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.
References
[1]
Nagajyothi F, Machado FS, Burleigh BA, Jelicks LA, Scherer PE, Mukherjee S, et al. Mechanisms of Trypanosoma cruzi persistence in Chagas disease. Cell Microbiol. 2012; 14(5): 634–643. doi: 10.1111/j.1462-5822.2012.01764.x. pmid:22309180
Coura JR, Vi？as PA. Chagas disease: a new worldwide challenge. Nature. 2010; 465(7301): S6–7. doi: 10.1038/nature09221. pmid:20571554
[4]
Machado FS, Dutra WO, Esper L, Gollob K, Teixeira MM, Factor SM, et al. Current understanding of immunity to Trypanosoma cruzi infection and pathogenesis of Chagas disease. Semin Immunopathol. 2012; 34(6): 753–770. doi: 10.1007/s00281-012-0351-7. pmid:23076807
[5]
Sathler-Avelar R, Vitelli-Avelar DM, Elói-Santos SM, Gontijo ED, Teixeira-Carvalho A, Martins-Filho OA. Blood leukocytes from benznidazole-treated indeterminate chagas disease patients display an overall type-1-modulated cytokine profile upon short-term in vitro stimulation with trypanosome cruzi antigens. BMC Infectious Diseases. 2012; 12: 123. doi: 10.1186/1471-2334-12-123. pmid:22625224
[6]
Machado-de-Assis GF, Diniz GA, Montoya RA, Dias JC, Coura JR, Machado-Coelho GL, et al. A serological, parasitological and clinical evaluation of untreated Chagas disease patients and those treated with benznidazole before and thirteen years after intervention. Mem Inst Oswaldo Cruz. 2013; 108(7): 873–880. doi: 10.1590/0074-0276130122. pmid:24037109
[7]
Diniz L de F, Caldas IS, Guedes PM, Crepalde G, de Lana M, Carneiro CM, et al. Effects of ravuconazole treatment on parasite load and immune response in dogs experimentally infected with Trypanosoma cruzi. Antimicrob Agents Chemother. 2010; 54(7): 2979–2986. doi: 10.1128/AAC.01742-09. pmid:20404124
[8]
Guedes PMM, Veloso VM, Mineo TWP, Santiago-Silva J, Crepalde G, Caldas IS et al. Hematological alterations during experimental canine infection by Trypanosoma cruzi. Rev. Bras. Parasitol. Vet. 2012; 21(2): 151–156. pmid:22832757 doi: 10.1590/s1984-29612012000200015
[9]
Romanha JA, Castro SL, Soeiro MNC, Lannes-Vieira J, Ribeiro I, Talvani A, et al. In vitro and in vivo experimental models for drug screening and development for Chagas disease. Mem Inst Oswaldo Cruz. 2010; 105(2): 233–238. pmid:20428688 doi: 10.1590/s0074-02762010000200022
[10]
Carvalho CME, Silverio JC, da Silva AA, Pereira IR, Coelho JMC, et al. Inducible nitric oxide synthase in heart tissue and nitric oxide in serum of Trypanosoma cruzi-infected rhesus monkeys: association with heart injury. PLoS Negl Trop Dis. 2012; 6(5): e1644. doi: 10.1371/journal.pntd.0001644. pmid:22590660
[11]
Pisharath H, Zao CL, Kreeger J, Portugal S, Kawabe T, Burton T, et al. Immunopathologic characterization of naturally acquired Trypanosoma cruzi infection and cardiac sequalae in cynomolgus macaques (Macaca fascicularis). J Am Assoc Lab Anim Sci. 2013; 52(5): 545–552. pmid:24041209
[12]
Williams JT, Mubiru JN, Schlabritz-Loutsevitch NE, Rubicz RC, VandeBerg JL, Dick EJ Jr, et al. Polymerase chain reaction detection of Trypanosoma cruzi in Macaca fascicularis using archived tissues. Am J Trop Med Hyg. 2009; 81(2): 228–234. pmid:19635875
[13]
Valet G. Cytomics: An entry to biomedical cell systems biology. Cytometry. 2005; 63A(2): 67–68. doi: 10.1002/cyto.a.20110
[14]
Herrera G, Diaz L, Martinez-Romero A, Gomes A, Villamón E, Callaghan RC, et al. Cytomics: a multiparametric, dynamic approach to cell research. Toxicol In Vitro. 2007; 21(2): 176–82. pmid:16934431 doi: 10.1016/j.tiv.2006.07.003
[15]
Dias JC. The indeterminate form of human chronic Chagas' disease: A clinical epidemiological review. Rev Soc Bras Med Trop. 1989; 22(3):147–156. pmid:2486527 doi: 10.1590/s0037-86821989000300007
[16]
Vitelli-Avelar DM, Sathler-Avelar R, Dias JCP, Pascoal VPM, Teixeira-Carvalho A, Lage PS, Elói-Santos SM, et al. Chagasic patients with indeterminate clinical form of the disease have high frequencies of circulating CD3+CD16-CD56+ natural killer T Cells and CD4+CD25High regulatory T lymphocytes. Scandinavian Journal of Immunology. 2005; 62(3): 297–308. pmid:16179017 doi: 10.1111/j.1365-3083.2005.01668.x
[17]
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–504. pmid:14597658 doi: 10.1101/gr.1239303
[18]
Taylor R. Interpretation of the correlation: a basic review. J. Diagn Med Sono. 1990; 6(1): 35–39.
[19]
Nelson M, Loveday M. Exploring the innate immunological response of an alternative nonhuman primate model of infectious disease; the common marmoset. J Immunol Res. 2014; 2014: 913632. doi: 10.1155/2014/913632. pmid:25170519
[20]
Gardner MB, Luciw PA. Macaque models of human infectious disease. ILAR J. 2008; 49(2): 220–255. pmid:18323583 doi: 10.1093/ilar.49.2.220
[21]
Zabalgoitia M, Ventura J, Anderson L, Carey KD, Williams JT, VandeBerg JL. Morphologic and functional characterization of chagasic heart disease in non-human primates. Am. J. Trop. Med. Hyg. 2003; 68(2): 248–252. pmid:12641420
[22]
Vitelli-Avelar DM, Sathler-Avelar R, Massara RL, Borges JD, Lage PS, Lana M, et al. Are increased frequency of macrophage-like and natural killer (NK) cells, together with high levels of NKT and CD4+CD25high T cells balancing activated CD8+ T cells, the key to control Chagas’ disease morbidity? Clin Exp Immunol. 2006; 145(1): 81–92. pmid:16792677 doi: 10.1111/j.1365-2249.2006.03123.x
[23]
Krasselt M, Baerwald C, Ulf Wagner U, Rossol M. CD56+ monocytes have a dysregulated cytokine response to lipopolysaccharide and accumulate in rheumatoid arthritis and immunosenescence. Arthritis Res Ther. 2013; 15(5): R139. doi: 10.1186/ar4321. pmid:24286519
[24]
Dambaeva SV, Breburda EE, Durning M, Garthwaite MA, Golos TG. Characterization of decidual leukocyte populations in cynomolgus and vervet monkeys. J Reprod Immunol. 2009; 80(1–2): 57–69. doi: 10.1016/j.jri.2008.12.006. pmid:19398130
[25]
Sconocchia G, Keyvanfar K, Ouriaghli FE, Grube M, Rezvani K, Fujiwara H, et al. Phenotype and function of a CD56+ peripheral blood monocyte. Leukemia. 2005; 19(1): 69–76. pmid:15526027 doi: 10.1038/sj.leu.2403550
[26]
Machado FS, Tyler KM, Brant F, Esper L, Teixeira MM, et al. Pathogenesis of Chagas disease: time to move on. Front Biosci (Elite Ed). 2012; 4: 1743–1758. doi: 10.2741/495
[27]
Lieke T, Graefe SEB, Klauenberg U, Fleischer B, Jacobs T. NK cells contribute to the control of Trypanosoma cruzi infection by killing free parasites by perforin-independent mechanisms. Infect Immun. 2004; 72(12): 6817–6825. pmid:15557602 doi: 10.1128/iai.72.12.6817-6825.2004
[28]
Dutra WO, Martins-Filho OA, Cancado JR, Pinto-Dias JC, Brener Z, Freeman Júnior GL, et al. Activated T and B lymphocytes in peripheral blood of patients with Chagas’ disease. Int Immunol. 1994; 6(4): 499–506. pmid:8018591 doi: 10.1093/intimm/6.4.499
[29]
Reis DD, Jones EM, Tostes S Jr, Lopes ER, Gazzinelli G, Colley DG, et al. Characterization of inflammatory infiltrates in chronic chagasic myocardial lesions: presence of tumor necrosis factor-alpha+ cells and dominance of granzyme A+, CD8+ lymphocytes. Am J Trop Med Hyg. 1993; 48(5): 637–644. pmid:8517482
[30]
Andrade MCR, Dick EJ Jr, Guardado-Mendoza R, Hohmann ML, Mejido DCP, VandeBerg JL, et al. Nonspecific lymphocytic myocarditis in baboons is associated with Trypanosoma cruzi infection. Am J Trop Med Hyg. 2009; 81(2): 235–239. pmid:19635876
[31]
Williams JT, Dick EJ Jr, VandeBerg JL, Hubbard GB. Natural Chagas disease in four baboons. J Med Primatol. 2009; 38(2): 107–113. doi: 10.1111/j.1600-0684.2008.00308.x. pmid:18671766
[32]
Grieves JL, Hubbard GB, Williams JT, VandeBerg JL, Dick EJ Jr, López-Alvarenga JC, et al. Trypanosoma cruzi in non-human primates with a history of stillbirths: a retrospective study (Papio hamadryas spp.) and case report (Macaca fascicularis). J Med Primatol. 2008; 37(6): 318–328. doi: 10.1111/j.1600-0684.2008.00302.x. pmid:18671769
[33]
Sullivan NL, Eickhoff CS, Zhang X, Giddings OK, Lane TE, Hoft DF. Importance of the CCR5-CCL5 axis for mucosal Trypanosoma cruzi protection and B cell activation. J Immunol. 2011; 187(3): 1358–1368. doi: 10.4049/jimmunol.1100033. pmid:21715689
[34]
Cardillo F, Postol E, Nihei J, Aroeira LS, Nomizo A, Mengel J. B cells modulate T cells so as to favour T helper type 1 and CD8+ T-cell responses in the acute phase of Trypanosoma cruzi infection. Immunology. 2007; 122(4): 584–595. pmid:17635611 doi: 10.1111/j.1365-2567.2007.02677.x
[35]
Sathler-Avelar R, Lemos EM, Reis DD, Medrano-Mercado N, Araújo-Jorge TC, Antas PRZ, et al. Phenotypic features of peripheral blood leucocytes during early stages of human infection with Trypanosoma cruzi. Scand J Immunol. 2003; 58(6): 655–663. pmid:14636422 doi: 10.1111/j.1365-3083.2003.01340.x
