Discovery of circulating miRNAs as biomarkers of chronic Chagas heart disease via a small RNA-Seq approach.

Chagas disease affects approximately 7 million people worldwide in Latin America and is a neglected tropical disease. Twenty to thirty percent of chronically infected patients develop chronic Chagas cardiomyopathy decades after acute infection. Identifying biomarkers of Chagas disease progression is necessary to develop better therapeutic and preventive strategies. Circulating microRNAs are increasingly reliable biomarkers of disease and therapeutic targets. To identify new circulating microRNAs for Chagas disease, we performed exploratory small RNA sequencing from the plasma of patients and performed de novo miRNA prediction, identifying potential new microRNAs. The levels of the new microRNAs temporarily named miR-Contig-1519 and miR-Contig-3244 and microRNAs that are biomarkers for nonchagasic cardiomyopathies, such as miR-148a-3p and miR-224-5p, were validated by quantitative reverse transcription. We found a specific circulating microRNA signature defined by low miR-Contig-3244, miR-Contig-1519, and miR-148a-3 levels but high miR-224-5p levels for patients with chronic Chagas disease. Finally, we predicted in silico that these altered circulating microRNAs could affect the expression of target genes involved in different cellular pathways and biological processes, which we will explore in the future.

Immune-neuroendocrine and metabolic disorders in human and experimental T. cruzi infection: New clues for understanding Chagas disease pathology.

Studies in mice undergoing acute Trypanosoma cruzi infection and patients with Chagas disease, led to identify several immune-neuroendocrine disturbances and metabolic disorders. Here, we review relevant findings concerning such abnormalities and discuss their possible influence on disease physiopathology.

Immune response triggered by Trypanosoma cruzi infection strikes adipose tissue homeostasis altering lipid storage, enzyme profile and adipokine expression.

Adipose tissue is a target of Trypanosoma cruzi infection being a parasite reservoir during the chronic phase in mice and humans. Previously, we reported that acute Trypanosoma cruzi infection in mice is linked to a severe adipose tissue loss, probably triggered by inflammation, as well as by the parasite itself. Here, we evaluated how infection affects adipose tissue homeostasis, considering adipocyte anabolic and catabolic pathways, the immune-endocrine pattern and the possible repercussion upon adipogenesis. During in vivo infection, both lipolytic and lipogenic pathways are profoundly affected, since the expression of lipolytic enzymes and lipogenic enzymes was intensely downregulated. A similar pattern was observed in isolated adipocytes from infected animals and in 3T3-L1 adipocytes infected in vitro with Trypanosoma cruzi. Moreover, 3T3-L1 adipocytes exposed to plasmas derived from infected animals also tend to downregulate lipolytic enzyme expression which was less evident regarding lipogenic enzymes. Moreover, in vivo-infected adipose tissue reveals a pro-inflammatory profile, with increased leucocyte infiltration accompanied by TNF and IL-6 overexpression, and adiponectin downregulation. Strikingly, the nuclear factor PPAR-γ is strongly decreased in adipocytes during in vivo infection. Attempts to favor PPAR-γ-mediated actions in the adipose tissue of infected animals using agonists failed, indicating that inflammation or parasite-derived factors are strongly involved in PPAR-γ inhibition. Here, we report that experimental acute Trypanosoma cruzi infection disrupts both adipocyte catabolic and anabolic metabolism secondary to PPAR-γ robust downregulation, tipping the balance towards to an adverse status compatible with the adipose tissue atrophy and the acquisition of an inflammatory phenotype.

Evidence in Favor of an Alternative Glucocorticoid Synthesis Pathway During Acute Experimental Chagas Disease.

It is well-established that infectious stress activates the hypothalamus-pituitary-adrenal axis leading to the production of pituitary adrenocorticotropin (ACTH) and adrenal glucocorticoids (GCs). Usually, GC synthesis is mediated by protein kinase A (PKA) signaling pathway triggered by ACTH. We previously demonstrated that acute murine Chagas disease courses with a marked increase of GC, with some data suggesting that GC synthesis may be ACTH-dissociated in the late phase of this parasitic infection. Alternative pathways of GC synthesis have been reported in sepsis or mental diseases, in which interleukin (IL)-1ß, prostaglandin E2 (PGE2), and/or cAMP-activated guanine nucleotide exchange factor 2 (EPAC2) are likely to play a role in this regard. Accordingly, we have searched for the existence of an ACTH-independent pathway in an experimental model of a major parasitic disease like Chagas disease, in addition to characterizing potential alternative pathways of GC synthesis. To this end, C57BL/6 male mice were infected with T. cruzi (Tc), and evaluated throughout the acute phase for several parameters, including the kinetic of GC and ACTH release, the adrenal level of MC2R (ACTH receptor) expression, the p-PKA/PKA ratio as ACTH-dependent mechanism of signal transduction, as well as adrenal expression of IL-1ß and its receptor, EPAC2 and PGE2 synthase. Our results reveal the existence of two phases involved in GC synthesis during Tc infection in mice, an initial one dealing with the well-known ACTH-dependent pathway, followed by a further ACTH-hyporesponsive phase. Furthermore, inflamed adrenal microenvironment may tune the production of intracellular mediators that also operate upon GC synthesis, like PGE2 synthase and EPAC2, as emerging driving forces for GC production in the advanced course of Tc infection. In essence, GC production seems to be associated with a biphasic action of PGE2, suggesting that the effect of PGE2/cAMP in the ACTH-independent second phase may be mediated by EPAC2.

Genetic Engineering of Lactococcus lactis Co-producing Antigen and the Mucosal Adjuvant 3' 5'- cyclic di Adenosine Monophosphate (c-di-AMP) as a Design Strategy to Develop a Mucosal Vaccine Prototype.

Lactococcus lactis is a promising candidate for the development of mucosal vaccines. More than 20 years of experimental research supports this immunization approach. In addition, 3' 5'- cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that plays a key role in the regulation of diverse physiological functions (potassium and cellular wall homeostasis, among others). Moreover, recent studies showed that c-di-AMP has a strong mucosal adjuvant activity that promotes both humoral and cellular immune responses. In this study, we report the development of a novel mucosal vaccine prototype based on a genetically engineered L. lactis strain. First, we demonstrate that homologous expression of cdaA gen in L. lactis is able to increase c-di-AMP levels. Thus, we hypothesized that in vivo synthesis of the adjuvant can be combined with production of an antigen of interest in a separate form or jointly in the same strain. Therefore, a specifically designed fragment of the trans-sialidase (TScf) enzyme from the Trypanosoma cruzi parasite, the etiological agent of Chagas disease, was selected to evaluate as proof of concept the immune response triggered by our vaccine prototypes. Consequently, we found that oral administration of a L. lactis strain expressing antigenic TScf combined with another L. lactis strain producing the adjuvant c-di-AMP could elicit a TS-specific immune response. Also, an additional L. lactis strain containing a single plasmid with both cdaA and tscf genes under the Pcit and Pnis promoters, respectively, was also able to elicit a specific immune response. Thus, the current report is the first one to describe an engineered L. lactis strain that simultaneously synthesizes the adjuvant c-di-AMP as well as a heterologous antigen in order to develop a simple and economical system for the formulation of vaccine prototypes using a food grade lactic acid bacterium.

Disruption of tumor necrosis factor alpha receptor 1 signaling accelerates NAFLD progression in mice upon a high-fat diet.

Obesity is accompanied by a low-grade inflammation state, characterized by increased proinflammatory cytokines levels such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1ß). In this regard, there exists a lack of studies in hepatic tissue about the role of TNFα receptor 1 (TNFR1) in the context of obesity and insulin resistance during the progression of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to evaluate the effects of high-caloric feeding (HFD) (40% fat, for 16 weeks) on liver inflammation-induced apoptosis, insulin resistance, hepatic lipid accumulation and its progression toward nonalcoholic steatohepatitis (NASH) in TNFR1 knock-out and wild-type mice. Mechanisms involved in HFD-derived IL-1ß release and impairment of insulin signaling are still unknown, so we determined whether IL-1ß affects liver insulin sensitivity and apoptosis through TNFα receptor 1 (TNFR1)-dependent pathways. We showed that knocking out TNFR1 induces an enhanced IL-1ß plasmatic release upon HFD feed. This was correlated with higher hepatic and epididymal white adipose tissue mRNA levels. In vivo and in vitro assays confirmed an impairment in hepatic insulin signaling, in part due to IL-1ß-induced decrease of AKT activation and diminution of IRS1 levels, followed by an increase in inflammation, macrophage (resident and recruited) accumulation, hepatocyte apoptotic process and finally hepatic damage. In addition, TNFR1 KO mice displayed higher levels of pro-fibrogenic markers. TNFR1 signaling disruption upon an HFD leads to an accelerated progression from simple steatosis to a more severe phenotype with many NASH features, pointing out a key role of TNFR1 in NAFLD progression.

Death of adrenocortical cells during murine acute T. cruzi infection is not associated with TNF-R1 signaling but mostly with the type II pathway of Fas-mediated apoptosis.

Earlier studies from our laboratory demonstrated that acute experimental Trypanosoma cruzi infection promotes an intense inflammation along with a sepsis-like dysregulated adrenal response characterized by normal levels of ACTH with raised glucocorticoid secretion. Inflammation was also known to result in adrenal cell apoptosis, which in turn may influence HPA axis uncoupling. To explore factors and pathways which may be involved in the apoptosis of adrenal cells, together with its impact on the functionality of the gland, we carried out a series of studies in mice lacking death receptors, such as TNF-R1 (C57BL/6-Tnfrsf1a tm1Imx or TNF-R1-/-) or Fas ligand (C57BL/6 Fas-deficient lpr mice), undergoing acute T. cruzi infection. Here we demonstrate that the late hypercorticosterolism seen in C57BL/6 mice during acute T. cruzi infection coexists with and hyperplasia and hypertrophy of zona fasciculata, paralleled by increased number of apoptotic cells. Apoptosis seems to be mediated mainly by the type II pathway of Fas-mediated apoptosis, which engages the mitochondrial pathway of apoptosis triggering the cytochrome c release to increase caspase-3 activation. Fas-induced apoptosis of adrenocortical cells is also related with an exacerbated production of intra-adrenal cytokines that probably maintain the late supply of adrenal hormones during host response. Present results shed light on the molecular mechanisms dealing with these phenomena which are crucial not only for the development of interventions attempting to avoid adrenal dysfunction, but also for its wide occurrence in other infectious-based critical illnesses.

Evasion and Immuno-Endocrine Regulation in Parasite Infection: Two Sides of the Same Coin in Chagas Disease?

Chagas disease is a serious illness caused by the protozoan parasite Trypanosoma cruzi. Nearly 30% of chronically infected people develop cardiac, digestive, or mixed alterations, suggesting a broad range of host-parasite interactions that finally impact upon chronic disease outcome. The ability of T. cruzi to persist and cause pathology seems to depend on diverse factors like T. cruzi strains, the infective load and the route of infection, presence of virulence factors, the parasite capacity to avoid protective immune response, the strength and type of host defense mechanisms and the genetic background of the host. The host-parasite interaction is subject to a constant neuro-endocrine regulation that is thought to influence the adaptive immune system, and as the infection proceeds it can lead to a broad range of outcomes, ranging from pathogen elimination to its continued persistence in the host. In this context, T. cruzi evasion strategies and host defense mechanisms can be envisioned as two sides of the same coin, influencing parasite persistence and different outcomes observed in Chagas disease. Understanding how T. cruzi evade host's innate and adaptive immune response will provide important clues to better dissect mechanisms underlying the pathophysiology of Chagas disease.

Immunoendocrine dysbalance during uncontrolled T. cruzi infection is associated with the acquisition of a Th-1-like phenotype by Foxp3(+) T cells.

We previously showed that Trypanosomacruzi infection in C57BL/6 mice results in a lethal infection linked to unbalanced pro- and anti-inflammatory mediators production. Here, we examined the dynamics of CD4(+)Foxp3(+) regulatory T (Treg) cells within this inflammatory and highly Th1-polarized environment. Treg cells showed a reduced proliferation rate and their frequency is progressively reduced along infection compared to effector T (Teff) cells. Also, a higher fraction of Treg cells showed a naïve phenotype, meanwhile Teff cells were mostly of the effector memory type. T. cruzi infection was associated with the production of pro- and anti-inflammatory cytokines, notably IL-27p28, and with the induction of T-bet and IFN-γ expression in Treg cells. Furthermore, endogenous glucocorticoids released in response to T. cruzi-driven immune activation were crucial to sustain the Treg/Teff cell balance. Notably, IL-2 plus dexamethasone combined treatment before infection was associated with increased Treg cell proliferation and expression of GATA-3, IL-4 and IL-10, and increased mice survival time. Overall, our results indicate that therapies aimed at specifically boosting Treg cells, which during T. cruzi infection are overwhelmed by the effector immune response, represent new opportunities for the treatment of Chagas disease, which is actually only based on parasite-targeted chemotherapy.

Tumor necrosis factor-α regulates glucocorticoid synthesis in the adrenal glands of Trypanosoma cruzi acutely-infected mice. the role of TNF-R1.

Adrenal steroidogenesis is under a complex regulation involving extrinsic and intrinsic adrenal factors. TNF-α is an inflammatory cytokine produced in response to tissue injury and several other stimuli. We have previously demonstrated that TNF-R1 knockout (TNF-R1(-/-)) mice have a dysregulated synthesis of glucocorticoids (GCs) during Trypanosoma cruzi acute infection. Since TNF-α may influence GCs production, not only through the hypothalamus-pituitary axis, but also at the adrenal level, we now investigated the role of this cytokine on the adrenal GCs production. Wild type (WT) and TNF-R1(-/-) mice undergoing acute infection (Tc-WT and Tc-TNF-R1(-/-) groups), displayed adrenal hyperplasia together with increased GCs levels. Notably, systemic ACTH remained unchanged in Tc-WT and Tc-TNF-R1(-/-) compared with uninfected mice, suggesting some degree of ACTH-independence of GCs synthesis. TNF-α expression was increased within the adrenal gland from both infected mouse groups, with Tc-WT mice showing an augmented TNF-R1 expression. Tc-WT mice showed increased levels of P-p38 and P-ERK compared to uninfected WT animals, whereas Tc-TNF-R1(-/-) mice had increased p38 and JNK phosphorylation respect to Tc-WT mice. Strikingly, adrenal NF-κB and AP-1 activation during infection was blunted in Tc-TNF-R1(-/-) mice. The accumulation of mRNAs for steroidogenic acute regulatory protein and cytochrome P450 were significantly increased in both Tc-WT and Tc-TNF-R1(-/-) mice; being much more augmented in the latter group, which also had remarkably increased GCs levels. TNF-α emerges as a potent modulator of steroidogenesis in adrenocortical cells during T. cruzi infection in which MAPK pathways, NF-κB and AP-1 seem to play a role in the adrenal synthesis of pro-inflammatory cytokines and enzymes regulating GCs synthesis. These results suggest the existence of an intrinsic immune-adrenal interaction involved in the dysregulated synthesis of GCs during murine Chagas disease.

Immunoendocrinology of the thymus in Chagas disease.

During immune response to infectious agents, the host develops an inflammatory response which could fail to eliminate the pathogen or may become dysregulated. In this case, the ongoing response acquires a new status and turns out to be detrimental. The same elements taking part in the establishment and regulation of the inflammatory response (cytokines, chemokines, regulatory T cells and counteracting compounds like glucocorticoids) may also mediate harmful effects. Thymic disturbances seen during Trypanosoma cruzi (T. cruzi) infection fit well with this conceptual framework. After infection, this organ suffers a severe atrophy due to apoptosis-induced thymocyte exhaustion, mainly affecting the immature double-positive (DP) CD4+CD8+ population. Thymus cellularity depletion, which occurs in the absence of main immunological mediators involved in anti-T. cruzi defense, seems to be linked to a systemic cytokine/hormonal imbalance, involving a dysregulated increase in Tumor Necrosis Factor alpha (TNF-α) and corticosterone hormone levels. Additionally, we have found an anomalous exit of potentially autoimmune DP cells to the periphery, in parallel to a shrinkage in the compartment of natural regulatory T cells. In this context, our data clearly point to the view that the thymus is a target organ of T. cruzi infection. Preserved thymus may be essential for the development of an effective immune response against T. cruzi, but this organ is severely affected by a dysregulated circuit of proinflammatory cytokines and glucocorticoids. Also, the alterations observed in the DP population might have potential implications for the autoimmune component of human Chagas disease.

Time course of organic anion excretion in rats with bilateral ureteral obstruction: role of organic anion transporters (Oat1 and Oat3).

BACKGROUND: Urinary tract obstruction is a common cause of renal failure. In this study, we evaluated the time course of P-aminohippurate (PAH) renal excretion and the cortical expression of organic anion transporters (Oat1 and Oat3) at 1 (BUO-1), 2 (BUO-2) and 7 (BUO-7) days after release of 24-hour bilateral ureteral obstruction (BUO) in the rat. METHODS: Conventional clearance technique, differential centrifugation, semiquantitative immunoblotting and immunohistochemical techniques have been employed. RESULTS: These studies showed that Oat1 and Oat3 in basolateral membranes were downregulated both at BUO-1 and BUO-2. Concomitantly, the rats developed a reduction in PAH renal elimination. In contrast, total recovery in PAH renal excretion and in the expression of Oat1 and Oat3 were observed at BUO-7, as compared with the sham group. A direct correlation was observed between the secretory clearance of PAH and Oat1 (r(2) = 0.88) and Oat3 (r(2) = 0.83) expression in basolateral membranes. CONCLUSION: These results indicate that the differential expression of organic anion transporters is one of the main molecular mechanisms contributing to the organic anion excretion modifications observed during the time course of obstructive nephropathy. This study provides evidence regarding the importance of adjusting the dose regimens of negatively charged drugs during the different time phases of this pathology.

Expression of rat renal cortical OAT1 and OAT3 in response to acute biliary obstruction.

Renal function in the course of obstructive jaundice has been the subject of great interest; however, little is known about the expression of renal organic anion transporters. The objective of this work was to study, in rats with acute extrahepatic cholestasis, the cortical renal expression of the organic anion transporter 1 (OAT1) and the organic anion transporter 3 (OAT3), in association with the pharmacokinetics and renal excretion of furosemide (FS). Male Wistar rats underwent bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. All studies were carried out 21 hours after surgery. Rats were anesthetized and the pharmacokinetic parameters of FS and the renal elimination of FS were determined. Afterwards, the kidneys were excised and processed for immunoblot (basolateral membrane and renal homogenates) or immunocytochemical (light microscopic and confocal immunofluorescence microscopic analysis) techniques. The systemic and renal clearance of FS as well as the excreted and secreted load of FS increased in BDL rats. In kidneys from BDL rats, immunoblotting showed a significant increase in the abundance of both OAT1 and OAT3 in homogenates from renal cortex. In basolateral membranes from kidney cortex of BDL rats, OATI abundance was also increased and OAT3 abundance was not modified. Immunocytochemical techniques confirmed these results. In conclusion, acute obstructive jaundice is associated with an upregulation of OAT1 and OAT3, which might explain, at least in part, the increased systemic and renal elimination of FS.

Altered expression of rat renal cortical OAT1 and OAT3 in response to bilateral ureteral obstruction.

BACKGROUND: Bilateral ureteral obstruction (BUO) is characterized by the development of hemodynamic and tubular lesions. However, little is known about the expression of organic anion renal transporters. The objective of this work was to study the renal excretion of p-aminohippurate (PAH) and the cortical renal expression of the organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) in BUO rats. METHODS: Male Wistar rats underwent bilateral obstruction of the proximal ureters for 24 hours (BUO) or sham operation. After 24 hours of ureteral releasing, the following studies were performed: PAH renal excretion employing conventional clearance techniques and OAT1 and OAT3 abundance (homogenates, intracellular and basolateral plasma membrane fractions from renal cortex) using immunoblotting and immunocytochemical techniques (light microscopic and confocal immunofluorescence microscopic analysis). RESULTS: BUO rats showed a lower renal excretion of PAH. In obstructed kidneys, immunoblotting revealed a significant decrease in the abundance of both OAT1 and OAT3 in basolateral plasma membranes from renal cortex. An increase of OAT1 expression was observed in homogenates and in intracellular membrane fractions in kidneys from BUO rats compared with sham-operated ones, indicating an internalization of this carrier. Immunocytochemical techniques confirmed these results. On the contrary, OAT3 expression was reduced both in homogenates and in intracellular membrane fractions in obstructed kidneys. CONCLUSION: BUO was associated with down-regulation of OAT1 and OAT3 in basolateral plasma membranes from proximal tubule cells, thus these carriers may play important roles in the impaired organic anion excretion displayed in the obstructed kidney.

Renal elimination of organic anions in rats with bilateral ureteral obstruction.

Urinary tract obstruction is an important cause of acute renal failure. Several abnormalities in renal tubular function may occur in obstructive nephropathy. The tubular secretion of organic anions is an important function of the kidney that eliminates potentially toxic organic anions from the body, however, the mechanisms involved in organic anions renal elimination in rats with bilateral ureteral obstruction (BUO) have not been elucidated. In this study, it was evaluated the renal handling of p-aminohippurate (PAH) in adult male Wistar rats with BUO. A diminished renal clearance of PAH was observed in BUO rats as consequence of a diminution in the secreted load of this organic anion. The increase in the abundance of organic anions transporter 1 (OAT1) and the absence of modification in cortical renal blood flow, measured with fluorescence microspheres, do not explain the altered secretion of PAH. The diminished Na,K-ATPase activity in cortex from obstructed kidneys might condition OAT1 function. Additionally, it is also possible to conclude that in the presence of BUO, PAH clearance is not a good estimate of renal plasma flow.

Haemodynamic and tubular renal dysfunction in rats with sustained arterial calcinosis.

1. In humans, two of the principal characteristics of vascular ageing are arterial wall calcification and decreased arterial distensibility, which induce organ damage. To amplify arterial calcium accumulation in laboratory animals, it is necessary to use an overdose of vitamin D(3). 2. The aim of the present study was to assess the impact of arterial calcium overload on renal function. 3. Adult male Wistar rats were randomly divided into two groups: control and treated rats. Treated rats were injected 10 days before the experiment with a single dose of vitamin D(3) (300 000 IU/kg, i.m.). 4. Treated rats showed a decrease in renal blood flow and glomerular filtration rate. Tubular parameters were not modified under basal conditions. In contrast, a statistically significant increase in the fractional excretion of Na, K, Ca and H(2)O were observed in treated rats after the acute increment of sodium distal delivery, suggesting that the reabsorptive capacity of the thick ascending limb may be altered in treated rats. 5. Thus, Na(+)/K(+)-ATPase activity was evaluated in homogenates from renal cortex and medulla. Rats with arterial calcinosis presented a diminished activity of Na(+)/K(+)-ATPase in medulla homogenates. 6. An increment in the abundance of the Na-K-2Cl cotransporter (NKCC2) was observed in renal medulla homogenates from treated rats. It is suggested that this may compensate for the inefficiency of Na(+)/K(+)-ATPase under basal conditions but, in the presence of acute distal sodium overload, the increment in NKCC2 abundance may not be sufficient to compensate for the decrease in Na(+)/K(+)-ATPase activity. 7. In summary, in our experimental model of arterial calcinosis, renal function is impaired, presenting a vascular compromise and altered function of the medullar thick ascending limb that becomes evident in the presence of acute high distal sodium delivery.

Gender-related differences in the pharmacodynamics of furosemide in rats.

The possibility of gender differences in the relationship between dose and efficacy of drugs has historically been limited. The aim of the present study was to evaluate the influence of sex on the pharmacodynamics of furosemide (FUR) in adult Wistar rats. Conventional renal clearance studies were performed in the absence and in the presence of a FUR dose necessary to produce identical urine excretion rates of FUR in both sexes. Fractional excretions of sodium, potassium, and water were similar in male and female rats in the absence of FUR. Natriuretic, kaliuretic, and diuretic responses to FUR, when expressed in fractional terms, were significantly increased in female rats. Diuretic, natriuretic, and kaliuretic efficiencies of FUR were also higher in female rats as compared with males. This might be explained by the lower abundance of the Na-K-2Cl cotransporter observed in homogenates from the medullae of female kidneys as compared with male ones.