Mariela N. González

Caspase-1/ASC Inflammasome-Mediated Activation of IL-1β–ROS–NF-κB Pathway for Control of Trypanosoma cruzi Replication and Survival Is Dispensable in NLRP3−/− Macrophages

In this study, we have utilized wild-type (WT), ASC−/−, and NLRP3−/− macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1β production in mφs in comparison to LPS-treated controls. When WT and ASC−/− macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3−/− macrophages, despite an inability to elicit IL-1β activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3−/− macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1β-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1β/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.

Trypanosoma cruzi: biological characterization of a isolate from an endemic area and its susceptibility to conventional drugs.

We describe some biological and molecular characteristics of a Trypanosoma cruzi isolate derived from a Triatomine captured in Nicaragua. PCR based typification showed that this isolate, named Nicaragua, belonged to the lineage Tc I. Nicaragua infected culture cells were treated with allopurinol, showing different behavior according to the cellular compartment, being cardiomyocyte primary cultures more resistant to this drug. The course of the infection in a mice experimental model and its susceptibility to benznidazole and allopurinol was analyzed. In benznidazole treatment, mice reverted the high lethal effect of parasites during the acute infection, however, a few parasites were detected in the heart of 88% of mice 1 year post-infection. Since T. cruzi is a heterogeneous species population it is important to study and characterize different parasites actually circulating in humans in endemic areas. In this work we show that T. cruzi Nicaragua isolate, is sensitive to early benznidazole treatment.

Elevated serum levels of macrophage migration inhibitory factor are associated with progressive chronic cardiomyopathy in patients with Chagas disease.

Clinical symptoms of chronic Chagas disease occur in around 30% of the individuals infected with Trypanosoma cruzi and are characterized by heart inflammation and dysfunction. The pathogenesis of chronic chagasic cardiomyopathy (CCC) is not completely understood yet, partially because disease evolution depends on complex host-parasite interactions. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that promotes numerous pathophysiological processes. In the current study, we investigated the link between MIF and CCC progression. Immunohistochemical analysis demonstrated MIF overexpression in the hearts from chronically T. cruzi-infected mice, particularly those showing intense inflammatory infiltration. We also found that MIF exogenously added to parasite-infected murine macrophage cultures is capable of enhancing the production of TNF-α and reactive oxygen species, both with pathogenic roles in CCC. Thus, the integrated action of MIF and other cytokines and chemokines may account for leukocyte influx to the infected myocardium, accompanied by enhanced local production of multiple inflammatory mediators. We further examined by ELISA the level of MIF in the sera from chronic indeterminate and cardiomyopathic chagasic patients, and healthy subjects. CCC patients displayed significantly higher MIF concentrations than those recorded in asymptomatic T. cruzi-infected and uninfected individuals. Interestingly, increased MIF levels were associated with severe progressive Chagas heart disease, in correlation with elevated serum concentration of high sensitivity C-reactive protein and also with several echocardiographic indicators of left ventricular dysfunction, one of the hallmarks of CCC. Our present findings represent the first evidence that enhanced MIF production is associated with progressive cardiac impairment in chronic human infection with T. cruzi, strengthening the relationship between inflammatory response and parasite-driven pathology. These observations contribute to unravel the elements involved in the pathogenesis of CCC and may also be helpful for the design of novel therapies aimed to control long-term morbidity in chagasic patients.

Granulocyte colony-stimulating factor partially repairs the damage provoked by Trypanosoma cruzi in murine myocardium

BACKGROUND The hallmark of Trypanosoma cruzi infection is cardiomyopathy that leads to end-stage heart failure. We investigated whether G-CSF, known to induce heart tissue repair by bone marrow stem cell mobilization, ameliorates T. cruzi-induced myocarditis. METHODS AND RESULTS T. cruzi-infected C3H/He mice were treated with G-CSF and monitored for parasite burden, BMSC mobilization, cytokine profile and cardiac remodeling. G-CSF increased the expression of CXCR4, CD34, and c-Kit, indicating mobilization and migration of BMSC, some of which differentiated to cardiomyocytes as evidenced by increased levels of GATA4(+)/MEF2C(+) cells and desmin expression in chagasic hearts. G-CSF enhanced a mixed cytokine response (IL-10+TGF-β>IFN-γ+TNF-α>IL-4) associated with increased heart inflammation and no beneficial effect on parasite control. Further, G-CSF controlled T. cruzi-induced extracellular-matrix alterations of collagens (Col I and Col llI), hydroxyproline, and glycosaminoglycan contents and promoted compensatory cardiac remodeling; however, these responses were not sufficient to control T. cruzi-induced cardiomyocyte atrophy. Benznidazole treatment prior to G-CSF resulted in the control of parasitism and parasite-induced inflammation, and subsequently, G-CSF was effective in executing the tissue repair, as evidenced by extracellular-matrix homeostasis and normalization of cardiomyocyte size in chagasic hearts. CONCLUSIONS G-CSF treatment after T. cruzi infection enhanced migration and homing of BMSC, some of which differentiated to cardiomyocytes. Yet, G-CSF promoted a mixed (Treg>Th1>Th2) immune response that contributed to persistent inflammation and limited improvement in cardiac homeostasis. Combinatorial therapy (BZ → G-CSF) was beneficial in arresting inflammatory processes and tissue damage in chagasic hearts.

Altered distribution of peripheral blood memory B cells in humans chronically infected with Trypanosoma cruzi.

Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.

Trypanosoma cruzi infection induces the expression of CD40 in murine cardiomyocytes favoring CD40 ligation-dependent production of cardiopathogenic IL-6

The inflammatory response in the myocardium is an important aspect of the pathogenesis of Chagas’ heart disease raised by Trypanosoma cruzi. CD40, a transmembrane type I receptor belonging to the tumor necrosis factor receptor (TNFR) family, is expressed in a broad spectrum of cell types and is crucial in several inflammatory and autoimmune diseases. Activation of CD40 through ligation to CD40L (CD154) induces multiple effects, including the secretion of proinflammatory molecules. In the present study, we examined the ability of T. cruzi to trigger the expression of CD40 in cardiac myocytes in vitro and in a murine model of chagasic cardiomyopathy. Our results indicate, for the first time, that T. cruzi is able to induce the expression of CD40 in HL-1 murine cardiomyocytes. Moreover, ligation of CD40 receptor upregulated interleukin-6 (IL-6), associated with inflammation. Furthermore, the induction of this costimulatory molecule was demonstrated in vivo in myocardium of mice infected with T. cruzi. This suggests that CD40-bearing cardiac muscle cells could interact with CD40L-expressing lymphocytes infiltrating the heart, thus contributing to inflammatory injury in chagasic cardiomyopathy.

Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates

Trypanosoma cruzi is a genetically heterogeneous group of organisms that cause Chagas disease. It has been long suspected that the clinical outcome of the disease and response to therapeutic agents are, at least in part, related to the genetic characteristics of the parasite. Herein, we sought to validate the significance of the genotype of T. cruzi isolates recovered from patients with different clinical forms of Chagas disease living in Argentina on their biological behaviour and susceptibility to drugs. Genotype identification of the newly established isolates confirmed the reported predominance of TcV, with a minor frequency of TcI. Epimastigote sensitivity assays demonstrated marked dissimilar responses to benznidazole, nifurtimox, pentamidine and dihydroartemisinin in vitro. Two TcV isolates exhibiting divergent response to benznidazole in epimastigote assays were further tested for the expression of anti-oxidant proteins. Benznidazole-resistant BOL-FC10A epimastigotes had decreased expression of Old Yellow Enzyme and cytosolic superoxide dismutase, and overexpression of mitochondrial superoxide dismutase and tryparedoxin- 1, compared to benznidazole-susceptible AR-SE23C parasites. Drug sensitivity assays on intracellular amastigotes and trypomastigotes reproduced the higher susceptibility of AR-SE23C over BOL-FC10A parasites to benznidazole observed in epimastigotes assays. However, the susceptibility/resistance profile of amastigotes and trypomastigotes to nifurtimox, pentamidine and dihydroartemisinin varied markedly with respect to that of epimastigotes. C3H/He mice infected with AR-SE23C trypomastigotes had higher levels of parasitemia and mortality rate during the acute phase of infection compared to mice infected with BOL-FC10A trypomastigotes. Treatment of infected mice with benznidazole or nifurtimox was efficient to reduce patent parasitemia induced by either isolate. Nevertheless, qPCR performed at 70 dpi revealed parasite DNA in the blood of mice infected with AR-SE23C but not in BOL-FC10A infected mice. These results demonstrate high level of intra-type diversity which may represent an important obstacle for the testing of chemotherapeutic agents.