Inhibitory Effects of Trypanosoma cruzi Sialoglycoproteins on CD4 + T Cells Are Associated with Increased Susceptibility to Infection Marise Pinheiro Nunes 1 *, Ba ´ rbara Fortes 2 , Joa ˜ o Luiz Silva-Filho 3 , Euge ˆ nia Terra-Granado 4 , Leonardo Santos 1 , Luciana Conde 1 , Isadora de Arau ´ jo Oliveira 3 , Leonardo Freire-de-Lima 3 , Marina Vieira Martins 1 , Ana Acacia Sa ´ Pinheiro 3 , Christina Maeda Takyia 3 , Ce ´ lio Geraldo Freire-de-Lima 3 , Adriane Regina Todeschini 3 , George Alexandre DosReis 3 , Alexandre Morrot 2 * 1 Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, 2 Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 3 Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 4 National Cancer Institute, Rio de Janeiro, Brazil Abstract Background: The Trypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens characterized by decreased IL-2 synthesis. Trypanosoma cruzi mucin (Tc Muc) has been implicated in this phenomenom. These molecules contain a unique type of glycosylation consisting of several sialylated O-glycans linked to the protein backbone via N-acetylglucosamine residues. Methodology/Principal Findings: In this study, we evaluated the ability of Tc Muc to modulate the activation of CD4 + T cells. Our data show that cross-linking of CD3 on naı ¨ve CD4 + T cells in the presence of Tc Muc resulted in the inhibition of both cytokine secretion and proliferation. We further show that the sialylated O-Linked Glycan residues from tc mucin potentiate the suppression of T cell response by inducing G1-phase cell cycle arrest associated with upregulation of mitogen inhibitor p27 kip1 . These inhibitory effects cannot be reversed by the addition of exogenous IL-2, rendering CD4 + T cells anergic when activated by TCR triggering. Additionally, in vivo administration of Tc Muc during T. cruzi infection enhanced parasitemia and aggravated heart damage. Analysis of recall responses during infection showed lower frequencies of IFN-c producing CD4 + T cells in the spleen of Tc Muc treated mice, compared to untreated controls. Conclusions/Significance: Our results indicate that Tc Muc mediates inhibitory efects on CD4 + T expansion and cytokine production, by blocking cell cycle progression in the G1 phase. We propose that the sialyl motif of Tc Muc is able to interact with sialic acid-binding Ig-like lectins (Siglecs) on CD4 + T cells, which may allow the parasite to modulate the immune system. Citation: Nunes MP, Fortes B, Silva-Filho JL, Terra-Granado E, Santos L, et al. (2013) Inhibitory Effects of Trypanosoma cruzi Sialoglycoproteins on CD4 + T Cells Are Associated with Increased Susceptibility to Infection. PLoS ONE 8(10): e77568. doi:10.1371/journal.pone.0077568 Editor: Herbert B. Tanowitz, Albert Einstein College of Medicine, United States of America Received May 15, 2013; Accepted September 3, 2013; Published October 28, 2013 Copyright: ß 2013 Nunes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´ gico do Brasil (CNPq), Instituto Nacional de Cie ˆncia e Tecnologia de Vacinas (INCTv/CNPq), Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundac ¸a ˜o Oswaldo Cruz. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mnunes@ioc.fiocruz.br (MPN); morrot@micro.ufrj.br (AM) Introduction Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and is an important endemic infection in Latin America. Lately, it has also become a health concern in the United States, Canada and Europe [1,2]. The parasite is transmitted via the faeces of insect vectors of the family Reduviidae [3]. When the parasite enters the host it evokes a strong immunological response that is able to control the parasitic multiplication but not eliminate it [4–6]. After a delay that can be as much as 20 years, about a third of infected patients enter the chronic phase, characterized by the symptoms of Chagas disease [7] It is not yet clear how the observed pathology is triggered, but there is considerable evidence that persistence of the parasite is associated with a chronic inflammatory response, a major cause of Chagas disease [8–13]. T. cruzi employs a variety of strategies to evade the immune system and maintain itself in the infected host. The main method involves inhibiting specific T-cell responses so that it frequently establishes chronic infections [12–19]. A number of both host- dependent and parasite-induced mechanisms accomplish this immune regulation [20]. The T cells of infected hosts are largely unresponsive to antigens and mitogens, and this results in reduced IL-2 synthesis and increased nitric oxide (NO) production. Although spleen cell responses to ConA were more apparent in infected IFN-cR 2/2 or inducible nitric oxide synthase PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e77568