TRIM24-Mediated Acetylation of STAT6 Suppresses Th2-Induced Allergic Rhinitis

Abstract

Purpose: Allergic rhinitis (AR) is a T helper type 2 (Th2)-mediated inflammatory disease. The E3 ligase tripartite motif-containing 24 (TRIM24) regulates the recruitment of acetyltransferase CREB-binding protein (CBP) to signal transducer and activator of transcription 6 (STAT6). CBP mediates the acetylation of STAT6 and decreases its activity. To date, the precise role of TRIM24 in AR has not been fully interpreted. Herein, our study aimed to explore the functions of TRIM24 in AR.

Methods: The expression of TRIM24 in peripheral blood mononuclear cells (PBMCs) and CD4⁺ T cells from patients with AR was measured. TRIM24-conditional knockout mice with TRIM24 deficiency in CD4⁺ T cells were generated. Wide-type (WT) AR mice and TRIM24-conditional knockout AR mice were established. Then, AR symptoms and interleukin (IL)-4 levels were compared. Further, the proliferation, activation and polarization of CD4⁺ T cells from WT mice and TRIM24 knockout mice after stimulation were determined. The effects of TRIM24 deficiency on STAT6 activities were also evaluated.

Results: Downregulated TRIM24 expression was detected in PBMCs and CD4⁺ T cells from patients with AR. TRIM24 conditional knockout mice had more sever AR symptoms with elevated IL-4 production. TRIM24-knockout CD4⁺ T cells had similar proliferation and activation when compared to WT CD4⁺ T cells, while they had enhanced Th2 polarization. TRIM24-knockout CD4⁺ T cells had decreased acetylation of STAT6 and enhanced STAT6 activities after IL-4 stimulation. The regulation of STAT6 activities by TRIM24 depended on TRIM24 N terminal RIGN domain and Lys383 acetylation site of STAT6.

Conclusions: TRIM24 suppresses Th2-mediated AR by regulating the acetylation of STAT6.

Keywords: STAT6; T cells; TRIM24; Th2; acetylation; allergic rhinitis; transcription factor.

Fig. 1. Low level of TRIM24 is observed in the CD4⁺ T cells of patients with AR. We collected PBMCs and CD4⁺ T cells from healthy donors (normal) and patients with AR. (A) qPCR analysis of TRIM24 in the PBMCs from healthy donors (n = 28) and the patients with AR (n = 26). (B) qPCR analysis of TRIM24, IL-4 and GATA3 in the CD4⁺ T cells from healthy donors (n = 15) and patients with AR (n = 15). Actin was used as loading control and for the relative normalization. (C, D) The protein level of TRIM24 was evaluated by western blot analysis (n = 3). (E) The correlation analysis between TRIM24 expression and the amount of serum IgE in patients with AR (n = 15). Simple linear regression was performed as a statistical method. Data are shown as the mean ± standard deviation. Two-tailed Student’s t-tests were performed in (A, B) panels.

TRIM24, tripartite motif-containing 24; AR, allergic rhinitis; PBMC, peripheral blood mononuclear cell; qPCR, quantitative polymerase chain reaction; IL, interleukin; IgE, immunoglobulin E. ^*P < 0.05; ^**P < 0.01.

Fig. 2. TRIM24 deficiency in T cells promotes the response to Derp1-induced allergic rhinitis. Mice were nasally sensitized with Derp1. Mice were nasally administered Derp1 on indicated consecutive days. (A) the genotype and TRIM24 protein level of WT and TRIM^fl/fl−CD4^cre mice. (B, C) The number of rubbing per day and numbers of sneezes within 1 minute (n = 10). (D) Serum immunoglobulin levels (n = 6). (E) The number of eosinophils, that of goblet cells in the respiratory epithelium, and the thickness of respiratory epithelium (n = 5). The count of eosinophils is based on its cytoplasm in the nasal lamina propria were stained red by H&E. The goblet cells in the nasal epithelial surface were stained red by PAS stain. (F) Production of IL-4, IL-5 and IL-13 in the serum was measured by ELISA. (G) The expressions of Il4, Il5 and Il13 in the nasal lymphoid tissues were measured by qPCR (n = 6). Actin was used as a loading control and for relative normalization. (H) The frequencies of Th1, Th2 and Th17 in total CD4⁺ T cells isolated from nasal lymphoid tissues of WT-and TRIM24-knockout mice were measured by flow cytometry (n=5). Data are presented as mean ± standard deviation.

TRIM24, tripartite motif-containing 24; WT, wide-type; H&E, hematoxylin and eosin; PAS, Periodic acid–Schiff; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction; Th, T helper. ^*P < 0.05; ^**P < 0.01.

Fig. 3. TRIM24 suppresses the polarization of Th2 cells. (A) Naive CD4⁺ T cells isolated from WT or TRIM^fl/fl−CD4^cre mice were stimulated with anti-CD3 (5 μg/mL)/CD28 (1 μg/mL) for the indicated time. Proliferation ratio of these CD4⁺ T cells measured by MTT assay (n = 3). (B) Representative flow cytometry dot plots with double annexin V-FITC/PI staining and summary data described as indicated (n = 3). (C) Naive CD4⁺ Th cells isolated from WT- or TRIM24-knockout mice were stimulated for 72 hours with anti-CD3 (5 μg/mL)/anti-CD28 (1 μg/mL) under the Th2-polarizing condition, followed by intracellular cytokine staining and flow cytometry (n = 4). The data are representative of at least 3 independent experiments with similar results. Data are presented as mean ± standard deviation.

TRIM24, tripartite motif-containing 24; Th, T helper; WT, wide-type; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FITC, fluorescein isothiocyanate; PI, propidium iodide. ^*P < 0.05.

Fig. 4. TRIM24 mediates the acetylation of STAT6 and its target genes. (A) The WT- and TRIM24-knockout T cells were stimulated with IL-4 (10 ng/mL) for indicated time points. Western blot analysis of the indicated phosphorylated (P-) and total STAT6 in whole-cell lysates. (B) Naive CD4⁺ Th cells isolated from WT- or TRIM24-knockout mice were stimulated for 72 hours with anti-CD3 (5 μg/mL)/anti-CD28 (1 μg/mL) under the Th2-polarizing condition. qPCR analysis of GATA3 in these CD4⁺ T cells (n = 6). Actin was used as a loading control and for relative normalization. (C) ChIP-qPCR analysis of Stat6 binding to the promoters of GATA3 in WT- and Trim24-deficient CD4⁺ T cells stimulated with or without IL-4 (10 ng/mL) (n = 5). (D, E) Luciferase assay of Stat6-induced transcriptional suppression of TRIM24 in HEK293 T cells that were transfected with the indicated expression vector, and then were stimulated with (+) or without (−) IL-4 (10 ng/mL) for 4 hours (n = 5). The data are representative of at least 3 independent experiments with similar results. Data are presented as mean ± standard deviation.

TRIM24, tripartite motif-containing 24; STAT6, signal transducer and activator of transcription 6; WT, wide-type; IL, interleukin; Th, T helper; qPCR, quantitative polymerase chain reaction; ChIP, chromatin immunoprecipitation. ^*P < 0.05.