Early investigations suggested that infection with was associated in both humans and mice with a severe T cell unresponsiveness to mitogens and antigens during the acute phase of the disease [37,38]. cells. We therefore sought to examine the role of GRAIL in CD4 T cell proliferation during contamination. Methodology/Principal Findings Balb/c mice were infected intraperitoneally with 500 blood-derived trypomastigotes of Tulahuen strain, and spleen cells from control non-infected or infected animals were obtained. CD4 T cell proliferation was assessed by CFSE staining, and the expression of GRAIL in splenic T cells was measured by real-time PCR, flow cytometry and Western blot. We found increased GRAIL expression at the early stages of contamination, coinciding with the peak of parasitemia, with these findings correlating with impaired proliferation and poor IL-2 and IFN- secretion in response to plate-bound antibodies. In addition, we showed that this expression of GRAIL E3-ubiquitin ligase in CD4 T cells PF-04937319 during the acute phase of contamination was complemented by a high expression of inhibitory receptors such PF-04937319 as PD-1 and CTLA-4. We exhibited that GRAIL expression during contamination was modulated by the mammalian target of the rapamycin (mTOR) pathway, since addition of PF-04937319 IL-2 or CTLA-4 blockade in splenocytes from mice 21 days post contamination led to a reduction in GRAIL expression. Furthermore, addition of IL-2 was able to activate the mTOR pathway, inducing Otubain-1 expression, which mediated GRAIL degradation and improved T cell PF-04937319 Mouse monoclonal to CD74(PE) proliferation. Conclusions We hypothesize that GRAIL expression induced by the parasite may be maintained by the increased expression of inhibitory molecules, which blocked mTOR activation and IL-2 secretion. Consequently, the GRAIL regulator Otubain-1 was not expressed and GRAIL maintained the brake on T cell proliferation. Our findings reveal a novel association between increased GRAIL expression and impaired CD4 T cell proliferation during contamination. Author Summary Chagas disease is usually caused by the protozoan parasite and is endemic in Central and South America, where it affects about 10 million people. In addition, migration has led to the disease being established in non-endemic countries. Contamination involves an acute stage that evolves to a chronic stage where infected individuals may or may not show clinical symptoms or suffer progressive heart disease. The relevance of T cells in the control of contamination has been exhibited in human contamination and in experimental models. However, the parasite employs different strategies to downregulate the T cell function. These mechanisms can act at the initial time of T cell activation, leading to a state of anergy where lymphocytes do not respond. However, the molecular components that regulate this process during contamination are not well comprehended. PF-04937319 Our findings demonstrate for the first time that this T cell hyporesponsiveness could be linked to an increased expression of GRAIL. We propose that GRAIL expression induced by the parasite could be maintained by increased expression of inhibitory molecules, which blocked mTOR activation and IL-2 secretion. GRAIL could then play a key role in downregulating T cell functions by allowing the parasites to establish the chronic disease. Introduction Chagas disease, caused by the intracellular protozoa is usually complex, requiring the generation of a substantial antibody response and the activation of both CD4 and CD8 T cell responses. Even in cases in which these responses are sufficiently stimulated to be able to control the acute contamination, is not completely eradicated, but instead persists in infected hosts for decades [3]. employs a variety of strategies to evade the immune system and remain in the infected host. The main method involves the inhibition of specific T-cell responses, and consequently, can favor the establishment of chronic infections [4,5,6,7,8]. Related to this, a number of both host-dependent and parasite-induced mechanisms have been previously shown to affect immune regulation [9,10]. Moreover, T cells from infected hosts are largely unresponsive to antigens and mitogens, resulting in reduced IL-2 synthesis [8]. IL-2 production initiates proliferation, effector functions, and clonal expansion via IL-2 receptor (IL-2R)-mediated signaling [11]. In the absence of a robust activation initiated by TCR and CD28 signaling, CD4 T cells fail to proliferate or to produce IL-2 and enter a state of unresponsiveness following immunogenic stimulation, referred to as anergy [11,12]. In the case of CD4 T cells, the development of anergy depends on the alteration of the expression of several genes [11,12,13]. Post-translational modification of proteins via ubiquitination also plays an essential role in the regulatory mechanism of CD4 T cell anergy [14,15]. GRAIL, also known as ring finger protein-128 (RNF-128), has been identified as a.
