Ischemic strokes have been implicated as a cause of death in Chagas disease patients. Inflammation has been recognized as a key component in all ischemic processes, including the intravascular events triggered by vessel interruption, brain damage and repair. In this study, we evaluated the association between inflammatory markers and the death risk (DR) and stroke risk (SR) of patients with different clinical forms of chronic Chagas disease. The mRNA expression levels of cytokines, transcription factors expressed in the adaptive immune response (Th1, Th2, Th9, Th17, Th22 and regulatory T cell), and iNOS were analyzed by real-time PCR in peripheral blood mononuclear cells of chagasic patients who exhibited the indeterminate, cardiac, digestive and cardiodigestive clinical forms of the disease, and the levels of these transcripts were correlated with the DR and SR. Cardiac patients exhibited lower mRNA expression levels of GATA-3, FoxP3, AHR, IL-4, IL-9, IL-10 and IL-22 but exhibited higher expression of IFN-γ and TNF-α compared with indeterminate patients. Digestive patients showed similar levels of GATA-3, IL-4 and IL-10 than indeterminate patients. Cardiodigestive patients exhibited higher levels of TNF-α compared with indeterminate and digestive patients. Furthermore, we demonstrated that patients with high DR and SR exhibited lower GATA-3, FoxP3, and IL-10 expression and higher IFN-γ, TNF-α and iNOS mRNA expression than patients with low DR and SR. A negative correlation was observed between Foxp3 and IL-10 mRNA expression and the DR and SR. Moreover, TNF-α and iNOS expression was positively correlated with DR and SR. Our data suggest that an inflammatory imbalance in chronic Chagas disease patients is associated with a high DR and SR. This study provides a better understanding of the stroke pathobiology in the general population and might aid the development of therapeutic strategies for controlling the morbidity and mortality of Chagas disease.

Chronic chagasic cardiomyopathy (CCC) is observed in 30% to 50% of the individuals infected by Trypanosoma cruzi and heart failure is the important cause of death among patients in the chronic phase of Chagas disease. Although some studies have elucidated the role of adaptive immune responses involving T and B lymphocytes in cardiac pathogenesis, the role of innate immunity receptors such as Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in CCC pathophysiology has not yet been determined. In this study, we evaluated the association among innate immune receptors (TLR1-9 and nucleotide-binding domain-like receptor protein 3/NLRP3), its adapter molecules (Myd88, TRIF, ASC and caspase-1) and cytokines (IL-1β, IL-6, IL-12, IL-18, IL-23, TNF-α, and IFN-β) with clinical manifestation, digestive and cardiac function in patients with different clinical forms of chronic Chagas disease. The TLR8 mRNA expression levels were enhanced in the peripheral blood mononuclear cells (PBMC) from digestive and cardiodigestive patients compared to indeterminate and cardiac patients. Furthermore, mRNA expression of IFN-β (cytokine produced after TLR8 activation) was higher in digestive and cardiodigestive patients when compared to indeterminate. Moreover, there was a positive correlation between TLR8 and IFN-β mRNA expression with sigmoid and rectum size. Cardiac and cardiodigestive patients presented higher TLR2, IL-12 and TNF-α mRNA expression than indeterminate and digestive patients. Moreover, cardiac patients also expressed higher levels of NLRP3, ASC and IL-1β mRNAs than indeterminate patients. In addition, we showed a negative correlation among TLR2, IL-1β, IL-12 and TNF-α levels with left ventricular ejection fraction, and positive correlation between NLRP3 with cardiothoracic index, and TLR2, IL-1β and IL-12 with left ventricular mass index. Together, our data suggest that high expression of innate immune receptors in cardiac and digestive patients may induce an enhancement of cytokine expression and participate of cardiac and digestive dysfunction.

Background Triatomines are responsible for the vector transmission of the protozoan parasite Trypanosoma cruzi, which causes Chagas disease. Triatoma brasiliensis is the main vector of the parasite in Brazil, and dogs are an important reservoir of the parasite. The aim of this study was to evaluate the insecticidal effect of fluralaner (Bravecto®) on T. brasiliensis after a blood meal in treated dogs. Methods Healthy mongrel dogs (n = 8) were recruited from the Zoonoses Control Center (ZCC) in the city of Natal, Rio Grande do Norte, Brazil, and randomized into two groups, a fluralaner (Bravecto®)-treated group (n = 4) and a control group (n = 4). Colony-reared third-, fourth- and fifth-instar nymphs of T. brasiliensis nymphs (n = 10) were allowed to feed on dogs from both groups for 30–40 min, once monthly, for up to 12 months. Bug mortality was observed up to 5 days after each blood meal. Results Mortality in triatomines which had a blood meal on fluralaner (Bravecto®)-treated dogs was 100% for up to 7 months after treatment, with mortality decreasing to 66.4% after 8 months, 57% after 9 months, 35% after 10 months, 10% after 11 months and 0% after 12 months. The mortality of triatomines that fed on non-treated control dogs was always ≤ 2.5%. Conclusions Our results suggest that fluralaner (Bravecto®) treatment of dogs induces long-term mortality of T. brasiliensis after the blood meal. This is a potential approach to be used to control vector transmission of T. cruzi, the etiological agent of Chagas disease, especially in endemic areas. Graphical Abstract

Background Leishmania infantum is the etiological agent of visceral leishmaniasis (VL) in the New World, where the sand fly Lutzomyia longipalpis and domestic dogs are considered the main vector and host reservoirs, respectively. Systemic insecticides have been studied as an alternative to control vector-borne diseases, including VL. Fluralaner, an isoxazoline class compound, is a systemic insecticide used in dogs, with proven efficiency against different species of phlebotomine sand flies. However, to date no studies have demonstrated the efficacy of fluralaner on Lu. longipalpis. The aim of this study was to evaluate the insecticidal effect of fluralaner (Bravecto®) on the sand fly Lu. longipalpis after blood meal in treated dogs. Methods Healthy mongrel dogs (n = 8) were recruited from the Zoonoses Control Center in the city of Natal, Rio Grande do Norte, Brazil, and randomized into two groups: fluralaner treated (n = 4) and non-treated control (n = 4). Colony-reared female specimens of Lu. longipalpis (n = 20) were allowed to feed on all dogs for 40 min before treatment (for fluralaner-treated dogs), at day 1 after treatment and then monthly until 1 year post-treatment. Results In the treatment group, there was 100% mortality of Lu. longipalpis for up to 5 months after treatment initiation, decreasing to 72.5% at 6 months post-treatment initiation. The efficacy of fluralaner ranged from 100% at day 1 (P = 0.0002) to 68% ( P = 0.0015) at 6 months, decreasing to 1.4% at 1 year post-treatment. Sand fly mortality carried out blood meal in non-treated control dogs remained constant at ≤ 15%. Conclusions Taken together, our results suggest that fluralaner may be used as a control strategy for VL in dogs in VL endemic areas.

Digestive and cardiodigestive forms of Chagas' disease are observed in 2% to 27% of the patients, depending on their geographic location, Trypanosoma cruzi strain and immunopathological responses. The aim of this work was to evaluate the role of NOD2 innate immune receptor in the pathogenesis of the digestive system in Chagas' disease. Patients with digestive form of the disease showed lower mRNA expression of NOD2, higher expression of RIP2 and α-defensin 6, compared to indeterminate form, detected by Real-time PCR in peripheral blood mononuclear cells. In addition, there was a negative correlation between the expression of NOD2 and the degree of dilation of the esophagus, sigmoid and rectum in those patients. The infection of NOD2-/mice with T. cruzi strain isolated from the digestive patient induced a decrease in intestinal motility. Histopathological analysis of the colon and jejunum of NOD2-/and wild type C57BL/6 animals revealed discrete inflammatory foci during the acute phase of infection. Interestingly, during the chronic phase of the infection there was inflammation and hypertrophy of the longitudinal and circular muscular layer more pronounced in the colon and jejunum from NOD2-/animals, when compared to wild type C57BL/6 mice. Together, our results suggest that NOD2 plays a protective role against the development of digestive form of Chagas' disease.

Resistance or susceptibility to T. cruzi infection is dependent on the host immunological profile. Innate immune receptors, such as Toll-like receptors (TLRs/TLR2, TLR4, TLR7, and TLR9) and Nod-like receptors (NLRs/NOD1 and NLRP3 inflammasome) are involved with the resistance against acute experimental T. cruzi infection. Here, we evaluated the impact of T. cruzi virulence on the expression of innate immune receptors and its products in mice. For that, we used six T. cruzi strains/isolates that showed low (AM64/TcIV and 3253/Tc-V), medium (PL1.10.14/TcIII and CL/TcVI), or high (Colombian/Tc-I and Y/TcII) virulence and pathogenicity to the vertebrate host and belonging to the six discrete typing units (DTUs)—TcI to TcVI. Parasitemia, mortality, and myocarditis were evaluated and correlated to the expression of TLRs, NLRs, adapter molecules, cytokines, and iNOS in myocardium by real time PCR. Cytokines (IL-1β, IL-12, TNF-α, and IFN-γ) were quantified in sera 15 days after infection. Our data indicate that high virulent strains of T. cruzi, which generate high parasitemia, severe myocarditis, and 100% mortality in infected mice, inhibit the expression of TLR2, TLR4, TLR9, TRIF, and Myd88 transcripts, leading to a low IL-12 production, when compared to medium and low virulent T. cruzi strains. On the other hand, the high virulent T. cruzi strains induce the upregulation of NLRP3, caspase-1, IL-1β, TNF-α, and iNOS mRNA in heart muscle, compared to low and medium virulent strains, which may contribute to myocarditis and death. Moreover, high virulent strains induce higher levels of IL-1β and TNF-α in sera compared to less virulent parasites. Altogether the data indicate that differential TLR and NLR expression in heart muscle is correlated with virulence and pathogenicity of T cruzi strains. A better knowledge of the immunological mechanisms involved in resistance to T. cruzi infection is important to understand the natural history of Chagas disease, can lead to identification of immunological markers and/or to serve as a basis for alternative therapies.

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