The RNA-Binding Protein HuR Posttranscriptionally Regulates IL-2 Homeostasis and CD4+ Th2 Differentiation

Abstract

Posttranscriptional gene regulation by RNA-binding proteins, such as HuR (elavl1), fine-tune gene expression in T cells, leading to powerful effects on immune responses. HuR can stabilize target mRNAs and/or promote translation by interacting with their 3' untranslated region adenylate and uridylate-rich elements. It was previously demonstrated that HuR facilitates Th2 cytokine expression by mRNA stabilization. However, its effects upon IL-2 homeostasis and CD4⁺ Th2 differentiation are not as well understood. We found that optimal translation of Il2ra (CD25) required interaction of its mRNA with HuR. Conditional HuR knockout in CD4⁺ T cells resulted in loss of IL-2 homeostasis and defects in JAK-STAT signaling, Th2 differentiation, and cytokine production. HuR-knockout CD4⁺ T cells from OVA-immunized mice also failed to proliferate in response to Ag. These results demonstrate that HuR plays a pivotal role in maintaining normal IL-2 homeostasis and initiating CD4⁺ Th2 differentiation.

FIGURE 1. Efficient HuR ablation in late-stage thymocytes does not alter thymic egress or peripheral T cell distribution

(A and B) Isolated CD4⁺ T cells from the SP and LNs of HuR-KO mice (distal lck-Cre ROSA HuR^fl/fl) were sorted according to their YFP expression (YFP⁺ versus YFP⁻); CD4⁺ T cells from WT HuR^fl/fl mice were used as control cells. HuR levels were assessed by RT-PCR (A) and Western blot (B). (C) Frequency of DP, CD4 single-positive (CD4⁺ single-positive), and CD8 single-positive (CD8⁺ single-positive) cells among total thymocytes in HuR-KO mice (upper panels) and in YFP⁺ thymocytes (lower panel). (D) Frequency of YFP⁺ (HuR-deficient) cells among DP, CD4⁺ single-positive, and CD8⁺ single-positive thymocytes (upper panel) and representative line graphs of YFP expression (lower panel). (E) Frequency of DP, CD4⁺ single-positive, and CD8⁺ single-positive cells among total splenocytes (upper panel) and YFP⁺ splenocytes (lower panel). (F) Frequency of YFP⁺ (HuR-deficient) cells among DP, CD4⁺ single-positive, and CD8⁺ single-positive splenocytes. Data in (A) and (B) are representative of two independent experiments using at least two mice per group in each experiment. Data in (C)–(F) are representative of four independent experiments consisting of at least two mice per group in each experiment, along with representative flow cytometry plots. Bars represent mean + SEM of two (A and B) or four (D and F) independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001, one-way ANOVA with the Tukey multiple-comparisons test or Student t test.

FIGURE 2. Increases in IL-2 and decreases in Th2 cytokine expression in HuR-ablated CD4⁺ T cells

(A) YFP⁺ (HuR-KO), YFP⁻ (endogenous control), or WT (HuR^fl/fl control with no Cre) CD4⁺ T cells were stimulated under nonpolarizing conditions with plate-bound anti-CD3 and anti-CD28. On day 5 of activation, cells were harvested, stimulated with PMA and ionomycin for 5 h, and stained for intracellular cytokines. (B) IL-2, IL-4, and IL-13 levels in the culture supernatant under nonpolarizing conditions on day 5 postactivation of YFP⁺, YFP⁻, and WT CD4⁺ T cells, as measured by ELISA. (C) IL-2 levels in activated YFP⁺, YFP⁻, and WT CD4⁺ T cells after restimulation with PMA and ionomycin for 5 h, as detected by ELISA. (D) RT-PCR analysis of Il2 mRNA in YFP⁺, YFP⁻, and WT CD4⁺ T cells activated under

FIGURE 3. HuR deficiency alters the expression of components of the IL-2 signaling pathway

(A) Flow cytometry of kinetic changes in CD25 protein expression of activated YFP⁺, YFP⁻, and WT CD4⁺ T cells on days 0–5. (B) p-Stat5, p-Stat6, total Stat5, and total Stat6 protein levels in activated YFP⁺, YFP⁻, and WT CD4⁺ T cells on day 4 postactivation. (C) Blimp1 protein expression in activated YFP⁺, YFP⁻, and WT CD4⁺ T cells on day 4 postactivation. Data are representative of two (B) or three (A and C) independent experiments. nonpolarizing conditions on days 0 to 5. (E) Transcriptional measurement using nascent RNA capture assay and RT-PCR analysis of Il2. Data are combined from three (B, C, and E) or four [(A), right panel and (D)] independent experiments, along with representative flow cytometry plots (A, left panel). Error bars represent mean + SEM of three (B, C, and E) or four [(A), right panel] independent experiments. The p values in (D) were calculated based on YFP⁺ versus YFP⁻ and WT. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with the Tukey multiple-comparisons test.

FIGURE 4. Gene expression downstream of IL-2/p-Stat5 signaling is altered in the absence of HuR

Transcriptional measurement using nascent RNA capture assay and RT-PCR analysis of Il2ra (A), Prdm1 (B), Il4 (C), and Gata3 (D) mRNAs in activated YFP⁺, YFP⁻, and WT CD4⁺ T cells. Data show relative amounts of nascent mRNAs on day 4 postactivation. (E) Steady-state Gata3 mRNA kinetics in activated YFP⁺, YFP⁻, and WT CD4⁺ T cells, as measured by RT-PCR. All data are from three or more independent experiments and represent mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with the Tukey multiple-comparisons test.

FIGURE 5. HuR physically interacts with the Il2ra 3′UTR mRNA and enhances its translational efficiency in activated CD4⁺ T cells

(A) RIP using HuR or IgG1 Ab, followed by RT-PCR to determine physical HuR mRNA targets. Data are fold enrichment of Il2ra mRNA in anti-HuR samples compared with IgG1 controls. (B) Putative HuR targets, ARE elements (gray), present in the 3′UTR of Il2ra mRNA (left panels) and biotin pull-down shows association of HuR with different portions of Il2ra mRNA (right panel). Data show HuR interaction with the second section of Il2ra 3′UTR mRNA containing the first two putative HuR binding sites. (C) Il2ra mRNA stability assay in activated YFP⁺, YFP⁻, and WT CD4⁺ T cells on day 4 postactivation. Data represent the percentage of mRNA remaining over time after actinomycin D treatment. (D) Absorbance profile for RNA separated by velocity sedimentation through a sucrose gradient. RNA was extracted from each fraction. Polysomal gradient analysis of Il2ra (E), Il2 (F), and Gapdh (G) mRNAs in activated YFP⁺, YFP⁻, and WT CD4⁺ T cells. Data are the percentage of Il2ra, Il2, and Gapdh mRNA distribution in 40S, 60S, 80S, and polysome fractions by RT-PCR. (H) RIP using HuR or IgG1 Ab, followed by RT-PCR to determine HuR mRNA targets. Data are fold enrichment of Il2 mRNA in anti-HuR samples compared with IgG1 controls. (I) Il2 mRNA stability assay in activated YFP⁺, YFP⁻, and WT CD4⁺ T cells on day 4 postactivation. Data are from three (A and H) or two (D, F, and G) independent experiments or are a representative of three (C and I) or two (B and D) independent experiments. Data are mean + SEM of three (A and H) or two (D, F, and G) independent experiments. *p < 0.05, **p < 0.01, two-tailed unpaired t test (A and H), one-way ANOVA with the Tukey multiple-comparisons test (E–G). N.S., not significant.

FIGURE 6. Exogenous IL-4 cannot rescue Th2 cytokine expression in HuR-deficient cells

(A) YFP⁺, YFP⁻, or WT CD4⁺ T cells were stimulated or not with 100 U/ml rIL-4. Five days postactivation, cells were harvested and restimulated with PMA and ionomycin for 5 h, and cytokine production was assessed by intracellular cytokine staining. (B) p-Stat5 and p-Stat6 levels in YFP⁺, YFP⁻, and WT CD4⁺ T cells activated in the presence of IL-4. (C) CD25 expression in YFP⁺, YFP⁻, and WT CD4⁺ T cells activated in the presence or absence of 1000 U/ml rIL-4 for 5 d. Data (mean + SEM) are a representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with the Tukey multiple-comparisons test.

FIGURE 7. HuR deficiency results in impaired CD4⁺ T cell Ag-induced proliferation

Mice were immunized with 50 μg of whole OVA protein and boosted with 50 μg of OVA protein on day 10. SP and LNs were harvested on day 17, and CD4⁺ T cells were stained with Cell Proliferation Dye eFluor 660 and cultured in the presence of OVA peptide–loaded APCs for 3 d. Data show the percentage proliferation of YFP⁺ or YFP⁻ CD4⁺ T cells in OVA protein–immunized or sham controls. Data are representative of three independent experiments (upper panel) or are combined from three experiments (lower panels). *p < 0.05, ****p < 0.0001, two-tailed unpaired t test.