Grace Kelly Silva

IL-17 Produced during Trypanosoma cruzi Infection Plays a Central Role in Regulating Parasite-Induced Myocarditis

Background Chagas disease is a neglected disease caused by the intracellular parasite Trypanosoma cruzi. Around 30% of the infected patients develop chronic cardiomyopathy or megasyndromes, which are high-cost morbid conditions. Immune response against myocardial self-antigens and exacerbated Th1 cytokine production has been associated with the pathogenesis of the disease. As IL-17 is involved in the pathogenesis of several autoimmune, inflammatory and infectious diseases, we investigated its role during the infection with T. cruzi. Methodology/Principal Findings First, we detected significant amounts of CD4, CD8 and NK cells producing IL-17 after incubating live parasites with spleen cells from normal BALB/c mice. IL-17 is also produced in vivo by CD4+, CD8+ and NK cells from BALB/c mice on the early acute phase of infection. Treatment of infected mice with anti-mouse IL-17 mAb resulted in increased myocarditis, premature mortality, and decreased parasite load in the heart. IL-17 neutralization resulted in increased production of IL-12, IFN-γ and TNF-α and enhanced specific type 1 chemokine and chemokine receptors expression. Moreover, the results showed that IL-17 regulates T-bet, RORγt and STAT-3 expression in the heart, showing that IL-17 controls the differentiation of Th1 cells in infected mice. Conclusion/Significance These results show that IL-17 controls the resistance to T. cruzi infection in mice regulating the Th1 cells differentiation, cytokine and chemokine production and control parasite-induced myocarditis, regulating the influx of inflammatory cells to the heart tissue. Correlations between the levels of IL-17, the extent of myocardial destruction, and the evolution of cardiac disease could identify a clinical marker of disease progression and may help in the design of alternative therapies for the control of chronic morbidity of chagasic patients.

Cutting Edge: Nucleotide-Binding Oligomerization Domain 1-Dependent Responses Account for Murine Resistance against Trypanosoma cruzi Infection

An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)-like receptor proteins in host response to T. cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1−/− mice showed an impaired induction of NF-κB–dependent products in response to infection and failed to restrict T. cruzi infection in presence of IFN-γ. Despite normal cytokine production in the sera, Nod1−/− mice were highly susceptible to T. cruzi infection, in a similar manner to MyD88−/− and NO synthase 2−/− mice. These studies indicate that Nod1-dependent responses account for host resistance against T. cruzi infection by mechanisms independent of cytokine production.

Current status of Chagas disease chemotherapy

Chagas disease affects 7.7 million people and 28 million people are at risk of acquiring the disease in 15 endemic countries of Latin America. Benznidazole and nifurtimox are drugs that have been used to treat the disease. However, both drugs induce severe side effects. Treatment with benznidazole has been recommended for the acute phase (0–4 months after infection), recent chronic phase (children 0–14 years of age, treated 4 months after infection) and congenital infection. Average cure rates for Chagas disease patients obtained from clinical trials were 97.9% (congenital infection, treatment performed 0–6 months of age), 71.5% (acute phase), 57.6% (recent chronic phase, children 0–13 years of age) and 5.9% (late chronic phase, great majority of patients between 15 and 69 years of age). Clinical evidence about the capacity of antiparasitic treatment to avoid, stop or revert heart pathology in indeterminate and cardiac chronic patients is contradictory. The investigation of novel therapeutic strategies against Chagas disease remains a priority in the research of tropical diseases. Unfortunately, Chagas disease remains neglected in the formulation of strategies toward control of this disease. This article focuses on current therapeutic approaches to Chagas disease.

Apoptosis-Associated Speck–like Protein Containing a Caspase Recruitment Domain Inflammasomes Mediate IL-1β Response and Host Resistance to Trypanosoma cruzi Infection

The innate immune response to Trypanosoma cruzi infection comprises several pattern recognition receptors (PRRs), including TLR-2, -4, -7, and -9, as well as the cytosolic receptor Nod1. However, there are additional PRRs that account for the host immune responses to T. cruzi. In this context, the nucleotide-binding oligomerization domain–like receptors (NLRs) that activate the inflammasomes are candidate receptors that deserve renewed investigation. Following pathogen infection, NLRs form large molecular platforms, termed inflammasomes, which activate caspase-1 and induce the production of active IL-1β and IL-18. In this study, we evaluated the involvement of inflammasomes in T. cruzi infection and demonstrated that apoptosis-associated speck–like protein containing a caspase recruitment domain (ASC) inflammasomes, including NLR family, pyrin domain–containing 3 (NLRP3), but not NLR family, caspase recruitment domain–containing 4 or NLR family, pyrin domain–containing 6, are required for triggering the activation of caspase-1 and the secretion of IL-1β. The mechanism by which T. cruzi mediates the activation of the ASC/NLRP3 pathway involves K+ efflux, lysosomal acidification, reactive oxygen species generation, and lysosomal damage. We also demonstrate that despite normal IFN-γ production in the heart, ASC−/− and caspase-1−/− infected mice exhibit a higher incidence of mortality, cardiac parasitism, and heart inflammation. These data suggest that ASC inflammasomes are critical determinants of host resistance to infection with T. cruzi.

IL-18 Triggered by the Nlrp3 Inflammasome Induces Host Innate Resistance in a Pulmonary Model of Fungal Infection

Pathogens are sensed by innate immune receptors that initiate an efficient adaptive immune response upon activation. The elements of the innate immune recognition process for Paracoccidioides brasiliensis include TLR-2, TLR-4, and dectin-1. However, there are additional receptors necessary for the host immune responses to P. brasiliensis. The nucleotide-binding oligomerization domain–like receptor (NLRs), which activate inflammasomes, are candidate receptors that deserve renewed investigation. After pathogen infection, the NLRs form large signaling platforms called inflammasomes, which lead to caspase-1 activation and maturation of proinflammatory cytokines (IL-18 and IL-1β). In this study, we showed that NLR family pyrin domain–containing 3 (Nlrp3) is required to induce caspase-1 activation and further secretion of IL-1β and IL-18 by P. brasiliensis–infected macrophages. Additionally, potassium efflux and lysosomal acidification induced by the fungus were important steps in the caspase-1 activation mechanism. Notably, Nlrp3 and caspase-1 knockout mice were more susceptible to infection than were the wild-type animals, suggesting that the Nlrp3-dependent inflammasomes contribute to host protection against P. brasiliensis. This protective effect occurred owing to the inflammatory response mediated by IL-18, as shown by an augmented fungus burden in IL-18 knockout mice. Taken together, our results show that the Nlrp3 inflammasome is essential for resistance against P. brasiliensis because it orchestrates robust caspase-1 activation and triggers an IL-18–dependent proinflammatory response.

IFN-γ Plays a Unique Role in Protection against Low Virulent Trypanosoma cruzi Strain

Background T. cruzi strains have been divided into six discrete typing units (DTUs) according to their genetic background. These groups are designated T. cruzi I to VI. In this context, amastigotes from G strain (T. cruzi I) are highly infective in vitro and show no parasitemia in vivo. Here we aimed to understand why amastigotes from G strain are highly infective in vitro and do not contribute for a patent in vivo infection. Methodology/Principal Findings Our in vitro studies demonstrated the first evidence that IFN-γ would be associated to the low virulence of G strain in vivo. After intraperitoneal amastigotes inoculation in wild-type and knockout mice for TNF-α, Nod2, Myd88, iNOS, IL-12p40, IL-18, CD4, CD8 and IFN-γ we found that the latter is crucial for controlling infection by G strain amastigotes. Conclusions/Significance Our results showed that amastigotes from G strain are highly infective in vitro but did not contribute for a patent infection in vivo due to its susceptibility to IFN-γ production by host immune cells. These data are useful to understand the mechanisms underlying the contrasting behavior of different T. cruzi groups for in vitro and in vivo infection.

The IL-33/ST2 Pathway Controls Coxsackievirus B5–Induced Experimental Pancreatitis

Coxsackievirus B (CVB) is a common cause of acute and chronic infectious myocarditis and pancreatitis. Th1 cells producing IFN-γ and TNF-α are important for CVB clearance, but they are also associated with the pathogenesis of inflammatory lesions, suggesting that the modulation of Th1 and Th2 balance is likely important in controlling CVB-induced pancreatitis. We investigated the role of IL-33, which is an important recently discovered cytokine for induction of Th2-associated responses, in experimental CVB5 infection. We found that mice deficient in IL-33R, T1/ST2, significantly developed more severe pancreatitis, had greater weight loss, and contained higher viral load compared with wild-type (WT) mice when infected with CVB5. Conversely, WT mice treated with rIL-33 developed significantly lower viral titers, and pancreatitis was attenuated. Mechanistic studies demonstrated that IL-33 enhances the degranulation and production of IFN-γ and TNF-α by CD8+ T and NK cells, which is associated with viral clearance. Furthermore, IL-33 triggers the production of IL-4 from mast cells, which results in enhanced differentiation of M2 macrophages and regulatory T cells, leading to the attenuation of inflammatory pancreatitis. Adoptively transferred mast cells or M2 macrophages reversed the heightened pancreatitis in the T1/ST2−/− mice. In contrast, inhibition of regulatory T cells exacerbated the disease in WT mice. Together, our findings reveal an unrecognized IL-33/ST2 functional pathway and a key mechanism for CVB5-induced pancreatitis. These data further suggest a novel approach in treating virus-induced pancreatitis, which is a major medical condition with unmet clinical needs.

Ruthenium complex with benznidazole and nitric oxide as a new candidate for the treatment of chagas disease.

Background Chagas disease remains a serious medical and social problem in Latin America and is an emerging concern in nonendemic countries as a result of population movement, transfusion of infected blood or organs and congenital transmission. The current treatment of infected patients is unsatisfactory due to strain-specific drug resistance and the side effects of the current medications. For this reason, the discovery of safer and more effective chemotherapy is mandatory for the successful treatment and future eradication of Chagas disease. Methods and Findings We investigated the effect of a ruthenium complex with benznidazole and nitric oxide (RuBzNO2) against Trypanosoma cruzi both in vitro and in vivo. Our results demonstrated that RuBzNO2 was more effective than the same concentrations of benznidazole (Bz) in eliminating both the extracellular trypomastigote and the intracellular amastigote forms of the parasite, with no cytotoxic effect in mouse cells. In vivo treatment with the compound improved the survival of infected mice, inhibiting heart damage more efficiently than Bz alone. Accordingly, tissue inflammation and parasitism was significantly diminished after treatment with RuBzNO2 in a more effective manner than that with the same concentrations of Bz. Conclusions The complexation of Bz with ruthenium and nitric oxide (RuBzNO2) increases its effectiveness against T. cruzi and enables treatment with lower concentrations of the compound, which may reduce the side effects of Bz. Our findings provide a new potential candidate for the treatment of Chagas disease.

A parent-of-origin effect determines the susceptibility of a non-informative F1 population to Trypanosoma cruzi infection in vivo.

The development of Chagas disease is determined by a complex interaction between the genetic traits of both the protozoan parasite, T. cruzi, and the infected host. This process is regulated by multiple genes that control different aspects of the host-parasite interaction. While determination of the relevant genes in humans is extremely difficult, it is feasible to use inbred mouse strains to determine the genes and loci responsible for host resistance to infection. In this study, we investigated the susceptibility of several inbred mouse strains to infection with the highly virulent Y strain of T. cruzi and found a considerable difference in susceptibility between A/J and C57BL/6 mice. We explored the differences between these two mouse strains and found that the A/J strain presented higher mortality, exacerbated and uncontrolled parasitemia and distinct histopathology in the target organs, which were associated with a higher parasite burden and more extensive tissue lesions. We then employed a genetic approach to assess the pattern of inheritance of the resistance phenotype in an F1 population and detected a strong parent-of-origin effect determining the susceptibility of the F1 male mice. This effect is unlikely to result from imprinted genes because the inheritance of this susceptibility was affected by the direction of the parental crossing. Collectively, our genetic approach of using the F1 population suggests that genes contained in the murine chromosome X contribute to the natural resistance against T. cruzi infection. Future linkage studies may reveal the locus and genes participating on the host resistance process reported herein.

TLR3 is required for survival following Coxsackievirus B3 infection by driving T lymphocyte activation and polarization: The role of dendritic cells

Type B coxsackievirus (CVB) is a common cause of acute and chronic myocarditis, meningitis and pancreatitis, often leading to heart failure and pancreatic deficiency. The polarization of CD4+ T lymphocytes and their cytokine milieu are key factors in the outcome of CVB-induced diseases. Thus, sensing the virus and driving the adaptive immune response are essential for the establishment of a protective immune response. TLR3 is a crucial virus recognition receptor that confers the host with resistance to CVB infection. In the current study, we found that TLR3 expression in dendritic cells plays a role in their activation upon CVB3 infection in vitro, as TLR3-deficient dendritic cells up-regulate CD80 and CD86 to a less degree than WT cells. Instead, they up-regulated the inhibitory molecule PD-L1 and secreted considerably lower levels of TNF-α and IL-10 and a higher level of IL-23. T lymphocyte proliferation in co-culture with CVB3-infected dendritic cells was increased by TLR3-expressing DCs and other cells. Furthermore, in the absence of TLR3, the T lymphocyte response was shifted toward a Th17 profile, which was previously reported to be deleterious for the host. TLR3-deficient mice were very susceptible to CVB3 infection, with increased pancreatic injury and extensive inflammatory infiltrate in the heart that was associated with uncontrolled viral replication. Adoptive transfer of TLR3+ dendritic cells slightly improved the survival of TLR-deficient mice following CVB3 infection. Therefore, our findings highlight the importance of TLR3 signaling in DCs and in other cells to induce activation and polarization of the CD4+ T lymphocyte response toward a Th1 profile and consequently for a better outcome of CVB3 infection. These data provide new insight into the immune-mediated mechanisms by which CVBs are recognized and cleared in order to prevent the development of myocarditis and pancreatitis and may contribute to the design of therapies for enteroviral infections.