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.

Quantitative analysis of proteins secreted by Leishmania (Viannia) braziliensis strains associated to distinct clinical manifestations of American Tegumentary Leishmaniasis.

The role of Leishmania braziliensis in the development of different clinical forms of American Tegumentary Leishmaniasis (ATL) is unclear, but it has been suggested that molecules secreted/released by parasites could modulate the clinical outcome. Here, we analyzed the infection rate and cytokine profile of macrophages pretreated with the secretome of two L. braziliensis strains associated with polar clinical forms of ATL: one associated with localized self-healing cutaneous leishmaniasis (LCL) and other associated with the disseminated form (DL). Besides, we use an iTRAQ-based quantitative proteomics approach to compare the abundance of proteins secreted by those strains. In vitro infection demonstrated that pretreatment with secretome resulted in higher number of infected macrophages, as well as higher number of amastigotes per cell. Additionally, macrophages pretreated with LCL secretome exhibited a proinflammatory profile, whereas those pretreated with the DL one did not. These findings suggest that secretomes made macrophages more susceptible to infection and that molecules secreted by each strain modulate, differentially, the macrophages' cytokine profile. Indeed, proteomics analysis showed that the DL secretome is rich in molecules involved in macrophage deactivation, while is poor in proteins that activate proinflammatory pathways. Together, our results reveal new molecules that may contribute to the infection, persistence and dissemination of the parasite. SIGNIFICANCE: Leishmania braziliensis is associated to localized self-healing cutaneous lesions (LCL), disseminated leishmaniasis (DL), and mucocutaneous lesions (MCL). To understand the role of the parasite in those distinct clinical manifestations we evaluated infection rates and cytokine profiles of macrophages pre-treated with secretomes of two L. braziliensis strains associated with DL and LCL, and quantitatively compared these secretomes. The infection index of macrophages pretreated with the DL secretome was significantly higher than that exhibited by non-treated cells. Interestingly, whereas the LCL secretome stimulated a proinflammatory setting, favoring an effector cell response that would explain the proper resolution of the disease caused by this strain, the DL strain was not able to elicit such response or has mechanisms to prevent this activation. Indeed, DL secretome is rich in peptidases that may deactivate cell pathways crucial for parasite elimination, while is poor in proteins that could activate proinflammatory pathways, favoring parasite infection and persistence.

Erratum: Protein malnutrition promotes dysregulation of molecules involved in T cell migration in the thymus of mice infected with Leishmania infantum.

This corrects the article DOI: 10.1038/srep45991.

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.

Protein malnutrition promotes dysregulation of molecules involved in T cell migration in the thymus of mice infected with Leishmania infantum.

Protein malnutrition, the most deleterious cause of malnutrition in developing countries, has been considered a primary risk factor for the development of clinical visceral leishmaniasis (VL). Protein malnutrition and infection with Leishmania infantum leads to lymphoid tissue disorganization, including changes in cellularity and lymphocyte subpopulations in the thymus and spleen. Here we report that protein malnutrition modifies thymic chemotactic factors by diminishing the CCL5, CXCL12, IGF1, CXCL9 and CXCL10 protein levels in infected animals. Nevertheless, T cells preserve their migratory capability, as they were able to migrate ex vivo in response to chemotactic stimuli, indicating that malnutrition may compromise the thymic microenvironment and alter in vivo thymocyte migration. Decrease in chemotactic factors protein levels was accompanied by an early increase in the parasite load of the spleen. These results suggest that the precondition of malnutrition is affecting the cell-mediated immune response to L. infantum by altering T cell migration and interfering with the capacity of protein-deprived animals to control parasite spreading and proliferation. Our data provide evidence for a disturbance of T lymphocyte migration involving both central and peripheral T-cells, which likely contribute to the pathophysiology of VL that occurs in malnourished individuals.

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 5x10(4) 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-ß 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.

Interações imunoendócrinas na migração de linfócitos na doença de chagas humana crônica: relação com a cardiopatia / immunendocrine interactions on lymphocyte migration in chronic human chagas disease: relationship to cardiopathy

A doença de Chagas permanece sendo um grave problema de saúde pública nas Américas. Na fisiopatologia da doença, é observada uma intensa resposta imune, com alta produção de citocinas inflamatórias e quimiocinas que contribuem para o tráfego de células ativadas para tecidos alvo. Este evento de migração celular pode estar relacionado à formação de cardiopatia chagásica nos indivíduos infectados. Um desequilíbrio na produção hormonal do eixo Hipotálamo-Pituitária-Adrenal (HPA) foi observado em pacientes chagásicos, e, esse processo pode influenciar o sistema imune e possivelmente a gênese da cardiopatia. Nesse estudo, foi avaliada a resposta migratória de células T de pacientes chagásicos com diferentes formas de cardiopatia, correlacionando esses eventos com a produção de cortisol e DHEA ocorrida na fase crônica da doença. Primeiramente foi observado que TNF, IL-6, IFN-gama, IL-17, IL-10 e TGF-beta são mais expressas de acordo com a gravidade da cardiopatia. Em paralelo a esse aumento de citocinas foi observado um desequilíbrio hormonal no eixo HPA com diminuição do hormônio DHEA sérico e aumento da razão Cortisol/DHEA circulantes. Além disso, foi observado um aumento da resposta migratória de células T, com fenótipo ativado (HLADR+/VLA-4+) sobre fibronectina, CXCL12 e TNF-alfa, e também combinados a um pré tratamento de DHEA e Cortisol destas células. Estes resultados indicam que distúrbios neuroendócrinos, correlacionados a um perfil inflamatório sistêmico, podem contribuir para aumentar o potencial migratório de células T aos sítios inflamatórios, incluindo tecido cardíaco, estando envolvidos na cardiopatia relatada na doença...

Chagas disease remains a serious public health problem in the Americas. In thepathophysiology of disease, it is observed an intense immune response with highexpression of inflammatory cytokines and chemokines that contribute to activated Tcells traffic, that target the inflamed tissue. These cell migration events may berelated to the formation of Chagas heart disease in infected individuals. Animbalance in hormone production of the Hypothalamus-Pituitary-Adrenal axis (HPA)was observed in patients with Chagas disease, and this process could influence theimmune system, including, cardiopathy formation. In this study, T cells migratoryresponse from chagasic patients with different forms of cardiopathy was performed tocorrelate these events with cortisol and DHEA production in the chronic phase of thedisease. Firstly, it was observed that inflammatory cytokines, such as TNF, IL-6, IFN-gama, IL-17, IL-10 e TGF-beta were expressed in terms of disease severity. In parallel withcytokine enhancement, it was observed a hormonal imbalance in the HPA axis,where a decrease in hormone DHEA resulted in increased cortisol ratio / circulatingDHEA. In addition, there was an increase in migratory response of T cells with anactivated phenotype (HLADR+/VLA-4+) over fibronectin, CXCL12 and TNF-alpha, andalso a combined pre treatment of DHEA and cortisol. These results indicate thatneuroendocrine disorders correlated to a systemic inflammatory profile, can increasemigration potential of T cells to inflammatory sites, including heart tissue and thusbeing involved in heart disease...

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.

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 10(2) 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.

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.

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.

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.

Impacto da superexpressão da poteína prion celular sobre o sistema imune: correlação com maior susceptibilidade à infecção experimental pelo Trypanosoma cruzi / Impact of cellular prion protein overexpression on immune system: correlation with higher susceptibility during experimental Trypanosoma cruzi infection

A proteína príon celular (PrPc) é uma glicoproteína constitutivamente expressa no sistema nervoso, bem como no sistema imune, porém, seu papel fisiológico e em processos infecciosos é pouco compreendido. Dados prévios da literatura sugerem disfunções fisiológicas no sistema imune em modelos murinos transgênicos que superexpressam essa proteína podendo levar os mesmos a um processo de imunodeficiência mediante infecções. Para testar essa hipótese, este projeto tem o objetivo de avaliar o impacto da infecção experimental pelo T. cruzi em camundongos que superexpressam (TG20) ou não expressam PrPc (KO), analisando parâmetros de infecção correlacionados com a infiltração linfocitária no tecido cardíaco. Nossos resultados demonstram que a superexpressão de PrPc em camundongos gera inicialmente susceptibilidade do modelo à infecção se comparado ao grupo que não expressa a proteína, conforme observado nas taxas de sobrevida e parasitemia. A presença de células T, B, bem como células CD11b(positivo) de infiltrado inflamatório é numericamente maior no grupo TG20, porém, esse evento não parece estar relacionado diretamente com alterações observadas no baço e linfonodos desses animais durante cinética de infecção; em relação à expressão de moléculas de ativação linfócitos nestes modelos, observamos aumento das mesmas nas subpopulações linfocitárias do grupo TG20. Nos timo dos modelos TG20 foram observadas alterações fenotípicas, bem como aumento de espressão de laminina e fibronectina e seus receptores nos timócitos. Os dados deste trabalho demonstram que Prpc tem um grande impacto na fisiologia da infecção pelo T. cruzi no modelo murino que superexpressa essa proteína, correlacionando esse evento a uma imunodeficiência previamente observada no mesmo modelo não infectado.

Is there a role for cellular prion protein in intrathymic T cell differentiation and migration?

The cellular prion protein (PrP(C)) is expressed in the nervous and immune systems. Functionally, PrP(C) has been suggested to participate in neuron survival, neuritogenesis and T lymphocyte activation. Moreover, PrP(C) interaction with laminin influences neuronal adhesion and neurite extension. Nevertheless, so far the physiological role of PrP(C) has not been completely elucidated, particularly in the immune system. The aim of the study was to evaluate the possible participation of PrP(C) in intrathymic T cell development. We evaluated T cell differentiation markers in thymocytes and peripheral lymphocytes, as well as thymocyte death in PrP(C)-null or PrP(C)-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 PrP(C)-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 PrP(C) overexpression alters intrathymic T cell development, a defect that likely has a negative impact in the formation of the T cell peripheral pool.