Effect of peroxisome proliferator-activated receptor-alpha ligands in the interaction between adipocytes and macrophages in obese adipose tissue

Objective: This study was designed to examine the effect of peroxisome proliferator‐activated receptor‐α (PPAR‐α) ligands on the inflammatory changes induced by the interaction between adipocytes and macrophages in obese adipose tissue. Methods and Procedures: PPAR‐α ligands (Wy‐14,643 and fenofibrate) were added to 3T3‐L1 adipocytes, RAW264 macrophages, or co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages in vitro, and monocyte chemoattractant protein‐1 (MCP‐1) and tumor necrosis factor‐α (TNF‐α) mRNA expression and secretion were examined. PPAR‐α ligands were administered to genetically obese ob/ob mice for 2 weeks. Moreover, the effect of PPAR‐α ligands was also evaluated in the adipose tissue explants and peritoneal macrophages obtained from PPAR‐α‐deficient mice. Results: In the co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages, PPAR‐α ligands reduced MCP‐1 and TNF‐α mRNA expression and secretion in vitro relative to vehicle‐treated group. The anti‐inflammatory effect of Wy‐14,643 was observed in adipocytes treated with macrophage‐conditioned media or mouse recombinant TNF‐α and in macrophages treated with adipocyte‐conditioned media or palmitate. Systemic administration of PPAR‐α ligands inhibited the inflammatory changes in adipose tissue from ob/ob mice. Wy‐14,643 also exerted an anti‐inflammatory effect in the adipose tissue explants but not in peritoneal macrophages obtained from PPAR‐α‐deficient mice. Discussion: This study provides evidence for the anti‐inflammatory effect of PPAR‐α ligands in the interaction between adipocytes and macrophages in obese adipose tissue, thereby improving the dysregulation of adipocytokine production and obesity‐related metabolic syndrome.     

Notch receptor expression in Trypanosoma cruzi‐infected human umbilical vein endothelial cells treated with benznidazole or simvastatin revealed by microarray analysis

Chagas disease is a vector‐borne disease caused by the protozoan parasite Trypanosoma cruzi. Current therapy involves benznidazole. Benznidazole and other drugs can modify gene expression patterns, improving the response to the inflammatory influx induced by T. cruzi and decreasing the endothelial activation or immune cell recruitment, among other effects. Here, we performed a microarray analysis of human umbilical vein endothelial cells (HUVECs) treated with benznidazole and the anti‐inflammatory drugs acetylsalicylic acid or simvastatin and infected with T. cruzi. Parasitic infection produces differential expression of a set of genes in HUVECs treated with benznidazole alone or a combination with simvastatin or acetylsalicylic acid. The differentially expressed genes were involved in inflammation, adhesion, cardiac function, and remodeling. Notch1 and high mobility group B1 were genes of interest in this analysis due to their importance in placental development, cardiac development, and inflammation. Quantitative polymerase chain reaction confirmation of these two genes indicated that both are upregulated in the presence of benznidazole.     

The adipocyte as an important target cell for Trypanosoma cruzi infection

Adipose tissue plays an active role in normal metabolic homeostasis as well as in the development of human disease. Beyond its obvious role as a depot for triglycerides, adipose tissue controls energy expenditure through secretion of several factors. Little attention has been given to the role of adipocytes in the pathogenesis of Chagas disease and the associated metabolic alterations. Our previous studies have indicated that hyperglycemia significantly increases parasitemia and mortality in mice infected with Trypanosoma cruzi. We determined the consequences of adipocyte infection in vitro and in vivo. Cultured 3T3-L1 adipocytes can be infected with high efficiency. Electron micrographs of infected cells revealed a large number of intracellular parasites that cluster around lipid droplets. Furthermore, infected adipocytes exhibited changes in expression levels of a number of different adipocyte-specific or adipocyte-enriched proteins. The adipocyte is therefore an important target cell during acute Chagas disease. Infection of adipocytes by T. cruzi profoundly influences the pattern of adipokines. During chronic infection, adipocytes may represent an important long-term reservoir for parasites from which relapse of infection can occur. We have demonstrated that acute infection has a unique metabolic profile with a high degree of local inflammation in adipose tissue, hypoadiponectinemia, hypoglycemia, and hypoinsulinemia but with relatively normal glucose disposal during an oral glucose tolerance test.     

High Fat Diet Modulates Trypanosoma cruzi Infection Associated Myocarditis

Background

Trypanosoma cruzi, the causative agent of Chagas disease, has high affinity for lipoproteins and adipose tissue. Infection results in myocarditis, fat loss and alterations in lipid homeostasis. This study was aimed at analyzing the effect of high fat diet (HFD) on regulating acute T. cruzi infection-induced myocarditis and to evaluate the effect of HFD on lipid metabolism in adipose tissue and heart during acute T. cruzi infection.

Methodology/Principal Findings

CD1 mice were infected with T. cruzi (Brazil strain) and fed either a regular control diet (RD) or HFD for 35 days following infection. Serum lipid profile, tissue cholesterol levels, blood parasitemia, and tissue parasite load were analyzed to evaluate the effect of diet on infection. MicroPET and MRI analysis were performed to examine the morphological and functional status of the heart during acute infection. qPCR and immunoblot analysis were carried out to analyze the effect of diet on the genes involved in the host lipid metabolism during infection. Oil red O staining of the adipose tissue demonstrated reduced lipolysis in HFD compared to RD fed mice. HFD reduced mortality, parasitemia and cardiac parasite load, but increased parasite load in adipocytes. HFD decreased lipolysis during acute infection. Both qPCR and protein analysis demonstrated alterations in lipid metabolic pathways in adipose tissue and heart in RD fed mice, which were further modulated by HFD. Both microPET and MRI analyses demonstrated changes in infected RD murine hearts which were ameliorated by HFD.

Conclusion/Significance

These studies indicate that Chagasic cardiomyopathy is associated with a cardiac lipidpathy and that both cardiac lipotoxicity and adipose tissue play a role in the pathogenesis of Chagas disease. HFD protected mice from T. cruzi infection-induced myocardial damage most likely due to the effects of HFD on both adipogenesis and T. cruzi infection-induced cardiac lipidopathy.     

Fat tissue regulates the pathogenesis and severity of cardiomyopathy in murine chagas disease

Chronic Chagas cardiomyopathy (CCC) caused by a parasite Trypanosoma cruzi is a life-threatening disease in Latin America, for which there is no effective drug or vaccine. The pathogenesis of CCC is complex and multifactorial. Previously, we demonstrated T. cruzi infected mice lose a significant amount of fat tissue which correlates with progression of CCC. Based on this an investigation was undertaken during both acute and chronic T. cruzi infection utilizing the FAT-ATTAC murine model (that allows modulation of fat mass) to understand the consequences of the loss of adipocytes in the regulation of cardiac parasite load, parasite persistence, inflammation, mitochondrial stress, ER stress, survival, CCC progression and CCC severity. Mice were infected intraperitoneally with 5x10⁴ and 10³ trypomastigotes to generate acute and chronic Chagas models, respectively. Ablation of adipocytes was carried out in uninfected and infected mice by treatment with AP21087 for 10 days starting at 15DPI (acute infection) and at 65DPI (indeterminate infection). During acute infection, cardiac ultrasound imaging, histological, and biochemical analyses demonstrated that fat ablation increased cardiac parasite load, cardiac pathology and right ventricular dilation and decreased survival. During chronic indeterminate infection ablation of fat cells increased cardiac pathology and caused bi-ventricular dilation. These data demonstrate that dysfunctional adipose tissue not only affects cardiac metabolism but also the inflammatory status, morphology and physiology of the myocardium and increases the risk of progression and severity of CCC in murine Chagas disease.     

Adipocytes in both brown and white adipose tissue of adult mice are functionally connected via gap junctions: implications for Chagas disease

Adipose tissue serves as a host reservoir for the protozoan Trypanosoma cruzi, the causative organism in Chagas disease. Gap junctions interconnect cells of most tissues, serving to synchronize cell activities including secretion in glandular tissue, and we have previously demonstrated that gap junctions are altered in various tissues and cells infected with T. cruzi. Herein, we examined the gap junction protein connexin 43 (Cx43) expression in infected adipose tissues. Adipose tissue is the largest endocrine organ of the body and is also involved in other physiological functions. In mammals, it is primarily composed of white adipocytes. Although gap junctions are a prominent feature of brown adipocytes, they have not been explored extensively in white adipocytes, especially in the setting of infection. Thus, we examined functional coupling in both white and brown adipocytes in mice. Injection of electrical current or the dye Lucifer Yellow into adipocytes within fat tissue spread to adjacent cells, which was reduced by treatment with agents known to block gap junctions. Moreover, Cx43 was detected in both brown and white fat tissue. At thirty and ninety days post-infection, Cx43 was downregulated in brown adipocytes and upregulated in white adipocytes. Gap junction-mediated intercellular communication likely contributes to hormone secretion and other functions in white adipose tissue and to nonshivering thermogenesis in brown fat, and modulation of the coupling by T. cruzi infection is expected to impact these functions.     

Regulation of host cell cyclin D1 by Trypanosoma cruzi in myoblasts

Infection with the parasite Trypanosoma cruzi causes Chagas disease. In this study we demonstrated that there was an increase in cyclin D1 expression in T. cruzi (Tulahuen strain)-infected myoblasts. To examine a possible mechanism for the increased cyclin D1 expression we transfected L6E9 myoblasts with cyclin D1 luciferase reporter constructs and infected with T. cruzi. There was no evidence of an increase in promoter activity. Additionally, quantitative PCR did not demonstrate any change in cyclin D1 message during infection. Moreover, we demonstrated that the cyclin D1 protein was significantly stabilized after infection. Collectively, these data indicate that infection with T. cruzi increases cyclin D1 protein abundance post-translationally.     

Role of CCL3/MIP-1α and CCL5/RANTES during acute Trypanosoma cruzi infection in rats

Chagas’ disease is caused by Trypanosoma cruzi infection and is characterized by chronic fibrogenic inflammation and heart dysfunction. Chemokines are produced during infection and drive tissue inflammation. In rats, acute infection is characterized by intense myocarditis and regression of inflammation after control of parasitism. We investigated the role of CCL3 and CCL5 during infection by using DNA vaccination encoding for each chemokine separately or simultaneously. MetRANTES treatment was used to evaluate the role of CCR1 and CCR5, the receptors for CCL3 and CCL5. Vaccination with CCL3 or CCL5 increased heart parasitism and decreased local IFN-γ production, but did not influence intensity of inflammation. Simultaneous treatment with both plasmids or treatment with MetRANTES enhanced cardiac inflammation, fibrosis and parasitism. In conclusion, chemokines CCL3 and CCL5 are relevant, but not essential, for control of T. cruzi infection in rats. On the other hand, combined blockade of these chemokines or their receptors enhanced tissue inflammation and fibrosis, clearly contrasting with available data in murine models of T. cruzi infection. These data reinforce the important role of chemokines during T. cruzi infection but suggest that caution must be taken when expanding the therapeutic modulation of the chemokine system in mice to the human infection.     

Modulation of inflammatory response and parasitism by 15-Deoxy-Δ(12,14) prostaglandin J(2) in Trypanosoma cruzi-infected cardiomyocytes

Trypanosoma cruzi infection produces an intense inflammatory response in diverse tissues including the heart. The inflammatory reaction is critical for the control of the parasites’ proliferation and evolution of Chagas disease. 15-Deoxy-Δ^12,14 prostaglandin J₂ (15dPGJ2) can repress the inflammatory response in many experimental models. However, the precise role of peroxisome proliferator-activated receptor γ (PPARγ) ligands in T. cruzi infection or in Chagas disease is poorly understood. This work reports the first evidence that 15dPGJ2 treatment increases the number of intracellular parasites as shown by fluorescence microscopy and it is also able to inhibit the expression and activity of different inflammatory enzymes such as inducible nitric oxide synthase (NOS-2), matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), as well as pro-inflammatory cytokine (TNF-α and IL-6) mRNA expression in neonatal mouse cardiomyocytes after T. cruzi infection. Transfection of cardiomyocytes with small interfering RNA (siRNA) induces silencing of PPARγ and impairs the effects of 15dPGJ2 on the modulation of pro-inflammatory enzymes. Moreover, transfection restores the ability of these cells to control the intracellular growth of T. cruzi. We also found that PPARγ-independent pathways are involved, since 15dPGJ2 also exerts its effect through extracellular signal-regulated kinases-mitogen-activated protein kinase (Erk-MAPK) and nuclear factor-κB (NF-κB). The use of specific pharmacological inhibitors confirmed these findings. Our data point out that 15dPGJ2 is a potent modulator of the inflammatory process and regulator of parasites growth through PPARγ-dependent and independent (Erk-MAPK- and NF-κB) pathways in T. cruzi infected neonatal cardiac cells.     

Temporal fluctuation in shrub species preferences of two native rodents: The effect of infection status on habitat use

Small mammals use plant species for gathering food resources and for shelter. Preferences for certain plant species are related to nutritional restrictions and behavioural patterns, which could be altered in the presence of an infectious disease. Several native small mammals are part of the wild cycle of the protozoan Trypanosoma cruzi, responsible for Chagas disease in humans. This is a vector‐borne disease transmitted by insects of the subfamily Triatominae. We examined the effect of T. cruzi infection status on the use and preference patterns of shrub species by two native rodent species: Octodon degus and Phyllotis darwini. This study was conducted during four sampling years (2010–2013) in a hyper‐endemic zone of Chagas disease located in a semiarid Mediterranean ecosystem. We captured individuals of 599 O. degus and 575 P. darwini (89% of the total captures), which were related to nine shrub species and examined for T. cruzi infection. In a community‐level analysis, infected and non‐infected O. degus used individual shrub species within the shrub community significantly non‐randomly relative to their availability; the same pattern was detected for non‐infected P. darwini individuals, whereas infected individuals used the shrub community according to the abundance of each shrub species. Examining individual preferences, both rodents showed a strong preference for Flourensia thurifera and Colliguaja odorifera regardless of their infection status. Preferences for specific shrub species were variable among years, showing a ‘core’ of preferred shrub species and variable levels of use of the remaining ones. Our results show that T. cruzi infection in wild small mammals can modify habitat use patterns and preferences for certain shrub species, probably affecting processes acting at community level.     

The Effect of the Interleukin‐1 Cytokine Family Members IL‐1F6 and IL‐1F8 on Adipocyte Differentiation

Obesity is characterized by chronic low‐grade inflammation originating from expanding adipose tissue. In the present study, we examined the adipogenic expression levels of IL‐1F6 and IL‐1F8, both members of the IL‐1 family of cytokines, and their effects on adipose tissue gene expression. Although IL‐1F6 is primarily present in adipose tissue resident macrophages and induced by inflammation, IL‐1F8 is absent. IL‐1F6, but not IL‐1F8, reduces adipocyte differentiation, as shown by a significant decrease in PPARγ gene expression. Finally, both IL‐1F6 and IL‐1F8 are able to induce inflammatory gene expression in mature adipocytes. In conclusion, we demonstrate for the first time that IL‐1F6 is present in adipose tissue and that IL‐1F6 and IL‐1F8 are involved in the regulation of adipose tissue gene expression. Importantly, IL‐1F6 inhibits PPARγ expression which may lead to reduced adipocyte differentiation suggesting metabolic effects of this cytokine.     

Targeting host mitochondria: A role for the Trypanosoma cruzi amastigote flagellum

Trypanosoma cruzi is the kinetoplastid protozoan parasite that causes human Chagas disease, a chronic disease with complex outcomes including severe cardiomyopathy and sudden death. In mammalian hosts, T. cruzi colonises a wide range of tissues and cell types where it replicates within the host cell cytoplasm. Like all intracellular pathogens, T. cruzi amastigotes must interact with its immediate host cell environment in a manner that facilitates access to nutrients and promotes a suitable niche for replication and survival. Although potentially exploitable to devise strategies for pathogen control, fundamental knowledge of the host pathways co‐opted by T. cruzi during infection is currently lacking. Here, we report that intracellular T. cruzi amastigotes establish close contact with host mitochondria via their single flagellum. Given the key bioenergetic and homeostatic roles of mitochondria, this striking finding suggests a functional role for host mitochondria in the infection process and points to the T. cruzi amastigote flagellum as an active participant in pathogenesis. Our study establishes the basis for future investigation of the molecular and functional consequences of this intriguing host–parasite interaction.     

Metallothionein-1 and nitric oxide expression are inversely correlated in a murine model of Chagas disease

Chagas disease, caused by Trypanosoma cruzi, represents an endemic among Latin America countries. The participation of free radicals, especially nitric oxide (NO), has been demonstrated in the pathophysiology of seropositive individuals with T. cruzi. In Chagas disease, increased NO contributes to the development of cardiomyopathy and megacolon. Metallothioneins (MTs) are efficient free radicals scavengers of NO in vitro and in vivo. Here, we developed a murine model of the chronic phase of Chagas disease using endemic T. cruzi RyCH1 in BALB/c mice, which were divided into four groups: infected non-treated (Inf), infected N-monomethyl-L-arginine treated (Inf L-NAME), non-infected L-NAME treated and non-infected vehicle-treated. We determined blood parasitaemia and NO levels, the extent of parasite nests in tissues and liver MT-I expression levels. It was observed that NO levels were increasing in Inf mice in a time-dependent manner. Inf L-NAME mice had fewer T. cruzi nests in cardiac and skeletal muscle with decreased blood NO levels at day 135 post infection. This affect was negatively correlated with an increase of MT-I expression (r = -0.8462, p < 0.0001). In conclusion, we determined that in Chagas disease, an unknown inhibitory mechanism reduces MT-I expression, allowing augmented NO levels.     

High fat diet aggravates cardiomyopathy in murine chronic Chagas disease

Infection with Trypanosoma cruzi, the etiologic agent in Chagas disease, may result in heart disease. Over the last decades, Chagas disease endemic areas in Latin America have seen a dietary transition from the traditional regional diet to a Western style, fat rich diet. Previously, we demonstrated that during acute infection high fat diet (HFD) protects mice from the consequences of infection-induced myocardial damage through effects on adipogenesis in adipose tissue and reduced cardiac lipidopathy. However, the effect of HFD on the subsequent stages of infection – the indeterminate and chronic stages – has not been investigated. To address this gap in knowledge, we studied the effect of HFD during indeterminate and chronic stages of Chagas disease in the mouse model. We report, for the first time, the effect of HFD on myocardial inflammation, vasculopathy, and other types of dysfunction observed during chronic T. cruzi infection. Our results show that HFD perturbs lipid metabolism and induces oxidative stress to exacerbate late chronic Chagas disease cardiac pathology.     

L-arginine supplementation increases cardiac collagenogenesis in mice chronically infected with Berenice-78 Trypanosoma cruzi strain

Chagas disease, caused by Trypanosoma cruzi, is a major neglected tropical disease that occurs mainly as chronic infection and systemic infection. Currently, there is no suitable and effective drug to treat this parasitic disease. Administration of nutrients with immunomodulatory properties, such as arginine and nitric oxide radicals, may be helpful as antiparasitic therapy. In this study, we evaluated the effects of arginine supplementation during the acute phase of infection under the development of chronic Chagas' heart disease in Swiss mice inoculated with the Berenice-78 strain of T. cruzi. The effectiveness of arginine was determined by daily detection of the parasite in the blood and long-term serum levels of nitric oxide and tumor necrosis factor-alpha, in addition to evaluation of heart tissue damage. Arginine could flatten parasitemia and prevent elevation of tumor necrosis factor-alpha in T. cruzi-infected mice. Regarding chronic inflammatory myocardial derangements, similar findings were verified among T. cruzi-infected groups. Arginine promoted collagenogenesis in the heart muscle tissue of T. cruzi-infected arginine-supplemented group. These data show the paradoxical benefits of arginine in improving the outcome of Chagas chronic cardiomyopathy.     

Trypanosoma cruzi: Cardiac mitochondrial alterations produced by different strains in the acute phase of the infection

The parasite Trypanosoma cruzi is the causative agent of Chagas disease. T. cruzi invasion and replication in cardiomyocytes induce cellular injuries and cytotoxic reactions, with the production of inflammatory cytokines and nitric oxide, both source of reactive oxygen species. The myocyte response to oxidative stress involves the progression of cellular changes primarily targeting mitochondria. We studied the cardiac mitochondrial structure and the enzymatic activity of citrate synthase and respiratory chain CI–CIV complexes, in Albino Swiss mice infected with T. cruzi, Tulahuen strain and SGO Z12 isolate, in two periods of the acute infection. Changes in the mitochondrial structure were detected in both infected groups, reaching values of 71% for Tulahuen and 88% for SGO Z12 infected mice, 30 days post infection. The citrate synthase activity was different according to the evolution of the infection and the parasite strain, but the respiratory chain alterations were similar with either strain.     

Endothelial Transmigration by Trypanosoma cruzi

Chagas heart disease, the leading cause of heart failure in Latin America, results from infection with the parasite Trypanosoma cruzi. Although T. cruzi disseminates intravascularly, how the parasite contends with the endothelial barrier to escape the bloodstream and infect tissues has not been described. Understanding the interaction between T. cruzi and the vascular endothelium, likely a key step in parasite dissemination, could inform future therapies to interrupt disease pathogenesis. We adapted systems useful in the study of leukocyte transmigration to investigate both the occurrence of parasite transmigration and its determinants in vitro. Here we provide the first evidence that T. cruzi can rapidly migrate across endothelial cells by a mechanism that is distinct from productive infection and does not disrupt monolayer integrity or alter permeability. Our results show that this process is facilitated by a known modulator of cellular infection and vascular permeability, bradykinin, and can be augmented by the chemokine CCL2. These represent novel findings in our understanding of parasite dissemination, and may help identify new therapeutic strategies to limit the dissemination of the parasite.