Cynomolgus macaques naturally infected with Trypanosoma cruzi-I exhibit an overall mixed pro-inflammatory/modulated cytokine signature characteristic of human Chagas disease

Background: Non-human primates have been shown to be useful models for Chagas disease. We previously reported that natural T. cruzi infection of cynomolgus macaques triggers clinical features and immunophenotypic changes of peripheral blood leukocytes resembling those observed in human Chagas disease. In the present study, we further characterize the cytokine-mediated microenvironment to provide supportive evidence of the utility of cynomolgus macaques as a model for drug development for human Chagas disease. Methods and findings: In this cross-sectional study design, flow cytometry and systems biology approaches were used to characterize the ex vivo and in vitro T. cruzi-specific functional cytokine signature of circulating leukocytes from TcI-T. cruzi naturally infected cynomolgus macaques (CH). Results showed that CH presented an overall CD4+-derived IFN-γ pattern regulated by IL-10-derived from CD4+ T-cells and B-cells, contrasting with the baseline profile observed in non-infected hosts (NI). Homologous TcI-T. cruzi-antigen recall in vitro induced a broad pro-inflammatory cytokine response in CH, mediated by TNF from innate/adaptive cells, counterbalanced by monocyte/B-cell-derived IL-10. TcIV-antigen triggered a more selective cytokine signature mediated by NK and T-cell-derived IFN-γ with modest regulation by IL-10 from T-cells. While NI presented a cytokine network comprised of small number of neighborhood connections, CH displayed a complex cross-talk amongst network elements. Noteworthy, was the ability of TcI-antigen to drive a complex global pro-inflammatory network mediated by TNF and IFN-γ from NK-cells, CD4+ and CD8+ T-cells, regulated by IL-10+CD8+ T-cells, in contrast to the TcIV-antigens that trigger a modest network, with moderate connecting edges. Conclusions: Altogether, our findings demonstrated that CH present a pro-inflammatory/regulatory cytokine signature similar to that observed in human Chagas disease. These data bring additional insights that further validate these non-human primates as experimental models for Chagas disease

Phenotypic Features of Circulating Leukocytes from Non-human Primates Naturally Infected with Trypanosoma cruzi Resemble the Major Immunological Findings Observed in Human Chagas Disease

Background: Cynomolgus macaques (Macaca fascicularis) represent a feasible model for research on Chagas disease since natural T. cruzi infection in these primates leads to clinical outcomes similar to those observed in humans. However, it is still unknown whether these clinical similarities are accompanied by equivalent immunological characteristics in the two species. We have performed a detailed immunophenotypic analysis of circulating leukocytes together with systems biology approaches from 15 cynomolgus macaques naturally infected with T. cruzi (CH) presenting the chronic phase of Chagas disease to identify biomarkers that might be useful for clinical investigations. Methods and findings: Our data established that CH displayed increased expression of CD32+ and CD56+ in monocytes and enhanced frequency of NK Granzyme A+ cells as compared to non-infected controls (NI). Moreover, higher expression of CD54 and HLA-DR by T-cells, especially within the CD8+ subset, was the hallmark of CH. A high level of expression of Granzyme A and Perforin underscored the enhanced cytotoxicity-linked pattern of CD8+ T-lymphocytes from CH. Increased frequency of B-cells with up-regulated expression of Fc-γRII was also observed in CH. Complex and imbricate biomarker networks demonstrated that CH showed a shift towards cross-talk among cells of the adaptive immune system. Systems biology analysis further established monocytes and NK-cell phenotypes and the T-cell activation status, along with the Granzyme A expression by CD8+ T-cells, as the most reliable biomarkers of potential use for clinical applications. Conclusions: Altogether, these findings demonstrated that the similarities in phenotypic features of circulating leukocytes observed in cynomolgus macaques and humans infected with T. cruzi further supports the use of these monkeys in preclinical toxicology and pharmacology studies applied to development and testing of new drugs for Chagas disease

Phenotypic and Functional Signatures of Peripheral Blood and Spleen Compartments of Cynomolgus Macaques Infected With T. cruzi: Associations With Cardiac Histopathological Characteristics

We performed a detailed analysis of immunophenotypic features of circulating leukocytes and spleen cells from cynomolgus macaques that had been naturally infected with Trypanosoma cruzi, identifying their unique and shared characteristics in relation to cardiac histopathological lesion status. T. cruzi-infected macaques were categorized into three groups: asymptomatic [CCC(-)], with mild chronic chagasic cardiopathy [CCC(+)], or with moderate chronic chagasic cardiopathy [CCC(++)]. Our findings demonstrated significant differences in innate and adaptive immunity cells of the peripheral blood and spleen compartments, by comparison with non-infected controls. CCC(+) and CCC(++) hosts exhibited decreased frequencies of monocytes, NK and NKT-cell subsets in both compartments, and increased frequencies of activated CD8+ T-cells and GranA+/GranB+ cells. While a balanced cytokine profile (TNF/IL-10) was observed in peripheral blood of CCC(-) macaques, a predominant pro-inflammatory profile (increased levels of TNF and IFN/IL-10) was observed in both CCC(+) and CCC(++) subgroups. Our data demonstrated that cardiac histopathological features of T. cruzi-infected cynomolgus macaques are associated with perturbations of the immune system similarly to those observed in chagasic humans. These results provide further support for the validity of the cynomolgus macaque model for pre-clinical research on Chagas disease, and provide insights pertaining to the underlying immunological mechanisms involved in the progression of cardiac Chagas disease

Reticulocyte count: comparison among methods

Introduction: The automated counting of reticulocytes has some advantages over the manual method routinely used in clinical laboratories. Technological innovations provide more statistically reliable results, while optimizing the time to perform this test. However, the cost for implementing the automated procedure in laboratory routines still constitutes a barrier to its use in small- and medium-size Brazilian laboratories. Objective: This study evaluated the performance of a new laboratory protocol for reticulocyte counting by flow cytometry using acridine orange (FC/AO), compared with the manual method and with another automated one by flow cytometry using the commercial kit BD Retic-Count (FC/RC) Conclusion: The results showed that, besides being comparable to the manual method, still considered standard, the evaluated new protocol is economically more advantageous than the automated methods currently available, and its cost is comparable to that of the manual method for laboratories that already have appropriate equipment and infrastructure

Regulatory T Cells Phenotype in Different Clinical Forms of Chagas' Disease

CD25High CD4+ regulatory T cells (Treg cells) have been described as key players in immune regulation, preventing infection-induced immune pathology and limiting collateral tissue damage caused by vigorous anti-parasite immune response. In this review, we summarize data obtained by the investigation of Treg cells in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood, as well as after stimulation with Trypanosoma cruzi antigens, demonstrated that individuals in the indeterminate (IND) clinical form of the disease have a higher frequency of Treg cells, suggesting that an expansion of those cells could be beneficial, possibly by limiting strong cytotoxic activity and tissue damage. Additional analysis demonstrated an activated status of Treg cells based on low expression of CD62L and high expression of CD40L, CD69, and CD54 by cells from all chagasic patients after T. cruzi antigenic stimulation. Moreover, there was an increase in the frequency of the population of Foxp3+ CD25HighCD4+ cells that was also IL-10+ in the IND group, whereas in the cardiac (CARD) group, there was an increase in the percentage of Foxp3+ CD25High CD4+ cells that expressed CTLA-4. These data suggest that IL-10 produced by Treg cells is effective in controlling disease development in IND patients. However, in CARD patients, the same regulatory mechanism, mediated by IL-10 and CTLA-4 expression is unlikely to be sufficient to control the progression of the disease. These data suggest that Treg cells may play an important role in controlling the immune response in Chagas' disease and the balance between regulatory and effector T cells may be important for the progression and development of the disease. Additional detailed analysis of the mechanisms on how these cells are activated and exert their function will certainly give insights for the rational design of procedure to achieve the appropriate balance between protection and pathology during parasite infections

Aspectos imunológicos celulares e humorais na fase crônica da doença de Chagas.

Submitted by Nuzia Santos ([email protected]) on 2017-09-15T18:57:45Z No. of bitstreams: 1 Danielle Marquete Vitelli Avelar.pdf: 2625792 bytes, checksum: 3798c3d0c9772e470825417574d9b8e8 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2017-09-15T19:02:07Z (GMT) No. of bitstreams: 1 Danielle Marquete Vitelli Avelar.pdf: 2625792 bytes, checksum: 3798c3d0c9772e470825417574d9b8e8 (MD5)Made available in DSpace on 2017-09-15T19:02:07Z (GMT). No. of bitstreams: 1 Danielle Marquete Vitelli Avelar.pdf: 2625792 bytes, checksum: 3798c3d0c9772e470825417574d9b8e8 (MD5) Previous issue date: 2008Fundação Oswaldo Cruz. Centro de Pesquisas Rene Rachou. Belo Horizonte, MG, Brasil.Diversos estudos que avaliam os mecanismos imunológ icos associados à manifestação de diferentes formas clínicas da doença de Chagas têm sugerido eventos multifatoriais. Com esse estudo, nosso objetivo foi adicionar novos elemento s à complexa rede imunológica que envolve a interação parasita-hospedeiro e as distintas manife stações clínicas da doença de Chagas crônica, indeterminada (IND), cardíaca (CARD) e digestiva (DI G). Para esse objetivo, realizamos uma análise detalhada de características fenotípicas da resposta imune com ênfase na análise imunofenotípica de células mononucleares do sangue periférico, no perfil de citocinas intracitoplasmáticas de leucócitos circulantes bem como na magnitude da resposta de IgG anti- Trypanosoma cruzi . Nossos principais achados demonstraram elevadas fr eqüências de células reguladoras CD4 + CD25 high e NKT, associadas com níveis aumentados de células NK citotóxicas circulantes em IND, enquanto percentuais aumentados de linfócitos T CD8 + HLA-DR + foram observados em CARD e DIG. Adicionalmente, nossos da dos demonstraram valores aumentados de células pré-NK, além de elevados valores de monóc itos pró-inflamatórios e linfócitos B ativados, contrastando com a ativação diminuída de células T e diminuição da freqüência de células T reguladoras CD4 + CD25 high marcos do estágio recente (E-IND) da doença de Chag as crônica. Análise comparativa transversal entre E-IND , IND e CARD ainda sugeriu que uma mudança em direção a altos valores de “macrófagos-like” e níveis basais de monócitos pró- inflamatórios além de elevados valores de células NK maduras, NKT e CD4 + CD25 high podem resultar na forma clínica IND. Por outro lado, altos níveis de células CD8 + HLA-DR + paralelo a níveis basais de células NK maduras, NKT e CD4 + CD25 high podem levar a doença cardíaca. Além disso, freqüência aumentada de linfócitos totais al to-produtores de IL-4, IL-10 e IL-13 e um perfil misto de citocinas derivadas de NK e neutrófi los foram marcos do grupo IND, enquanto freqüências aumentadas de monócitos alto-produtores de TNF- α e baixas freqüências de linfócitos T CD8 + IL-10 + foram as principais características de CARD. A aval iação do perfil de citocinas de leucócitos circulantes aplicando a est ratégia de visão panorâmica demonstrou que enquanto a maioria dos IND apresentam um perfil de c itocinas reguladoras, a maioria dos CARD apresentam um micro-ambiente de citocinas inflamató rias. Apesar dos nossos achados corroborarem com relatos prévios de perfis distinto s de anticorpos IgG anti- T. cruzi entre IND e CARD, eles indicaram que marcadores da resposta imu ne humoral são, sem dúvida, mais aplicáveis para diagnóstico sorológico e monitoraçã o de critério de cura. Em suma, nossos dados demonstram que, mais que uma mudança em direção a u m padrão polarizado, um fino balanço é relevante para direcionar os mecanismos imuno-media dos essenciais para a definição da doença de Chagas.Studies on immunological mechanisms associated with different clinical forms of Chagas disease have suggested multifactorial events for the diseas e development. Herein, we intended to add new elements to the complex immunological network u nderlying the parasite-host relationship and the distinct clinical outcome of chronic Chagas disease, referred as indeterminate (IND), cardiac (CARD) and digestive (DIG) clinical forms. For this purpose, we have performed a detailed analysis of phenotypic features of the imm une response emphasizing the e x vivo immunophenotype of peripheral blood mononuclear cel ls, the intracitoplasmatic cytokine profile of circulating leukocyte subsets as well as the mag nitude of anti- Trypanosoma cruzi IgG immune response. Our major findings demonstrated that high er frequency of both CD4 + CD25 high and NKT regulatory cells, associated with increased leve ls of circulating cytotoxic NK cells was observed in IND whereas increased percentage of CD8 + HLA-DR + T-lymphocytes subset was exclusively associated with CARD and DIG. Additiona lly, our data demonstrated that increased values of pre-NK-cells besides higher values of proi nflammatory monocytes and activated B lymphocytes contrasting with the impaired T- lympho cytes activation and decreased frequency of CD4 + CD25 high regulatory T lymphocytes was the hallmark of early stage (E-IND) of chronic Chagas disease. Comparative cross-sectional analysi s among E-IND, IND and CARD further suggested that a shift toward high values of macrop hage-like cells and basal levels of proinflammatory monocytes besides high values of ma ture NK cells, NKT and regulatory T cells may account for IND clinical form outcome. On the ot her hand, the upsurge of high levels of CD8 + HLA-DR + cells parallel with basal levels of mature NK cells , NKT and CD4 + CD25 high cells might lead to cardiac disease. Moreover, enhanced f requency of total lymphocytes high producers of IL-4, IL-10 and IL-13 besides a mixed pattern of NK and neutrophil-derived cytokines was the hallmark of IND, whereas enhanced frequency of TNF- α producing monocytes and lower frequency of IL-10 producing CD8 + T-cells was the major features of CARD. The assessment of the cytokine profile of circulating l eukocytes applying the panoramic overview strategy demonstrated that while most IND presented a regulatory cytokine profile, the majority of CARD displayed a particular inflammatory cytokin e milieu. Despite our findings corroborate previous reports of distinct profiles of anti- T. cruzi IgG between IND and CARD, they indicated that the humoral immune response biomarkers are ind eed more applicable to the serological diagnosis and cure criteria monitoring. Altogether, our data demonstrated that more than shift toward a polarized pattern, a fine balance is relev ant to drive the resultant immune-mediated mechanisms underlying the chronic Chagas disease ou tcome

Innate Immunity and Regulatory T-Cells in Human Chagas Disease: What Must be Understood?

There is a general consensus that during chronic Trypanosoma cruzi infection, the host immune system induces complex processes to ensure the control of parasite growth while preserving the potential to mount and maintain a life-long controlled humoral and cellular immune response against the invading pathogen. This review summarises evidence in an attempt to elucidate “what must be understood” to further clarify the role of innate immunity in the development/maintenance of clinical Chagas disease and the impact of etiological treatment on host immunity, highlighting the contributions of the innate immunity and regulatory T (Treg) cells. Recently, increasing focus on innate immunity suggest that chronic T. cruzi infection may cause morbidity when innate effector functions, or the down-regulation of adaptive regulatory mechanisms are lacking. In this context, stable asymptomatic host-parasite interactions seem to be influenced by the effector/regulatory balance with the participation of macrophages, natural killer (NK) and CD8+ T cells in parallel with the establishment of regulatory mechanisms mediated by NKT and Treg cells. Moreover, a balanced innate immune activation state, apart from Treg cells, may play a role in controlling the adverse events triggered by the massive antigen release induced by trypanosomicidal agents during Chagas disease etiological treatment