Luiz Ricardo Berbert

Immunoneuroendocrine alterations in patients with progressive forms of chronic Chagas disease

We studied the features of parallel immunoneuroendocrine responses in patients with different degrees of chronic Chagas myocarditis (indeterminate, mild/moderate or severe). A systemic inflammatory scenario was evident in patients with severe myocarditis compared to healthy subjects. This was paralleled by a disrupted activation of the hypothalamus-pituitary-adrenal axis, characterized by decreased concentrations of dehydroepiandrosterone-sulfate (DHEA-s) and an unbalanced cortisol/DHEA-s ratio, reinforcing the view that severe Chagas disease is devoid of an adequate anti-inflammatory milieu, likely involved in pathology. Our study constitutes the first demonstration of neuroendocrine disturbances, in parallel to a systemic inflammatory profile, during progressive human Chagas disease.

Chagasic Thymic Atrophy Does Not Affect Negative Selection but Results in the Export of Activated CD4+CD8+ T Cells in Severe Forms of Human Disease

Extrathymic CD4+CD8+ double-positive (DP) T cells are increased in some pathophysiological conditions, including infectious diseases. In the murine model of Chagas disease, it has been shown that the protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironment and the lymphoid compartment. In the acute phase, this results in a severe atrophy of the organ and early release of DP cells into the periphery. To date, the effect of the changes promoted by the parasite infection on thymic central tolerance has remained elusive. Herein we show that the intrathymic key elements that are necessary to promote the negative selection of thymocytes undergoing maturation during the thymopoiesis remains functional during the acute chagasic thymic atrophy. Intrathymic expression of the autoimmune regulator factor (Aire) and tissue-restricted antigen (TRA) genes is normal. In addition, the expression of the proapoptotic Bim protein in thymocytes was not changed, revealing that the parasite infection-induced thymus atrophy has no effect on these marker genes necessary to promote clonal deletion of T cells. In a chicken egg ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic system, the administration of OVA peptide into infected mice with thymic atrophy promoted OVA-specific thymocyte apoptosis, further indicating normal negative selection process during the infection. Yet, although the intrathymic checkpoints necessary for thymic negative selection are present in the acute phase of Chagas disease, we found that the DP cells released into the periphery acquire an activated phenotype similar to what is described for activated effector or memory single-positive T cells. Most interestingly, we also demonstrate that increased percentages of peripheral blood subset of DP cells exhibiting an activated HLA-DR+ phenotype are associated with severe cardiac forms of human chronic Chagas disease. These cells may contribute to the immunopathological events seen in the Chagas disease.

Caspase-8 and caspase-9 mediate thymocyte apoptosis in Trypanosoma cruzi acutely infected mice

Trypanosoma cruzi acute infection leads to thymic atrophy, largely as a result of death of immature DP T cells. In a second vein, the glucocorticoid hormone imbalance promotes DP T cell apoptosis in infected mice. Herein, we assessed the involvement of caspase signaling in thymocyte death during T. cruzi acute infection. BALB/c mice were infected i.p. with 102 trypomastigote forms of T. cruzi and analyzed from 7 to 19 dpi. Thymocyte apoptosis was observed in early stages of infection, increasing along with time postinfection. Immature DN and DP as well as CD4+ and CD8+ thymocytes from infected mice showed increased activation of caspase‐8, ‐9, and ‐3. In vitro treatment of thymocytes from infected mice with a general caspase inhibitor or the combination of caspase‐8‐ and caspase‐9‐specific inhibitors increased the number of living thymocytes. Intrathymic injection of the general caspase inhibitor, but not caspase‐8 or ‐9 inhibitors individually, prevented thymic atrophy and thymocyte depletion in infected mice. Moreover, blockade of glucocorticoid receptor activity with RU486 prevented DP thymocyte apoptosis, together with caspase‐8 and ‐9 activation. These findings indicate that DP T cell apoptosis following experimental T. cruzi acute infection is dependent on glucocorticoid stimulation, promoting caspase‐8 and ‐9 activation.

TNF-α is involved in the abnormal thymocyte migration during experimental Trypanosoma cruzi infection and favors the export of immature cells.

Previous studies revealed a significant production of inflammatory cytokines together with severe thymic atrophy and thymocyte migratory disturbances during experimental Chagas disease. Migratory activity of thymocytes and mature T cells seem to be finely tuned by cytokines, chemokines and extracellular matrix (ECM) components. Systemic TNF-α is enhanced during infection and appears to be crucial in the response against the parasite. However, it also seems to be involved in disease pathology, since it is implicated in the arrival of T cells to effector sites, including the myocardium. Herein, we analyzed the role of TNF-α in the migratory activity of thymocytes in Trypanosoma cruzi (T. cruzi) acutely-infected mice. We found increased expression and deposition of TNF-α in the thymus of infected animals compared to controls, accompanied by increased co-localization of fibronectin, a cell migration-related ECM molecule, whose contents in the thymus of infected mice is also augmented. In-vivo studies showed an enhanced export of thymocytes in T. cruzi-infected mice, as ascertained by intrathymic injection of FITC alone or in combination with TNF-α. The increase of immature CD4+CD8+ T cells in secondary lymphoid organs was even more clear-cut when TNF-α was co-injected with FITC. Ex-vivo transmigration assays also revealed higher number of migrating cells when TNF-α was added onto fibronectin lattices, with higher input of all thymocyte subsets, including immature CD4+CD8+. Infected animals also exhibit enhanced levels of expression of both mRNA TNF-α receptors in the CD4+CD8+ subpopulation. Our findings suggest that in T. cruzi acute infection, when TNF-α is complexed with fibronectin, it favours the altered migration of thymocytes, promoting the release of mature and immature T cells to different compartments of the immune system. Conceptually, this work reinforces the notion that thymocyte migration is a multivectorial biological event in health and disease, and that TNF-α is a further player in the process.

Trypanosoma cruzi Infection through the Oral Route Promotes a Severe Infection in Mice: New Disease Form from an Old Infection?

Oral transmission of Chagas disease has been documented in Latin American countries. Nevertheless, significant studies on the pathophysiology of this form of infection are largely lacking. The few studies investigating oral route infection disregard that inoculation in the oral cavity (Oral infection, OI) or by gavage (Gastrointestinal infection, GI) represent different infection routes, yet both show clear-cut parasitemia and heart parasitism during the acute infection. Herein, BALB/c mice were subjected to acute OI or GI infection using 5x104 culture-derived Trypanosoma cruzi trypomastigotes. OI mice displayed higher parasitemia and mortality rates than their GI counterparts. Heart histopathology showed larger areas of infiltration in the GI mice, whereas liver lesions were more severe in the OI animals, accompanied by higher Alanine Transaminase and Aspartate Transaminase serum contents. A differential cytokine pattern was also observed because OI mice presented higher pro-inflammatory cytokine (IFN-γ, TNF) serum levels than GI animals. Real-time PCR confirmed a higher TNF, IFN-γ, as well as IL-10 expression in the cardiac tissue from the OI group compared with GI. Conversely, TGF-β and IL-17 serum levels were greater in the GI animals. Immunolabeling revealed macrophages as the main tissue source of TNF in infected mice. The high mortality rate observed in the OI mice paralleled the TNF serum rise, with its inhibition by an anti-TNF treatment. Moreover, differences in susceptibility between GI versus OI mice were more clearly related to the host response than to the effect of gastric pH on parasites, since infection in magnesium hydroxide-treated mice showed similar results. Overall, the present study provides conclusive evidence that the initial site of parasite entrance critically affects host immune response and disease outcome. In light of the occurrence of oral Chagas disease outbreaks, our results raise important implications in terms of the current view of the natural disease course and host-parasite relationship.

Extrathymic CD4+CD8+ lymphocytes in Chagas disease: possible relationship with an immunoendocrine imbalance.

Double‐positive (DP) CD4+CD8+ T cells normally represent a thymic subpopulation that is developed in the thymus as a precursor of CD4+ or CD8+ single‐positive T cells. Recent evidence has shown that DP cells with an activated phenotype can be tracked in secondary lymph organs. The detection of an activated DP population in the periphery, a population that expresses T cell receptors unselected during thymic negative selection in murine models of Trypanosoma cruzi infection and in humans with Chagas disease, raise new questions about the relevance of this population in the pathogenesis of this major parasitic disease and its possible link with immunoendocrine alterations.

Trypanosoma cruzi Entrance through Systemic or Mucosal Infection Sites Differentially Modulates Regional Immune Response Following Acute Infection in Mice

Acute Chagas disease is characterized by a systemic infection that leads to the strong activation of the adaptive immune response. Outbreaks of oral contamination by the infective protozoan Trypanosoma cruzi are frequent in Brazil and other Latin American countries, and an increased severity of clinical manifestations and mortality is observed in infected patients. These findings have elicited questions about the specific responses triggered after T. cruzi entry via mucosal sites, possibly modulating local immune mechanisms, and further impacting regional and systemic immunity. Here, we provide evidence for the existence of differential lymphoid organ responses in experimental models of acute T. cruzi infection.

Dynamics of Lymphocyte Populations during Trypanosoma cruzi Infection: From Thymocyte Depletion to Differential Cell Expansion/Contraction in Peripheral Lymphoid Organs.

The comprehension of the immune responses in infectious diseases is crucial for developing novel therapeutic strategies. Here, we review current findings on the dynamics of lymphocyte subpopulations following experimental acute infection by Trypanosoma cruzi, the causative agent of Chagas disease. In the thymus, although the negative selection process of the T-cell repertoire remains operational, there is a massive thymocyte depletion and abnormal release of immature CD4(+)CD8(+) cells to peripheral lymphoid organs, where they acquire an activated phenotype similar to activated effector or memory T cells. These cells apparently bypassed the negative selection process, and some of them are potentially autoimmune. In infected animals, an atrophy of mesenteric lymph nodes is also observed, in contrast with the lymphocyte expansion in spleen and subcutaneous lymph nodes, illustrating a complex and organ specific dynamics of lymphocyte subpopulations. Accordingly, T- and B-cell activation is seen in subcutaneous lymph nodes and spleen, but not in mesenteric lymph nodes. Lastly, although the function of peripheral CD4(+)CD8(+) T-cell population remains to be defined in vivo, their presence may contribute to the immunopathological events found in both murine and human Chagas disease.

Unraveling Chagas disease transmission through the oral route: Gateways to Trypanosoma cruzi infection and target tissues

Oral transmission of Trypanosoma cruzi, the causative agent of Chagas disease, is the most important route of infection in Brazilian Amazon and Venezuela. Other South American countries have also reported outbreaks associated with food consumption. A recent study showed the importance of parasite contact with oral cavity to induce a highly severe acute disease in mice. However, it remains uncertain the primary site of parasite entry and multiplication due to an oral infection. Here, we evaluated the presence of T. cruzi Dm28c luciferase (Dm28c-luc) parasites in orally infected mice, by bioluminescence and quantitative real-time PCR. In vivo bioluminescent images indicated the nasomaxillary region as the site of parasite invasion in the host, becoming consistently infected throughout the acute phase. At later moments, 7 and 21 days post-infection (dpi), luminescent signal is denser in the thorax, abdomen and genital region, because of parasite dissemination in different tissues. Ex vivo analysis demonstrated that the nasomaxillary region, heart, mandibular lymph nodes, liver, spleen, brain, epididymal fat associated to male sex organs, salivary glands, cheek muscle, mesenteric fat and lymph nodes, stomach, esophagus, small and large intestine are target tissues at latter moments of infection. In the same line, amastigote nests of Dm28c GFP T. cruzi were detected in the nasal cavity of 6 dpi mice. Parasite quantification by real-time qPCR at 7 and 21 dpi showed predominant T. cruzi detection and expansion in mouse nasal cavity. Moreover, T. cruzi DNA was also observed in the mandibular lymph nodes, pituitary gland, heart, liver, small intestine and spleen at 7 dpi, and further, disseminated to other tissues, such as the brain, stomach, esophagus and large intestine at 21 dpi. Our results clearly demonstrated that oral cavity and adjacent compartments is the main target region in oral T. cruzi infection leading to parasite multiplication at the nasal cavity.

Is There a Role for Cellular Prion Protein in Intrathymic T Cell Differentiation and Migration

The cellular prion protein (PrPC) is expressed in the nervous and immune systems. Functionally, PrPC has been suggested to participate in neuron survival, neuritogenesis and T lymphocyte activation. Moreover, PrPC interaction with laminin influences neuronal adhesion and neurite extension. Nevertheless, so far the physiological role of PrPC has not been completely elucidated, particularly in the immune system. The aim of the study was to evaluate the possible participation of PrPC in intrathymic T cell development. We evaluated T cell differentiation markers in thymocytes and peripheral lymphocytes, as well as thymocyte death in PrPC-null or PrPC-overexpressing (Tga20) mice, compared to wild-type controls. In these same animals, we ascertained laminin-driven thymocyte migration. Compared to controls, only marginal differences were found in PrPC-null animals. However, Tga20 mice exhibited a severe thymic hypoplasia, with 10–20% lymphocytes compared to wild-type counterparts. In particular, the frequency of CD4+CD8+ cells was largely reduced, and this was accompanied by a dramatic increase in the frequency of CD4–CD8– thymocytes, which could be as high as 60–65% of the whole-cell suspensions. Moreover, Tga20 mice exhibited an increase in thymocyte death, comprising the CD4+CD8+, as well as CD4+ and CD8+ single-positive cells. Additionally, laminin-driven migration was largely impaired in Tga20 mice, in which we also found a significant decrease in total T lymphocytes in the spleen and lymph nodes. Our results show that PrPC overexpression alters intrathymic T cell development, a defect that likely has a negative impact in the formation of the T cell peripheral pool.