Arid5a exacerbates IFN-γ-mediated septic shock by stabilizing T-bet mRNA

Abstract

Adenine-thymine (AT)-rich interactive domain containing protein 5a (Arid5a) is an RNA-binding protein that has been shown to play an important immune regulatory function via the stabilization of IL-6 and STAT3 mRNA. However, the role of Arid5a in the overwhelming and uncontrolled immune response that leads to septic shock is unknown. Here, we report that Arid5a-deficient mice are highly resistant to lipopolysaccharide (LPS)-induced endotoxic shock and secrete lower levels of major proinflammatory cytokines, including IFN-γ, IL-6, and TNF-α, than WT mice in response to LPS. Arid5a deficiency resulted in decreased levels of IFN-γ under Th1 cell conditions, in which T-box expressed in T cells (T-bet) mRNA expression was inhibited. Arid5a bound to the conserved stem loop structure of the 3'UTR of T-bet and stabilized its mRNA. Arid5a-deficient mice were also resistant to Propionibacterium acnes-primed LPS injection, which is considered to be a T-cell-mediated IFN-γ dependent endotoxic shock mouse model. Thus, regulation of IFN-γ by Arid5a via the stabilization of T-bet mRNA in Th1 cells contributes to the development of septic shock in mice. In addition, our previous study suggests that Arid5a control the IL-6 level in vivo in response to LPS by stabilization of IL-6 mRNA. We also observed that neutralization of IFN-γ and IL-6 significantly recovered the mice from endotoxic shock. Taken together, we conclude that Arid5a regulates the augmentation of IL-6 and IFN-γ in response to LPS, which possibly works synergistically for amplification of various other cytokines that ultimately cause the development of septic shock in mice.

Keywords: Arid5a; IFN-γ; IL-6; T-bet; septic shock.

Fig. 1.

Arid5a-deficient mice are resistant to LPS-induced endotoxic shock. (A) 8-wk-old WT (n = 15) and Arid5a-deficient (n = 10) mice were injected with E. coli LPS (15 mg/kg weight), and survival was monitored for 7 d. (B) H&E staining of the liver, lung, and spleen sections of WT and Arid5a-deficient mice 48 h after LPS challenge.

Fig. 2.

Serum proinflammatory cytokines were significantly decreased in Arid5a-deficient mice after LPS challenge. (A–C) The serum concentrations of IFN-γ (A), IL-6 (B), and TNF-α (C) in Arid5a-deficient mice (n = 5) were measured at the indicated points after LPS (5 mg/kg) injection. Error bars show the means ± SEM *P < 0.05; **P < 0.01 (Student’s t test).

Fig. S1.

Expression of the proinflammatory cytokines IL-6 and IFN-γ was significantly decreased in Arid5a-deficient splenocytes after LPS challenge. Twenty-four hours after LPS injection (5 mg/kg; n = 3), spleens were collected from WT and deficient mice, homogenates were prepared, and IL-6, TNF-α and IFN-γ levels were measured by ELISA. Error bars show the means ± SD. *P < 0.05; **P < 0.01; (Student’s t test); N.D., not detected.

Fig. 3.

Anti–IFN-γ and Anti–IL-6R treatment recover the mice from endotoxic shock. Age-matched WT B6 mice were administered E. coli LPS (25 mg/kg). Four hours later, the mice were treated with PBS (n = 10), anti–IL-6R (n = 13), anti–IFN-γ (n = 10), or anti–TNF-α (n = 5) antibodies, and survival was monitored for 7 d.

Fig. 4.

Arid5a-deficient Th1 cells expressed decreased levels of IFN-γ and T-bet in vitro and in vivo. (A) Naive CD4⁺ T cells from WT and Arid5a-deficient mice were stimulated under Th0 conditions (stimulated with CD3ε and CD28) or Th1 conditions (stimulated with CD3ε, CD28, and IL-12). Forty-eight hours after differentiation, IFN-γ, T-bet, and STAT4 mRNA expression was measured by RT-PCR. (B) Immunoblot analysis of phosphorylated STAT4 and total STAT4 in WT or Arid5a-deficient CD4⁺ T cells was stimulated for 12 h under Th0 and Th1 cell conditions. (C) Quantitative real-time PCR analysis of IFN-γ, T-bet, and STAT4 mRNA in CD4⁺ T cells isolated from the spleens of LPS (5 μg/kg)-challenged WT or Arid5a-deficient mice. The data are representative of three independent experiments (A and C). Error bars show the means ± SD (A–D). *P < 0.05; **P < 0.01 (Student’s t test).

Fig. S2.

IL-12 and IL-18 expression were comparable between WT and Arid5a-deficient mice (n = 3) in peritoneal macrophages after LPS administration. Peritoneal macrophages were treated with LPS (500 ng/mL) at the indicated points. Total RNA was isolated, and equal amounts of RNA were used for the quantitative RT-PCR assay. Error bars show the means ± SD.

Fig. S3.

Expression of Arid5a and T-bet in CD4⁺ T cells isolated from LPS-treated mice. Four hours after LPS injection, spleens were collected, and naive and effector CD4⁺ T cells were isolated using MACS microbeads. Total RNA was isolated, and equal amounts of RNA were used for the quantitative RT-PCR assay. Error bars show the means ± SD. *P < 0.05; **P < 0.01 (Student’s t test).

Fig. 5.

Arid5a stabilizes T-bet, but not IFN-γ, mRNA. (A and B) HEK 293T cells were transfected with a luciferase vector encoding the full-length 3′UTR and promoter regions of IFN-γ (A) or T-bet (B) together with an empty vector (EV) or an Arid5a expression vector. Luciferase activity was measured 48 h after transfection. The values were normalized to those obtained with transfection with EV. (C) HEK293 Tet-off cells were transfected with pTREtight-T-bet-CDS + 3′UTR together with an Arid5a expression vector or an empty vector. The cells were uniformly divided 3 h after transfection and incubated overnight. Total RNA was prepared after doxycycline (1 μg/mL) treatment, and T-bet mRNA levels were determined by RT-PCR. (D) Association of Arid5a with T-bet mRNA by RIP assay. HEK293T cells were transfected with the T-bet (CDS + 3′UTR) and a Flag-tagged Arid5a expression vector. The cell lysates were immunoprecipitated with Flag or IgG antibody, and immunoprecipitates were analyzed using primers specific to T-bet and GAPDH mRNA. The data are representative of three independent experiments (A–D). Error bars show the means ± SD (A–D). *P < 0.05; **P < 0.01; N.S., not significant (Student’s t test).

Fig. 6.

Arid5a associates with the stem loop-like structure located near the ARE region of T-bet mRNA. (A) Schematic diagram of the luciferase vectors encoding T-bet 3′UTR region (1–701), T-bet 3′UTR region (1–302), or T-bet 3′UTR region (321–701). The black circle shows AU-rich elements (AREs). (B) Luciferase activity of the pGL3 vectors encoding T-bet 3′UTR region (1–701), T-bet 3′UTR region (1–302), or T-bet 3′UTR region (321–701) cotransfected for 48 h with an Arid5a expression vector or empty vector. The data are representative of three independent experiments. Error bars show the means ± SD (B). *P < 0.05; **P < 0.01; N.S., not significant (Student’s t test). (C) Schematic diagram shows the conserved stem loop-like element (616–641) between humans and mice near the ARE region (closed circle) of T-bet 3′UTR mRNA. (D) Diagram of the stem loop structure (616–641) of the T-bet 3′UTR. (E) EMSA evaluating the interaction of recombinant Arid5a protein with 3′ biotinylated nucleotides, as in D.

Fig. 7.

Arid5a-deficient mice are protected from T-cell–mediated, IFN-γ–dependent endotoxic shock. (A) Schematic diagram shows the experimental design. Mice were administered heat-killed P. acnes (1 mg in 200 μL PBS) i.p. On day 5, the mice were treated with low-dose LPS (5 μg/mouse). Serum cytokines and the mortality of the mice were analyzed after LPS treatment. (B) Survival analysis of WT (n = 7) and Arid5a-deficient mice (n = 7) after P. acnes-primed LPS treatment (C) Serum was collected 4 h after LPS treatment from WT (n = 4) and Arid5a-deficient mice (n = 4), and the proinflammatory cytokines IFN-γ, IL-6, and TNF-α were measured by ELISA. **P < 0.01, (Student’s t test).