves. From the second day after inoculation until the end of the observation period (day 18 21), levels of parasitaemia were measured every 2 days from the peripheral tail blood of all inoculated animals, according to Brener s technique (Brener, 1962), to confirm infection. All subsequent surgical procedures and experimental protocols were performed between the 18th and 21st day of infection during the acute phase of infection, which is characterized by marked tissue invasion and substantial mortality. Experimental groups The experimental groups were divided according to the proposed objective for each study described below. The numbers of animals used in each group are indicated in the Results section. Study I: evaluation of the parasympathetic autonomic system impairment in KDVAChT mice during acute Chagas infection. The following groups were studied: (a) WT- CON (wild-type C57BL/6j mice injected with vehicle as a control, CON); (b) KDVAChT-CON (KDVAChT mice injected with vehicle); (c) WT-CHG (wild-type C57BL/6j mice inoculated with 1000 trypomastigote forms of the Y strain of T. cruzi for the induction of Chagas disease, CHG); and (d) KDVAChT-CHG (KDVAChT mice inoculated with 1000 trypomastigote forms of the Y strain of T. cruzi). Study II: evaluation of the effect of parasympathetic nervous system stimulation by pyridostigmine bromide on acute Chagas disease. The following groups were investigated: (a) CON-NT (wild-type FVB mice injected with vehicle as a control, CON, and not treated with pyridostigmine bromide, NT); (b) CHG-NT (wild-type FVB mice inoculated with 15,000 trypomastigote forms of the Romildo strain of T. cruzi and not treated with pyridostigmine bromide); and (c) CHG-PYR (wild-type FVB mice inoculated with 15,000 trypomastigote forms of the Romildo strain of T. cruzi and treated with 30 mg kg 1 of pyridostigmine bromide, PYR, an anticholinesterase agent, dissolved in tap water). The solution drinking volume was monitored daily, and the pyridostigmine bromide concentration was adjusted according to the drinking volume. Study III: evaluation of the effect of the absence of β 2 - adrenergic sympathetic signalling in KOβ2 mice during the acute phase of Chagas disease. The following groups were analysed: (a) WT-CON (wild-type FVB mice injected with vehicle as a control); (b) KOβ2-CON (KOβ2 mice injected with vehicle as a control); (c) WT-CHG (wildtype FVB mice inoculated with 15,000 trypomastigote forms of the Romildo strain of T. cruzi); and (d) KOβ2- CHG (KOβ2miceinoculatedwith15,000trypomastigote forms of the Romildo strain of T. cruzi). Study IV: evaluation of the effect of β 2 -adrenergic sympathetic stimulation with salbutamol during the acute phase of Chagas disease. The following groups were evaluated: (a) CON-NT (wild-type FVB mice injected with vehicle as a control and not treated with salbutamol); (b) CHG-NT (wild-type FVB mice inoculated with 15,000 trypomastigote forms of the Romildo strain of T. cruzi and not treated with salbutamol); and (c) CHG-SALB (wild-type FVB mice inoculated with 15,000 trypomastigote forms of the Romildo strain of T. cruzi and administered daily intraperitoneal doses of 200 μg of salbutamol, a β 2 - adrenergic agonist, during the entire observation period).

4 Exp Physiol (2012) pp Autonomic neuroimmunomodulation during acute Chagas disease 1189 Conventional ECG monitoring Immediately prior to inoculation and after days of observation, all of the animals were submitted to a conventional ECG study under general anaesthesia with isoflurane 2%, administred using an anaesthetic vaporizer. Needle electrodes were placed under the skin to record the conventional bipolar limb leads (I, II and III), the unipolar limb leads (avr, avl and avf) and the unipolar precordial (chest) leads (VA, with the needle placed immediately to the right of the sternum in the fourth intercostal space; VB, with the needle placed immediately to the left of the sternum in the fourth intercostal space; and VC, with the needle positioned in the fifth intercostal space at the midaxillary line). To avoid errors in the positioning of the leads, the electrodes were consistently put in place by the same individual (M.P.R.M.). The ECG was recorded using a six-channel digital ECG recorder (ER-661; Medikor, Budapest, Hungary) coupled to a 12-bit analog-to-digital interface (CAD12/36; Lynx Tecnologia Eletrônica Ltda, São Paulo, Brazil) on an IBM personal computer (sampling rate of 3 khz). The ECG was recorded for 2 min. The intervals and wave durations (in milliseconds) were calculated automatically using customized software following wave identification and cursor placement. The ECG traces were consistently analysed by the same individual (M.P.R.M.) who was blinded to the study protocol. The following ECG parameters were examined: (a) RR interval; (b) P wave duration; (c) PR interval; (d) QRS duration; (e) QT interval; and (f) corrected QT interval (QTc, defined as the QT interval corrected for heart rate using Bazett s equation, where the corrected QTc = QT (in seconds)/rr (in seconds) 1/2. In contrast to humans, the T wave in small rodents is not well defined and appears as a shoulder of the QRS complex. Accordingly, to measure the QT interval, we used the apex of the T wave, which can be determined with high accuracy. The ECG parameters were determined from each lead and were averaged. Chronic ECG recordings After the second ECG recording, the animals were reanaesthetized with a mixture of ketamine and xylazine (100 and 10 mg kg 1, respectively, I.P.; Virbac Brasil S.A., São Paulo, SP, Brazil), and a pair of stainless-steel electrodes were implanted in the subcutaneous tissue to collect chronic recordings of conventional bipolar limb ECG lead II. The animals were also cannulated with polyethylene tubing placed in the jugular vein for drug administration. After the surgical procedures, the animals were left to recover in individual cages for at least h. After h of surgical recovery and in the absence of anaesthesia, the electrodes were connected to a bioelectric amplifier (model 8811A; Hewlett Packard, Waltham, MA, USA), and the baseline ECG was continuously sampled (3 khz) for a period of 30 min with a personal computer (IBM/PC) equipped with a 12-bit analog-todigital interface (CAD12/36; Lynx Tecnologia Eletrônica Ltda). The time series of RR intervals derived from these chronic ECG recordings were used to study the cardiac autonomic modulation present in heart rate variability. Heart rate variability analysis From the baseline chronic ECG recordings (30 min), the RR interval time series were derived automatically through the detection of the peak of the R waves using customized linear analysis software, which was kindly provided by Dr Alberto Porta (University of Milan, Italy). The time series of the RR intervals were divided into contiguous segments of 300 beats overlapping by half (Welch protocol). After calculating the mean and variance for each segment, a model-based autoregressive spectral analysis was performed, as described elsewhere (Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology, 1996; Montano et al. 2009). Briefly, a model of the oscillatory components present in the stationary segments of the beat-to-beat time series of the RR intervals was calculated based on the Levinson Durbin recursion, and the order for the model was chosen according to Akaike s criterion (Montano et al. 2009). This procedure allows for the automatic quantification of the centre frequency and power of each relevant oscillatory component present in the time series. The oscillatory components were labelled as very low (VLF), low (LF) or high frequency (HF) when the central frequencies were within the bands of , or Hz, respectively. The power of the LF and HF components of heart rate variability was also expressed in normalized units (n.u.), which were obtained by calculating the percentage of the LF and HF variability with respect to the total power after subtracting the power of the VLF component (frequencies <0.10 Hz). The normalization procedure tends to minimize the effect of changes in the total power on the absolute values of the LF and HF variabilities (Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology, 1996; Montano et al. 2009). Pharmacological autonomic blockade After 30 min of baseline chronic ECG recording, onehalf of the animals were given an intravenous injection of atropine sulfate (1 mg kg 1, I.V.) followed by an injection of propranolol (1 mg kg 1, I.V.) 15 min later, while the other half received injections in the reverse sequence (propranolol followed by atropine sulphate). This procedure allowed for the quantification of the cardiac sympathetic and parasympathetic autonomic tones, respectively, measured as the difference between the

5 1190 M. P. R. Machado and others Exp Physiol (2012) pp RR interval after atropine and the RR interval after double blockade (sympathetic tone) or as the difference between the RR interval after propranolol and the RR interval after double blockade (parasympathetic tone), which were finally adjusted as percentile changes of baseline RR intervals. At the end of this protocol, all animals were killed with an overdose of ketamine plus xylazine (400 and 40 mg kg 1, respectively, I.P.), and the chest cavity was then opened to remove the heart for histopathological analysis. In order to check the accuracy of use of propranolol, a nonspecific β-blocker rather than a selective β 1 -antagonist like atenolol, during evaluation of autonomic tones by means of pharmacological blockades, an additional set of 15 age-matched non-infected control C57Bl/6j mice were instrumented under anaesthesia with ECG electrodes and venous catheters. After 48 h, all these animals were submitted in conscious, freely moving conditions to one of the following two experimental protocols: (a) sequential injections of propranolol (1 mg kg 1, I.V.) and atropine (1 mg kg 1, I.V.) followed 24 h after by sequential injections of atenolol (1 mg kg 1, I.V.) and atropine (1 mg kg 1, I.V.) in order to quantify and compare vagal tones (n = 7 animals); or (b) sequential injections of atropine (1 mg kg 1, I.V.) and propranolol (1 mg kg 1, I.V.) followed 24 h after by sequential injections of atropine (1 mg kg 1, I.V.) and atenolol (1 mg kg 1, I.V.) in order to quantify and compare sympathetic tones (n = 8 animals). Linear regression analysis between autonomic tones quantified using propranolol or atenolol were performed. Histopathological examination To evaluate the extent of inflammatory infiltration, tissue damage and parasite nests, hearts from all animals were excised, cleaned in 0.9% saline solution and fixed in phosphate-buffered 10% formalin solution for 48 h. After embedding the samples in paraffin, 5-μmthick longitudinal sections of the hearts were stained with Haematoxylin and Eosin, mounted on slides and analysed using an upright light microscope (Axiolab; Carl Zeiss Inc., Oberkochen, Germany). All regions of the hearts, including the atrial and ventricular myocardium, endocardium and pericardium, as well as the intrinsic cardiac neural ganglions, were examined by two blinded observers (M.P.R.M., L.E.R). The inflammatory infiltrate and parasite nests were characterized using a semiquantitative approach and the scoring system described by Chapadeiro et al. (1988). Statistical analysis All numerical data were expressed as the means (±SEM), whereas the semi-quantitative data from the histological examinations were expressed as the medians and 25th (P25%) and 75th percentiles (P75%). The Kaplan Meier survival curves for the chagasic mice were compared between groups using the Log-rank test. According to the normality and variance homogeneity of the distribution, parametric statistics, such as one-way- or two-way ANOVA followed by Tukey s multiple comparison test, or non-parametric tests, such as the Kruskall Wallis ANOVA followed by Dunn s multiple comparison test or the Mann Whitney U test, were performed using SigmaStat software (SPSS Inc., Chicago, IL, USA). The differences were considered significant when P < Results Study I: evaluation of the effect of parasympathetic autonomic system impairment in KDVAChT mice during acute Chagas infection During the observational period, similar mortality rates (Fig. 1A) were observed in both the KDVAChT-CHG (33%) and WT-CHG mice (20%, n.s.), and no deaths occurred in uninfected animals. The parasitaemia curves presented in Fig. 1B demonstrate a reduced number of circulating parasites in the KDVAChT-CHG mice in comparison to the WT-CHG animals. The ECG parameters measured at the end of the acute phase are presented in Table 1. Note that the WT-CHG mice exhibited a significant reduction in the PR interval as well as an enlargement of the QRS duration and QaT and QaTc intervals, which are suggestive of lesions in the cardiac ventricles. Chagasic infection in KDVAChT- CHG mice not only induced similar changes in the PR, QRS, QaT and QaTc intervals but also induced significant alterations in atrial ECG parameters, such as the P wave duration and PR interval enlargement and tachycardia. These results were suggestive of worsening and widening of the myocardial lesions, including the atria, which were innervated by an acetylcholine-deficient parasympathetic nervous system in the KDVAChT-CHG animals. In fact, data from the heart rate variability analysis (Table 1) showed a significant reduction in the RR variance, which represents a marker of total variability, in the WT-CHG mice compared with the WT-CON animals. The reduced acetylcholine release in the KDVAChT- CHG animals promoted further worsening of the HRV parameters, including the HF power parameter, which is a marker of cardiac vagal modulation (Table 1). These findings concerning cardiac autonomic modulation were confirmed following pharmacological blockade, which resulted in a marked reduction in the cardiac vagal tone in the KOVAChT-CHG group (Fig. 1C) without any change to the cardiac sympathetic tone (Fig. 1D). The histological findings revealed diffuse and light inflammatory infiltration (average inflammatory score = 1, P25% = 1 and P75% = 1) in areas including the atria, ventricles and neural ganglia of the hearts of WT-CHG mice (Fig. 1F and I). Similarly diffuse but more

6 Exp Physiol (2012) pp Autonomic neuroimmunomodulation during acute Chagas disease 1191

7 1192 M. P. R. Machado and others Exp Physiol (2012) pp Table 1. Mean values (±SEM) of the ECG and heart rate variability (HRV) parameters in wild-type (WT) or vesicular acetylcholine transporter gene (KDVAChT) knockdown C57BL/6j male mice collected days after inoculation with Trypanosoma cruzi (chagasic infection; CHG) or vehicle (non-infected control; CON) WT-CON KDVAChT-CON WT-CHG KDVAChT-CHG ECG parameters No. of animals RR (ms) ± ± ± ± 2.8 P(ms) 11.7 ± ± ± ± 1.1 PR (ms) 46.6 ± ± ± ± 1.0 QRS (ms) 11.5 ± ± ± ± 1.9 QaT (ms) 16.0 ± ± ± ± 2.2 QaTc (ms 1/2 ) 46.7 ± ± ± ± 7.3 HRV parameters No. of animals RR (ms) ± ± ± ± 2.2 Variance (ms 2 ) 43.7 ± ± ± ± 0.5 VLF (ms 2 ) 15.6 ± ± ± ± 0.2 LF (ms 2 ) 15.4 ± ± ± ± 0.4 LF (n.u.) 76.2 ± ± ± ± 13.4 HF (ms 2 ) 1.7 ± ± ± ± 0.1 HF (n.u.) 23.8 ± ± ± ± 13.4 LF/HF 1.7 ± ± ± ± 1.9 Abbreviations: HF, high-frequency oscillation power; LF, low-frequency oscillation power; LF/HF, LF/HF ratio; n.u., normalized units; P, P wave duration; PR, PR interval; QaT, QaT interval; QaTc, QaT interval corrected using the Bazett equation; QRS, QRS duration; RR, RR interval; and VLF, very low-frequency oscillation power. P < 0.05 compared with wild-type non-infected control (WT-CON) animals. P < 0.05 compared with VAChT knockdown non-infected (KDVAChT-CON) animals. P < 0.05 compared with wild-type chagasic infected (WT-CHG) animals. intense inflammatory infiltration (average inflammatory score = 2, P25% = 2 and P75% = 2, P < 0.01), also involving all of the cardiac structures, was observed in the KDVAChT-CHG mice (Fig. 1G and J).No histological changeswereobservedinthecontrolnon-infectedanimals (Fig. 1E and H). A paucity of parasite nests (0 ± 0per slide) was observed in the KDVAChT-CHG mice, in comparison to 4.1 ± 0.4 parasite nests per slide (P < 0.01) in the WT-CHG animals (Fig. 1K). Study II: evaluation of the effect of parasympathetic nervous system stimulation with pyridostigmine bromide on acute Chagas disease The follow-up of non-treated chagasic animals (CHG-NT group) revealed a survival rate of 73% after 21 days of infection. There was a trend towards a reduced survival rate (for 40%, 6 survived out of all 15 mice) that approached significance in chagasic mice treated with pyridostigmine bromide (CHG-PYR group) during the entire acute phase (Fig. 2A). No mortality was observed in the normal control animals. The increased mortality rate of the CHG-PYR mice was accompanied by a significant increase in the circulating parasite levels (Fig. 2B). The electrocardiographic parameters measured at the end of the 21-day follow-up are presented in Table 2. All of the ECG parameters demonstrated significant elongation in the CHG-NT animals, and this result was suggestive of global cardiac effects. The chagasic mice treated with pyridostigmine bromide showed a trend towards a reduced PR interval and a significant decrease in P wave duration without any improvement Figure 1. Evaluation of the effect of parasympathetic autonomic system impairment in vesicular acetylcholine transporter gene knockdown (KDVAChT) mice on acute Chagas infection A, survival curves. B, parasitaemia curves (means ± SEM, P < 0.05 versus wild-type chagasic (WT-CHG) group). C, cardiac vagal parasympathetic tone measured as the tachycardic response to atropine (means ± SEM, P < 0.05 versus wild-type control (WT-CON), #P < 0.05 versus KDVAChT-CON and &P < 0.05 versus WT-CHG). D, cardiac sympathetic tone measured as the bradycardic response to propranolol (means ± SEM). E and H, light photomicrographs of Haematoxylin and Eosin stained tissue from the atrium (E) and ventricle (H) of a WT-CON mouse ( 400). F, I and L, light photomicrographs of Haematoxylin and Eosin stained tissue from the atrium (F) and ventricle (I and L) of a WT-CHG mouse ( 400). Note the light inflammatory infiltration. The arrow in L indicates an amastigote nest. G, J and M, light photomicrographs of Haematoxylin and Eosin stained tissue from the atrium (G) and ventricle (J and M) of a KDVAChT-CHG mouse ( 400). Note the moderate to intense inflammatory infiltration ( 400). K, intrinsic autonomic ganglion of a KDVAChT-CHG mouse ( 400). Note the intense inflammation around the ganglionic neurons (one of which is indicated by the arrow).

8 Exp Physiol (2012) pp Autonomic neuroimmunomodulation during acute Chagas disease 1193

9 1194 M. P. R. Machado and others Exp Physiol (2012) pp Table 2. Mean values (±SEM) of the ECG and HRV parameters collected days after inoculation with T. cruzi (chagasic infection; CHG) or vehicle (uninfected control; CON) in male wild-type FVB/N mice left untreated (NT) or treated with pyridostigmine bromide (PYR) CON-NT CHG-NT CHG-PYR ECG parameters No. of animals RR (ms) ± ± ± 7.9 P(ms) 12.9 ± ± ± 0.5 PR (ms) 31.1 ± ± ± 1.0 QRS (ms) 10.4 ± ± ± 1.2 QaT (ms) 13.6 ± ± ± 2.8 QaTc (ms 1/2 ) 42.4 ± ± ± 7.8 HRV parameters No. of animals RR (ms) ± ± ± 2.7 Variance (ms 2 ) 13.0 ± ± ± 3.9 VLF (ms 2 ) 5.6 ± ± ± 2.0 LF (ms 2 ) 2.7 ± ± ± 1.3 LF (n.u.) 22.6 ± ± ± 5.8 HF (ms 2 ) 4.7 ± ± ± 0.9 HF (n.u.) 66.0 ± ± ± 5.8 LF/HF 1.9 ± ± ± 1.1 Abbreviations are as for Table 1. P < 0.05 compared with non-infected non-treated control (CON-NT) animals. P < 0.05 compared with chagasic infected non-treated (CHG-NT) animals. in the ventricular ECG parameters (QRS, QaT and iqatc) in comparison to untreated chagasic animals (Table 2). The evaluation of autonomic changes according to the time-domain and frequency-domain heart rate variability analysis indicated that the CHG-NT animals presented a marked reduction in the RR variance and HF component of the RR oscillations, in comparison to normal control animals (CON-NT group). This finding was not observed in the chagasic animals treated with pyridostigmine bromide (CHG-PYR), because these animals presented values similar to the CON-NT mice. No changes were observed in the low-frequency HRV components, which are related to sympathetic autonomic modulation. These data obtained from the HRV analysis were consistent with the finding of significantly reduced cardiac vagal tone in CHG-NT mice and an augmented, rather than normal, cardiac vagal tone in CHG-PYR animals in response to autonomic blockade (Fig. 2C). As shown in Table 2, these animals also exhibited a trend towards reduced baseline heart rate in comparison to non-chagasic and untreated chagasic mice. No changes in the cardiac sympathetic tone were seen in any of the three groups (Fig. 2D). A semi-quantitative examination of the cardiac inflammatory infiltration indicated moderate to intense myocarditis (average inflammatory score = 3.0, p25% = 2.5 and P75% = 3.0) in the CHG-NT mice (Fig. 2F and I), which appeared to be more dramatic in the atria (Fig. 2F) than the ventricles (Fig.2I) in comparison to normal CON-NT animals (Fig. 2E and H). In mice treated with pyridostigmine bromide, reduced inflammatory infiltration, which was categorized as light to moderate, was observed in the atria (Fig. 2G), the ventricles (Fig. 2J) and the neural ganglia (Fig. 2K). Interestingly, the reduced myocarditis observed in the CHG-PYR mice was associated with a significantly increased number of amastigote nests in the myocardium (40.8± 5.1 versus 18.1± 2.1 parasite nests per slide, P < 0.05) in comparison to the number of nests in the CHG-NT animals (Fig. 2L and M). Study III: evaluation of the effect of the lack of β 2 -adrenergic sympathetic signalling in KOβ2 mice during the acute phase of Chagas disease There were no deaths among KOβ2 mice infected with T. cruzi during the 21 day follow-up period, whereas 27% of the chagasic wild-type mice had died by the end of the of observation period (Fig. 3A). The survival rate of the uninfected wild-type and KOβ2 mice was 100%. Moreover, the KOβ2-CHG group exhibited increased levels of parasitaemia in comparison to the WT-CHG group (Fig. 3B). The ECG analysis (Table 3) demonstrated that the mean RR, PR, QRS, QaT and QaTc values were significantly higher in WT-CHG animals compared with the WT-CON animals, and these elevated levels indicated global cardiac effects with limited extension into the atria (normal P wave Figure 2. Evaluation of the effect parasympathetic nervous system stimulation with pyridostigmine bromide (PYR) on acute Chagas disease A, survival curves. B, parasitaemia curves (means ± SEM, P < 0.05 versus chagasic non-treated (CHG- NT) group). C, cardiac vagal parasympathetic tone measured as the tachycardic response to atropine (means ± SEM, P < 0.05 versus control non-treated (CON-NT) and &P < 0.05 versus CHG-NT), D, cardiac sympathetic tone measured as the bradycardic response to propranolol (means ± SEM). E and H, light photomicrographs of Haematoxylin and Eosin stained tissue from the atrium (E) and ventricle (H) of a CON-NT mouse ( 400). F, I and L, light photomicrographs of Haematoxylin and Eosin stained tissue from the atrium (F) and ventricle (I and L) of a CHG-NT mouse ( 400). Note the light to moderate inflammatory infiltration. G, J and M, light photomicrographs of Haematoxylin and Eosin stained tissue from the atrium (G) and ventricle (J and M) of a chagasic pyridostigmine bromide-treated (CHG-PYR) mouse ( 400). Note the near absence of inflammatory infiltration ( 400). K, intrinsic autonomic ganglion of a CHG-PYR mouse. Note the light inflammation with the preservation of neurons. The arrows in J, L and M indicate amastigote nests.

10 Exp Physiol (2012) pp Autonomic neuroimmunomodulation during acute Chagas disease 1195

11 1196 M. P. R. Machado and others Exp Physiol (2012) pp Table 3. Mean values (±SEM) of the ECG and HRV parameters in WT or β 2 -adrenergic receptor gene knockout (KOβ2) FVB/N male mice collected days after inoculation with T. cruzi (chagasic infection; CHG) or vehicle (uninfected control; CON) WT-CON KOβ2-CON WT-CHG KOβ2-CHG ECG parameters No. of animals RR (ms) ± ± ± ± 5.9 P(ms) 12.9 ± ± ± ± 0.3 PR (ms) 31.1 ± ± ± ± 0.9 QRS (ms) 10.4 ± ± ± ± 0.4 QaT (ms) 13.6 ± ± ± ± 2.6 QaTc (ms 1/2 ) 42.4 ± ± ± ± 7.6 HRV parameters No. of animals RR (ms) ± ± ± ± 2.8 Variance (ms 2 ) 13.0 ± ± ± ± 1.1 VLF (ms 2 ) 5.6 ± ± ± ± 0.7 LF (ms 2 ) 2.7 ± ± ± ± 0.4 LF (n.u.) 22.6 ± ± ± ± 3.0 HF (ms 2 ) 4.7 ± ± ± ± 0.3 HF (n.u.) 66.0 ± ± ± ± 3.0 LF/HF 1.9 ± ± ± ± 0.3 Abbreviations are as for Table 1. P < 0.05 compared with wild-type non-infected control (WT-CON) animals. P < 0.05 compared with β 2 -adrenergic receptor knockout non-infected (KOβ2-CON) animals. P < 0.05 compared with wild-type chagasic infected (WT-CHG) animals. duration). The KOβ2-CHG mice exhibited significantly reduced ECG parameters in comparison to the WT- CHG animals, although the RR interval remained elevated (Table 3) in the KOβ2-CHG animals, suggesting that in the absence of β 2 -adrenergic receptors, the cardiac lesions were less intense than in the WT-CHG animals. Additionally, there was a marked reduction in the variance and LF and HF components of the HRV in WT- CHG animals compared with WT-CON animals (Table 3). The lack of expression of β 2 -adrenergic receptors in the KOβ2-CHG group was associated with significantly elevated mean values for the variance and LF and HF components in comparison to the WT-CHG group, although these values were similar to those observed in the WT-CON mice (Table 3). Interestingly, pharmacological blockade with atropine in non-infected KOβ2 animals revealed enhanced cardiac vagaltonecomparedwiththewt-conmice(fig.3c), indicating that in normal uninfected conditions, KOβ2 mice demonstrate augmented cardiac vagal tone without any modification of the cardiac sympathetic tone (Fig. 3D). Infection of wild-type animals with T. cruzi (WT-CHG group) led to a marked reduction in the cardiac vagal effect of atropine on the sinus node that was accompanied by a less intense decrease in the sympathetic tone (Fig. 3C and D).The KOβ2-CHG mice demonstrated a cardiac vagal tone that was similar to that observed in non-infected wild-type animals, higher than that in WT- CHG mice, and significantly less than that in the KOβ2- CON group. These data suggest that even after acute Chagas infection, KOβ2-CHG mice display an apparent cardiac vagal tone. The diffuse myocarditis was light to moderate in the WT-CHG group (average inflammatory score = 2.0, p25% = 1.5 and P75% = 2.0) in comparison to the WT- CON and KOβ2-CON groups (Fig. 3), which presented no Figure 3. Evaluation of the effect of the absence of β 2 -adrenergic sympathetic signalling in β 2 - adrenergic receptor gene knockout (KOβ2) mice on the acute phase of Chagas disease A, survival curves. B, parasitaemia curves (means ± SEM, P < 0.05 versus wild-type chagasic (WT-CHG) group). C, cardiac vagal parasympathetic tone measured as the tachycardic response to atropine (means ± SEM, P < 0.05 versus wild-type control (WT-CON), #P < 0.05 versus β 2 -adrenergic receptor gene knockout control (KOβ2-CON) and &P < 0.05 versus WT-CHG). D, cardiac sympathetic tone measured as the bradycardic response to propranolol (means ± SEM, P < 0.05 versus WT-CON and #P < 0.05 versus KOβ2-CON). E and H, light photomicrographs of Haematoxylin and Eosin stained tissue from the atrium (E) and ventricle (H) of a KOβ2-CON mouse ( 400). F, I and L, light photomicrographs of Haematoxylin and Eosin stained tissue from the atrium (F) and ventricle (I and L) ofawt-chg mouse ( 400). Note the moderate inflammatory infiltration. G, J and M, light photomicrographs of Haematoxylin and Eosin stained tissue from the atrium (G) and ventricle (J and M) ofaβ 2 -adrenergic receptor gene knockout chagasic (KOβ2-CHG) mouse ( 400). Note the near absence of inflammatory infiltration ( 400). K, intrinsic autonomic ganglion of a KOβ2-CHG mouse. Note the slight inflammation with the preservation of neurons. The arrows in K and L indicate amastigote nests.

12 Exp Physiol (2012) pp Autonomic neuroimmunomodulation during acute Chagas disease 1197

13 1198 M. P. R. Machado and others Exp Physiol (2012) pp inflammation. Moreover, the KOβ2-CHG mice presented less inflammatory infiltration (average inflammatory score = 2.0, p25% = 1.5 and P75% = 2.0, P < 0.05 versus WT-CHG) in the atria (Fig. 3G), ventricles (Fig. 3J) and neural ganglia (Fig. 3K),andthereducedmyocarditis observed in these animals was associated with a significantly greater number of tissue amastigote nests in the heart (29.6 ± 2.8 versus 11.3 ± 1.3 parasite nests per slide, P < 0.05) compared with the WT-CHG mice (Fig. 3L and M). Study IV: evaluation of the effect of β 2 -adrenergic sympathetic stimulation with salbutamol on the acute phase of Chagas disease Upon follow-up, the untreated chagasic animals (CHG- NT group) demonstrated a mortality rate of 27% after 21 days of infection. Additionally, there was trend towards decreased mortality (for 60%, 9 survived out of all 15 mice) among chagasic mice treated with salbutamol (CHG- SALB group) during the acute phase of disease (Fig. 4A). No mortality was observed in the normal control animals. The increased mortality rate in the CHG-SALB mice was accompanied by a significant but slight increase in the circulating parasite levels (Fig. 4B). Except for the PR interval, all of the ECG parameters were prolonged in the CHG-NT animals (Table 4); however, treatment with salbutamol improved or even normalized many of the ECG parameters in the CHG- SALB mice (Table 4). These results suggest that salbutamol treatment led to global improvements in acute chagasic myocarditis. Reduced variance and a decreased LF component were observed in CHG-NT animals, whereas these parameters were similar between the CHG-SALB and CON-NT animals (Table 4). These findings were confirmed by pharmacological blockade, which resulted in lower cardiac vagal and sympathetic tone in the CHG-NT animals and increased cardiac vagal and sympathetic tone, Table 4. Mean values (±SEM) of the ECG and HRV parameters collected days after inoculation with T. cruzi (chagasic infection; CHG) or vehicle (uninfected control; CON) in male wildtype FVB/N mice left untreated (NT) or treated with salbutamol (SALB) CON-NT CHG-NT CHG-SALB ECG parameters No. of animals RR (ms) ± ± ± 7.9 P(ms) 12.9 ± ± ± 0.3 PR (ms) 31.1 ± ± ± 0.7 QRS (ms) 10.4 ± ± ± 0.4 QaT (ms) 13.6 ± ± ± 0.7 QaTc (ms 1/2 ) 42.4 ± ± ± 2.0 HRV parameters No. of animals RR (ms) ± ± ± 3.0 Variance (ms 2 ) 13.0 ± ± ± 2.5 VLF (ms 2 ) 5.6 ± ± ± 1.4 LF (ms 2 ) 2.7 ± ± ± 0.5 LF (n.u.) 22.6 ± ± ± 4.0 HF (ms 2 ) 4.7 ± ± ± 1.0 HF (n.u.) 66.0 ± ± ± 4.0 LF/HF 1.9 ± ± ± 0.5 Abbreviations are as for Table 1. P < 0.05 compared with non-infected non-treated control (CON-NT) animals. P < 0.05 compared with chagasic infected non-treated (CHG-NT) animals. approximating normal levels, in the CHG-SALB mice (Fig. 4C and D). Moderate to severe inflammatory infiltration (average inflammatory score = 3.0, p25% = 3.0 and P75% = 3.0) was observed in the CHG-NT mice (Fig. 4F and I), while no inflammation was present in the CON-NT group. The chagasic animals treated with salbutamol presented less severe myocarditis (average inflammatory score = 1.0, p25% = 1.0 and P75% = 1.0, P < 0.05) of the atria, ventricles and ganglia (Fig. 4G and J) compared with the CHG-NT mice. The number of tissue amastigote nests was significantly higher in the CHG-SALB mice (106.8 ± 8.7 versus 25.9 ± 3.8 parasite nests per slide, P < 0.01) than in the CHG-NT mice (Fig. 4L andm). Figure 4. Evaluation of the effect of β 2 -adrenergic sympathetic stimulation with salbutamol (SALB) on the acute phase of Chagas disease A, survival curves. B, parasitaemia curves (means ± SEM, P < 0.05 versus CHG-NT group). C, cardiac vagal parasympathetic tone measured as the tachycardic response to atropine (means ± SEM, P < 0.05 versus control non-treated (CON-NT) and &P < 0.05 versus chagasic non-treated (CHG-NT)). D, cardiac sympathetic tone measured as the bradycardic response to propranolol (means ± SEM, P < 0.05 versus CON-NT). E and H, light photomicrographs of Haematoxylin and Eosin stained tissue from the atrium (E) and ventricle (H) of a CON-NT mouse ( 400). F, I and L, light photomicrographs of Haematoxylin and Eosin stained tissue from the atrium (F) and ventricle (I and L) of a CHG-NT mouse ( 400). Note the light to moderate inflammatory infiltration. G, J and M, light photomicrograph of Haematoxylin and Eosin stained tissue from the atrium (G) and ventricle (J and M) of a chagasic salbutamol-treated (CHG- SALB) mouse ( 400). Note the near absence of inflammatory infiltration ( 400). K, intrinsic autonomic ganglion of a CHG-SALB mouse. Note the light inflammation. The arrows in J, L and M indicate amastigote nests.

14 Exp Physiol (2012) pp Autonomic neuroimmunomodulation during acute Chagas disease 1199 Comparisons of autonomic tones quantified using propranolol or atenolol No statistically significant differences were observed between parasympathetic (Fig. S1) or sympathetic tone (Fig. S2) calculated using the β-blockers propranolol or atenolol. In addition, significant positive correlations were found between parasympathetic tones calculated using the propranolol atropine or atenolol atropine approach (Fig. S1C) or between sympathetic tones calculated using the atropine propranolol or atropine atenolol approaches (Fig. S2C). Discussion To our knowledge, the present study is the first to have analysed the effects of deliberate modifications to autonomic nervous system signalling during the acute phase of experimental Chagas disease in mice. Furthermore, this study evaluated the effects of these changes on electrocardiographic, autonomic, histopathological, immune-inflammatory and parasitological parameters associated with the pathogenesis of the disease. The present study demonstrated the existence of electrocardiographic changes associated with diffuse inflammatory infiltration of mononuclear cells in the atrial and ventricular myocardium in wild-type and untreated chagasic C57BL/6j and FVB/N mice, which confirms the presence of acute myocarditis days following infection with T. cruzi, as previously shown in different strains of mice (Soares & Santos, 1999). In addition, to our knowledge, this is the first demonstration of autonomic dysfunction induced by acute Chagas disease in mice. This dysfunction was characterized by a marked reduction in heart rate variability, affecting mainly the HF components, as well as a concurrent reduction in cardiac vagal tone (and to a lesser extent sympathetic tone), as measured by pharmacological blockade in chagasic animals. Only morphological reports describing the ganglia and nerve lesions in chagasic mice have been published in the literature (Ribeiro et al. 2002). As our findings are consistent with previous observations in hamsters, rats and humans during the acute phase of Chagas disease (Soares & Santos, 1999; Dias da Silva et al. 2003), the mouse model used here represents a suitable tool for use in future studies. Considering the anti-inflammatory role played by both the sympathetic (Elenkov & Chrousos, 1999) and the vagal parasympathetic nervous systems (Tracey, 2009) in shifting the Th1 Th2 balance of the immune response towards a predominantly Th2 anti-inflammatory response, deliberate changes in the expression of neurotransmitters or receptors in the autonomic nervous system should have a profound impact on the inflammatory immune response and parasite load in the heart during acute Chagas disease. In fact, there were significantly lower levels of parasitaemia in KDVAChT-CHG mice compared with WT-CHG mice, which is consistent with the idea that reduced acetylcholine levels in the tissues could shift the immune response towards a predominantly inflammatory Th1 response with a consequent reduction in the circulating parasite levels. In addition, the significantly increased levels of parasitaemia observed in chagasic mice treated with pyridostigmine bromide and salbutamol may have been due to a blunted Th1 response in these animals, which would have led to a subsequent decrease in the clearance of circulating parasites and an increased level of parasitaemia. Our findings related to the numbers of amastigote parasite nests in the myocardium in the different chagasic groups analysed also suggest that a reduced inflammatory response is associated with elevated tissue parasitism and decreased local inflammatory infiltration. In the KDVAChT-CHG animals, no amastigote nests were found in any of the cardiac histological slides, although a marked inflammatory infiltrate consisting predominantly of mononuclear cells was observed in large sections of myocardial tissue, specifically in the atria. This diffuse myocarditis was associated with marked electrocardiographic changes, mainly in the atrial (P wave and RR and PR intervals) ECG parameters. Concurrently, severe cardiac autonomic dysfunction was also present but did not have additional effects on mortality. As a decrease of approximately 30 40% in the tissue acetylcholine levels has been observed in KDVAChT mice (Prado et al. 2006) and several reports (Wang et al. 2003) have indicated a role for acetylcholine in negatively modulating the Th1 response profile, our data from the KOVAChT-CHG animals suggest an exacerbation of the Th1 response due to the reduced inhibitory action of acetylcholine on effector Th1 cells. On the contrary, opposing results related to tissue parasitism and myocardial inflammation were observed in chagasic animals treated with pyridostigmine bromide, KOβ2 mice and salbutamol-treated mice, reinforcing the idea of a modulatory role of the autonomic nervous system on the Th1 pro-inflammatory response. Regarding pyridostigmine bromide treatment, the observed increase in tissue parasitism and reduction in myocardial inflammation may have been due to the antiinflammatory effect of acetylcholine (Wang et al. 2003; Tracey, 2009). In particular, this effect was probably greater in the heart tissue, because there were observed increases in the HF component of the HRV and the cardiac vagal tone. A direct toxic effect of pyridostigmine bromide on immune cells seems less likely because this drug was shown not to provoke changes in human immune cells in an in vitro study (Telford et al. 2004)