HIV-1 Infection-Induced Suppression of the Let-7i/IL-2 Axis Contributes to CD4(+) T Cell Death

Abstract

The mechanisms underlying HIV-1-mediated CD4(+) T cell depletion are highly complicated. Interleukin-2 (IL-2) is a key cytokine that maintains the survival and proliferation of activated CD4(+) T cells. IL-2 levels are disturbed during HIV-1 infection, but the underlying mechanism(s) requires further investigation. We have reported that cellular microRNA (miRNA) let-7i upregulates IL-2 expression by targeting the promoter TATA-box region, which functions as a positive regulator. In this study, we found that HIV-1 infection decreases the expression of let-7i in CD4(+) T cells by attenuating its promoter activity. The reduced let-7i miRNA expression led to a decline in IL-2 levels. A let-7i mimic increased IL-2 expression and subsequently enhanced the resistance of CD4(+) T cells to HIV-1-induced apoptosis. By contrast, the blockage of let-7i with a specific inhibitor resulted in elevated CD4(+) T cell apoptosis during HIV-1 infection. Furthermore, by knocking down the expression of IL-2, we found that the let-7i-mediated CD4(+) T cell resistance to apoptosis during HIV-1 infection was dependent on IL-2 signaling rather than an alternative CD95-mediated cell-death pathway. Taken together, our findings reveal a novel pathway for HIV-1-induced dysregulation of IL-2 cytokines and depletion of CD4(+) T-lymphocytes.

Figure 1. IL-2 expression significantly decreased in HIV-1-infected CD4⁺ T cells.

Activated primary blood CD4⁺ T cells (1 × 10⁶) were infected with HIV-1_NL4-3 viruses at equivalent to 5 ng of p24. (A) P24 levels in the supernatants of HIV-1_NL4-3 infected or uninfected CD4⁺ T cell cultures were examined by ELISA at multiple time points post-infection as indicated. (B) IL-2 mRNA levels in HIV-1-infected or -uninfected CD4⁺ T cells were measured by real-time quantitative RT-PCR at multiple time points post-infection as indicated. A combination of GAPDH, β-actin, RPL13A and IPO8 reference gene mRNA was used as internal control. The IL-2 mRNA level at each time point was normalized to the uninfected sample of D0. These data represent three independent experiments. (C) Intracellular IL-2 protein levels in HIV-1-infected or -uninfected CD4⁺ T cells at day 3-post infection were analyzed by flow cytometry (FCM). The IL-2 positive cells were gated by unstained cell control. (D) Statistic analysis of (C) was done with data from 6 independent experiments. Paired, two-tailed student’s t test: *p < 0.05. (E) Apoptosis of HIV-1_NL4-3 infected or uninfected CD4⁺ T cells was measured at day 3-post infection using Annexin-V staining method. The Annexin V positive cells were gated by unstained cell control. (F) Statistic analysis of (E) was done with data from 6 independent experiments. Paired, two-tailed student’s t test: *p < 0.05.

Figure 2. Let-7i upregulates IL-2 expression by targeting the TATA-box region of promoter.

(A) Predicted binding between let-7i and the TATA box region of human IL-2 gene promoter. MFE: minimum free energy. (B) Intracellular IL-2 levels in HIV-1-infected CD4⁺ T cells transfected with let-7i mimics were measured by FCM. The IL-2 positive cells were gated by unstained cell control. (C) Statistical analysis of (B) was done with data from 6 independent experiments. Paired, two-tailed student’s t test: *p < 0.05. (D) Intracellular IL-2 levels in HIV-1-infected CD4⁺ T cells transfected with let-7i inhibitors were measured by FCM. (E) Statistical analysis of (D) was done with data from 6 independent experiments. Paired, two-tailed student’s t test: *p < 0.05. (F,G) HEK293T cells were transfected with a plasmid containing firefly luciferase with let-7i binding site at the 3′ UTR (pMIR-REPORT-let-7i_bindingsite) and a renilla luciferase-expressing plasmid. Cells were co-transfected with let-7i mimic (F) or let-7i inhibitor (G). Dual luciferase activity was measured at 48 h after transfection. Statistical analysis was done with data from three independent experiments with triplicate samples. Paired, two-tailed student’s t test: **p < 0.01. Mm-, miRNA mimic; ih-, miRNA inhibitor. NC, negative control.

Figure 3. HIV-1 infection significantly reduces primary, precursor and mature let-7i expression in CD4⁺ T cells.

(A) Mature let-7i levels in HIV-1_NL4-3 infected or uninfected CD4⁺ T cells were measured by stem-loop real-time RT-PCR. A combination of GAPDH, β-actin, RPL13A and IPO8 mRNA was use as internal control. The let-7i level at each time point was normalized to that of D0 uninfected sample. (B) Primary let-7i levels during HIV-1 infection were analyzed by real-time RT-PCR, and normalized by the same way as (A). (C) Precursor let-7i levels measured during HIV-1 infection by real-time RT-PCR, and normalized by the same way as (A). (D) HeLa-CD4 cells were transfected with let-7i promoter reporter (pGL4-let-7i) and a renilla luciferase-expressing plasmid at 48 hrs post HIV-1_NL4-3 infection. After another 48 hrs, the let-7i promoter activity was detected by dual luciferase assay. Statistical analysis was done with data from three independent experiments. Paired, two-tailed student’s t test: *p < 0.05. All these data represent at least three independent experiments with triplicate samples.

Figure 4. Let-7i increases resistance to apoptosis in HIV-1-infected CD4⁺ T cells.

Activated human CD4⁺ T cells were treated with IL-2 or transfected with let-7i mimic/control or let-7i inhibitor/control oligonucleotides. Cells were then infected with HIV-1_NL4-3 as described above. (A) Annexin V staining for apoptosis of IL-2 treated CD4⁺ T cells during HIV-1_NL4-3 infection were analyzed by FCM 24-48 hrs post infection. (B) Statistical analysis of (A) was done with data from 9 independent experiments. (C) Annexin V staining for apoptosis of HIV-1-infected CD4⁺ T cells transfected with let-7i mimic or negative control (mm-NC) were measured by FCM 24-48 hrs post infection. (D) Statistical analysis of (C) was done with data from 8 independent experiments. (E) Annexin V staining for apoptosis of HIV-1-infected CD4⁺ T cells treated with let-7i inhibitor or negative control (ih-NC) were analyzed by FCM 24-48 hrs post infection. (F) Statistical analysis of (E) was done with data from 5 independent experiments. All the statistical analysis was done with paired, two-tailed student’s t test: *p < 0.05.

Figure 5. The resistance to apoptosis in HIV-1 infected CD4⁺ T cells endowed by let-7i is dependent on IL-2 expression.

(A) Silencing of IL-2 by IL-2-specific siRNAs was confirmed by FCM analysis on IL-2-producing CD4⁺ T cells. (B) Statistical analysis of (A) was done with data from three independent experiments. Paired, two-tailed student’s t test, **p < 0.01. (C) Human CD4⁺ T cells were transfected with let-7i mimic, siRNA against IL-2 or respective controls (mm-NC, si-NC). Cells were then infected with HIV-1_NL4-3 as described above. Apoptosis of CD4⁺ T cells were measured by Annexin V staining. (D) Statistical analysis of (C) was done with data from three independent experiments. Paired, two-tailed student’s t test: *p < 0.05; **p < 0.01, ***p < 0.001. (E) Human CD4⁺ T cells were treated as in (C), and CD95 expression was measured by FCM. Statistical analysis was done with data from three independent experiments. Paired, two-tailed student’s t test. NS, non-significant.