Brain-Thymus Connections in Chagas Disease.

BACKGROUND: The brain and the immune systems represent the two primary adaptive systems within the body. Both are involved in a dynamic process of communication, vital for the preservation of mammalian homeostasis. This interplay involves two major pathways: the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. SUMMARY: The establishment of infection can affect immunoneuroendocrine interactions, with functional consequences for immune organs, particularly the thymus. Interestingly, the physiology of this primary organ is not only under the control of the central nervous system (CNS) but also exhibits autocrine/paracrine regulatory circuitries mediated by hormones and neuropeptides that can be altered in situations of infectious stress or chronic inflammation. In particular, Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), impacts upon immunoneuroendocrine circuits disrupting thymus physiology. Here, we discuss the most relevant findings reported in relation to brain-thymic connections during T. cruzi infection, as well as their possible implications for the immunopathology of human Chagas disease. KEY MESSAGES: During T. cruzi infection, the CNS influences thymus physiology through an intricate network involving hormones, neuropeptides, and pro-inflammatory cytokines. Despite some uncertainties in the mechanisms and the fact that the link between these abnormalities and chronic Chagasic cardiomyopathy is still unknown, it is evident that the precise control exerted by the brain over the thymus is markedly disrupted throughout the course of T. cruzi infection.

The high identity of the Trypanosoma cruzi Group-I of trans-sialidases points them as promising vaccine immunogens.

Trans-sialidase (TS) superfamily of proteins comprises eight subgroups, being the proteins of Group-I (TS-GI) promising immunogens in vaccine approaches against Trypanosoma cruzi. Strikingly, TS-GI antigenic variability among parasite lineages and their influence on vaccine development has not been previously analyzed. Here, a search in GenBank detects 49 TS-GI indexed sequences, whereas the main infecting human different parasite discrete typing units (DTU) are represented. In silico comparison among these sequences indicate that they share an identity above 92%. Moreover, the antigenic regions (T-cell and B-cell epitopes) are conserved in most sequences or present amino acid substitutions that scarcely may alter the antigenicity. Additionally, since the generic term TS is usually used to refer to different immunogens of this broad family, a further in silico analysis of the TS-GI-derived fragments tested in preclinical vaccines was done to determine the coverage and identity among them, showing that overall amino acid identity of vaccine immunogens is high, but the segment coverage varies widely. Accordingly, strong H-2K, H-2I, and B-cell epitopes are dissimilarly represented among vaccine TS-derived fragments depending on the extension of the TG-GI sequence used. Moreover, bioinformatic analysis detected a set of 150 T-cell strong epitopes among the DTU-indexed sequences that strongly bind human HLA-I supertypes. In all currently reported experimental vaccines based on TS-GI fragments, mapping these 150 epitopes showed that they are moderately represented. However, despite vaccine epitopes do not present all the substitutions observed in the DTUs, these regions of the proteins are equally recognized by the same HLAs.  Interestingly, the predictions regarding global and South American population coverage estimated in these 150 epitopes are similar to the estimations in experimental vaccines when the complete sequence of TS-GI is used as an antigen. In silico prediction also shows that a number of these MHC-I restricted T-cell strong epitopes could be also cross-recognized by HLA-I supertypes and H-2Kb or H-2Kd backgrounds, indicating that these mice may be used to improve and facilitate the development of new TS-based vaccines and suggesting an immunogenic and protective potential in humans. Further molecular docking analyses were performed to strengthen these results. Taken together, different strategies that would cover more or eventually fully of these T-cell and also B-cell epitopes to reach a high level of coverage are considered.

Diminished Prolinemia in Chronic Chagasic Patients: A New Clue for Disease Pathology?

Trypanosoma cruzi, the etiological agent of Chagas disease, is dependent on proline for a variety of processes, such as energy metabolism, host cell invasion, differentiation, and resistance to osmotic, metabolic, and oxidative stress. On this basis, we investigated a possible relationship between prolinemia and severity of T. cruzi infection in chronic patients, as reported here. The study population consisted of 112 subjects, separated into 83 chronically T. cruzi-infected patients and 29 age-matched healthy volunteers (control) of both sexes, recruited at the Chagas Disease Service from the Department of Cardiology, Hospital Provincial del Centenario de Rosario (Rosario, Argentina). Chagasic patients were separated into three groups: chronic asymptomatic, mild/moderate, and severe chronic chagasic cardiomyopathy (CCC) subjects. We observed a significant decrease of 11.7% in prolinemia in chagasic patients when compared to controls. Further analysis within the three groups of chagasic patients also revealed a statistically significant decrease of prolinemia in severe CCC patients compared to controls, showing a relative difference of 13.6% in proline concentrations. These data point to the possibility that collagen-which participates in the healing process of cardiac tissue-and proline metabolism in the myocardium could constitute new factors affecting the evolution of Chagas disease.

Dysregulated Network of Immune, Endocrine and Metabolic Markers is Associated to More Severe Human Chronic Chagas Cardiomyopathy.

Individuals who are infected with Trypanosoma cruzi develop chronic Chagas cardiomyopathy (CCC), which is a complication involving a series of immune pathogenetic mechanisms, although an association between immune and metabolic alterations was more recently proposed. Accordingly, we investigated the immuno-metabolic response in chagasic patients and their possible influence on CCC pathogenesis. To this end, T. cruzi-seropositive (asymptomatic or with CCC) and sero-negative individuals were studied. Serum tumour necrosis factor (TNF)-α, interleukin (IL)-6, adipocytokines and the expression of their receptors in peripheral blood mononuclear cell (PBMC) were evaluated, together with other factors influencing the immune response. CCC patients showed major metabolic and hormonal abnormalities, in parallel with increased IL-6 and leptin serum levels. TNF-α receptor s, leptin and adiponectin receptors (ObR and Adipo-Rs respectively), as well as PPAR-γ expression in PBMCs from CCC patients were compatible with a counteracting response leading to an unfavourable immune-metabolic profile. These results suggest that persistently increased levels of immune-metabolic pro-inflammatory mediators along with the adverse endocrine anti-inflammatory response of CCC individuals, may contribute to the underlying mechanisms dealing with myocardial tissue damage.

The sympathetic nervous system affects the susceptibility and course of Trypanosoma cruzi infection.

Trypanosoma cruzi (T. cruzi) is an intracellular parasite that causes Chagas' disease, a major health problem in Latin America. Using a murine model of infection with this parasite, we have previously shown that corticosterone blood levels are markedly elevated during the course of the disease in C57Bl/6 male mice and that this increase is protective for the host by restricting the production of pro-inflammatory cytokines. Since the hypothalamus-pituitary-adrenal (HPA) axis usually operates in a concerted way with the sympathetic nervous system (SNS), we have now studied whether noradrenergic nerves can affect the course of T. cruzi infection and the sexual dimorphism observed in the disease. We found a decreased splenic noradrenaline concentration and content, paralleled by a reduction in noradrenergic nerve fibers in the spleen of infected mice, and increased HPA axis activity. These alterations were more marked in males than in females. When the spontaneous loss of noradrenergic nerve fibers was advanced by chemical sympathectomy prior to infection, males died earlier and mortality significantly increased in females. Chemical denervation did not significantly affect the concentration of specific IgM and IgG2a antibodies to T. cruzi, and did not worsen myocarditis, but resulted in increased parasitemia and IL-6 and IFN-γ blood levels. The results obtained in this model of parasitic disease provide further indications of the relevance of interactions between the immune system and the SNS for host defense.

Intranasal Trans-Sialidase Vaccine Mitigates Acute and Chronic Pathology in a Preclinical Oral Chagas Disease Model.

Chagas disease, caused by Trypanosoma cruzi, leads to severe complications in 30% of infected individuals, including acute myocarditis and chronic fibrosing cardiomyopathy. Despite the significant burden of this disease, there is currently no licensed vaccine available to prevent it. This study aimed to evaluate the mucosal and systemic immunogenicity as well as the prophylactic efficacy of a mucosal vaccine candidate and its impact on both acute and chronic cardiomyopathy. The results showed that the nasal administration of trans-sialidase (TS) plus c-di-AMP (TS+A) vaccine elicited a NALT expression of IFN-γ, IL-17a and IL-4 mRNA as well as a nasal-specific production of IgA. An in vivo challenge with TS also triggered increased proliferation of lymphocytes from the NALT, sentinel cervical lymph node, and spleen. TS+A immunization increased the plasma levels of Th1/Th2/Th17 cytokines and elicited an evident cellular response by which to judge enhanced delayed-type hypersensitivity responses following a TS footpad challenge. After oral infection, TS+A-vaccinated mice showed significantly reduced parasitemia and parasite load in the heart, muscles and intestines, while markers of hepatic and muscle damage as well as clinical manifestations of acute infection were strongly diminished. TS+A also attenuated acute myocarditis and the expression of inflammatory markers in the heart. The protection conferred by TS+A extended into the chronic phase, where it resulted in a clear reduction in chronic myocarditis, fibrosis and functional electrocardiographic abnormalities, associated with a decreased expression of the pro-fibrotic TGF-ß. These results revealed that it is possible to develop a mucosal vaccine against T. cruzi based on TS and c-di-AMP that is capable of reducing the development of Chagas cardiomyopathy, the hallmark of Chagas disease.

Systemic inflammatory Th1 cytokines during Trypanosoma cruzi infection disrupt the typical anatomical cell distribution and phenotypic/functional characteristics of various cell subsets within the thymus.

The thymus plays a crucial role in T cell differentiation, a complex process influenced by various factors such as antigens, the microenvironment and thymic architecture. The way the thymus resolves infections is critical, as chronic persistence of microbes or inflammatory mediators can obstruct the differentiation. Here, we illustrate that following inflammatory T helper 1 infectious processes like those caused by Candida albicans or Trypanosoma cruzi, single positive thymocytes adopt a mature phenotype. Further investigations focused on T. cruzi infection, reveal a substantial existence of CD44+ cells in both the cortical and medullary areas of the thymus at the onset of infection. This disturbance coincides with heightened interferon gamma (IFNγ) production by thymocytes and an increased cytotoxic capacity against T. cruzi-infected macrophages. Additionally, we observe a reduced exportation capacity in T. cruzi-infected mice. Some alterations can be reversed in IFNγ knockout mice (KO). Notably, the majority of these effects can be replicated by systemic expression of interleukin (IL)-12+IL-18, underlining the predominantly inflammatory rather than pathogen-specific nature of these phenomena. Understanding the mechanisms through which systemic inflammation disrupts normal T cell development, as well as subsequent T cell exportation to secondary lymphoid organs (SLO) is pivotal for comprehending susceptibility to diseases in different pathological scenarios.

Reciprocal influences between leptin and glucocorticoids during acute Trypanosoma cruzi infection.

Leptin and glucocorticoids (GCs) are involved in metabolic functions, thymic homeostasis and immune activity through complex interactions. We recently showed that C57BL/6 mice infected with Trypanosoma cruzi revealed a fatal disease associated with a dysregulated immune-endocrine response characterized by weight loss, deleterious synthesis of pro-inflammatory cytokines and GCs-driven thymus atrophy. Extending this study, we now explored the relationship between leptin and GCs, in terms of infection outcome, thymic and metabolic changes. T. cruzi-infected mice showed a food intake reduction, together with hypoglycemia and lipolysis-related changes. Infected animals also displayed a reduction in systemic and adipose tissue levels of leptin, paralleled by a down-regulation of their receptor (ObR) in the hypothalamus. Studies in infected mice subjected to adrenalectomy (Adx) showed a worsened course of infection accompanied by even more diminished systemic and intrathymic leptin levels, for which GCs are necessary not only to decrease inflammation but also to sustain leptin secretion. Adx also protected from thymic atrophy, independently of the reduced leptin contents. Leptin administration to infected mice aggravated inflammation, lowered parasite burden and attenuated GCs release, but did not normalize thymic atrophy or metabolic parameters. Acute T. cruzi infection in C57BL/6 mice coexists with a dysregulation of leptin/hypothalamic ObR circuitry dissociated from body weight and food intake control. Endogenous GCs production attempted to reestablish systemic leptin concentrations, but failed to improve leptin-protective activities at the thymic level, suggesting that the leptin/GCs intrathymic relationship is also altered during this infection.

Trypanosoma cruzi, Chagas disease and cancer: putting together the pieces of a complex puzzle.

Considering the extensive and widespread impact on individuals, cancer can presently be categorized as a pandemic. In many instances, the development of tumors has been linked to endemic microbe infections. Among parasitic infections, Trypanosoma cruzi stands out as one of the most extensively discussed protozoans in the literature that explores the association between diseases of parasite origin and cancer. However, the effective association remains an unsolved paradox. Both the parasite, along with protozoan-derived molecules, and the associated antiparasitic immune response can induce alterations in various host cell pathways, leading to modifications in cell cycle, metabolism, glycosylation, DNA mutations, or changes in neuronal signaling. Furthermore, the presence of the parasite can trigger cell death or a senescent phenotype and modulate the immune system, the metastatic cascade, and the formation of new blood vessels. The interaction among the parasite (and its molecules), the host, and cancer undoubtedly encompasses various mechanisms that operate differentially depending on the context. Remarkably, contrary to expectations, the evidence tilts the balance toward inhibiting tumor growth or resisting tumor development. This effect is primarily observed in malignant cells, rather than normal cells, indicating a selective or specific component. Nevertheless, nonspecific bystander mechanisms, such as T. cruzi's adjuvancy or the presence of proinflammatory cytokines, may also play a significant role in this phenomenon. This work aims to elucidate this complex scenario by synthesizing the main findings presented in the literature and by proposing new questions and answers, thereby adding pieces to this challenging puzzle.

Association between altered tryptophan metabolism, plasma aryl hydrocarbon receptor agonists, and inflammatory Chagas disease.

Introduction: Chagas disease causes a cardiac illness characterized by immunoinflammatory reactions leading to myocardial fibrosis and remodeling. The development of Chronic Chagas Cardiomyopathy (CCC) in some patients while others remain asymptomatic is not fully understood, but dysregulated inflammatory responses are implicated. The Aryl hydrocarbon receptor (AhR) plays a crucial role in regulating inflammation. Certain tryptophan (Trp) metabolites have been identified as AhR ligands with regulatory functions. Methods results and discussion: We investigated AhR expression, agonist response, ligand production, and AhR-dependent responses, such as IDO activation and regulatory T (Treg) cells induction, in two T. cruzi-infected mouse strains (B6 and Balb/c) showing different polymorphisms in AhR. Furthermore, we assessed the metabolic profile of Trp catabolites and AhR agonistic activity levels in plasma samples from patients with chronic Chagas disease (CCD) and healthy donors (HD) using a luciferase reporter assay and liquid chromatography-mass spectrophotometry (LC-MS) analysis. T. cruzi-infected B6 mice showed impaired AhR-dependent responses compared to Balb/c mice, including reduced IDO activity, kynurenine levels, Treg cell induction, CYP1A1 up-regulation, and AhR expression following agonist activation. Additionally, B6 mice exhibited no detectable AhR agonist activity in plasma and displayed lower CYP1A1 up-regulation and AhR expression upon agonist activation. Similarly, CCC patients had decreased AhR agonistic activity in plasma compared to HD patients and exhibited dysregulation in Trp metabolic pathways, resulting in altered plasma metabolite profiles. Notably, patients with severe CCC specifically showed increased N-acetylserotonin levels in their plasma. The methods and findings presented here contribute to a better understanding of CCC development mechanisms and may identify potential specific biomarkers for T. cruzi infection and the severity of associated heart disease. These insights could be valuable in designing new therapeutic strategies. Ultimately, this research aims to establish the AhR agonistic activity and Trp metabolic profile in plasma as an innovative, non-invasive predictor of prognosis for chronic Chagas disease.

Central nervous system commitment in Chagas disease.

The involvement of the central nervous system (CNS) during human acute and chronic Chagas disease (CD) has been largely reported. Meningoencephalitis is a frequent finding during the acute infection, while during chronic phase the CNS involvement is often accompanied by behavioral and cognitive impairments. In the same vein, several studies have shown that rodents infected with Trypanosoma cruzi (T. cruzi) display behavior abnormalities, accompanied by brain inflammation, in situ production of pro-inflammatory cytokines and parasitism in diverse cerebral areas, with involvement of microglia, macrophages, astrocytes, and neurons. However, the mechanisms used by the parasite to reach the brain remain now largely unknown. Herein we discuss the evidence unravelling the CNS involvement and complexity of neuroimmune interactions that take place in acute and chronic CD. Also, we provide some clues to hypothesize brain infections routes in human and experimental acute CD following oral infection by T. cruzi, an infection route that became a major CD related public health issue in Brazil.

Assessment of a combined treatment with a therapeutic vaccine and benznidazole for the Trypanosoma cruzi chronic infection.

The difficulties encountered in achieving treatments for chronic Chagas disease have promoted the investigation of new therapeutic strategies. In this study, we used two murine models of Trypanosoma cruzi chronic infection to determine the usefulness of applying a therapeutic vaccine alone or followed by benznidazole (Bz) chemotherapy. A vaccine formulation based on an N-terminal fragment of Trans-sialidase (TS) and Immunostimulant Particle Adjuvant (ISPA) - TSNt-ISPA was obtained. Firstly, the immunogenicity and protective capacity of TSNt-ISPA was demonstrated as a prophylactic formulation in an acute model of infection. Later, the formulation was assessed as a therapeutic vaccine alone or combined with (Bz) using two models of chronic infection. BALB/c mice chronically infected with Sylvio X10/4 or Tulahuen cl2 T. cruzi strains were not treated as control or treated only with the therapeutic vaccine TSNt-ISPA, with a combined treatment TSNt-ISPA+Bz (Bz applied after the vaccine), or only with Bz. The vaccination schedule consisted of TSNt-ISPA administration at days110, 120, and 130 post-infection (pi) and Bz administration was performed daily from day 140 to 170 pi. At day 273 pi, electrocardiographic (ECG) parameters, heart parasite load, myocarditis, and heart fibrosis were assessed. In both models, therapeutic administration of TSNt-ISPA reduced ECG alterations and the cardiac tissue damage observed in the chronic phase. Moreover, vaccine treatment significantly decreased heart parasite load in both Sylvio X10/4 and Tulahuen cl2 infected mice. The combined treatment, but not Bz or vaccine administration alone, allowed to restore ECG parameters in Tulahuen cl2 infected mice. The results indicate the usefulness of the therapeutic TSNt-ISPA formulation in BALB/c mice chronically infected with Sylvio X10/4 or Tulahuen cl2 strain. For the mice infected with T. cruzi Tulahuen cl2 strain, the combined treatment with the vaccine and Bz had a more positive effect on the course of heart disease than the individual treatments with the vaccine or Bz alone.

Immunization With Lipopolysaccharide-Activated Dendritic Cells Generates a Specific CD8+ T Cell Response That Confers Partial Protection Against Infection With Trypanosoma cruzi.

Lipopolysaccharide (LPS) induces the activation of dendritic cells (DCs) throughout the engagement of toll-like receptor 4. LPS-activated DCs show increased capacity to process and present pathogen-derived antigens to activate naïve T cells. DCs-based vaccines have been successfully used to treat some cancer types, and lately transferred to the field of infectious diseases, in particular against HIV. However, there is no vaccine or DC therapy for any parasitic disease that is currently available. The immune response against Trypanosoma cruzi substantially relies on T cells, and both CD4+ and CD8+ T lymphocytes are required to control parasite growth. Here, we develop a vaccination strategy based on DCs derived from bone marrow, activated with LPS and loaded with TsKb20, an immunodominant epitope of the trans-sialidase family of proteins. We extensively characterized the CD8+ T cell response generated after immunization and compared three different readouts: a tetramer staining, ELISpot and Activation-Induced Marker (AIM) assays. To our knowledge, this work shows for the first time a proper set of T cell markers to evaluate specific CD8+ T cell responses in mice. We also show that our immunization scheme confers protection against T. cruzi, augmenting survival and reducing parasite burden in female but not male mice. We conclude that the immunization with LPS-activated DCs has the potential to prime significant CD8+ T cell responses in C57BL/6 mice independently of the sex, but this response will only be effective in female, possibly due to mice sexual dimorphisms in the response generated against T. cruzi.

Nasal immunization with a L. lactis-derived trans-sialidase antigen plus c-di-AMP protects against acute oral T. cruzi infection.

The new generation of vaccines for Chagas disease, are focused to induce both humoral and cellular response to effectively control Trypanosoma cruzi parasites. The administration of vaccine formulations intranasally has the advantage over parenteral routes that can induce a specific response at mucosal and systemic levels. This study aimed to evaluate and compare the immunogenicity and prophylactic effectiveness of two Trans-sialidase (TS)-based mucosal vaccines against T. cruzi administered intranasally. Vaccines consisted of a recombinant fragment of TS expressed in Lactococcus lactis formulated in two different adjuvants. The first, was an immunostimulant particle (ISPA, an ISCOMATRIX-like adjuvant), while the second was the dinucleotide c-di-AMP, which have shown immunostimulant properties at the mucosal level. BALB/c mice were immunized intranasally (3 doses, one every two weeks) with each formulation (TS + ISPA or TS + c-di-AMP) and with TS alone or vehicle (saline solution) as controls. Fifteen days after the last immunization, both TS + ISPA or TS + c-di-AMP induced an evident systemic humoral and cellular response, as judged by the increased plasma anti-TS IgG2a titers and IgG2a/IgG1 ratio and enhanced cellular response against TS. Plasma derived antibodies from TS + c-di-AMP also inhibit in vitro the invasion capacity of T. cruzi. Furthermore, specific secretory IgA was more enhanced in TS + c-di-AMP group. Protective efficacy was proved in vaccinated animals by an oral T. cruzi-challenge. Parasitemia control was only achieved by animals vaccinated with TS + c-di-AMP, despite all vaccinates groups showed enhanced CD8+IFN-γ+ T cell numbers. In addition, it was reflected during the acute phase in a significant reduction of tissue parasite load, clinical manifestations and diminished tissue damage. The better prophylactic capacity elicited by TS + c-di-AMP was related to the induction of neutralizing plasma antibodies and augmented levels of mucosal IgA since TS + ISPA and TS + c-di-AMP groups displayed similar immunogenicity and CD8+IFN-γ+ T-cell response. Therefore, TS + c-di-AMP formulation appears as a promising strategy for prophylaxis against T. cruzi.

Immunoendocrinology of the thymus in Chagas disease.

During immune response to infectious agents, the host develops an inflammatory response which could fail to eliminate the pathogen or may become dysregulated. In this case, the ongoing response acquires a new status and turns out to be detrimental. The same elements taking part in the establishment and regulation of the inflammatory response (cytokines, chemokines, regulatory T cells and counteracting compounds like glucocorticoids) may also mediate harmful effects. Thymic disturbances seen during Trypanosoma cruzi (T. cruzi) infection fit well with this conceptual framework. After infection, this organ suffers a severe atrophy due to apoptosis-induced thymocyte exhaustion, mainly affecting the immature double-positive (DP) CD4+CD8+ population. Thymus cellularity depletion, which occurs in the absence of main immunological mediators involved in anti-T. cruzi defense, seems to be linked to a systemic cytokine/hormonal imbalance, involving a dysregulated increase in Tumor Necrosis Factor alpha (TNF-α) and corticosterone hormone levels. Additionally, we have found an anomalous exit of potentially autoimmune DP cells to the periphery, in parallel to a shrinkage in the compartment of natural regulatory T cells. In this context, our data clearly point to the view that the thymus is a target organ of T. cruzi infection. Preserved thymus may be essential for the development of an effective immune response against T. cruzi, but this organ is severely affected by a dysregulated circuit of proinflammatory cytokines and glucocorticoids. Also, the alterations observed in the DP population might have potential implications for the autoimmune component of human Chagas disease.

A high corticosterone/DHEA-s ratio in young rats infected with Trypanosoma cruzi is associated with increased susceptibility.

We have previously established that young male rats are more susceptible to the effects of Trypanosoma cruzi infection than adult rats. To explore underlying age-associated differences in disease outcome, we simultaneously assessed hormone levels and cytokine release throughout the acute infection period in young and adult rats infected with T. cruzi. Young rats were inoculated with 1 x 10(6) and adult rats with 7 x 10(6) blood trypomastigotes, according to their relative body weight. At zero, seven, 14, 21 and 28 days after infection, blood was collected for the determination of gonadal and adrenal hormones, tumor necrosis factor α (TNF-α), interleukin (IL)-10 and specific IgM and IgG subtypes. Young animals displayed significantly higher parasitaemia values and an endocrine pattern that was characterised by elevated values in corticosterone (CT) and the CT/dehydroepiandrosterone-sulfate ratio, which favours immunosuppression and susceptibility. In contrast, adult male rats were able to restrict the parasite burden, which likely resulted from increased IgG antibody synthesis and oestradiol levels. Adult rats also showed a reduced TNF-α/IL-10 ratio and less tissue damage. We conclude that young animals exhibited increased vulnerability to T. cruzi infection compared with adults and this is associated with an unsuitable immunoendocrine milieu.

Targeting Myeloid-Derived Suppressor Cells to Enhance a Trans-Sialidase-Based Vaccine Against Trypanosoma cruzi.

Trypanosoma cruzi (T. cruzi) is a hemoflagellate protozoan parasite that causes Chagas disease, a neglected tropical disease that affects more than 6 million people around the world, mostly in Latin America. Despite intensive research, there is no vaccine available; therefore, new approaches are needed to further improve vaccine efficacy. It is well established that experimental T. cruzi infection induces a marked immunosuppressed state, which includes notably increases of CD11b+ GR-1+ myeloid-derived suppressor cells (MDSCs) in the spleen, liver and heart of infected mice. We previously showed that a trans-sialidase based vaccine (TSf-ISPA) is able to confer protection against a virulent T. cruzi strain, stimulating the effector immune response and decreasing CD11b+ GR-1+ splenocytes significantly. Here, we show that even in the immunological context elicited by the TSf-ISPA vaccine, the remaining MDSCs are still able to influence several immune populations. Depletion of MDSCs with 5 fluorouracil (5FU) at day 15 post-infection notably reshaped the immune response, as evidenced by flow cytometry of spleen cells obtained from mice after 21 days post-infection. After infection, TSf-ISPA-vaccinated and 5FU-treated mice showed a marked increase of the CD8 response, which included an increased expression of CD107a and CD44 markers in CD8+ cultured splenocytes. In addition, vaccinated and MDSC depleted mice showed an increase in the percentage and number of CD4+ Foxp3+ regulatory T cells (Tregs) as well as in the expression of Foxp3+ in CD4+ splenocytes. Furthermore, depletion of MDSCs also caused changes in the percentage and number of CD11chigh CD8α+ dendritic cells as well as in activation/maturation markers such as CD80, CD40 and MHCII. Thus, the obtained results suggest that MDSCs not only play a role suppressing the effector response during T. cruzi infection, but also strongly modulate the immune response in vaccinated mice, even when the vaccine formulation has a significant protective capacity. Although MDSC depletion at day 15 post-infection did not ameliorated survival or parasitemia levels, depletion of MDSCs during the first week of infection caused a beneficial trend in parasitemia and mice survival of vaccinated mice, supporting the possibility to target MDSCs from different approaches to enhance vaccine efficacy. Finally, since we previously showed that TSf-ISPA immunization causes a slight but significant increase of CD11b+ GR-1+ splenocytes, here we also targeted those cells at the stage of immunization, prior to T. cruzi challenge. Notably, 5FU administration before each dose of TSf-ISPA vaccine was able to significantly ameliorate survival and decrease parasitemia levels of TSf-ISPA-vaccinated and infected mice. Overall, this work supports that targeting MDSCs may be a valuable tool during vaccine design against T. cruzi, and likely for other pathologies that are characterized by the subversion of the immune system.

Enhanced Migratory Capacity of T Lymphocytes in Severe Chagasic Patients Is Correlated With VLA-4 and TNF-α Expression.

Trypanosoma cruzi infection in humans leads to progression to chronic chagasic myocarditis (CCM) in 30% of infected individuals, paralleling T cell inflammatory infiltrates in the heart tissue. T-cell trafficking into the hearts of CCM patients may be modulated by in situ expression of chemotactic or haptotactic molecules, as the chemokine CXCL12, the cytokine tumor necrosis factor-alpha (TNF-α), and extracellular matrix proteins (ECM), such as fibronectin. Herein we evaluated the expression of fibronectin, CXCL12, and TNF-α in the myocardial tissue of T. cruzi seropositive (asymptomatic or with CCM), as well as seronegative individuals as healthy controls. Hearts from CCM patients exhibited enhanced expression of these three molecules. CXCL12 and TNF-α serum levels were also increased in CCM individuals. We then evaluated T lymphocytes from chronic chagasic patients by cytofluorometry, in terms of membrane expression levels of molecules involved in cell activation and cell migration, respectively, HLA-DR and the VLA-4 (very late antigen-4, being one integrin-type fibronectin receptor). Indeed, the expression of HLA-DR and VLA-4 was enhanced on T lymphocytes from chagasic patients, especially in the CCM group. To further approach the dynamics of T cell migratory events, we performed fibronectin-, TNF-α-, and CXCL12-driven migration. Peripheral blood mononuclear cells (PBMCs) and T cells from CCM patients presented an ex vivo enhanced migratory capacity driven by fibronectin alone when this ECM protein was placed in the membrane of transwell migration chambers. When TNF-α was previously placed upon fibronectin, we observed a further and significant increase in the migratory response of both PBMCs and T lymphocytes. Overall, these data suggest the existence in patients with chronic Chagas disease of a cardiac inflammatory infiltrate vector that promotes the recruitment and accumulation of activated T cells, driven in part by enhanced tissue expression of fibronectin and TNF-α, as well as the respective corresponding VLA-4 and TNF receptors.

Chagas disease reactivation in rheumatologic patients: association with immunosuppressive therapy and humoral response.

Evidence for Chagas disease reactivation (CDR) in rheumatologic patients under rheumatologic treatments (RTs) is scarce. To screen and follow-up patients with rheumatic diseases and concomitant Chagas disease under RT to detect CDR and to describe a possible relationship between CDR and specific RT. An observational, longitudinal, prospective, consecutive study was carried out between 2018 and 2020. Included patients were evaluated during the follow-up for clinical and laboratorial manifestations of CDR. Direct blood parasitological examination (Strout method) and polymerase chain reaction (PCR) were employed to diagnose CDR. The dynamic of anti-T. cruzi-specific antibodies was also assessed by IHA and ELISA (total IgG and Anti-SAPA). Fifty-one patients were included (86% women). Rheumatoid arthritis was the predominant disease (57%). Classic DMARDs (86.3%) and corticosteroids (61%) were the most frequent RT. CDR was developed in 6 patients (11.7%), exhibiting both positive Strout and PCR. Symptomatic reactivation of CD (fever, asthenia, arthralgias, myalgias) occurred in two patients who had previously been diagnosed with it. Regardless of the different RT, all patients who experienced CDR had previously received more than ≥ 20 mg/day of prednisone equivalent. Despite immunosuppression, patients with CDR exhibited increased levels of specific anti-T. cruzi and anti-SAPA antibodies, which decreased after anti-parasitic treatment. CDR is possible in rheumatologic patients, especially after receiving high doses of corticosteroids. Since CDR symptoms may mimic rheumatic disease activity, monitoring of Chagas disease is highly recommended before, during and after immunosuppression. Key Points • Chagas disease reactivation (CDR) in the context of rheumatological treatment was associated to high doses of corticosteroids. • CDR was associated with an increase in anti-T. cruzi antibodies despite the immunosuppressive treatment. • Suspecting and anticipating CDR is mandatory in this patient population to diagnose and treat it.

Cytokines and cell adhesion receptors in the regulation of immunity to Trypanosoma cruzi.

Pathophysiology of Chagas' disease is not completely defined, although innate and adaptative immune responses are crucial. In acute infection some parasite antigens can activate macrophages, and this may result in pro-inflammatory cytokine production, nitric oxide synthesis, and consequent control of parasitemia and mortality. Cell-mediated immunity in Trypanosoma cruzi infection is also modulated by cytokines, but in addition to parasite-specific responses, autoimmunity can be also triggered. Importantly, cytokines may also play a role in the cell-mediated immunity of infected subjects. Finally, leukocyte influx towards target tissues is regulated by cytokines, chemokines, and extracellular matrix components which may represent potential therapeutic targets in infected patients. Here we will discuss recent findings on the role of cytokines, chemokines and extracellular matrix components in the regulation of innate and adaptive immunity during T. cruzi infection.