Désio Aurélio Farias-de-Oliveira

Galectin-3 modulates carbohydrate-dependent thymocyte interactions with the thymic microenvironment

The process of thymocyte differentiation occurs within the context of the thymic microenvironment, in which T cell precursors interact with thymic microenvironmental cells and extracellular matrix. Here we studied the expression of galectin‐3, a β‐galactoside binding lectin, in the thymus of young adult mice. Galectin‐3 was found mainly in the medulla and to a lesser extent in the cortex. We further showed that distinct microenvironmental elements, such as thymic epithelial cells, the epithelial component of thymic nurse complexes and phagocytic cells of the thymic reticulum produce, secrete and accumulate galectin‐3 on the cell surface. Functionally, galectin‐3‐enriched medium inhibited in vitro thymocyte interactions with thymic microenvironmental cells, accelerated the release of thymocytes from thymic nurse cells and inhibited the reconstitution of these lymphoepithelial complexes. These effects were blocked by exogenous lactose (Galβ1–4Glc), but not melibiose (Galα1–6Glc), and by a monospecific anti‐galectin‐3 antibody. Recombinant galectin‐3 also inhibited thymocyte/thymic epithelial cell interactions. Our data indicate that intrathymically produced galectin‐3 disrupts thymocyte/microenvironmental cell interactions, thus acting as a de‐adhesion molecule.

Functional Insulin-Like Growth Factor-1/Insulin-Like Growth Factor-1 Receptor-Mediated Circuit in Human and Murine Thymic Epithelial Cells

Interactions between thymocytes and thymic epithelial cell (TEC) can be modulated by growth hormone via insulin-like growth factor-1 (IGF-1). In this study, we showed IGF-1 and IGF-1 receptor mRNA expression by human and murine TEC and thymocytes. Functionally, IGF-1 stimulates extracellular matrix production by human TEC. Moreover, pretreatment of murine TEC with IGF-1 increases their adhesion to thymocytes. Interestingly, we observed an increase in the frequency of CD4–CD8–CD90+ T cells which adhered to pretreated TEC, supporting the concept that IGF-1 may also act indirectly on intrathymic T cell differentiation and migration through the thymic epithelium.

Differential Regional Immune Response in Chagas Disease

Following infection, lymphocytes expand exponentially and differentiate into effector cells to control infection and coordinate the multiple effector arms of the immune response. Soon after this expansion, the majority of antigen-specific lymphocytes die, thus keeping homeostasis, and a small pool of memory cells develops, providing long-term immunity to subsequent reinfection. The extent of infection and rate of pathogen clearance are thought to determine both the magnitude of cell expansion and the homeostatic contraction to a stable number of memory cells. This straight correlation between the kinetics of T cell response and the dynamics of lymphoid tissue cell numbers is a constant feature in acute infections yielded by pathogens that are cleared during the course of response. However, the regional dynamics of the immune response mounted against pathogens that are able to establish a persistent infection remain poorly understood. Herein we discuss the differential lymphocyte dynamics in distinct central and peripheral lymphoid organs following acute infection by Trypanosoma cruzi, the causative agent of Chagas disease. While the thymus and mesenteric lymph nodes undergo a severe atrophy with massive lymphocyte depletion, the spleen and subcutaneous lymph nodes expand due to T and B cell activation/proliferation. These events are regulated by cytokines, as well as parasite-derived moieties. In this regard, identifying the molecular mechanisms underlying regional lymphocyte dynamics secondary to T. cruzi infection may hopefully contribute to the design of novel immune intervention strategies to control pathology in this infection.

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.

Triiodothyronine Modulates Extracellular Matrix-Mediated Interactions between Thymocytes and Thymic Microenvironmental Cells

Objectives: Thyroid hormones exert immunomodulatory activities and the thymus is one of their target organs. We previously showed that triiodothyronine (T3) modulates thymic hormone production and extracellular matrix (ECM) expression by mouse thymic epithelial cells (TEC). This concept is enlarged herein by studying the effects of T3 in human TEC preparations including primary cultures derived from thymic nurse cell complexes, as well as human and murine TEC lines. Methods and Results: We observed that in all cases, ECM ligands and receptors (such as fibronectin, laminin, VLA-5 and VLA-6) are enhanced in vitro, as ascertained by immunocytochemistry, ELISA and cytofluorometry. Moreover, thymocyte adhesion to these TEC preparations is augmented by T3. Interestingly, TEC-thymocyte adhesion is also upregulated when thymocytes from T3-treated mice adhere to untreated TEC cultures. Such an enhancing effect of T3 upon TEC-thymocyte interactions is likely due to the increase in the expression of ECM ligands and receptors, since it is prevented when T3-treated TEC cultures are incubated with anti-ECM antibodies prior to the adhesion assay. We then tested whether T3 could modulate interactions between thymocytes and nonepithelial microenvironmental cells, exemplified herein by the phagocytic cells of the mouse thymic reticulum. In fact, in vitro treatment of these cells with T3 increases ECM ligands and receptors and augments their ability to adhere to thymocytes. Lastly, using immunochemistry-based assays, we showed the presence of the nuclear T3 receptor in all thymic microenvironmental cell preparations. Conclusion: Our data show that T3 upregulates ECM-mediated heterocellular interactions of thymocytes with distinct thymic microenvironmental cells, in both humans and mice.

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.

Thymus Atrophy and Double-Positive Escape Are Common Features in Infectious Diseases

The thymus is a primary lymphoid organ in which bone marrow-derived T-cell precursors undergo differentiation, leading to migration of positively selected thymocytes to the T-cell-dependent areas of secondary lymphoid organs. This organ can undergo atrophy, caused by several endogenous and exogenous factors such as ageing, hormone fluctuations, and infectious agents. This paper will focus on emerging data on the thymic atrophy caused by infectious agents. We present data on the dynamics of thymus lymphocytes during acute Trypanosoma cruzi infection, showing that the resulting thymus atrophy comprises the abnormal release of thymic-derived T cells and may have an impact on host immune response.

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.

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.

EphB2 and EphB3 play an important role in the lymphoid seeding of murine adult thymus

Adult thymuses lacking either ephrin type B receptor 2 (EphB2) or EphB3, or expressing a truncated form of EphB2, the forward signal‐deficient EphB2LacZ, have low numbers of early thymic progenitors (ETPs) and are colonized in vivo by reduced numbers of injected bone marrow (BM) lineage‐negative (Lin−) cells. Hematopoietic progenitors from these EphB mutants showed decreased capacities to colonize wild type (WT) thymuses compared with WT precursors, with EphB2−/− cells exhibiting the greatest reduction. WT BM Lin− cells also showed decreased colonizing capacity into mutant thymuses. The reduction was also more severe in EphB2−/− host thymuses, with a less severe phenotype in the EphB2LacZ thymus. These results suggest a major function for forward signaling through EphB2 and, to a lesser extent, EphB3, in either colonizing progenitor cells or thymic stromal cells, for in vivo adult thymus recruitment. Furthermore, the altered expression of the molecules involved in thymic colonization that occurs in the mutant thymus correlates with the observed colonizing capacities of different mutant mice. Reduced production of CCL21 and CCL25 occurred in the thymus of the 3 EphB‐deficient mice, but their expression, similar to that of P‐selectin, on blood vessels, the method of entry of progenitor cells into the vascular thymus, only showed a significant reduction in EphB2−/− and EphB3−/− thymuses. Decreased migration into the EphB2−/− thymuses correlated also with reduced expression of both ephrinB1 and ephrinB2, without changes in the EphB2LacZ thymuses. In the EphB3−/− thymuses, only ephrinB1 expression appeared significantly diminished, confirming the relevance of forward signals mediated by the EphB2‐ephrinB1 pair in cell recruitment into the adult thymus.