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.

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-kappaB-dependent products in response to infection and failed to restrict T. cruzi infection in presence of IFN-gamma. 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.

Regulation of Trypanosoma cruzi-induced myocarditis by programmed death cell receptor 1.

Trypanosoma cruzi infection causes intense myocarditis, leading to cardiomyopathy and severe cardiac dysfunction. Protective adaptive immunity depends on balanced signaling through a T cell receptor and coreceptors expressed on the T cell surface. Such coreceptors can trigger stimulatory or inhibitory signals after binding to their ligands in antigen-presenting cells (APC). T. cruzi modulates the expression of coreceptors in lymphocytes after infection. Deregulated inflammation may be due to unbalanced expression of these molecules. Programmed death cell receptor 1 (PD-1) is a negative T cell coreceptor that has been associated with T cell anergy or exhaustion and persistent intracellular infections. We aimed to study the role of PD-1 during T. cruzi-induced acute myocarditis in mice. Cytometry assays showed that PD-1 and its ligands are strongly upregulated in lymphocytes and APC in response to T. cruzi infection in vivo and in vitro. Lymphocytes infiltrating the myocardium exhibited high levels of expression of these molecules. An increased cardiac inflammatory response was found in mice treated with blocking antibodies against PD-1, PD-L1, and to a lesser extent, PD-L2, compared to that found in mice treated with rat IgG. Similar results in PD-1(-/-) mice were obtained. Moreover, the PD-1 blockade/deficiency led to reduced parasitemia and tissue parasitism but increased mortality. These results suggest the participation of a PD-1 signaling pathway in the control of acute myocarditis induced by T. cruzi and provide additional insight into the regulatory mechanisms in the pathogenesis of Chagas' disease.

Canonical PI3Kγ signaling in myeloid cells restricts Trypanosoma cruzi infection and dampens chagasic myocarditis.

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi (T. cruzi) and is an important cause of severe inflammatory heart disease. However, the mechanisms driving Chagas disease cardiomyopathy have not been completely elucidated. Here, we show that the canonical PI3Kγ pathway is upregulated in both human chagasic hearts and hearts of acutely infected mice. PI3Kγ-deficient mice and mutant mice carrying catalytically inactive PI3Kγ are more susceptible to T. cruzi infection. The canonical PI3Kγ signaling in myeloid cells is essential to restrict T. cruzi heart parasitism and ultimately to avoid myocarditis, heart damage, and death of mice. Furthermore, high PIK3CG expression correlates with low parasitism in human Chagas' hearts. In conclusion, these results indicate an essential role of the canonical PI3Kγ signaling pathway in the control of T. cruzi infection, providing further insight into the molecular mechanisms involved in the pathophysiology of chagasic heart disease.

The use of a heterogeneously controlled mouse population reveals a significant correlation of acute phase parasitemia with mortality in Chagas disease.

Chagas disease develops upon infection with the protozoan parasite Trypanosoma cruzi and undergoes an acute phase characterized by massive parasite replication and the presence of parasites in the blood. This condition is known as acute phase parasitemia. This initial stage may result in a cure, in the development of the chronic stages of the disease or in the death of the infected host. Despite intensive investigation related to the characterization of the acute and chronic phases of the disease, the cause-effect relationship of acute phase parasitemia to the outcome of the disease is still poorly understood. In this study, we artificially generated a heterogeneously controlled mouse population by intercrossing F1 mice obtained from a parental breeding of highly susceptible A/J with highly resistant C57BL/6 mouse strains. This F2 population was infected and used to assess the correlation of acute phase parasitemia with the longevity of the animals. We used nonparametric statistical analyses and found a significant association between parasitemia and mortality. If males and females were evaluated separately, we found that the former were more susceptible to death, although parasitemia was similar in males and females. In females, we found a strong negative correlation between parasitemia and longevity. In males, however, additional factors independent of parasitemia may favor mouse mortality during the development of the disease. The correlations of acute phase parasitemia with mortality reported in this study may facilitate an appropriate prognostic approach to the disease in humans. Moreover, these results illustrate the complexity of the mammalian genetic traits that regulate host resistance during Chagas disease.

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.

Haeme oxygenase activity protects the host against excessive cardiac inflammation during experimental Trypanosoma cruzi infection.

The infection with Trypanosoma cruzi induces a robust cardiac inflammation that plays a pathogenic role in the development of Chagas heart disease. In this study, we aimed at investigating the effects of Haem Oxygenase (HO) during experimental infection by T. cruzi in BALB/c and C57BL/6 mice. HO has recently emerged as a key factor modulating the immune response in diverse models of inflammatory diseases. In mice with two different genetic backgrounds, the pharmacologic inhibition of HO activity with zinc-protoporphyrin IX (ZnPPIX) induced enhanced myocarditis and reduced parasitaemia, which was accompanied by an amplified production of nitric oxide and increased influx of CD4(+), CD8(+) and IFN-γ(+) cells to the myocardium in comparison with the control group. Conversely, treatment with haemin (an activator of HO) lead to a decreased number of intracardiac CD4(+) (but not CD8(+)) cells compared to the control group. The mechanism involved in these observations is a modulation of the induction of regulatory T cells, because the stimulation or inhibition of HO was parallelled by an enhanced or reduced frequency of regulatory T cells, respectively. Hence, HO may be involved in the regulation of heart tissue inflammation and could be a potential target in conceiving future therapeutic approaches for 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.

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.

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.

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-gamma and TNF-alpha and enhanced specific type 1 chemokine and chemokine receptors expression. Moreover, the results showed that IL-17 regulates T-bet, RORgammat 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.