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

Abstract

Background: Trypanosoma 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 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 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.

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 Trypanosoma cruzi infection.

Fig 1. Proliferation and cytokine production are impaired in CD4 T cells during the acute phase of T. cruzi infection.

CD4+ T cells from spleen control or infected animals at various days post-infection were purified by magnetic beads. Then, cells were stained with CFSE and cultured in 96-well plates with bound anti-CD3 (1 ug/ml) plus CD28 (1 ug/ml). After 72 h incubation, proliferation was assessed by flow cytometry, and cytokine production was evaluated by ELISA. CD4+ T cell proliferation at different times post-infection was compared to proliferation in control cells and quantitatively analyzed as a percentage of proliferation by FlowJo software (p< 0.05 * versus control) (A). A representative histogram with CFSE dye dilution in dividing cells is shown (B). Supernatants were harvested and the amount of IFN-γ (C) and IL-2 (D) were determined by ELISA sandwich **p = 0.0044, ***p = 0.0001, paired t- test versus control. Parasitemia was determined at different time points post T. cruzi infection (E), n = 6. IFN-γ, IL-2 and the proliferation data are the mean ± SEM of three independent experiments (n = 4 mice/group).

Fig 2. Increased expression of inhibitory molecules on CD4 T cells in the acute phase of T. cruzi infection.

Spleen cells isolated from control and infected animals were stained with anti-CD4, anti-CD3, anti-PD-1 or anti-CTLA-4 mAbs. Then, cells were analyzed by flow cytometry. Graphs show the percentages of CD4+ CTLA-4+ (A, * p< 0.05 versus control) or CD4+ PD1+ cells (C, *** p< 0.0001 versus control). The data are mean ± SEM (n = 3) with representative dot plot graphs of at least three independent experiments being shown (B and D).

Fig 3. Increased GRAIL E3-ubiquitin ligase expression in spleen T cells during T. cruzi infection.

Cells were isolated from spleens of control or infected animals at different time points after infection, then these cells were used to extract mRNA and subsequent real-time PCR was performed to analyze GRAIL (RNF128) (A) or Cbl-b (B) expression. The expression of 18S served as an internal control and the data are expressed as fold induction compared to control animals. This experiment is representative of two independent experiments (*** p <0.0001; * p <0.05). GRAIL expression was evaluated by FACS in spleen cells isolated from control and infected animals at different days p.i. (C) and in CD4 T cells in vitro cultured with two different T. cruzi strains at different T cell/parasite ratios (D). Additionally, GRAIL expression in HEK 293 cells was used as a positive control (C). Then, 2x10⁶ cells were stained with anti-CD4 and anti-CD3 antibodies. After cells were fixed and permeabilized, GRAIL expression was detected using an anti-GRAIL antibody followed by an anti-rabbit IgG-PE. Cells were analyzed by flow cytometry on CD3+CD4+ gated cells. Representative histograms are shown (C and D) and bars display GRAIL mean fluorescence intensity (MFI, D) (n = 4, *p <0.05; **p = 0.0019).

Fig 4. GRAIL expression is regulated by the IL2/mTOR/Otubain-1 axis in CD4 T cells during T. cruzi infection.

CD4+ T cells were purified from the spleens of control or infected mice at different days post-infection. Then, cells were lysed and the expression of GRAIL, and Otub-1 and the phosphorylation of 4EBP1 were determined by Western blot using specific antibodies, with protein loading being evaluated by actin expression (A). Right panels show the densitometric analysis using GelPro software, n = 2 experiments *p< 0.05; **p< 0.005; ***p< 0.001. splenocytes of 21- day-p.i. animals were cultured in the presence of CTLA-4 blocking or control antibody (10 ug/ml) and GRAIL intracellular expression was evaluated by flow cytometry on CD3+CD4+ gated cells 48 h later. Representative histograms are shown and bars display GRAIL mean fluorescence intensity (MFI) *** p = 0.0006 (B). Additionally, CD4+ T cells from the spleens of control or 21-day p.i. animals were stained with CFSE (5 mM) and then cultured for 72 h in plate-bound anti-CD3/CD28 (1 μg/ml) in the presence or absence of exogenous rmIL-2 (20 ng/ml). The proliferation was evaluated by flow cytometry analysis and representative histograms indicating the CD4 T cell proliferation are shown (C). CD4+ T cells from spleen of 21 or 42-day p.i. animals were cultured in the presence or absence of exogenous rmIL-2 (20 ng/ml), and the expression of GRAIL, Otub-1 and 4EBP1 phosphorylation were evaluated by western blot. Right panels show the densitometric analysis using GelPro software, n = 2 experiments *p<0.05; ** p = 0.0053; (D). Intracellular GRAIL expression was evaluated by flow cytometry in CD4+ T cells from 21-day p.i. animals cultured in the presence or absence of exogenous rmIL-2 (20 ng/ml), with representative histograms being shown in (E). The data are representative of two independent experiments (n = 3 mice/group).