Memory-like NK Cells Are a Critical Component of Vaccine-Induced Immunity to Trypanosoma cruzi Infection.

Chagas disease by Trypanosoma cruzi infection is a major public health issue. The available therapeutic agents have limited efficacy and significant side effects. A reliable vaccine would reduce the threat of T. cruzi infections and prevent Chagas disease. Understanding the immune response to this infection would improve vaccine design. We previously demonstrated that adoptively transferred NK cells from mice immunized with highly attenuated T. cruzi, GFP-DDDHA strain, provided potent protection in naive recipients against secondary lethal challenge with various wild-type (WT) strains. To understand the importance of NK cells in protecting mice against T. cruzi infection, we performed an in-depth characterization of NK cell phenotype, responses, and memory-like traits during acute infections due to GFP-DDDHA and WT strains and in immunized mice during a recall response to a WT lethal challenge. NK cells robustly expanded and became more mature and cytolytic during the GFP-DDDHA strain immunization. NK cells in immunized mice responded more robustly after WT lethal challenge than during an acute primary WT infection. In addition, protection by immunization with the GFP-DDDHA strain is significantly weakened in NK cell-deficient mice and did not prevent parasitemia from WT lethal challenge, indicating that NK cells with memory-like traits were a critical component for early control of WT lethal challenge. Prior T. cruzi vaccine development studies have not included studies of this rapid NK response. These findings provide insights into overcoming existing challenges in developing a safe and effective vaccine to prevent this infection.

In vitro tripanocidal effect of 1,8-dioxooctahydroxanthenes (xanthenodiones) and tetraketones and improvement of cardiac parameters in vivo.

OBJECTIVE: Trypanosoma cruzi infection affects millions of people worldwide, and the drugs available for its treatment have limited efficacy. 1,8-Dioxooctahydroxanthenes and tetraketones are compounds with important biological applications. The aim of this study was to assess the trypanocidal and inflammatory activities of nine 1,8-dioxooctahydroxanthenes (1-9) and three tetraketones (10-12). METHODS AND RESULTS: By in vitro killing assay, three compounds were able to eliminate CL TdTomato expressing strain of T. cruzi, 9 (IC50=30.65µM), 10 (IC50=14.11µM), and 11 (IC50=26.43µM). However, only 9 was not toxic to Vero cells. Next, to evaluate the in vivo antitrypanosomal and immunological efficacy of 9, Swiss mice were infected with the Y and CL strains of T. cruzi and treated for 10 days with 50mg/kg of 9. This compound reduced the cardiac inflammatory infiltration in animals infected with both strains. Rank's ligand (RankL), CCL2, and interferon (IFN)-γ were measured in the cardiac tissue homogenate of the Y-strain-infected animals, and no interference of 9 was observed. However, compound 9 increased the RankL and interleukin (IL)-10 levels in CL-infected mice. No hepatic and renal toxicity was observed. CONCLUSION: Our findings showed that 1,8-dioxooctahydroxanthene has antiparasitic effect and ameliorates the cardiac inflammatory parameters related to T. cruzi infection.

Resolvin D1 Administration Is Beneficial in Trypanosoma cruzi Infection.

Chagas disease is a major public health issue, affecting ∼10 million people worldwide. Transmitted by a protozoan named Trypanosoma cruzi, this infection triggers a chronic inflammatory process that can lead to cardiomyopathy (Chagas disease). Resolvin D1 (RvD1) is a novel proresolution lipid mediator whose effects on inflammatory diseases dampens pathological inflammatory responses and can restore tissue homeostasis. Current therapies are not effective in altering the outcome of T. cruzi infection, and as RvD1 has been evaluated as a therapeutic agent in various inflammatory diseases, we examined if exogenous RvD1 could modulate the pathogenesis of Chagas disease in a murine model. CD-1 mice infected with the T. cruzi Brazil strain were treated with RvD1. Mice were administered 3 µg/kg of body weight RvD1 intraperitoneally on days 5, 10, and 15 to examine the effect of RvD1 on acute disease or administered the same dose on days 60, 65, and 70 to examine its effects on chronic infection. RvD1 therapy increased the survival rate and controlled parasite replication in mice with acute infection and reduced the levels of interferon gamma and transforming growth factor ß (TGF-ß) in mice with chronic infection. In addition, there was an increase in interleukin-10 levels with RvD1 therapy in both mice with acute infection and mice with chronic infection and a decrease in TGF-ß levels and collagen content in cardiac tissue. Together, these data indicate that RvD1 therapy can dampen the inflammatory response, promote the resolution of T. cruzi infection, and prevent cardiac fibrosis.

The Toxoplasma gondii Cyst Wall Interactome.

A characteristic of the latent cyst stage of Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole. Previously, our laboratory group published a proteomic analysis of purified in vitro cyst wall fragments that identified an inventory of cyst wall components. To further refine our understanding of the composition of the cyst wall, several cyst wall proteins were tagged with a promiscuous biotin ligase (BirA*), and their interacting partners were screened by streptavidin affinity purification. Within the cyst wall pulldowns, previously described cyst wall proteins, dense granule proteins, and uncharacterized hypothetical proteins were identified. Several of the newly identified hypothetical proteins were validated to be novel components of the cyst wall and tagged with BirA* to expand the model of the cyst wall interactome. Community detection of the cyst wall interactome model revealed three distinct clusters: a dense granule, a cyst matrix, and a cyst wall cluster. Characterization of several of the identified cyst wall proteins using genetic strategies revealed that MCP3 affects in vivo cyst sizes. This study provides a model of the potential protein interactions within the cyst wall and the groundwork to understand cyst wall formation.IMPORTANCE A model of the cyst wall interactome was constructed using proteins identified through BioID. The proteins within this cyst wall interactome model encompass several proteins identified in a prior characterization of the cyst wall proteome. This model provides a more comprehensive understanding of the composition of the cyst wall and may lead to insights on how the cyst wall is formed.

Induction of Effective Immunity against Trypanosoma cruzi.

Chagas disease, caused by Trypanosoma cruzi, is a major public health issue. Limitations in immune responses to natural T. cruzi infection usually result in parasite persistence with significant complications. A safe, effective, and reliable vaccine would reduce the threat of T. cruzi infections; however, no suitable vaccine is currently available due to a lack of understanding of the requirements for induction of fully protective immunity. We established a T. cruzi strain expressing green fluorescent protein (GFP) under the control of dihydrofolate reductase degradation domain (DDD) with a hemagglutinin (HA) tag, GFP-DDDHA, which was induced by trimethoprim-lactate (TMP-lactate), which results in the death of intracellular parasites. This attenuated strain induces very strong protection against reinfection. Using this GFP-DDDHA strain, we investigated the mechanisms underlying the protective immune response in mice. Immunization with this strain led to a response that included high levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), as well as a rapid expansion of effector and memory T cells in the spleen. More CD8+ T cells differentiate to memory cells following GFP-DDDHA infection than after infection with a wild-type (WT) strain. The GFP-DDDHA strain also provides cross-protection against another T. cruzi isolate. IFN-γ is important in mediating the protection, as IFN-γ knockout (KO) mice failed to acquire protection when infected with the GFP-DDDHA strain. Immune cells demonstrated earlier and stronger protective responses in immunized mice after reinfection with T. cruzi than those in naive mice. Adoptive transfers with several types of immune cells or with serum revealed that several branches of the immune system mediated protection. A combination of serum and natural killer cells provided the most effective protection against infection in these transfer experiments.

Microsporidia Interact with Host Cell Mitochondria via Voltage-Dependent Anion Channels Using Sporoplasm Surface Protein 1.

Microsporidia are opportunistic intracellular pathogens that can infect a wide variety of hosts ranging from invertebrates to vertebrates. During invasion, the microsporidian polar tube pushes into the host cell, creating a protective microenvironment, the invasion synapse, into which the sporoplasm extrudes. Within the synapse, the sporoplasm then invades the host cell, forming a parasitophorous vacuole (PV). Using a proteomic approach, we identified Encephalitozoon hellem sporoplasm surface protein 1 (EhSSP1), which localized to the surface of extruded sporoplasms. EhSSP1 was also found to interact with polar tube protein 4 (PTP4). Recombinant EhSSP1 (rEhSSP1) bound to human foreskin fibroblasts, and both anti-EhSSP1 and rEhSSP1 caused decreased levels of host cell invasion, suggesting that interaction of SSP1 with the host cell was involved in invasion. Coimmunoprecipitation (Co-IP) followed by proteomic analysis identified host cell voltage-dependent anion channels (VDACs) as EhSSP1 interacting proteins. Yeast two-hybrid assays demonstrated that EhSSP1 was able to interact with VDAC1, VDAC2, and VDAC3. rEhSSP1 colocalized with the host mitochondria which were associated with microsporidian PVs in infected cells. Transmission electron microscopy revealed that the outer mitochondrial membrane interacted with meronts and the PV membrane, mitochondria clustered around meronts, and the VDACs were concentrated at the interface of mitochondria and parasite. Knockdown of VDAC1, VDAC2, and VDAC3 in host cells resulted in significant decreases in the number and size of the PVs and a decrease in mitochondrial PV association. The interaction of EhSSP1 with VDAC probably plays an important part in energy acquisition by microsporidia via its role in the association of mitochondria with the PV.IMPORTANCE Microsporidia are important opportunistic human pathogens in immune-suppressed individuals, such as those with HIV/AIDS and recipients of organ transplants. The sporoplasm is critical for establishing microsporidian infection. Despite the biological importance of this structure for transmission, there is limited information about its structure and composition that could be targeted for therapeutic intervention. Here, we identified a novel E. hellem sporoplasm surface protein, EhSSP1, and demonstrated that it can bind to host cell mitochondria via host VDAC. Our data strongly suggest that the interaction between SSP1 and VDAC is important for the association of mitochondria with the parasitophorous vacuole during microsporidian infection. In addition, binding of SSP1 to the host cell is associated with the final steps of invasion in the invasion synapse.

The ß-blocker carvedilol and the benznidazole modulate the cardiac immune response in the acute infection induced by Colombian strain of the Trypanosoma cruzi.

BACKGROUND The infection led by Trypanosoma cruzi persists in mammalian tissues causing an inflammatory imbalance. Carvedilol (Cv), a non-selective beta blocker drug indicated to treat heart failure and antihypertensive has shown to promote antioxidant and immunomodulatory properties which might improve the inflammation induced by T. cruzi. OBJECTIVES Evaluate the role of Cv on the inflammatory response of C57BL/6 mice acutely infected with the Colombian strain of T. cruzi. METHODS Animals were infected with the Colombian strain of T. cruzi and treated with Cv (25 mg/kg/day), benznidazole (Bz) (100 mg/kg/day) or their combination. On the 28th day of infection and 23 days of treatment, the euthanasia occurred, and the heart preserved for histopathological, oxidative stress (SOD, catalase, TBARs, carbonylated proteins) and plasma (CCL2, CCL5, TNF, IL-10) analyses. Parasitaemia and survival were assessed along the infection. FINDINGS Cv decreased TBARs, but increased the mortality rate, the parasitaemia and the levels of CCL2, CCL5, catalase and the inflammatory infiltrate in the cardiac tissue. Bz led the reduction of the inflammatory infiltrate and circulating levels of oxidative stress and inflammatory mediators in the infected mice. MAIN CONCLUSIONS Our data suggest that Cv, in this experimental model using the Colombian strain of T. cruzi, caused damage to the host.

The B-blocker carvedilol and the benznidazole modulate the cardiac immune response in the acute infection induced by Colombian strain of the Trypanosoma cruzi

BACKGROUND The infection led by Trypanosoma cruzi persists in mammalian tissues causing an inflammatory imbalance. Carvedilol (Cv), a non-selective beta blocker drug indicated to treat heart failure and antihypertensive has shown to promote antioxidant and immunomodulatory properties which might improve the inflammation induced by T. cruzi. OBJECTIVES Evaluate the role of Cv on the inflammatory response of C57BL/6 mice acutely infected with the Colombian strain of T. cruzi. METHODS Animals were infected with the Colombian strain of T. cruzi and treated with Cv (25 mg/kg/day), benznidazole (Bz) (100 mg/kg/day) or their combination. On the 28th day of infection and 23 days of treatment, the euthanasia occurred, and the heart preserved for histopathological, oxidative stress (SOD, catalase, TBARs, carbonylated proteins) and plasma (CCL2, CCL5, TNF, IL-10) analyses. Parasitaemia and survival were assessed along the infection. FINDINGS Cv decreased TBARs, but increased the mortality rate, the parasitaemia and the levels of CCL2, CCL5, catalase and the inflammatory infiltrate in the cardiac tissue. Bz led the reduction of the inflammatory infiltrate and circulating levels of oxidative stress and inflammatory mediators in the infected mice. MAIN CONCLUSIONS Our data suggest that Cv, in this experimental model using the Colombian strain of T. cruzi, caused damage to the host.

Potential Role of Carvedilol in the Cardiac Immune Response Induced by Experimental Infection with Trypanosoma cruzi.

Trypanosoma cruzi causes a cardiac infection characterized by an inflammatory imbalance that could become the inciting factor of the illness. To this end, we evaluated the role of carvedilol, a beta-blocker with potential immunomodulatory properties, on the immune response in C57BL/6 mice infected with VL-10 strain of T. cruzi in the acute phase. Animals (n = 40) were grouped: (i) not infected, (ii) infected, (iii) infected + carvedilol, and (iv) not infected + carvedilol. We analyzed parameters related to parasitemia, plasma levels of TNF, IL-10, and CCL2, and cardiac histopathology after the administration of carvedilol for 30 days. We did not observe differences in the maximum peaks of parasitemia in the day of their detection among the groups. The plasma TNF was elevated at 60 days of infection in mice treated or not with carvedilol. However, we observed a decreased CCL2 level and increased IL-10 levels in those infected animals treated with carvedilol, which impacted the reduction of the inflammatory infiltration in cardiac tissue. For this experimental model, carvedilol therapy was not able to alter the levels of circulating parasites but modulates the pattern of CCL2 and IL-10 mediators when the VL10 strain of T. cruzi was used in C57BL6 mice.

Doxycycline and Benznidazole Reduce the Profile of Th1, Th2, and Th17 Chemokines and Chemokine Receptors in Cardiac Tissue from Chronic Trypanosoma cruzi-Infected Dogs.

Chemokines (CKs) and chemokine receptors (CKR) promote leukocyte recruitment into cardiac tissue infected by the Trypanosoma cruzi. This study investigated the long-term treatment with subantimicrobial doses of doxycycline (Dox) in association, or not, with benznidazole (Bz) on the expression of CK and CKR in cardiac tissue. Thirty mongrel dogs were infected, or not, with the Berenice-78 strain of T. cruzi and grouped according their treatments: (i) two months after infection, Dox (50 mg/kg) 2x/day for 12 months; (ii) nine months after infection, Bz (3,5 mg/kg) 2x/day for 60 days; (iii) Dox + Bz; and (iv) vehicle. After 14 months of infection, hearts were excised and processed for qPCR analysis of Th1 (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL11), Th2 (CCL1, CCL17, CCL24, and CCL26), Th17 (CCL20) CKs, Th1 (CCR5, CCR6, and CXCR3), and Th2/Th17 (CCR3, CCR4, and CCR8) CKR, as well as IL-17. T. cruzi infection increases CCL1, CCL2, CCL4, CCL5, CCL17, CXCL10, and CCR5 expression in the heart. Dox, Bz, or Dox + Bz treatments cause a reversal of CK and CKR and reduce the expression of CCL20, IL-17, CCR6, and CXCR3. Our data reveal an immune modulatory effect of Dox with Bz, during the chronic phase of infection suggesting a promising therapy for cardiac protection.

Enalapril in Combination with Benznidazole Reduces Cardiac Inflammation and Creatine Kinases in Mice Chronically Infected with Trypanosoma cruzi.

The protozoan Trypanosoma cruzi triggers an inflammatory process in mammalian heart causing events such as fibrosis, changes in the architecture and functionality in this organ. Enalapril, an angiotensin II-converting enzyme inhibitor, is a drug prescribed to ameliorate this heart dysfunction, and appears to exert a potential role in immune system regulation. Our aim was to evaluate the chronic cardiac inflammatory parameters after therapeutic treatment with enalapril and benznidazole in C57BL/6 mice infected with the VL-10 strain of T. cruzi. After infection, animals were treated with oral doses of enalapril (25 mg/kg), benznidazole (100 mg/kg), or both during 30 days. Morphometric parameters and levels of chemokines (CCL2, CCL5), IL-10, creatine kinases (CKs), and C-reactive protein were evaluated in the heart and serum at the 120th day of infection. Enalapril alone or in combination with benznidazole did not change the number of circulating parasites, but reduced cardiac leukocyte recruitment and total collagen in the cardiac tissue. Interestingly, the combination therapy (enalapril/benznidazole) also reduced the levels of chemokines, CK and CK-MB, and C-reactive proteins in chronic phase. In conclusion, during the chronic experimental T. cruzi infection, the combination therapy using enalapril plus benznidazole potentiated their immunomodulatory effects, resulting in a low production of biomarkers of cardiac lesions.