GRAIL and Otubain-1 are Related to T Cell Hyporesponsiveness during Trypanosoma cruzi Infection

Abstract

BackgroundTrypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens and is characterized by decreased IL-2 synthesis. In addition, the acquisition of the anergic phenotype is correlated with upregulation of “gene related to anergy in lymphocytes” (GRAIL) protein in CD4 T cells. We therefore sought to examine the role of GRAIL in CD4 T cell proliferation during T. cruzi infection.Methodology/Principal FindingsBalb/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 infection, coinciding with the peak of parasitemia, with these findings correlating with impaired proliferation and poor IL-2 and IFN-γ secretion in response to plate-bound antibodies. In addition, we showed that the expression of GRAIL E3-ubiquitin ligase in CD4 T cells during the acute phase of infection was complemented by a high expression of inhibitory receptors such as PD-1 and CTLA-4. We demonstrated that GRAIL expression during infection was modulated by the mammalian target of the rapamycin (mTOR) pathway, since addition of IL-2 or CTLA-4 blockade in splenocytes from mice 21 days post infection 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 proliferation.ConclusionsWe 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 Trypanosoma cruzi infection.

Limited Foxp3+ Regulatory T Cells Response During Acute Trypanosoma cruzi Infection Is Required to Allow the Emergence of Robust Parasite-Specific CD8+ T Cell Immunity

“…Live T. cruzi trypomastigotes (Tulahuén) were obtained from the extracellular medium of infected monolayers of Vero cells, as previously described ( 47 ).…”

Mammalian Target of Rapamycin Inhibition in Trypanosoma cruzi-Infected Macrophages Leads to an Intracellular Profile That Is Detrimental for Infection

“…The T. cruzi infection protocols were performed as described ( 29 ). Briefly, the infection was maintained through intraperitoneal inoculations every 11 days.…”

“…BALB/c mice underwent intraperitoneal infection as described previously ( 29 ), then PM were extracted and processed using several techniques. Non-infected animals were processed in parallel as control.…”

“…After that, BMDM were incubated with 20 µM DAF-FM diacetate probe for 30 min at 37°C for NO detection; 20 µM H2DCFDA probe for 20 min at 37°C for cROS detection; or 5 µM of a MitoSOX TM Red probe 20 min at 37°C for mtROS detection. Finally, these cells were analyzed by FACS as described previously ( 29 ).…”

“…Cells were processed as previously described ( 29 ). Lysates were prepared by protein measurement using the Bradford micro-technique ( 32 ).…”