Trypanosoma cruzi Infection in Non-Human Primates

For decades, non-human primates (NHPs) have been employed as experimental models to study many aspects of human diseases. They are the closest genetically to humans of any of the models applied in biomedical research; therefore, many authors have published scientific work regarding these animals and infectious diseases, including tuberculosis, AIDS, and tropical diseases. Among these, Chagas disease has caught the attention of many researchers all over the world. Recent studies have demonstrated great similarities with the human pathology, including cardiomyopathy and exacerbated pro-inflammatory response. Besides being genetically close to humans, NHP have a great probability to be naturally infected by Trypanosoma cruzi, which turns them into more interesting models to study Chagas disease mechanisms

Physiology and Pathology of Infectious Diseases: The Autoimmune Hypothesis of Chagas Disease

Infectious pathologies are a group of diseases that contribute with great impact on public health worldwide. Among the various diseases, some have a higher epidemiological importance, since their morbidity and mortality are very significant. In addition to the usual immune response, mounted against noxious agents, there is still the concept of infection-induced autoimmunity. Autoimmune diseases are defined as illnesses in which the evolution from benign to pathogenic autoimmunity takes place. However, proving a disease to be of autoimmune etiology is not a simple task. It is well known that both genetic influences and environmental factors trigger autoimmune disorders. However, some theories are still under great discussion. One of the most intriguing self-induced disorders is the hypothesis of autoimmunity during Chagas disease. Since the mid-1970s, the Chagas autoimmunity hypothesis has been considered an important contributor to the complex immune response developed by the host and triggered by Trypanosoma cruzi. New ideas and findings have strengthened this hypothesis, which has been reported in a series of publications from different groups around the world. The aim of this chapter is to discuss the mechanisms involving autoimmunity development during Chagas disease

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

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

Submitted by Nuzia Santos ([email protected]) on 2016-07-13T16:37:04Z No. of bitstreams: 1 ve_Avelar_Renato_Phenotypic_CPqRR_2016.pdf: 1907004 bytes, checksum: 99db081393078b71079f49a88cae660f (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-07-13T16:45:28Z (GMT) No. of bitstreams: 1 ve_Avelar_Renato_Phenotypic_CPqRR_2016.pdf: 1907004 bytes, checksum: 99db081393078b71079f49a88cae660f (MD5)Made available in DSpace on 2016-07-13T16:45:28Z (GMT). No. of bitstreams: 1 ve_Avelar_Renato_Phenotypic_CPqRR_2016.pdf: 1907004 bytes, checksum: 99db081393078b71079f49a88cae660f (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo Integrado de Pesquisas em Biomarcadores. Belo Horizonte, Minas Gerais, Brazil/Centro Universitário Newton Paiva. Belo Horizonte, MG, Brasil/Universidade Federal de Minas Gerais. Faculdade de Medicina. Belo Horizonte, MG, Brasil/Texas Biomedical Research Institute. San Antonio, TX, United States of AmericaFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo Integrado de Pesquisas em Biomarcadores. Belo Horizonte, Minas Gerais, Brazil/Texas Biomedical Research Institute. San Antonio, TX, United States of America/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo Integrado de Pesquisas em Biomarcadores. Belo Horizonte, Minas Gerais, Brazil/Centro Universitário Newton Paiva. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo Integrado de Pesquisas em Biomarcadores. Belo Horizonte, Minas Gerais, Brazil/Centro Universitário Newton Paiva. Belo Horizonte, MG, BrasilCentro Universitário Newton Paiva. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Faculdade de Medicina. Departamento de Propedêutica Complementar. Belo Horizonte, MG, BrasilLaboratório de Bioinformática e Análise Molecular, Instituto de Genética e Bioquímica Universidade Federal de Uberlândia, Campus Patos de Minas, Patos de Minas, Minas Gerais, BrazilUniversidade Federal de Uberlândia. Faculdade de Ciência da Computação. Laboratório de Bioinformática e Análise Molecular. Patos de Minas, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo Integrado de Pesquisas em Biomarcadores. Belo Horizonte, Minas Gerais, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo Integrado de Pesquisas em Biomarcadores. Belo Horizonte, Minas Gerais, BrasilTexas Biomedical Research Institute. San Antonio, TX, United States of AmericaTexas Biomedical Research Institute. San Antonio, TX, United States of AmericaTexas Biomedical Research Institute. San Antonio, TX, United States of America/University of Texas Health Science Center. South Texas Diabetes and Obesity Institute. San Antonio – Regional Academic Health Center. Edinburg, TX, United States of AmericaTexas Biomedical Research Institute. San Antonio, TX, United States of America/University of Texas Health Science Center. South Texas Diabetes and Obesity Institute. San Antonio – Regional Academic Health Center. Edinburg, TX, United States of AmericaBACKGROUND: 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

Systems biology strategy for analyzing adaptive immunity flow-cytometry data by heatmap and decision-tree analysis.

<p>(A) Bioinformatics tool applied for single-cell data mining using heatmap computational method to preprocess flow cytometry data and to identify the adaptive immunity cell attributes. (B) Decision tree analysis identifies “root” (CD3⁺HLA-DR⁺) and “secondary” (CD8⁺HLA-DR⁺ and CD8⁺ Granzyme A⁺) cell attributes with higher accuracy to distinguish between non-human primates naturally infected with <i>T</i>. <i>cruzi</i> and non-infected controls. (C) Scatter distribution plots show the potential of selected biomarkers to discriminate infected from non-infected individuals. White rectangles indicate true positive (Chagas disease) and true negative (non-infected subjects) classifications. Gray rectangles indicate subjects that require the analysis of additional characteristics for accurate classification by the algorithm sequence proposed by the decision tree. (C) ROC curve analysis illustrating the cut-off points, the global accuracy (area under the curve–AUC) and performance indexes (sensitivity–Se, specificity–Sp and likelihood ratio–LR) for each selected biomarker.</p

Adaptive immunity features from cynomolgus macaques naturally infected with <i>T</i>. <i>cruzi</i> (CH) and non-infected controls (NI).

<p>(A) The frequencies of CD3⁺ lymphocytes and T-cell subsets (CD4⁺ and CD8⁺), the expression of adhesion molecule (CD54) and activation status (CD69 and HLADR) were performed by multicolor flow cytometry. (B) The expression of cytotoxicity markers (Granzyme A, Granzyme B and Perforin) of CD8⁺ T-cells was investigated by intracellular staining flow cytometry. (C) Analysis of B-cells, the activation status (CD69), and the expression of the regulatory FcγR (CD32) were evaluated by three-color flow cytometry. The results are expressed as mean percentage with standard error. Significant differences at <i>p<</i>0.05 are identified by (*).</p

Systems biology strategy for analyzing innate immunity flow-cytometry data by heatmap and decision-tree analysis.

<p>(A) Bioinformatics tool applied for single-cell data mining using heatmap computational method to preprocess flow cytometry data and to identify the innate immunity cell attributes. (B) Decision tree analysis identifies “root” (CD14⁺CD56⁺) and “secondary” (NK Granzyme A⁺ and NK CD16⁺CD56^-) cell attributes with higher accuracy to distinguish between non-human primates naturally infected with <i>T</i>. <i>cruzi</i> and non-infected controls. (C) Scatter distribution plots show the potential of selected biomarkers to discriminate infected from non-infected individuals. White rectangles indicate true positive (Chagas disease) and true negative (non-infected subjects) classifications. Gray rectangles indicate subjects that require the analysis of additional characteristics for accurate classification by the algorithm sequence proposed by the decision tree. (C) ROC curve analysis illustrating the cut-off points, the global accuracy (area under the curve–AUC) and performance indexes (sensitivity–Se, specificity–Sp and likelihood ratio–LR) for each selected biomarker.</p