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Innate immune receptors over expression correlate with chronic chagasic cardiomyopathy and digestive damage in patients

  • Nathalie de Sena Pereira,

    Roles Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft

    Affiliations Department of Parasitology, Federal University of Minas Gerais, Minas Gerais, Belo Horizonte, Brazil, Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Rio Grande do Norte, Natal, Brazil, School of Health, Potiguar University, Natal, RN, Brazil

  • Tamyres Bernadete Dantas Queiroga,

    Roles Data curation, Formal analysis, Investigation

    Affiliation Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Rio Grande do Norte, Natal, Brazil

  • Daniela Ferreira Nunes,

    Roles Data curation, Formal analysis

    Affiliation Department of Parasitology, Federal University of Minas Gerais, Minas Gerais, Belo Horizonte, Brazil

  • Cléber de Mesquita Andrade,

    Roles Investigation, Writing – review & editing

    Affiliation Department of Biomedical Sciences, University of Rio Grande do Norte State, Rio Grande do Norte, Mossoró, Brazil

  • Manuela Sales Lima Nascimento,

    Roles Investigation, Writing – original draft, Writing – review & editing

    Affiliation Edmond and Lily Safra International Institute of Neurosciences, Rio Grande do Norte, Macaíba, Brazil

  • Maria Adelaide Do-Valle-Matta,

    Roles Investigation, Writing – original draft, Writing – review & editing

    Affiliation Laboratory of Cellular Ultrastructure, Oswaldo Cruz Institute/FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil

  • Antônia Cláudia Jácome da Câmara,

    Roles Funding acquisition, Investigation, Supervision, Writing – review & editing

    Affiliation Department of Clinical and Toxicological Analyses, Federal University of Rio Grande do Norte, Natal, Brazil

  • Lúcia Maria da Cunha Galvão,

    Roles Conceptualization, Funding acquisition, Supervision, Writing – review & editing

    Affiliation Department of Parasitology, Federal University of Minas Gerais, Minas Gerais, Belo Horizonte, Brazil

  • Paulo Marcos Matta Guedes ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing

    pauloguedes@cb.ufrn.br

    Affiliation Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Rio Grande do Norte, Natal, Brazil

  • Egler Chiari

    Roles Funding acquisition, Project administration, Supervision, Writing – review & editing

    Affiliation Department of Parasitology, Federal University of Minas Gerais, Minas Gerais, Belo Horizonte, Brazil

Innate immune receptors over expression correlate with chronic chagasic cardiomyopathy and digestive damage in patients

  • Nathalie de Sena Pereira, 
  • Tamyres Bernadete Dantas Queiroga, 
  • Daniela Ferreira Nunes, 
  • Cléber de Mesquita Andrade, 
  • Manuela Sales Lima Nascimento, 
  • Maria Adelaide Do-Valle-Matta, 
  • Antônia Cláudia Jácome da Câmara, 
  • Lúcia Maria da Cunha Galvão, 
  • Paulo Marcos Matta Guedes, 
  • Egler Chiari
PLOS
x

Abstract

Chronic chagasic cardiomyopathy (CCC) is observed in 30% to 50% of the individuals infected by Trypanosoma cruzi and heart failure is the important cause of death among patients in the chronic phase of Chagas disease. Although some studies have elucidated the role of adaptive immune responses involving T and B lymphocytes in cardiac pathogenesis, the role of innate immunity receptors such as Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in CCC pathophysiology has not yet been determined. In this study, we evaluated the association among innate immune receptors (TLR1-9 and nucleotide-binding domain-like receptor protein 3/NLRP3), its adapter molecules (Myd88, TRIF, ASC and caspase-1) and cytokines (IL-1β, IL-6, IL-12, IL-18, IL-23, TNF-α, and IFN-β) with clinical manifestation, digestive and cardiac function in patients with different clinical forms of chronic Chagas disease. The TLR8 mRNA expression levels were enhanced in the peripheral blood mononuclear cells (PBMC) from digestive and cardiodigestive patients compared to indeterminate and cardiac patients. Furthermore, mRNA expression of IFN-β (cytokine produced after TLR8 activation) was higher in digestive and cardiodigestive patients when compared to indeterminate. Moreover, there was a positive correlation between TLR8 and IFN-β mRNA expression with sigmoid and rectum size. Cardiac and cardiodigestive patients presented higher TLR2, IL-12 and TNF-α mRNA expression than indeterminate and digestive patients. Moreover, cardiac patients also expressed higher levels of NLRP3, ASC and IL-1β mRNAs than indeterminate patients. In addition, we showed a negative correlation among TLR2, IL-1β, IL-12 and TNF-α levels with left ventricular ejection fraction, and positive correlation between NLRP3 with cardiothoracic index, and TLR2, IL-1β and IL-12 with left ventricular mass index. Together, our data suggest that high expression of innate immune receptors in cardiac and digestive patients may induce an enhancement of cytokine expression and participate of cardiac and digestive dysfunction.

Author summary

Chronic chagasic cardiomyopathy (CCC) is the main cause of death during Trypanosoma cruzi (T. cruzi) infection in patients. Individuals with CCC have high production of inflammatory mediators such as IFN-γ, TNF-α, IL-1β and nitric oxide (NO) which are involved with myocarditis, fibrosis, and myocardial hypertrophy. Yet the role of innate immunity receptors in CCC pathophysiology has not been addressed. Activation of TLRs and NLRs is fundamental to activate the innate immune system and also to modulate adaptive responses. Herein we have evaluated the association between innate immune receptors and innate cytokines with the clinical manifestation, and with the cardiac function in patients with different clinical forms of chronic Chagas disease. Our data suggest that high TLR2 and NLRP3 expression in cardiac patients may induce an enhancement of proinflammatory cytokine expression such as IL-1β, IL-12, TNF-α and participate of the pathophysiology of CCC. A better understanding of immunological mechanisms involved in CCC may lead to reduced morbidity and mortality associated with the most lethal clinical manifestation of Chagas Disease.

Introduction

Chagas disease is caused by the Trypanosoma cruzi (T. cruzi) parasite, and affects about 6 million to 7 million people in Latin America; moreover, 1.2 million people have chronic chagasic cardiomyopathy (CCC), which is the main cause of 12,000 deaths annually [1,2]. In the chronic phase of Chagas disease approximately 30 to 50% of patients develop cardiac disease, 5 to 15% develop digestive disease and 2 to 10% develop cardiodigestive form [24]. Neuronal depopulation and changes in cardiac and myenteric plexus conduction systems are fundamental for the pathophysiology of CCC, megaesophagus and megacolon. Cardiac conduction system may be diffusely affected from the sinus node to the distal third of His bundle. Chagasic patients with CCC have right bundle branch block (present in 13 to 35% of patients) as an electrocardiographic alteration most frequently suggestive of Chagas' disease, often associated with anterosuperior left bundle branch block [5]. Ventricular extrasystole occurs (15% to 55% of patients) usually isolated, but when complex or associated with other electrocardiographic alterations are correlate with left ventricular systolic and diastolic function, and diastolic diameter [68]. The severity of ventricular arrhythmia is often correlated with the degree of left ventricular dysfunction, although some patients with CCC and ventricular tachycardia or ventricular atrial block have preserved global ventricular function [4,6]. Sudden death is more frequent in males (mainly between 30 and 50 years of age). Other electrocardiographic changes observed in Chagas' disease are represented by low voltage of the QRS complex, notches and abnormal thickenings, low amplitude or absence of R wave in precordial derivations [9,10]. Inflammatory process involves fibrosing and progressive chronic myocarditis is also the key substrate for impairment of the conduction system in Chagas disease [5]. Inflammatory cytokines (IL-12, IFN-γ and TNF-α), nitric oxide, autoantibodies, CD8+ T lymphocyte are possible correlated with neuronal depopulation [1115].

Cardiac disease has been correlated with immunological unbalance. Patients with CCC have high production of inflammatory cytokines such as IFN-γ, TNF-α, IL-1β and nitric oxide (NO) which are involved with myocarditis, fibrosis and myocardial hypertrophy. In contrast, asymptomatic patients produce high levels of IL-10 which support control of the inflammatory mechanism in the heart [1619]. The cardiac form of Chagas disease is related to an increase of T helper (Th) type 1 cells and a decrease of Th2, Th9, Th17, Th22 and regulatory T cell response [11,16]. In fact, exacerbated inflammatory process in cardiac patients has been associated to enhancing the risk of stroke and death [11]. Autoantibodies and CD8+ T lymphocytes have also been related to CCC immunopathogenic mechanism [2022].

TLRs and NLRs are families of pattern recognition receptors (PRRs) located in the plasma membrane, endosomes and cytosol, and are mainly expressed by professional antigen presenting cells (APC), endothelial cells and fibroblasts. PRRs are responsible for recognizing different chemical structures highly conserved in microorganisms known as Pathogen-Associated Molecular Patterns (PAMPs). Signaling through TLRs and NLRs induce the transcription of genes involved in inflammatory response, and its role in the experimental T. cruzi infection has been investigated. T. cruzi contains a variety of ligands such as glycosylphosphatidylinositol (GPI) anchors of mucin-like glycoproteins, glycoinositolphospholipid (GIPL) and nucleic acids which activate different PRRs [2326]. In fact, a deficiency of Myd88, TLR4, TLR7 and TLR9 lead mice to being more susceptible to T. cruzi infection [2729]. However, TLR2 signaling and NF-κβ activation induce pro-IL-1β production, which triggers cardiomyocyte hypertrophy in T. cruzi infected rats [30]. Chagasic patients with a decrease in signal transduction upon ligation of TLR2 or TLR4 to their respective ligand may exhibit low NF-κβ activation and have a low risk of developing CCC [31]. NLRs were extensively characterized as PRRs for bacterial and viral infection [3234], and their role in recognizing intracellular parasites has been studied. Knockout mice for NOD1 are more susceptible to T. cruzi infection. Bone marrow-derived macrophages from NOD1 knockout mice show a reduction of products dependent on NF-kB activation and fail to control the infection in the presence of IFN-γ [35]. NLRP3 inflammasome signaling activates apoptosis-associated speck–like protein containing a caspase recruitment domain (ASC) and caspase-1, thereby inducing the cleavage of pro-IL-1β and pro-IL-18 in their active forms [3638]. ASC inflammasomes are critical determinants of host resistance to infection with T. cruzi. NLRP3-/-, ASC-/- and caspase-1-/- mice exhibit a higher mortality, cardiac parasitism, and myocarditis than wide type mice [39,40]. However, T. cruzi NLRs agonists are not known.

Although several studies have elucidated the role of TLRs and NLRs in experimental infection by T. cruzi, the role in human CCC pathophysiology has not yet been determined. The activation of TLRs and NLRs is important in directing adaptive responses, thus resulting in macrophage activation which are important cells involved in heart disease [31,41]. In this study, we have described an increase in several innate components such as NLRP3, ASC, TLR2, IL-1β, IL-12 and TNF-α associated with the pathophysiology of CCC in humans. Moreover, the digestive form of chronic Chagas disease was correlated to high TLR8 and IFN-β mRNA expression. A better understanding of immunological mechanisms involved in CCC may lead to reduced morbidity and mortality associated with the cardiac form of the disease.

Methods

Study population and ethics statement

The population was composed of 65 individuals aged between 18 and 79 years old from an endemic area of Chagas disease in Rio Grande do Norte, Northeast, Brazil, as described previously [11]. The individuals were selected using two different serological methods (Chagatest, recombinant ELISA and HAI, and indirect immunofluorescence assay) in accordance with recommendations of the World Health Organization and the Brazilian Consensus of Chagas Disease II [42]. Western blot (TESAcruzi®, BioMérieux, Brazil) confirmatory sorological test was performed [43]. Informed consent was obtained from the participants and the study was approved by the Research Ethics Committee of the State University of Rio Grande do Norte (UERN) under protocol number 027.201, and a Certificate of National System of Ethics in Research (CAEE—SISNEP) with protocol number 0021.0.428.000–11. The study was performed according to human experimental guidelines of the Brazilian Ministry of Health and the Helsinki Declaration.

Clinical evaluations

Individuals with confirmed positive serology to Chagas disease were clinically evaluated including electrocardiogram (ECG) mapping and chest X-ray, 2D-echocardiogram (ECHO) and 24h Holter examination. Chagasic patients (Table 1) were classified as indeterminate (n = 18), cardiac (n = 17), digestive (n = 15) and cardiodigestive (n = 15) clinical forms, according to the World Health Organization and Brazilian Consensus of Chagas Disease [42]. Uninfected healthy individuals (n = 15) were used as controls. Clinical evaluations were performed in all chagasic patients as previously described [3]. First, plain posteroanterior and lateral chest radiography were performed to evaluate the cardiothoracic index, and which was considered abnormal if attaining a value >0.5 [3]. Esophageal contrast radiography was performed in right anterior oblique position using barium sulfate (Bariogel®, Cristália Laboratory, Brazil) classifying the esophagus changes into four groups [44]. Contrasted colon radiographs were performed in the supine, ventral and right lateral position [45] using barium sulfate solution (Bariogel ®, Cristália Laboratory, Brazil) via the rectum without prior bowel preparation or double contrast use. The sigmoid was classified into four grades (zero to three) according to Silva et al. [46] modified by Andrade and coworkers [3]. Radiographic examinations were performed using radiology equipment with X-ray penetration to deep parts (VMI®, Brazil). Electrocardiographic alterations were determined using a portable EP3 2008 electrocardiograph (Dixtal, Brazil) with three channels and 12-lead; electrocardiographic recording was based on the Minnesota Code modified, adapted for Chagas disease [10]. Next, conventional, parasternal, supra sternal, apical, subcostal transthoracic echocardiogram and its variations were performed in all patients to calculate cardiac dimension and volumes in accordance with the recommendations of the American Society of Echocardiography [47] and using echocardiography with color flow mapping performed in standard views (General Electric Healthcare, USA). The left ventricular ejection fraction (LVEF) was calculated according to the modified Simpson's rule (biplane method) [47]. The left ventricular mass index (LVMI) was calculate by the formula LVMI = heart mass (g)/ patient's body surface (m2). Patients with cardiomegaly, electrocardiographic or echocardiographic alterations suggestive of Chagas disease underwent electrocardiographic monitoring for 24 hours (24-Holter) using a Cardiolight Digital Recorder (Cardios, São Paulo, Brazil).

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Table 1. Clinical data of chronic chagasic patients from the Northeast of Brazil included in this investigation.

https://doi.org/10.1371/journal.pntd.0006589.t001

Real time PCR

Innate immune receptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and NLRP3), signaling molecules (Myd88, TRIF, ASC, Caspase-1) and cytokine (IL-1β, IL-6, IL- 12, IL-18 and TNF-α) mRNA expression were detected by Real-Time PCR (qPCR) in peripheral blood mononuclear cells (PBMC) obtained from chagasic patients. Total RNA was obtained using Trizol reagent (Invitrogen™, Carlsbad, CA, USA) and SV Total RNA Isolation System (Promega, Madison, WI, USA) with DNase treatment step. cDNA synthesis was performed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, USA) using the Eppendorf Mastercycler gradient set (Eppendorf, USA). The qPCR reactions were performed using SYBR Green (Applied Biosystems, USA) supported by 7500 Fast Real time thermocycler (Applied Biosystems, Warrington, USA). The reactions were performed in 96 well plates (MicroAmp®, Applied Biosystems, USA) and the standard PCR conditions were as follows: 50°C (2 min) and 95°C (10 min) followed by 40 cycles of 94°C (30 s), variable annealing primer temperature (Table 2) (30 s), and 72°C (1 min). Specific primers (Table 2) were obtained by the Primer Express software (Applied Biosystems, USA). The mRNA expression levels of the innate immune receptors, adapter molecules and cytokines were determined using the mean Ct values from triplicate measurements to calculate the relative expression levels of the target genes in the Chagas disease patients compared to healthy controls, and were normalized to the housekeeping gene β-actin using the 2–ΔΔCt formula.

Enzyme linked immunosorbent assay (ELISA)

Cytokine quantification was performed in sera from indeterminate (n = 18), cardiac (n = 17), cardiodigestive (n = 15) and digestive (n = 15) chagasic patients. Uninfected individuals were used as a control (n = 15). The ELISA sets were IL-1β, IL-12 (p70) and TNF-α (BD OptEIATM, BD Bioscience), and procedures were performed according to the manufactures`instructions. Optical densities were measure at 450ηm.

Statistical analysis

Data are reported as mean ± standard deviation (SD). Kolmogorov-Smirnov test was used to verify parametric or non-parametric data distribution. The mRNA expression levels were compared using the Kruskal-Wallis test. Correlations among left ventricular ejection fraction, esophagus and colon dilation, innate immune receptors and cytokines were performed using the Spearman test. Differences were considered significant when p <0.05. Our analyses were performed using PRISM 5.0 software (GraphPad, CA, USA).

Results

Clinical evaluations

Chagasic patients (n = 65) were classified as indeterminate (n = 18), cardiac (n = 17), digestive (n = 15) and cardiodigestive (n = 15) clinical forms. Chest X-ray demonstrated cardiomegaly in approximately 10% of cardiac and cardiodigestive patients. Electrocardiographic changes were not always associated with cardiac symptoms. Three cardiac patients had right bundle branch block, two also had anterosuperior divisional block. Four cardiodigestive patients presented right bundle branch block, two also presented anterosuperior divisional block. All ventricular atrial blocks were first degree, except for one patient with ventricular atrial blockade who received pacemaker implantation. The echocardiogram showed similar diastolic diameters and left ventricular mass index in indeterminate, cardiac, digestive and cardiodigestive chagasic patients (Table 1).

Chronic chagasic cardiomyopathy is correlated with high TLR2, IL-12 and TNF-α expression

In an attempt to elucidate the inflammatory mechanism involved in CCC development we analyzed the mRNA expression of innate immune receptors in chagasic individuals grouped according to clinical forms as indeterminate, cardiac, digestive and cardiodigestive (Table 1). Patients with different clinical manifestations of Chagas disease showed similar expression of TLR1, TLR3, TLR4, TLR5, TLR6, TLR7 and TLR9 mRNA (Fig 1). Interestingly, cardiac and cardiodigestive patients presented higher TLR2 mRNA expression than indeterminate and digestive patients (Fig 1B). Furthermore, cardiodigestive patients presented higher Myd88 mRNA expression than indeterminate and cardiac patients (Fig 2A). Cardiac patients showed higher mRNA expression of IL-12 and TNF-α transcripts (cytokines produced upon TLR activation) than indeterminate patients (Fig 2B and 2C). We observed similar expression of TRIF, IL-6, IL-23 and IFN-α in chagasic patients with different clinical manifestations of Chagas disease (Fig 2D–2G). Moreover, there was higher production of inflammatory cytokines (TNF-α and IL-12) induced by the TLRs activation in sera in cardiac patients than in indeterminate and uninfected controls (Fig 3A and 3B). However, no significant difference was observed between the levels of IL-1β between the different groups of patients (Fig 3C). Together, these data indicate that TLR2 expression in cardiac patients may induce an enhancement of IL-12 and TNF-α expression and correlate to cardiac dysfunction.

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Fig 1. Chronic chagasic cardiomyopathy is correlated with high TLR2 mRNA expression and digestive form is correlated with high TLR8 expression.

The mRNA expression levels of TLR1 (A), TLR2 (B), TLR3 (C), TLR4 (D), TLR5 (E), TLR6 (F), TLR7 (G), TLR8 (H) and TLR9 (I) were determined by real-time PCR in peripheral blood mononuclear cells of patients with the indeterminate (n = 18), cardiac (n = 17), cardiodigestive (n = 15) and digestive (n = 15) clinical forms of Chagas disease. The expression levels were normalized to the expression level of β-actin. The results are expressed as the means ± standard errors. *p < 0.05. UCI: Uninfected control individuals (n = 15).

https://doi.org/10.1371/journal.pntd.0006589.g001

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Fig 2. Chronic chagasic cardiomyopathy is correlated with high IL-12, TNF-α and IFN-β mRNA expression.

The mRNA expression levels of Myd88 (A), IL-12 (B), TNF-α (C), TRIF (D), IL-23 (E), IL-6 (F), IFN-α (G) and IFN-β (H) were determined by real-time PCR in peripheral blood mononuclear cells of patients with the indeterminate (n = 18), cardiac (n = 17), digestive (n = 15) and cardiodigestive (n = 15) clinical forms of Chagas disease. The expression levels were normalized to the expression level of β-actin. The results are expressed as the means ± standard errors. *p < 0.05. UCI: Uninfected control individuals (n = 15).

https://doi.org/10.1371/journal.pntd.0006589.g002

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Fig 3. Chronic chagasic cardiomyopathy is correlated with high IL-12 and TNF-α levels in sera.

Levels of the cytokines IL-12 (A), TNF-α (B) and IL-1β (C) was analyzed by ELISA in the sera of indeterminate/IND (n = 18), cardiac/CARD (n = 17), digestive/DIG (n = 15) and cardiodigestive/CARDIG (n = 15) patients. The results are expressed as means ± standard errors. *p < 0.05. UCI: Uninfected control individuals (n = 15).

https://doi.org/10.1371/journal.pntd.0006589.g003

Digestive and cardiodigestive clinical forms are correlated with high TLR8 and IFN-β mRNA expression

The mRNA expression of TLR8 was enhanced in digestive and cardiodigestive patients compared to indeterminate and cardiac patients. Furthermore, the mRNA expression of IFN-β (cytokine produced after TLR8 activation) was higher in digestive and cardiodigestive patients when compared with indeterminate (Fig 2H). In attempt to evaluate the TLR8 and IFN-β participation in the development of the digestive form of Chagas disease, we analyzed the correlation between the mRNA expression of TLR8 and IFN-β with the rectum and sigmoid size, resulting in a positive correlation observed between TLR80020and IFN-β and rectum and sigmoid size (Fig 4A–4D).

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Fig 4. High TLR8 and IFN-β expression are correlated with digestive form of Chagas disease.

The mRNA expression levels of TLR8 (A and B) and IFN-β (C and D) were determined by real-time PCR in peripheral blood mononuclear cells of patients with the indeterminate (n = 18), cardiac (n = 17), digestive (n = 15) and cardiodigestive (n = 15) clinical forms of Chagas disease and correlated with sigmoid and rectum size. The expression levels were normalized to the expression level of β-actin. Spearman test was used.

https://doi.org/10.1371/journal.pntd.0006589.g004

Enhanced expression of NLRP3, ASC and IL-1β transcripts correlates with the chronic chagasic cardiomyopathy in patients

We subsequently analyzed the expression of NLRP3 inflammossome, its signaling molecules (ASC and caspase-1) and cytokines produced after its activation (such as IL-1β and IL-18). Cardiac patients showed relevantly higher mRNA expression of NLRP3, ASC and IL-1β than indeterminate patients (Fig 5A–5C). Similar levels of caspase-1 and IL-18 mRNA expression were observed in patients with different clinical forms of chronic Chagas disease (Fig 5D and 5E). We then posteriorly analyzed the correlation between the mRNA expression of TLR2, NLRP3, IL-1β, IL-12 and TNF-α with the left ventricular ejection fraction (LVEF) and cardiothoracic index (CI). We found a negative correlation among NLRP3, TLR2, IL-12 and IL-1β mRNA expression with LVEF (Fig 6A–6D), and positive correlation between NLRP3 with CI (Fig 7A). No correlation was observed between TLR2, IL-1β and IL-12 with CI (Fig 7B–7D), and between NLRP3 with left ventricular mass index (LVMI) (Fig 8A). We also observed a positive correlation between LVMI with TLR2, IL-1β and IL-12 (Fig 8B–8D).

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Fig 5. Cardiac patients exhibited higher NLRP3, ASC and IL-1β mRNA expression than indeterminate patients.

The mRNA expression levels of NLRP3 (A), ASC (B), Caspase-1 (C), IL-1β (D) and IL-18 (E) were determined by real-time PCR in peripheral blood mononuclear cells of patients with the indeterminate (n = 18), cardiac (n = 17), digestive (n = 15) and cardiodigestive (n = 15) clinical forms of Chagas disease. The expression levels were normalized to the expression level of β-actin. The results are expressed as the means ± standard errors. *p < 0.05. UC: Uninfected control individuals (n = 15).

https://doi.org/10.1371/journal.pntd.0006589.g005

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Fig 6. High NLRP3, TLR2, IL-12 and IL-1β expression are correlated with low left ventricular ejection fraction (LVEF).

The mRNA expression levels of NLRP3 (A), TLR2 (B), IL-1β (C) and IL-12 (D) were determined by real-time PCR in peripheral blood mononuclear cells of patients with the indeterminate (n = 18), cardiac (n = 17), digestive (n = 15) and cardiodigestive (n = 15) clinical forms of Chagas disease and correlated with left ventricular ejection fraction (LVEF). The expression levels were normalized to the expression level of β-actin. Spearman test was used.

https://doi.org/10.1371/journal.pntd.0006589.g006

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Fig 7. High NLRP3 expression is correlated with high cardiothoracic index (CI).

The mRNA expression levels of NLRP3 (A), TLR2 (B), IL-1β (C) and IL-12 (D) were determined by real-time PCR in peripheral blood mononuclear cells of patients with the indeterminate (n = 18), cardiac (n = 17), digestive (n = 15) and cardiodigestive (n = 15) clinical forms of Chagas disease and correlated with cardiothoracic index (CI). The expression levels were normalized to the expression level of β-actin. Spearman test was used.

https://doi.org/10.1371/journal.pntd.0006589.g007

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Fig 8. High left ventricle mass index (LVMI) is correlated with high TLR-2, IL-1β and TNF-α mRNA expression.

The mRNA expression levels of NLRP3 (A), TLR2 (B), IL-1β (C) and IL-12 (D) were determined by real-time PCR in PBMC of indeterminate (n = 18), cardiac (n = 17), digestive (n = 15) and cardiodigestive (n = 15) patients and correlated with LVMI. Spearman test was used.

https://doi.org/10.1371/journal.pntd.0006589.g008

Discussion

Pathophysiological mechanisms involved in the development of chronic chagasic cardiomyopathy (CCC) have been studied in chagasic patients and several immunopathogenic mechanisms involving the participation of adaptive immune response such as CD4+ T helper response [11,1618,48,49], CD8+ T cells [5053], and autoantibodies production [20,21,5456] have been elucidated. However, the role of innate immunity receptors in the CCC pathophysiology has not been elicited. In this study, we have assessed the expression of Toll-like Receptors and Nod-like Receptors, their adapter molecules and induced cytokines in cardiac patients, and compared them to indeterminate, digestive and cardiodigestive clinical forms of the disease.

We initially analyzed the mRNA expression of TLRs in PBMCs obtained from the same chagasic patients described in previous study [11]. Patients who showed digestive and cardiodigestive clinical forms presented higher TLR8 mRNA expression when compared to cardiac and indeterminate patients. Human TLR8 recognizes single-stranded RNAs from RNA viruses, as well as detecting RNAs from bacteria in endosomes of dendritic cells [57]. Patients with the digestive clinical form are mainly characterized by the presence of a megaesophagus and megacolon which are caused by the destruction of intramural autonomic ganglia [58]. Gastrointestinal dysfunction can change the feed flow and is associated with bowel inflammatory lesions which distort epithelium gastrointestinal homeostasis, which in turn could allow bacteria penetration and TLR8 activation. This phenomenon could be associated with the development of digestive pathology in chronic chagasic patients. In fact, patients with ulcerative colitis have higher TLR8 mRNA in colon biopsies than healthy subjects, probably due to bacterial RNA of gut microbiota resulting from microbiota dysbiosis [59,60]. Intestinal inflammation intensity in the ulcerative colitis is positively correlated with TLR8 and inflammatory cytokines such as IL-6 and TNF-α [60]. Susceptibility to Crohn's disease (another intestinal inflammatory disease) has also been associated to high TLR8 levels [61,62]. TLR8 activation induces pro-inflammatory cytokine production such as IL-1β, IFN-α, IFN-β, TNF-α, IL-6, and IL-12 in PBMCs, monocytes and dendritic cells in patients [57,63]. Furthermore, in this study we observed higher mRNA expression of TLR8 and IFN-β in digestive and cardiodigestive patients when compared to indeterminate patients. Pathophysiologic alterations in the digestive system during Chagas disease result from the destruction of the enteric nervous system, mainly Auerbach's myenteric plexus. The inflammatory process around the neurons leads to degenerative phenomena, thereby reducing nervous cell numbers and leading to the development of megacolon and megaesophagus [6466].

Cardiac and cardiodigestive patients showed higher TLR2 mRNA expression than indeterminate and digestive patients. On the other hand, patients with different clinical manifestations of Chagas disease showed similar mRNA expression levels of TLR1, TLR3, TLR4, TLR5, TLR6, TLR7 and TLR9. Literature data has demonstrated that T. cruzi-infected individuals who have indeterminate clinical form of Chagas disease are heterozygous for the MAL/TIRAP S180L variant that leads to a decrease in signal transduction upon ligation of TLR2 or TLR4, probably leading to reduced inflammatory response in the heart [31,67]. Thus, low TLR2 and TLR4 signaling have been associated with a lower risk of developing CCC. TLR2 and TLR4 activations in dendritic cells and macrophages conducing Myd88 and TRIF signaling activate NF-kB and lead to the production of pro-inflammatory cytokines such as IL-6, IL12 and TNF-α [57]. CCC development has been correlated to immunological imbalance involving high IFN-γ and TNF-α production associated with low IL-10 and IL-17 secretion [11,1618,49,68]. In our study we also observed that cardiac patients showed higher mRNA expression of NLRP3, ASC, CASPASE-1, IL-1β, IL-12 and TNF-α than indeterminate patients. Furthermore, a negative correlation among TLR2, NLRP3, IL-1β and TNF-β mRNA expression with LVEF, and positive correlation of NLRP3 mRNA expression with CI was observed in chagasic patients. NLRP3 inflammasome and apoptosis-associated speck–like protein containing a caspase recruitment domain (ASC) activates caspase-1 in experimental T. cruzi infection, and induce the production of active IL-1β and IL-18 [39]. Pro-inflammatory cytokines (IL-1β, IL-6 and TNF-β) regulate cell death of inflammatory tissues, modify vascular endothelial permeability, recruit blood cells to inflamed tissues, and induce the production of acute-phase proteins [69]. NLRP3 inflammasome is activated by prokaryotic RNA and different agents that trigger damage-associated molecular patterns (DAMPs) such as UVB irradiation [70] pore-forming toxins [71], urate crystals and silica [72]. Moreover, several host-derived molecules indicative of damage activate the NLRP3 inflammasome, including reactive oxygen species [73], extracellular ATP adenosine [74], uric acid [75,76] and hyaluronan [77] which are released by injured cells. Thus, the NLRP3 activation mechanism observed during experimental T. cruzi infection [39] by an possible unknown parasite ligand may act together with DAMPs generated by injured cells [69], thereby participating in the immunophysiological mechanisms involved in CCC development in chronic chagasic patients. Pro-inflammatory cytokines such as TNF-α, TNF-β, IL-1α, IL1β, IL-6, IFN-α, IFN-γ and IL-8 induce acute phase protein productions which can opsonize parasites, activate complements, recruit immune cells and induce enzymes which degrade the extra cellular matrix. Acute phase proteins such as C-reactive protein, Serum amyloid A, Serum amyloid P component, Complement factors, Mannan-binding lectin, Fibrinogen, prothrombin, Plasminogen, Alpha 2-macroglobulin, Ferritin, Ceruloplasmin, Haptoglobin, Alpha 1-antitrypsin and α1-antichymotrypsin enhance the inflammatory process [78]. Increased levels of acute phase proteins are associated with increased risk for cardiovascular events in healthy individuals and coronary heart disease patients [79]. C-reactive protein levels are non-specific markers of systemic inflammatory processes, which reflect a vascular inflammation state and they are associated with cardiovascular damage [80]. High C-reactive protein levels have been described in non-chagasic cardiomyopathy [81] and also during CCC [8285]. Thus, NLRP3 and TLR2 recognize parasite antigens and molecules associated with cell damage, leading to an inflammatory process with cytokines and other inflammatory mediator production that might participate of CCC development.

CCC development is initiated by the presence of the parasite causing cardiomyocyte destruction due to parasite multiplication and inflammation [86]. However, CCC development depends on the parasite’s genetics [8789] and the genetic background of the patients [67,9092] which can induce different cardiac tropism patterns by the parasite and influence the immune response [87,93,94]. CCC involves persistent myocarditis, development of conduction disturbances, dysautonomia, cardiomegaly, fibrosis, ventricular wall thinning, microvascular damage, increased platelet activity, microthrombi, myocytolysis, myocardial fibrosis and death [4,95,96]. Persistent myocarditis is responsible for progressive neuronal damage, microcirculatory alterations, heart matrix deformations and consequent organ failure [97]. In this context the important inflammatory process in the myocardium which is responsible for CCC development seems to also be maintained by the innate immunity receptor activation such as TLR2 and NLRP3, which induce the production of inflammatory cytokines (IL-1β, IL-12 and TNF-α), thus amplifying the described inflammatory mechanisms and involving elements of adaptive immunity components such as T CD4 and CD8 lymphocytes and antibodies [57]. Our findings suggest that a high TLR2 and NLRP3 expression in chagasic cardiac patients may induce an enhancement of IL-1β, IL-12, and TNF-α, thereby increasing cardiac inflammation and contributing to the heart dysfunction. One limitation of this study concerns on the widely clinical presentation of patients with CCC, according to the extent of myocardial damage and the relative small sample size. Untreated patients samples are very rare and difficult to obtain but are essential for the understanding of immunological mechanisms of Chagas disease pathophysiology. A better knowledge of the immune response involved in CCC development, the main factor correlated to death related to Chagas disease, may also contribute to reducing mortality and morbidity. The present study generated important data about the disease pathophysiology understanding, suggesting that distinct pattern recognition receptors may contribute differentially to the development of clinical forms of Chagas disease.

References

  1. 1. WHO (2015) World Health Organization. Chagas disease in Latin America: na epidemiological update based on 2010 estimates. 90: 12.
  2. 2. WHO (2017) Chagas disease (American trypanosomiasis). Geneva, Switzerland. http://wwwwhoint/mediacentre/factsheets/fs340/en/.
  3. 3. Andrade Cde M, Camara AC, Nunes DF, Guedes PM, Pereira WO, et al. (2015) Chagas disease: morbidity profile in an endemic area of Northeastern Brazil. Rev Soc Bras Med Trop 48: 706–715. pmid:26676495
  4. 4. Rassi A Jr., Rassi A, Marin-Neto JA (2010) Chagas disease. Lancet 375: 1388–1402. pmid:20399979
  5. 5. Marin-Neto JA, Cunha-Neto E, Maciel BC, Simoes MV (2007) Pathogenesis of chronic Chagas heart disease. Circulation 115: 1109–1123. pmid:17339569
  6. 6. Rocha MO, Ribeiro AL, Teixeira MM (2003) Clinical management of chronic Chagas cardiomyopathy. Front Biosci 8: e44–54. pmid:12456332
  7. 7. Carrasco HA, Guerrero L, Parada H, Molina C, Vegas E, et al. (1990) Ventricular arrhythmias and left ventricular myocardial function in chronic chagasic patients. Int J Cardiol 28: 35–41. pmid:2365530
  8. 8. Garzon SA, Lorga AM, Nicolau JC (1995) Electrocardiography in Chagas' heart disease. Sao Paulo Med J 113: 802–813. pmid:8650480
  9. 9. Blackburn H, Keys A, Simonson E, Rautaharju P, Punsar S (1960) The electrocardiogram in population studies. A classification system. Circulation 21: 1160–1175. pmid:13849070
  10. 10. Maguire JH, Mott KE, Souza JA, Almeida EC, Ramos NB, et al. (1982) Electrocardiographic classification and abbreviated lead system for population-based studies of Chagas' disease. Bull Pan Am Health Organ 16: 47–58. pmid:7074255
  11. 11. Guedes PM, Andrade CM, Nunes DF, de Sena Pereira N, Queiroga TB, et al. (2016) Inflammation Enhances the Risks of Stroke and Death in Chronic Chagas Disease Patients. PLoS Negl Trop Dis 10: e0004669. pmid:27115869
  12. 12. Alcântara FG (1970) Desnervação dos gânglios cardíacos intramurais e cervicotorácicos na moléstia de Chagas. Revista Goiana de Medicina 16: 18.
  13. 13. Masuda MO, Levin M, De Oliveira SF, Dos Santos Costa PC, Bergami PL, et al. (1998) Functionally active cardiac antibodies in chronic Chagas' disease are specifically blocked by Trypanosoma cruzi antigens. FASEB J 12: 1551–1558. pmid:9806764
  14. 14. de Oliveira SF, Pedrosa RC, Nascimento JH, Campos de Carvalho AC, Masuda MO (1997) Sera from chronic chagasic patients with complex cardiac arrhythmias depress electrogenesis and conduction in isolated rabbit hearts. Circulation 96: 2031–2037. pmid:9323096
  15. 15. Costa PC, Fortes FS, Machado AB, Almeida NA, Olivares EL, et al. (2000) Sera from chronic chagasic patients depress cardiac electrogenesis and conduction. Braz J Med Biol Res 33: 439–446. pmid:10775309
  16. 16. Guedes PM, Gutierrez FR, Silva GK, Dellalibera-Joviliano R, Rodrigues GJ, et al. (2012) Deficient regulatory T cell activity and low frequency of IL-17-producing T cells correlate with the extent of cardiomyopathy in human Chagas' disease. PLoS Neglected Tropical Diseases 6: e1630. pmid:22545173
  17. 17. Abel LC, Rizzo LV, Ianni B, Albuquerque F, Bacal F, et al. (2001) Chronic Chagas' disease cardiomyopathy patients display an increased IFN-gamma response to Trypanosoma cruzi infection. Journal of autoimmunity 17: 99–107. pmid:11488642
  18. 18. Ribeirao M, Pereira-Chioccola VL, Renia L, Augusto Fragata Filho A, Schenkman S, et al. (2000) Chagasic patients develop a type 1 immune response to Trypanosoma cruzi trans-sialidase. Parasite Immunol 22: 49–53. pmid:10607290
  19. 19. Rocha Rodrigues DB, dos Reis MA, Romano A, Pereira SA, Teixeira Vde P, et al. (2012) In situ expression of regulatory cytokines by heart inflammatory cells in Chagas' disease patients with heart failure. Clin Dev Immunol 2012: 361730. pmid:22811738
  20. 20. Nunes DF, Guedes PM, Andrade Cde M, Camara AC, Chiari E, et al. (2013) Troponin T autoantibodies correlate with chronic cardiomyopathy in human Chagas disease. Trop Med Int Health 18: 1180–1192. pmid:23906320
  21. 21. Cunha-Neto E, Kalil J (1995) Autoimmunity in Chagas' heart disease. Sao Paulo Med J 113: 757–766. pmid:8650474
  22. 22. Rizzo LV, Cunha-Neto E, Teixeira AR (1989) Autoimmunity in Chagas' disease: specific inhibition of reactivity of CD4+ T cells against myosin in mice chronically infected with Trypanosoma cruzi. Infect Immun 57: 2640–2644. pmid:2474498
  23. 23. Campos MA, Almeida IC, Takeuchi O, Akira S, Valente EP, et al. (2001) Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite. Journal of immunology 167: 416–423.
  24. 24. Bellio M, Liveira AC, Mermelstein CS, Capella MA, Viola JP, et al. (1999) Costimulatory action of glycoinositolphospholipids from Trypanosoma cruzi: increased interleukin 2 secretion and induction of nuclear translocation of the nuclear factor of activated T cells 1. FASEB J 13: 1627–1636. pmid:10463955
  25. 25. DosReis GA, Pecanha LM, Bellio M, Previato JO, Mendonca-Previato L (2002) Glycoinositol phospholipids from Trypanosoma cruzi transmit signals to the cells of the host immune system through both ceramide and glycan chains. Microbes Infect 4: 1007–1013. pmid:12106795
  26. 26. Oliveira AC, Peixoto JR, de Arruda LB, Campos MA, Gazzinelli RT, et al. (2004) Expression of functional TLR4 confers proinflammatory responsiveness to Trypanosoma cruzi glycoinositolphospholipids and higher resistance to infection with T. cruzi. Journal of immunology 173: 5688–5696.
  27. 27. Caetano LC, Brazao V, Filipin Mdel V, Santello FH, Toldo MP, et al. (2011) Corticosterone evaluation in Wistar rats infected with the Y strain of Trypanosoma cruzi during the chronic phase. Exp Parasitol 127: 31–35. pmid:20599998
  28. 28. Bafica A, Santiago HC, Goldszmid R, Ropert C, Gazzinelli RT, et al. (2006) Cutting edge: TLR9 and TLR2 signaling together account for MyD88-dependent control of parasitemia in Trypanosoma cruzi infection. Journal of immunology 177: 3515–3519.
  29. 29. Bartholomeu DC, Ropert C, Melo MB, Parroche P, Junqueira CF, et al. (2008) Recruitment and endo-lysosomal activation of TLR9 in dendritic cells infected with Trypanosoma cruzi. Journal of immunology 181: 1333–1344.
  30. 30. Petersen CA, Krumholz KA, Burleigh BA (2005) Toll-like receptor 2 regulates interleukin-1beta-dependent cardiomyocyte hypertrophy triggered by Trypanosoma cruzi. Infect Immun 73: 6974–6980. pmid:16177377
  31. 31. Ramasawmy R, Cunha-Neto E, Fae KC, Borba SC, Teixeira PC, et al. (2009) Heterozygosity for the S180L variant of MAL/TIRAP, a gene expressing an adaptor protein in the Toll-like receptor pathway, is associated with lower risk of developing chronic Chagas cardiomyopathy. J Infect Dis 199: 1838–1845. pmid:19456234
  32. 32. Shaw MH, Reimer T, Kim YG, Nunez G (2008) NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Curr Opin Immunol 20: 377–382. pmid:18585455
  33. 33. Girardin SE, Tournebize R, Mavris M, Page AL, Li X, et al. (2001) CARD4/Nod1 mediates NF-kappaB and JNK activation by invasive Shigella flexneri. EMBO Rep 2: 736–742. pmid:11463746
  34. 34. Inohara N, Koseki T, del Peso L, Hu Y, Yee C, et al. (1999) Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J Biol Chem 274: 14560–14567. pmid:10329646
  35. 35. Silva GK, Gutierrez FR, Guedes PM, Horta CV, Cunha LD, et al. (2010) Cutting edge: nucleotide-binding oligomerization domain 1-dependent responses account for murine resistance against Trypanosoma cruzi infection. Journal of immunology 184: 1148–1152.
  36. 36. Martinon F, Tschopp J (2007) Inflammatory caspases and inflammasomes: master switches of inflammation. Cell Death Differ 14: 10–22. pmid:16977329
  37. 37. Lamkanfi M, Dixit VM (2009) The inflammasomes. PLoS Pathog 5: e1000510. pmid:20041168
  38. 38. Harder J, Franchi L, Munoz-Planillo R, Park JH, Reimer T, et al. (2009) Activation of the Nlrp3 inflammasome by Streptococcus pyogenes requires streptolysin O and NF-kappa B activation but proceeds independently of TLR signaling and P2X7 receptor. J Immunol 183: 5823–5829. pmid:19812205
  39. 39. Silva GK, Costa RS, Silveira TN, Caetano BC, Horta CV, et al. (2013) Apoptosis-associated speck-like protein containing a caspase recruitment domain inflammasomes mediate IL-1beta response and host resistance to Trypanosoma cruzi infection. J Immunol 191: 3373–3383. pmid:23966627
  40. 40. Goncalves VM, Matteucci KC, Buzzo CL, Miollo BH, Ferrante D, et al. (2013) NLRP3 controls Trypanosoma cruzi infection through a caspase-1-dependent IL-1R-independent NO production. PLoS Negl Trop Dis 7: e2469. pmid:24098823
  41. 41. Trinchieri G, Sher A (2007) Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol 7: 179–190. pmid:17318230
  42. 42. Dias JC, Ramos AN Jr., Gontijo ED, Luquetti A, Shikanai-Yasuda MA, et al. (2016) [Brazilian Consensus on Chagas Disease, 2015]. Epidemiol Serv Saude 25: 7–86.
  43. 43. Umezawa ES, Nascimento MS, Kesper N Jr., Coura JR, Borges-Pereira J, et al. (1996) Immunoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas' disease. J Clin Microbiol 34: 2143–2147. pmid:8862574
  44. 44. Rezende J, Lauar KM, de OA (1960) [Clinical and radiological aspects of aperistalsis of the esophagus]. Rev Bras Gastroenterol 12: 247–262. pmid:13741121
  45. 45. Ximenes CA, Rezende JM, Moreira H, Vaz MGM (1984) Técnica simplificada para diagnóstico radiológico do megacolón chagásico. Revista da Sociedade Brasileira de Medicina Tropical 17: 6.
  46. 46. Silva AL, Giacomin RT, Quirino VA, Miranda ES (2003) Proposta de Classificação do megacolón chagásico através do enema opaco. Rev Col Bras Cir 30: 6.
  47. 47. Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, et al. (2005) Recommendations for chamber quantification: a report from the American Society of Echocardiography's Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology. J Am Soc Echocardiogr 18: 1440–1463. pmid:16376782
  48. 48. Reis DD, Jones EM, Tostes S Jr., Lopes ER, Gazzinelli G, et al. (1993) Characterization of inflammatory infiltrates in chronic chagasic myocardial lesions: presence of tumor necrosis factor-alpha+ cells and dominance of granzyme A+, CD8+ lymphocytes. Am J Trop Med Hyg 48: 637–644. pmid:8517482
  49. 49. Gomes JA, Bahia-Oliveira LM, Rocha MO, Martins-Filho OA, Gazzinelli G, et al. (2003) Evidence that development of severe cardiomyopathy in human Chagas' disease is due to a Th1-specific immune response. Infect Immun 71: 1185–1193. pmid:12595431
  50. 50. Higuchi MD, Ries MM, Aiello VD, Benvenuti LA, Gutierrez PS, et al. (1997) Association of an increase in CD8+ T cells with the presence of Trypanosoma cruzi antigens in chronic, human, chagasic myocarditis. Am J Trop Med Hyg 56: 485–489. pmid:9180594
  51. 51. Higuchi Mde L, De Brito T, Martins Reis M, Barbosa A, Bellotti G, et al. (1993) Correlation between Trypanosoma cruzi parasitism and myocardial inflammatory infiltrate in human chronic chagasic myocarditis: Light microscopy and immunohistochemical findings. Cardiovasc Pathol 2: 101–106. pmid:25990604
  52. 52. Reis MM, Higuchi Mde L, Benvenuti LA, Aiello VD, Gutierrez PS, et al. (1997) An in situ quantitative immunohistochemical study of cytokines and IL-2R+ in chronic human chagasic myocarditis: correlation with the presence of myocardial Trypanosoma cruzi antigens. Clin Immunol Immunopathol 83: 165–172. pmid:9143377
  53. 53. Padilla AM, Bustamante JM, Tarleton RL (2009) CD8+ T cells in Trypanosoma cruzi infection. Curr Opin Immunol 21: 385–390. pmid:19646853
  54. 54. Levin MJ, Mesri E, Benarous R, Levitus G, Schijman A, et al. (1989) Identification of major Trypanosoma cruzi antigenic determinants in chronic Chagas' heart disease. Am J Trop Med Hyg 41: 530–538. pmid:2479275
  55. 55. Lopez Bergami P, Scaglione J, Levin MJ (2001) Antibodies against the carboxyl-terminal end of the Trypanosoma cruzi ribosomal P proteins are pathogenic. FASEB J 15: 2602–2612. pmid:11726536
  56. 56. Girones N, Rodriguez CI, Basso B, Bellon JM, Resino S, et al. (2001) Antibodies to an epitope from the Cha human autoantigen are markers of Chagas' disease. Clin Diagn Lab Immunol 8: 1039–1043. pmid:11687436
  57. 57. Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140: 805–820. pmid:20303872
  58. 58. Koberle F (1968) Chagas' disease and Chagas' syndromes: the pathology of American trypanosomiasis. Adv Parasitol 6: 63–116. pmid:4239747
  59. 59. Steenholdt C, Andresen L, Pedersen G, Hansen A, Brynskov J (2009) Expression and function of toll-like receptor 8 and Tollip in colonic epithelial cells from patients with inflammatory bowel disease. Scand J Gastroenterol 44: 195–204. pmid:18985539
  60. 60. Sanchez-Munoz F, Fonseca-Camarillo G, Villeda-Ramirez MA, Miranda-Perez E, Mendivil EJ, et al. (2011) Transcript levels of Toll-Like Receptors 5, 8 and 9 correlate with inflammatory activity in Ulcerative Colitis. BMC Gastroenterol 11: 138. pmid:22185629
  61. 61. Ortiz-Fernandez L, Garcia-Lozano JR, Montes-Cano MA, Conde-Jaldon M, Leo E, et al. (2015) Association of haplotypes of the TLR8 locus with susceptibility to Crohn's and Behcet's diseases. Clin Exp Rheumatol 33: S117–122.
  62. 62. Saruta M, Targan SR, Mei L, Ippoliti AF, Taylor KD, et al. (2009) High-frequency haplotypes in the X chromosome locus TLR8 are associated with both CD and UC in females. Inflamm Bowel Dis 15: 321–327. pmid:18942751
  63. 63. Gorden KB, Gorski KS, Gibson SJ, Kedl RM, Kieper WC, et al. (2005) Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J Immunol 174: 1259–1268. pmid:15661881
  64. 64. Koberle F (1962) [Quantitative pathology of the vegetative nervous system]. Wien Klin Wochenschr 74: 144–151. pmid:14457498
  65. 65. Andrade SG, Andrade ZA (1966) [Chagas' disease and neuron changes in Auerbach's plexus. (Experimental study in mice)]. Rev Inst Med Trop Sao Paulo 8: 219–224. pmid:4967920
  66. 66. Tafuri WL (1987) [Pathogenesis of Chagas' disease]. Rev Inst Med Trop Sao Paulo 29: 194–199. pmid:3130653
  67. 67. Frade AF, Pissetti CW, Ianni BM, Saba B, Lin-Wang HT, et al. (2013) Genetic susceptibility to Chagas disease cardiomyopathy: involvement of several genes of the innate immunity and chemokine-dependent migration pathways. BMC Infect Dis 13: 587. pmid:24330528
  68. 68. Correa-Oliveira R, Gomes J, Lemos EM, Cardoso GM, Reis DD, et al. (1999) The role of the immune response on the development of severe clinical forms of human Chagas disease. Mem Inst Oswaldo Cruz 94 Suppl 1: 253–255.
  69. 69. Schroder K, Tschopp J (2010) The inflammasomes. Cell 140: 821–832. pmid:20303873
  70. 70. Feldmeyer L, Keller M, Niklaus G, Hohl D, Werner S, et al. (2007) The inflammasome mediates UVB-induced activation and secretion of interleukin-1beta by keratinocytes. Curr Biol 17: 1140–1145. pmid:17600714
  71. 71. McCoy AJ, Koizumi Y, Toma C, Higa N, Dixit V, et al. (2010) Cytotoxins of the human pathogen Aeromonas hydrophila trigger, via the NLRP3 inflammasome, caspase-1 activation in macrophages. Eur J Immunol 40: 2797–2803. pmid:20722078
  72. 72. Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, et al. (2008) Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 9: 847–856. pmid:18604214
  73. 73. Minutoli L, Puzzolo D, Rinaldi M, Irrera N, Marini H, et al. (2016) ROS-Mediated NLRP3 Inflammasome Activation in Brain, Heart, Kidney, and Testis Ischemia/Reperfusion Injury. Oxid Med Cell Longev 2016: 2183026. pmid:27127546
  74. 74. Mariathasan S, Weiss DS, Newton K, McBride J, O'Rourke K, et al. (2006) Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440: 228–232. pmid:16407890
  75. 75. Griffith JW, Sun T, McIntosh MT, Bucala R (2009) Pure Hemozoin is inflammatory in vivo and activates the NALP3 inflammasome via release of uric acid. J Immunol 183: 5208–5220. pmid:19783673
  76. 76. Gasse P, Riteau N, Charron S, Girre S, Fick L, et al. (2009) Uric acid is a danger signal activating NALP3 inflammasome in lung injury inflammation and fibrosis. Am J Respir Crit Care Med 179: 903–913. pmid:19218193
  77. 77. Yamasaki K, Muto J, Taylor KR, Cogen AL, Audish D, et al. (2009) NLRP3/cryopyrin is necessary for interleukin-1beta (IL-1beta) release in response to hyaluronan, an endogenous trigger of inflammation in response to injury. J Biol Chem 284: 12762–12771. pmid:19258328
  78. 78. Ebersole JL, Cappelli D (2000) Acute-phase reactants in infections and inflammatory diseases. Periodontol 2000 23: 19–49.
  79. 79. Keles GC, Cetinkaya BO, Simsek SB, Koprulu D, Kahraman H (2007) The role of periodontal disease on acute phase proteins in patients with coronary heart disease and diabetes. Turk J Med Sci 37: 6.
  80. 80. Bisoendial RJ, Boekholdt SM, Vergeer M, Stroes ES, Kastelein JJ (2010) C-reactive protein is a mediator of cardiovascular disease. Eur Heart J 31: 2087–2091. pmid:20685682
  81. 81. Biasucci LM, Giubilato G, Biondi-Zoccai G, Sanna T, Liuzzo G, et al. (2006) C reactive protein is associated with malignant ventricular arrhythmias in patients with ischaemia with implantable cardioverter-defibrillator. Heart 92: 1147–1148. pmid:16844868
  82. 82. Bravo-Tobar ID, Nello-Perez C, Fernandez A, Mogollon N, Perez MC, et al. (2015) Adenosine Deaminase Activity and Serum C-Reactive Protein as Prognostic Markers of Chagas Disease Severity. Rev Inst Med Trop Sao Paulo 57: 385–392. pmid:26603224
  83. 83. Melo Coutinho CM, Cavalcanti GH, Bonaldo MC, Mortensen RF, Araujo-Jorge TC (1998) Trypanosoma cruzi: detection of a surface antigen cross-reactive to human C-reactive protein. Exp Parasitol 90: 143–153. pmid:9769244
  84. 84. Aparecida da Silva C, Fattori A, Sousa AL, Mazon SB, Monte Alegre S, et al. (2010) Determining the C-reactive protein level in patients with different clinical forms of chagas disease. Rev Esp Cardiol 63: 1096–1099. pmid:20804707
  85. 85. Lopez L, Arai K, Gimenez E, Jimenez M, Pascuzo C, et al. (2006) [C-reactive protein and interleukin-6 serum levels increase as Chagas disease progresses towards cardiac failure]. Rev Esp Cardiol 59: 50–56.
  86. 86. Gutierrez FR, Guedes PM, Gazzinelli RT, Silva JS (2009) The role of parasite persistence in pathogenesis of Chagas heart disease. Parasite Immunol 31: 673–685. pmid:19825107
  87. 87. Guedes PM, Veloso VM, Caliari MV, Carneiro CM, Souza SM, et al. (2007) Trypanosoma cruzi high infectivity in vitro is related to cardiac lesions during long-term infection in Beagle dogs. Mem Inst Oswaldo Cruz 102: 141–147. pmid:17426876
  88. 88. Zingales B, Miles MA, Campbell DA, Tibayrenc M, Macedo AM, et al. (2012) The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect Genet Evol 12: 240–253. pmid:22226704
  89. 89. Hasne MP, Soysa R, Ullman B (2016) The Trypanosoma cruzi Diamine Transporter Is Essential for Robust Infection of Mammalian Cells. PLoS One 11: e0152715. pmid:27050410
  90. 90. Dias FC, Medina Tda S, Mendes-Junior CT, Dantas RO, Pissetti CW, et al. (2013) Polymorphic sites at the immunoregulatory CTLA-4 gene are associated with chronic chagas disease and its clinical manifestations. PLoS One 8: e78367. pmid:24205212
  91. 91. Dias FC, Mendes-Junior CT, Silva MC, Tristao FS, Dellalibera-Joviliano R, et al. (2015) Human leucocyte antigen-G (HLA-G) and its murine functional homolog Qa2 in the Trypanosoma cruzi Infection. Mediators Inflamm 2015: 595829. pmid:25688175
  92. 92. Nogueira LG, Frade AF, Ianni BM, Laugier L, Pissetti CW, et al. (2015) Functional IL18 polymorphism and susceptibility to Chronic Chagas Disease. Cytokine 73: 79–83. pmid:25743241
  93. 93. Andrade LO, Machado CR, Chiari E, Pena SD, Macedo AM (1999) Differential tissue distribution of diverse clones of Trypanosoma cruzi in infected mice. Mol Biochem Parasitol 100: 163–172. pmid:10391378
  94. 94. Guedes PM, Veloso VM, Afonso LC, Caliari MV, Carneiro CM, et al. (2009) Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-gamma, TNF-alpha, and low IL-10 production during the acute infection phase. Vet Immunol Immunopathol 130: 43–52. pmid:19211152
  95. 95. Andrade ZA, Andrade SG (1955) [Pathogenesis of Chagas' chronic myocarditis; importance of ischemic lesions]. Arq Bras Med 45: 279–288. pmid:13283896
  96. 96. Rossi MA, Goncalves S, Ribeiro-dos-Santos R (1984) Experimental Trypanosoma cruzi cardiomyopathy in BALB/c mice. The potential role of intravascular platelet aggregation in its genesis. Am J Pathol 114: 209–216. pmid:6230012
  97. 97. Higuchi Mde L, Benvenuti LA, Martins Reis M, Metzger M (2003) Pathophysiology of the heart in Chagas' disease: current status and new developments. Cardiovasc Res 60: 96–107. pmid:14522411