Andrea Pellegrini

Myeloid-derived suppressor cells are key players in the resolution of inflammation during a model of acute infection

Myeloid‐derived suppressor cells (MDSCs) are key players in the immune suppressive network. During acute infection with the causative agent of Chagas disease, Trypanosoma cruzi, BALB/c mice show less inflammation and better survival than C57BL/6 (B6) mice. In this comparative study, we found a higher number of MDSCs in the spleens and livers of infected BALB/c mice compared with infected B6 mice. An analysis of the two major MDSCs subsets revealed a greater number of granulocytic cells in the spleens and livers of BALB/c mice when compared with that in B6 mice. Moreover, splenic MDSCs purified from infected BALB/c mice inhibited ConA‐induced splenocyte proliferation. Mechanistic studies demonstrated that ROS and nitric oxide were involved in the suppressive activity of MDSCs, with a higher number of infected CD8+ T cells suffering surface‐nitration compared to uninfected controls. An upregulation of NADPH oxidase p47 phox subunit and p‐STAT3 occurred in MDSCs and infected IL‐6 KO mice showed less recruitment of MDSCs and impaired survival. Remarkably, in vivo depletion of MDSCs led to increased production of IL‐6, IFN‐γ, and a Th17 response with very high parasitemia and mortality. These findings demonstrate a new facet of MDSCs as crucial regulators of inflammation during T. cruzi infection.

Importance of TLR2 on hepatic immune and non-immune cells to attenuate the strong inflammatory liver response during Trypanosoma cruzi acute infection.

Background Toll-like receptors (TLR) and cytokines play a central role in the pathogen clearance as well as in pathological processes. Recently, we reported that TLR2, TLR4 and TLR9 are differentially modulated in injured livers from BALB/c and C57BL/6 (B6) mice during Trypanosoma cruzi infection. However, the molecular and cellular mechanisms involved in local immune response remain unclear. Methodology/Principal Findings In this study, we demonstrate that hepatic leukocytes from infected B6 mice produced higher amounts of pro-inflammatory cytokines than BALB/c mice, whereas IL10 and TGFβ were only released by hepatic leukocytes from BALB/c. Strikingly, a higher expression of TLR2 and TLR4 was observed in hepatocytes of infected BALB/c mice. However, in infected B6 mice, the strong pro-inflammatory response was associated with a high and sustained expression of TLR9 and iNOS in leukocytes and hepatic tissue respectively. Additionally, co-expression of gp91- and p47-phox NADPH oxidase subunits were detected in liver tissue of infected B6 mice. Notably, the pre-treatment previous to infection with Pam3CSK4, TLR2-agonist, induced a significant reduction of transaminase activity levels and inflammatory foci number in livers of infected B6 mice. Moreover, lower pro-inflammatory cytokines and increased TGFβ levels were detected in purified hepatic leukocytes from TLR2-agonist pre-treated B6 mice. Conclusions/Significance Our results describe some of the main injurious signals involved in liver immune response during the T. cruzi acute infection. Additionally we show that the administration of Pam3CSk4, previous to infection, can attenuate the exacerbated inflammatory response of livers in B6 mice. These results could be useful to understand and design novel immune strategies in controlling liver pathologies.

Induction of NADPH oxidase activity and reactive oxygen species production by a single Trypanosoma cruzi antigen.

Trypanosoma cruzi is an intracellular protozoan parasite that predominantly invades mononuclear phagocytes and is able to establish a persistent infection. The production of reactive oxygen species (ROS) by phagocytes is an innate defence mechanism against microorganisms. It has been postulated that ROS such as superoxide anion (O(2)), hydrogen peroxide and peroxynitrite, may play a crucial role in the control of pathogen growth. However, information on parasite molecules able to trigger ROS production is scarce. In this work, we investigated whether cruzipain, an immunogenic glycoprotein from T. cruzi, was able to trigger the oxidative burst by murine cells. By employing chemiluminiscense and flow-cytometric analysis, we demonstrated that cruzipain induced ROS production in splenocytes from non-immune and cruzipain immune C57BL/6 mice and in a Raw 264.7 macrophage cell line. We also identified an O(2)(-) molecule as one of the ROS produced after antigen stimulation. Cruzipain stimulation induced NOX2 (gp91(phox)) and p47(phox) expression, as well as the co-localisation of both NADPH oxidase enzyme subunits. In the current study, we provide evidence that cruzipain not only increased ROS production but also promoted IL-6 and IL-1β cytokine production. Taken together, we believe these results demonstrate for the first time that cruzipain, a single parasite molecule, in the absence of infection, favors oxidative burst in murine cells. This represents an important advance in the knowledge of parasite molecules that interact with the phagocyte defence mechanism.

The role of Toll-like receptors and adaptive immunity in the development of protective or pathological immune response triggered by the Trypanosoma cruzi protozoan

Trypanosoma cruzi, the causal agent of Chagas disease, is an intracellular protozoan parasite that predominantly invades macrophages and cardiomyocytes, leading to persistent infection. Several members of the Toll-like receptor family are crucial for innate immunity to infection and are involved in maintaining tissue homeostasis. This review focuses on recent experimental findings of the innate and adaptive immune response in controlling the parasite and/or in generating heart and liver tissue injury. We also describe the importance of the hosts genetic background in the outcome of the disease and emphasize the importance of studying the response to specific parasite antigens. Understanding the dual participation of the immune response may contribute to the design of new therapies for Chagas disease.

In vitro activity of N-benzenesulfonylbenzotriazole on Trypanosoma cruzi epimastigote and trypomastigote forms.

Chagas disease is still an important health problem in Central and South America. However, the only drugs currently available for specific treatment of this disease may induce toxic side effects in the host. The aim of this work was to determine the activity of N-benzenesulfonylbenzotriazole (BSBZT) against the protozoan parasite Trypanosoma cruzi. The effects of BSBZT and benzotriazole (BZT) were compared to those of benznidazole (BZL) on epimastigote and trypomastigote forms. BSBZT was found to have an in vitro growth inhibitory dose-dependent activity against epimastigotes, with flow cytometry analysis confirming that the treated parasites presented size reduction. BSBZT showed an IC(50) of 21.56 μg/mL (81.07 μM) against epimastigotes at 72 h of incubation, whereas BZT did not affect the growth of this parasite form. Furthermore, the toxic effect of BSBZT, was stronger and appeared earlier (at 24h) in trypomastigotes than in epimastigotes, with the LC(50) of this compound being 28.40 μg/mL (106.79 μM) against trypomastigotes. The concentrations of BSBZT used in this study presented low hemolytic activity and cytotoxicity. Consequently, at concentrations near IC(50) and LC(50) (25μg/mL), BSBZT caused only 2.4% hemolysis and 15% of RAW 264.7 cell cytotoxicity. These results reveal the potential of BSBZT as a prototype in drug design for developing new anti-T. cruzi compounds.

Trypanosoma cruzi antigen immunization induces a higher B cell survival in BALB/c mice, a susceptible strain, compared to C57BL/6 B lymphocytes, a resistant strain to cardiac autoimmunity

Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and represents the most common infectious myocarditis worldwide. Autoimmunity is one of the mechanisms contributing to its pathogenesis. Although the cellular interactions that promote this autoimmune response are still poorly understood, several studies have demonstrated a key role for B lymphocytes since they secrete antibodies, cytokines and present antigens. Recently, we reported that immunization with cruzipain, an immunodominant T. cruzi antigen, induces a higher activation state in B cells from BALB/c mice (susceptible to cardiac autoimmunity) than B lymphocytes from C57BL/6 (a resistant strain). Here, we focused on the study of B cell survival in both mouse strains after cruzipain immunization and demonstrated an increased survival rate of B cells from BALB/c compared to C57BL/6 mice. This phenomenon was associated with a decreased expression of Fas/FasL and an increased expression of anti-apoptotic Bcl-2/Bcl-xL proteins. With the purpose to gain more knowledge about the mechanisms involved, we found that IL-4 produced by BALB/c B cells played a key role in the survival in an autocrine way whereas the addition of this bioactive cytokine rescued C57BL/6 B lymphocytes from apoptosis. Our findings suggest that in the absence of infection, both enhanced B cell activation induced by the immunization with a single parasite antigen and insufficient negative regulation can potentially contribute to autoimmunity seen in cruzipain immune BALB/c mice.

Trypanosoma cruzi, the causative agent of Chagas disease, modulates interleukin-6-induced STAT3 phosphorylation via gp130 cleavage in different host cells

Interleukin-6 mediates host defense and cell survival mainly through the activation of the transcription factor STAT3 via the glycoprotein gp130, a shared signal-transducing receptor for several IL-6-type cytokines. We have reported that the cardiotrophic parasite Trypanosoma cruzi protects murine cardiomyocytes from apoptosis. In agreement, an intense induction of the anti-apoptotic factor Bcl-2 is found in cardiac fibers during the acute phase of infection, establishing a higher threshold against apoptosis. We report here that inactive cruzipain, the main cysteine protease secreted by the parasite, specifically triggered TLR2 and the subsequent release of IL-6, which acted as an essential anti-apoptotic factor for cardiomyocyte cultures. Although comparable IL-6 levels were found under active cruzipain stimulation, starved cardiac cell monolayers could not be rescued from apoptosis. Moreover, cardiomyocytes treated with active cruzipain completely abrogated the STAT3 phosphorylation and nuclear translocation induced by recombinant IL-6. This inhibition was also observed on splenocytes, but it was reverted when the enzyme was complexed with chagasin, a parasite cysteine protease inhibitor. Furthermore, the inhibition of IL-6-induced p-STAT3 was evidenced in spleen cells stimulated with pre-activated supernatants derived from trypomastigotes. To account for these observations, we found that cruzipain enzymatically cleaved recombinant gp130 ectodomain, and induced the release of membrane-distal N-terminal domain of this receptor on human peripheral blood mononuclear cells. These results demonstrate, for the first time, that the parasite may modify the IL-6-induced response through the modulation of its cysteine protease activity, suggesting that specific inhibitors may help to improve the immune cell activation and cardioprotective effects.