Accepted Manuscript Trypanosoma cruzi: Do different sylvatic strains trigger distinct immune re��� sponses? Leony Cristina Caetano, Jos�� Cl��vis do Prado Jr, Miriam Paula Alonso Toldo, Ana Am��lia Carraro Abrah��o PII: S0014-4894(09)00273-2 DOI: 10.1016/j.exppara.2009.09.018 Reference: YEXPR 5836 To appear in: Experimental Parasitology Received Date: 21 September 2008 Revised Date: 17 September 2009 Accepted Date: 18 September 2009 Please cite this article as: Caetano, L.C., do Prado Jr, J.C., Toldo, M.P.A., Abrah��o, A.A.C., Trypanosoma cruzi: Do different sylvatic strains trigger distinct immune responses?, Experimental Parasitology (2009), doi: 10.1016/ j.exppara.2009.09.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Trypanosoma cruzi: Do different sylvatic strains trigger distinct immune responses? 1 2 Leony Cristina Caetano Jos�� Cl��vis do Prado Jr Miriam Paula Alonso Toldo and Ana 3 Am��lia Carraro Abrah��o 4 1 ��� Departamento de An��lises Cl��nicas, Toxicol��gicas e Bromatol��gicas, Faculdade de 5 Ci��ncias Farmac��uticas de Ribeir��o Preto, Universidade de S��o Paulo (FCFRP-USP). 6 7 Corresponding author. Present address: Leony Cristina Caetano, Departamento de An��lises 8 Cl��nicas, Toxicol��gicas e Bromatol��gicas, Faculdade de Ci��ncias Farmac��uticas de 9 Ribeir��o Preto FCFRP-USP, Universidade de S��o Paulo. Avenida do Caf�� s/n��, 14040-903, 10 Ribeir��o Preto, SP, Brasil. 11 Telephone: 55-16-3602-4721 12 Fax: 55-16-3602-4163 13 E-mail address: leony@fcfrp.usp.br 14 15 16 17 18 19 20 21 22 23 24

ACCEPTED MANUSCRIPT Abstract 25 Strains of T. cruzi are multiclonal populations that can be classified in groups or 26 genotypes, differing in pathogenicity, virulence, and histotropism. In this experiment the 27 distinct behavior of two strains of T. cruzi, MORC-1 and MORC-2, was documented. 28 Blood parasitemia, spleen proliferation, nitric oxide, hystopathology of the spleen and heart 29 were used as tools to evaluate parasite persistence. Groups of male mice were separated 30 and divided in two groups: Control (M-1) and (M-2), Infected (IM-1) and (IM-2). The peak 31 of parasitemia occurred on 10 days post infection for both strains. LPS stimulated animals, 32 infected MORC-2 group displayed significant higher concentrations of NO when compared 33 to infected MORC-1 group (P0.05). For ConA stimulated lymphoproliferation, infected 34 MORC-1 group displayed higher proliferation index as compared to infected MORC-2 35 group. An opposite behavior for IL-4 and TNF-�� was observed according to the strain. For 36 MORC-1 enhanced concentrations of IL-4 were present with concomitant reduced levels of 37 TNF-��, while for MORC-2 enhanced concentrations of TNF-�� and reduced levels of IL-4 38 were found. The histopathology of heart and spleen showed important differences in which 39 MORC-1 displayed statistically enhanced number of amastigote in the heart and spleen as 40 compared to MORC-2. Concluding, each strain triggered a distinct immune response with 41 enhanced cytokine TH-1 profile for MORC-2 and TH-2 for MORC-1. 42 Keywords: Trypanosoma cruzi Nitric oxide Spleen proliferation Histopathology mice. 43 44 45 46 47

ACCEPTED MANUSCRIPT 1. Introduction 48 49 Parasitic diseases represent a major health problem in Latin America. Chagas' 50 disease, or American Trypanosomiasis, caused by the protozoan parasite Trypanosoma 51 cruzi (T. cruzi), is particularly important. Thirty per cent of people infected with T. cruzi 52 develop Chagas��� disease, a chronic and long lasting inflammatory illness that causes 53 significant morbidity and mortality, with an estimated 8 million people infected (Dias, 54 2006 WHO, 2007). It is endemic throughout Latin America, from the southern United 55 States to southern Chile (Schofield et al., 2006). The natural acute infection is rarely lethal, 56 and once infected, individuals can remain symptom-less for their lifetimes. Murine models 57 show a similar course of experimentally induced disease, and have been employed 58 extensively as a means of investigating the pathogenesis of this trypanosomal infection. 59 Interestingly, the patterns of the lesions determined in different experimental models 60 by the inoculation with different strains of T. cruzi have varied depending on the combined 61 influence of parasite strain and animal species (de Diego et al., 1991). 62 Strains of T. cruzi are multiclonal populations that can be grouped into three major 63 groups or genotypes, differing in pathogenicity, virulence, histotropism and response to 64 chemotherapy. These characteristics define the different Biodemes Types I, II and III 65 (Andrade, 1974 Andrade and Magalh��es, 1996), and Zymodemes Z1, Z2 and Z3 (Miles et 66 al., 1980). 67 Pathophysiological concentrations of NO produced during the initial phase of the 68 acute infection participate in the killing of the parasites by macrophages through NO- 69 dependent mechanisms (Vespa et al., 1994 Petray et al., 1995 Rodrigues et al., 2000) 70 especially through gamma-interferon (IFN-��) and TNF-�� production (Oswald et al., 1994 71

ACCEPTED MANUSCRIPT Silva et al., 1995). Evidence has accumulated over the years showing that there is intense 72 suppression of lymphocyte proliferation during the acute phase of T. cruzi murine infection 73 (Kierszenbaum and Sztein, 1994). Several cytokines or mediators are responsible for this 74 suppression: nitric oxide (NO), whose synthesis by macrophages is stimulated by IFN-�� 75 and/or TNF-��, prostaglandins (PG), insufficient IL-2 synthesis, and/or reduced expression 76 of the IL-2 receptor (Rottenberg et al., 1989 Abrahamsohn and Coffman, 1995 Pinge 77 Filho et al., 1999). 78 The experiment described here was carried out to compare the behavior of two 79 different strains of T. cruzi, and the persistence of blood parasites throughout the infection 80 in mice. Parasitemia, spleen proliferation, nitric oxide, histopathology of the heart and 81 spleen were used to evaluate parasite persistence. 82 83 2. Material and methods 84 2.1. Animals 85 Male (n=25) Swiss mice (Mus musculus) aged 30 days and weighing 25g were 86 separated for NO quantification, lymphoproliferation, parasitemia and histopathology, 87 performed during the acute phase of infection, on peak of parasitemia. 88 2.2. Experimental Design 89 Animals were divided in groups: Control (C) (n=5), Infected MORC-1 (IM-1) 90 (n=10) and Infected MORC-2 (IM-2) (n=10). For parasitemia measurements 5 animals 91 were used for each strain. At the peak of parasitemia (10 days post infection) 5 animals of 92 each group were used for for determination of NO, lymphoproliferation and 93 hystopathology. Mice were separated in groups of 5 in plastic cages, and commercial 94 rodent diet and water were available ad libitum. Animal pads were changed three 95