Arid5a exacerbates IFN-γ–mediated septic shock bystabilizing T-bet mRNAMohammad Mahabub-Uz Zamana, Kazuya Masudaa, Kishan Kumar Nyatia,1, Praveen Kumar Dubeya,1, Barry Ripleya,Kai Wanga, Jaya Prakash Chalisea, Mitsuru Higaa, Hamza Haniehb, and Tadamitsu Kishimotoa,2

aLaboratory of Immune Regulation, World Premier International Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan;and bBiological Sciences Department, College of Science, King Faisal University, Al-Ahsa 31982, Saudi Arabia

Contributed by Tadamitsu Kishimoto, August 15, 2016 (sent for review July 11, 2016; reviewed by Abul K. Abbas, Ruslan Medzhitov, and Kenji Nakanishi)

Adenine-thymine (AT)-rich interactive domain containing protein 5a(Arid5a) is an RNA-binding protein that has been shown to play animportant immune regulatory function via the stabilization of IL-6and STAT3 mRNA. However, the role of Arid5a in the overwhelmingand uncontrolled immune response that leads to septic shock is un-known. Here, we report that Arid5a-deficient mice are highly resistantto lipopolysaccharide (LPS)-induced endotoxic shock and secrete lowerlevels of major proinflammatory cytokines, including IFN-γ, IL-6, andTNF-α, than WT mice in response to LPS. Arid5a deficiency resulted indecreased levels of IFN-γ under Th1 cell conditions, in which T-boxexpressed in T cells (T-bet) mRNA expression was inhibited. Arid5abound to the conserved stem loop structure of the 3′UTR of T-betand stabilized its mRNA. Arid5a-deficient mice were also resistant toPropionibacterium acnes-primed LPS injection, which is considered tobe a T-cell–mediated IFN-γ dependent endotoxic shock mouse model.Thus, regulation of IFN-γ by Arid5a via the stabilization of T-bet mRNAin Th1 cells contributes to the development of septic shock in mice. Inaddition, our previous study suggests that Arid5a control the IL-6 levelin vivo in response to LPS by stabilization of IL-6 mRNA. We also ob-served that neutralization of IFN-γ and IL-6 significantly recovered themice from endotoxic shock. Taken together, we conclude that Arid5aregulates the augmentation of IL-6 and IFN-γ in response to LPS, whichpossibly works synergistically for amplification of various other cyto-kines that ultimately cause the development of septic shock in mice.

septic shock | Arid5a | IFN-γ | T-bet | IL-6

Sepsis is a frequent and severe systemic inflammatory responseto infection associated with the excessive production of cyto-kines. The Gram-negative bacterial wall constituent endotoxin(LPS) is the major active agent in the pathogenesis of septic shock(1). IFN-γ plays an essential role in the development of septicshock. IFN-γ is produced by activated T lymphocytes and naturalkiller (NK) cells and mediates its action by binding a unique cellsurface receptor (2). Treatment of mice with IFN-γ increasessusceptibility to endotoxic shock (3), and IFN-γ receptor knockoutmice are resistant to endotoxic shock (4), clearly indicating itsimportance in the pathogenesis of septic shock.T cells play an important role in sepsis by secreting IFN-γ. Mice

depleted of T cells are resistant to polymicrobial sepsis and Escher-ichia coli infection (5, 6). T-box expressed in T cells (T-bet) is atranscription factor that plays a major role in regulating IFN-γ pro-duction in T lymphocytes (7, 8). The expression of T-bet in primaryT cells or developing Th2 cells results in the activation of IFN-γproduction (7), whereas mice lacking T-bet fail to develop Th1 cellsand display a dramatic reduction of IFN-γ production (8). T-betdysregulation has been associated with several Th1-driven immu-nopathological disorders, suggesting that regulation of T-bet is es-sential to control the innate immunity (9, 10).AT-rich interactive domain containing protein 5a (Arid5a) is a

member of the Arid protein family that functions as a uniqueRNA-binding protein (11, 12). It is expressed highly in macrophagein response to LPS and controls posttranscriptional regulation ofIL-6 by stabilizing IL-6 mRNA through binding to its 3′UTR (11).Arid5a deficiency was shown to inhibit the elevation of IL-6 serum

levels in LPS-treated mice (11). Our recent study also showed an in-trinsic role for Arid5a in T cells, where Arid5a was found to direct thedifferentiation of naive CD4+ T cells into inflammatory CD4+ T cellsby the stabilization of STAT3 mRNA (12). Overall, these findingssuggest Arid5a has an important immune regulatory function. How-ever, the role of Arid5a in the overwhelming and uncontrolled im-mune response that leads to endotoxic shock is unknown.In this study, we identified an essential role for Arid5a in LPS-

induced endotoxic shock. We report here that Arid5a augmentsIFN-γ secretion in Th1 cells via stabilization of the mRNA of itstranscription factor, T-bet, which in turn contributes to IFN-γ–mediated endotoxic shock in mice.

ResultsArid5a-Deficient Mice Are Highly Resistant to Endotoxic Shock. Todetermine the role of Arid5a in the innate immune response, wefirst investigated the role of Arid5a in a murine model of endotoxicshock, using Arid5a-deficient mice. Arid5a-deficient andWT controlmice were injected intraperitoneally (i.p.) with LPS, and survival wasmonitored. As shown in Fig. 1A, all the WTmice died within 96 h ofLPS administration, whereas 100% of the Arid5a-deficient micesurvived under similar conditions. As endotoxemia in septic shock isassociated with multiple organ damage, which leads to death, wenext evaluated histopathology of the liver, lung, and spleen of theWT and Arid5a-deficient mice, using H&E staining (Fig. 1B). TheWT mice exhibited widespread hemorrhaging in the liver, severefibrosis in the lung, and tissue disorganization in the spleen, whereasthe Arid5a-deficient mice were protected from tissue damage afterendotoxic shock (Fig. 1B). Thus, our data suggest Arid5a plays anessential role in the progression of endotoxic shock.

Significance

Septic shock is one of the major causes of morbidity and mortalitythroughout the world. Despite extensive efforts, septic shockremains a major medical challenge. In this study, using Arid5a-deficient mice, we demonstrated a critical role of this protein inlipopolysaccharide (LPS)-induced shock through regulation ofIFN-γ expression in T cells by preventing the mRNA degradation ofits major transcription factor T-bet. Further, we identified theconserved stem loop structure at 3′UTR of T-bet mRNA requiredfor binding Arid5a during its action. Our study indicates thetherapeutic potential of anti-agonistic agents for Arid5a in theprevention of pathological conditions of septic shock.

Author contributions: M.M.Z., K.M., and T.K. designed research; M.M.Z. performed re-search; K.M., K.K.N., P.K.D., B.R., K.W., J.P.C., M.H., and H.H. contributed new reagents/analytic tools; M.M.Z. analyzed data; and M.M.Z. and T.K. wrote the paper.

Reviewers: A.K.A., University of California; R.M., Yale University School of Medicine; andK.N., Hyogo College of Medicine.

The authors declare no conflict of interest.1K.K.N. and P.K.D. contributed equally to this work.2To whom correspondence should be addressed. Email: kishimoto@ifrec.osaka-u.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1613307113/-/DCSupplemental.

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Arid5a-Deficient Mice Produce Significantly Decreased Levels ofProinflammatory Cytokines in Response to Endotoxic Shock. Thetoxic effects of LPS are mainly mediated by proinflammatory cy-tokines such as TNF-α, IL-6, and IFN-γ (13). Therefore, to de-termine the influence of Arid5a on proinflammatory cytokinesduring LPS-induced shock, WT and Arid5a-deficient mice wereinjected i.p. with LPS, and serum cytokine levels were determinedby ELISA at various times after LPS injection. As shown in Fig. 2A–C, the production of major proinflammatory cytokines, in-cluding IFN-γ, IL-6, and TNF-α, was significantly decreased inArid5a-deficient mice compared with WT mice after in vivo in-jection of LPS. Consistent with these data, splenocytes collectedfrom LPS-challenged Arid5a-deficient mice expressed signifi-cantly decreased levels of IL-6 and IFN-γ compared with controlmice, whereas TNF-α was not detected in either WT or Arid5a-deficient splenocytes 24 h after LPS challenge (Fig. S1). Takentogether, these results suggest that the resistance of Arid5a nullmice to endotoxic shock is the result of a diminished response toproinflammatory cytokines.

Neutralization of IFN-γ and IL-6 Recovers Mice from Endotoxic Shock.Our results suggest that Arid5a-deficient mice are defective atproducing IFN-γ, IL-6, and TNF-α in response to LPS. Next, weasked which cytokine among these three proinflammatory cyto-kines has the most profound effect on endotoxemia. To answerthis question, we compared the effects of neutralizing antibodies

against IFN-γ, IL-6, and TNF-α 4 h after LPS treatment. Asshown in Fig. 3, anti–IFN-γ treatment rescued all the mice fromLPS-mediated septic shock, whereas anti–IL-6 receptor antibodymoderately rescued (78%) and anti–TNF-α treatment partiallyrescued (30%) the mice. Collectively, these results demonstratethat IFN-γ and IL-6 play an important role in endotoxic shock.

IFN-γ and T-bet Expression Is Significantly Decreased in Arid5a-DeficientActivated T Cells. Having confirmed that IFN-γ and IL-6 play aprofound role in endotoxemia, we next focused on the mechanismby which Arid5a augments these proinflammatory cytokines in theserum in response to LPS. Our previous study already clarified thatArid5a controls the IL-6 level in vivo by preventing the decay ofIL-6 mRNA (11). Therefore, in this study we emphasized themechanism by which Arid5a regulates the IFN-γ during endotoxicshock. IFN-γ is not expressed directly in response to LPS. Theinteraction of LPS with Toll-like receptor 4 (TLR4) on macro-phages and monocytes first triggers the production of variousinitial proinflammatory cytokines, such as IL-12 and IL-18, whichin turn activate T cells or NK cells to release IFN-γ (14). Initially,we evaluated the expression of IL-12 and IL-18 in peritonealmacrophages in WT and Arid5a-deficient mice. These cytokinesare directly affected by LPS and trigger the release of IFN-γ inother immunological cells, such as activated T cells. However, theirexpression levels were found to be comparable between WT andArid5a-deficient mice (Fig. S2), suggesting Arid5a may not directly

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).

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affect the expression of IL-12 and IL-18 for IFN-γ induction. T cellshave been demonstrated to play an important role in acute sepsis viathe increased production of IFN-γ (5, 6, 15). Moreover, injection ofmice with LPS significantly increases Arid5a expression in both naiveand effector CD4+ T cells (Fig. S3). Thus, to examine the possiblerole of Arid5a in controlling IFN-γ, we next examined the intrinsicrole of Arid5a in T cells. When naive CD4+ T cells collected fromWT and Arid5a-deficient spleens were stimulated under Th1 differ-entiation conditions, Arid5a-deficient Th1 cells were found to expresssignificantly lower levels of IFN-γ (Fig. 4A) compared with WT Th1cells. To determine whether Arid5a-deficient CD4+ T cells alsoexpressed low levels of IFN-γ under endotoxic shock conditions, weisolated CD4+ T cells fromWT andArid5a-deficient splenocytes afterin vivo injection of LPS and measured the IFN-γ expression byquantitative PCR. Consistent with the in vitro data, we also observedthat Arid5a-deficient T cells expressed severely reduced levels ofIFN-γ (Fig. 4C) after endotoxic shock.T-bet and STAT4 are the major transcription factors that

regulate IFN-γ expression in Th1 cells (10, 16). To determinewhether defective production of IFN-γ in Arid5a-deficient Th1cells is a result of the abnormal production of T-bet and STAT4,

we measured their expression under Th1-promoting conditions.Interestingly, we found that T-bet expression, but not STAT4,was significantly impaired in Arid5a-deficient Th1 cells (Fig. 4A)compared with WT cells. As Arid5a and T-bet expression weresignificantly increased in CD4+ T cells after LPS injection (Fig.S3), we next determined whether the depletion of Arid5a in CD4+

T cells affected T-bet expression under endotoxic shock conditions.Similar to the in vitro data, we observed that T-bet expression, butnot STAT4, was severely decreased in Arid5a-deficient CD4+

T cells under endotoxic shock conditions (Fig. 4C). STAT4 isknown to be activated via phosphorylation under Th1-promotingconditions, contributing to IFN-γ production (17). To determinewhether Arid5a contributes to IFN-γ production in Th1 cells throughSTAT4 activation, we compared the phosphorylation of STAT4 inWT and Arid5a-deficient mice under Th1-promoting conditions byimmunoblotting (Fig. 4B). However, no significant differences wereobserved in STAT4 phosphorylation or in total STAT4 levels be-tween WT and Arid5a-deficient Th1 cells (Fig. 4B), which suggestsArid5a is not involved in STAT4 activation. Thus, our in vitro andin vivo results indicate that Arid5a plays an important role in acti-vated T cells by regulating the expression of T-bet and IFN-γ.

Arid5a Binds to and Stabilizes the 3′UTR Region of T-bet mRNA.Having confirmed that Arid5a regulates T-bet and IFN-γ expres-sion in Th1 cells, we next asked how Arid5a controls their expres-sion. Arid family proteins are believed to have DNA-bindingproperties (18), but our group demonstrated that Arid5a can func-tion as an RNA-binding protein (11, 12). Thus, there is a possibilitythat Arid5a could regulate T-bet and IFN-γ transcriptionally, byactivating their promoters, or posttranscriptionally, by activatingtheir UTRs. To evaluate both possibilities, we constructed luciferasevectors encoding the promoters or 3′UTR regions of T-bet andIFN-γ and performed luciferase reporter assays by overexpressingArid5a or empty vector in HEK293T cells (Fig. 5 A and B). Over-expression of Arid5a affected neither the promoter nor the UTRactivity of IFN-γ (Fig. 5A). However, as shown in Fig. 5B, Arid5aoverexpression significantly augmented the luciferase activity of thepGL3 vector encoding the T-bet 3′UTR compared with that of thecontrol vector. No effect of Arid5a was observed on the T-betpromoter region (Fig. 5B), suggesting Arid5a is essential for T-betmRNA stabilization, but not for IFN-γ. This result was further con-firmed by measurement of the T-bet mRNA half-life after over-expressing Arid5a in HEKTet-off cells stably expressing the enhancerof a tetracycline-responsive promoter (TRE). Overexpression ofArid5a was shown to prevent the T-bet mRNA degradation afterstimulation with tetracycline derivative doxycycline compared with thecontrol cells transfected with empty vector (Fig. 5C). To evaluate the

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 micewere stimulated under Th0 conditions (stimulated with CD3e and CD28) or Th1 conditions (stimulated with CD3e, CD28, and IL-12). Forty-eight hours afterdifferentiation, IFN-γ, T-bet, and STAT4 mRNA expression was measured by RT-PCR. (B) Immunoblot analysis of phosphorylated STAT4 and total STAT4 in WTor 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 STAT4mRNA 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 independentexperiments (A and C). Error bars show the means ± SD (A–D). *P < 0.05; **P < 0.01 (Student’s t test).

Fig. 3. Anti–IFN-γ and Anti–IL-6R treatment recover the mice from endotoxicshock. Age-matched WT B6 mice were administered E. coli LPS (25 mg/kg). Fourhours later, the mice were treatedwith 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.

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association of T-bet mRNA with Arid5a protein, we next performedan RNA immunoprecipitation assay after overexpression of the T-bet(CDS + 3′UTR) plasmid together with Flag-tagged Arid5a or controlvector in HEK293T cells. Arid5a was immunoprecipitated with T-betmRNA, but not GAPDH (Fig. 5D). These results indicate thephysical association of Arid5a with T-bet 3′UTR mRNA. Takentogether, our results demonstrate that Arid5a stabilizes T-betmRNA by associating with its 3′UTR region.

The Conserved Stem Loop Region of the T-bet 3′UTR (616-641) IsRequired for Arid5a Binding. After confirming that Arid5a controlsT-bet 3′UTR mRNA stability via the 3′UTR, we next attempted toidentify which portions of the T-bet 3′UTR are critical for the stabi-lization of T-bet mRNA by Arid5a. Many RNA-binding proteins havebeen shown to associate with adenine-uridine-rich element (ARE)

regions (19). Mouse T-bet mRNA contains one ARE in its 3′UTR.To investigate regions of the T-bet 3′UTR that are critical for con-ferring Arid5a responsiveness, we prepared constructs of the pGL3luciferase vector encoding the non-ARE-containing fraction of theT-bet 3′UTR (1–320) or the ARE-containing fraction of the T-bet3′UTR (321–701), as shown in Fig. 6A. Overexpression of Arid5aenhanced the luciferase activity of the pGL3 vector encoding theARE-containing region (321–701) of the T-bet 3′UTR (Fig. 6B) morethan twofold compared with the control, whereas Arid5a was lessresponsive to the non-ARE region (1–301) (Fig. 6C). This resultsuggests that Arid5a stabilizes T-bet mRNA by binding to the ARE-containing fraction of the 3′UTR of T-bet.Having confirmed that Arid5a controls T-bet mRNA stability

via binding the ARE-containing region of the 3′UTR, we nextattempted to identify which elements in the ARE fraction of the

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 andpromoter 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 aftertransfection. The values were normalized to those obtained with transfection with EV. (C) HEK293 Tet-off cells were transfected with pTREtight-T-bet-CDS + 3′UTRtogether with an Arid5a expression vector or an empty vector. The cells were uniformly divided 3 h after transfection and incubated overnight. Total RNA wasprepared 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 orIgG antibody, and immunoprecipitates were analyzed using primers specific to T-bet and GAPDH mRNA. The data are representative of three independent ex-periments (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 vectorsencoding 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) Lu-ciferase 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 hwith 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 andmice 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 theinteraction of recombinant Arid5a protein with 3′ biotinylated nucleotides, as in D.

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T-bet 3′UTR are critical for the stabilization of T-bet mRNA byArid5a. Our group recently reported that Arid5a can recognize stemloop structure of the 3′UTR region of STAT3mRNA (12). Thus, welooked for the stem loop-like structure in the ARE-containingfraction of the 3′UTR of T-bet (321–701). Interestingly, amino acids616–641 of the T-bet 3′UTR were found to form a stem loop-likestructure (Fig. 6D) and were highly conserved between humans andmice (Fig. 6C). To confirm whether this conserved stem loop-likeelement (616-GCUAUUUAUUGUAGAGAGUGGU-641) in theT-bet 3′UTR is physically associated with Arid5a, we performed anelectrophoretic mobility shift assay (EMSA), using the biotinylatedRNA sequence of the conserved element (616–641) and recombi-nant Arid5a protein. As shown in Fig. 5D, Arid5a tightly bound withthe stem loop region (616–641) of the T-bet 3′UTR. Overall, ourdata suggest that Arid5a physically associates with the conservedstem loop region near the ARE site of the T-bet 3′UTR mRNA.

Arid5a-Deficient Mice Are Resistant to Propionibacterium acnes-PrimedEndotoxic Shock. Our results demonstrated that Arid5a controlsIFN-γ by stabilizing its master regulator, T-bet, in Th1 cells. Toconfirm whether this phenomenon is related to endotoxic shock,we evaluated the sensitivity of Arid5a-deficient mice in a P. acnes-primed endotoxic shock mouse model. Mice deficient in T cells(nu/nu), Th1 cells (IL-12p40−/−), or IFN-γ are highly resistant toP. acnes-primed, LPS-induced septic shock (15). Thus, it is believedthat the P. acnes-primed endotoxic shock mouse model is T-cell–mediated and IFN-γ–dependent. Previously, it was proposed thatpriming mice with heat-killed P. acnes results in the induction ofIL-12 by macrophages, which causes increased IFN-γ production byTh1 cells and primes the macrophages to be susceptible to LPS (15).Thus, to evaluate whether the regulation of IFN-γ expression in Th1cells by Arid5a affects endotoxic shock in mice, we challenged WTand Arid5a-deficient mice with P. acnes for 5 d, followed by asublethal dose of LPS (Fig. 7A). As shown in Fig. 7B, 24 h after LPSadministration, 50% of the WT mice died, whereas all of theArid5a-deficient mice survived under similar conditions. The serumproinflammatory cytokine levels were measured 4 h after LPStreatment. The serum IFN-γ, IL-6, and TNF-α levels were significantlydecreased in Arid5a-deficient mice compared with WTmice (Fig. 7C)after LPS administration. Because IFN-γ is known to play a key role inthe pathogenesis of disease in this mouse model, this is likely to bemediated by Arid5a regulation of IFN-γ expression.

DiscussionThe pathology of endotoxemia is believed to be jointly mediated bymultiple cytokines, including TNF-α, IL-6, and IFN-γ (13). How-ever, to the best of our knowledge, their effects have not yet beencompared under similar experimental conditions by using neutral-izing antibodies after the development of LPS-induced shock. Inthis study, we showed that anti–IFN-γ and anti–IL-6 receptor an-tibody treatment at 4 h after LPS treatment had the most pro-nounced effect in recovering mice from endotoxic shock comparedwith anti–TNF-α treatment (Fig. 3), suggesting IFN-γ and IL-6 playthe most significant role at late points after endotoxic shock in mice.Arid5a is an RNA-binding protein that was shown to regulate the

IL-6 level in vivo by stabilization of its mRNA (11). In this study, wedemonstrated a new and critical role of this protein in LPS-inducedshock through regulation of IFN-γ expression in Th1 cells. Theinteraction of LPS with TLR4 on macrophages and monocytes firsttriggers the production of various initial proinflammatory cytokinessuch as IL-12 and IL-18 that activate the Th1 cells to release theIFN-γ (14), which in turn trigger the release of additional immu-nomodulatory factors that together contribute to the developmentof endotoxic shock (20). Our studies showed that Arid5a did notinhibit LPS-dependent production of IL-12 or IL-18 in peritonealmacrophage (Fig. S2). However, IL-12–induced activated Arid5a-deficient Th1 cells expressed a significantly low level of IFN-γcompared with WT (Fig. 3A). Arid5a-deficient T cells isolated fromthe LPS-injected mice also showed a significantly low expression ofIFN-γ in comparison with WT (Fig. 3C), suggesting that activationof T cells after LPS injection was defective in Arid5a-deficient mice,which possibly contributes to low expression of IFN-γ.The transcription factor T-bet is a master regulator of the gen-

eration of IFN-γ–producing Th1 cells (7, 8). IFN-γ secretion wasreported to be impaired in T-bet-deficient mice (9). Recent studieshave strongly indicated that regulation of T-bet in Th1 cells is re-quired for combating bacterial and viral infection (21, 22). In thisreport, we showed that the expression of T-bet is impaired in Arid5a-deficient Th1 cells (Fig. 3A). T-bet expression was also significantlydecreased in Arid5a-deficient T cells isolated from endotoxic shockmice compared withWT T cells (Fig. 3C). We reasoned that Arid5a-deficient Th1 cells produce decreased levels of IFN-γ because of thedefective expression of T-bet, which contributes to the decreasedserum levels of IFN-γ in Arid5a-deficient mice after LPS injection.The combination of signals from antigens, costimulation, and

proinflammatory cytokines such as IL-12 promotes the activationof the TCR, IL-12–STAT4, and IFN-γ–STAT1 signaling pathwaysin T cells, which induces the expression of T-bet and results in the

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 themortality 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 LPStreatment (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).

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differentiation of naive T cells into Th1 cells (10). T-bet also pos-itively regulates its own expression through an autoregulator loopinvolving Hlx, a homeobox gene (10). Although transcriptionalregulation of T-bet has already been well studied, the posttran-scriptional regulation of T-bet is not well understood. In this study,we show that Arid5a regulates T-bet expression by protecting thedegradation of its mRNA via binding to the stem loop structure at3′UTR region (Figs. 5 and 6). This mechanism of Arid5a is con-sistent with our previous observations, where Arid5a was shown towork as a stabilizer of IL-6 and STAT3 mRNA (11, 12).Although the posttranscriptional degradation mechanism of

T-bet mRNA is not clearly known, previous study showed thatRNA-binding protein Regnase-1 is highly expressed in T cells,and overexpression of Regnase-1 degraded the 3′UTR region ofT-bet (Tbx21) mRNA (23). Moreover, transcriptome analysisalso showed that Tbx21 mRNA is highly upregulated in Regnase-1-deficient CD4+ T-cells (23), suggesting Regnase-1 is the poten-tial candidate for posttranscriptional destabilization of T-bet mRNA.In addition, our previous study suggests that Arid5a counteract thedestabilizing effect of Regnase-1 by preventing its binding to thestem loop structure of 3′UTR mRNA of IL-6 and STAT3 (11, 12).On the basis of those observations, we speculate that Arid5a stabi-lizes the T-bet mRNA, possibly by counteracting with Regnase-1.In this report, we did not focus on the role of Arid5a in NK cells,

which have also been shown to play important roles in endotoxemiaby secreting IFN-γ (24). We cannot ignore the possibility that Arid5amight also have a similar role in NK cells, as well as in the regulationof IFN-γ. In fact, T-bet is also expressed in NK cells (10). Furtherstudies are required by generating cell-specific Arid5a-deficient miceto reveal the complete mechanism of Arid5a in endotoxic shock.In summary, we conclude that Arid5a regulates the augmentation

of IFN-γ via stabilization of T-bet mRNA in Th1 cell, which in turncontributes to development of LPS-induced septic shock in mice.However, the role of Arid5a in the development of septic shock isnot solely dependent on its regulation of IFN-γ. The previouslydescribed role of Arid5a in regulation of IL-6 (11) also possiblyparticipates in the establishment of septic shock, as Arid5a-deficientmice secreted less IL-6 in response to LPS (Fig. 2) and anti–IL-6receptor antibody treatment showed a significant effect in re-covering the mice from septic shock (Fig. 3). So, it is reasonable tospeculate that Arid5a regulates the augmentation of both IL-6 andIFN-γ in response to LPS, which possibly works synergisticallyfor the amplification of various other cytokines that ultimately causethe development of septic shock in mice. Thus, our study indicatesthe therapeutic potential of anti-agonistic agents for Arid5a in theprevention of pathological conditions of sepsis.

Materials and MethodsMice. C57BL/6J WT mice (8–9 wk) were obtained from CLEA Japan, Inc.Arid5a−/− mice were generated as previously described (11). All animal experi-ments were performed in accordance with protocols approved by the InstitutionalAnimal Care and Use Committees of the Graduate School of Frontier Bioscience(Osaka University, Osaka, Japan).

LPS-Induced Endotoxic Shock. Arid5a−/− and WT female mice (body weight,18–20 g) were i.p. injected with LPS (15 mg/kg or 25 mg/kg) from E. coli (Sigma).The survival of the mice was monitored over the next 7 d. The roles of TNF-α,IL-6, and IFN-γ in endotoxic shock were investigated by neutralizing endoge-nous TNF-α, IL-6, and IFN-γ with their respective monoclonal antibodies. Anti–TNF-α (MP6-XT22 BioLegend, 10 mg/kg), anti–IL-6R (MR16-1, 250 mg/kg), andanti–IFN-γ (R4-6A2 BioLegend, 25 mg/kg) were injected into the mice 4 h afterLPS injection, and mortality was monitored over the next 7 d.

Cell Culture and in Vitro Th1 Cell Differentiation. Cell culture and in vitro Th1cell differentiation were performed according to the method describedpreviously (11, 12). Details are given in SI Materials and Methods.

Luciferase Assay. HEK293T cells were transfectedwith a pGL3 vector encodingthe IFN-γ UTR, T-bet 3′UTR region (1–701), T-bet 3′UTR region (1–302), andT-bet 3′UTR region (321–701) or a pLUC-MCS vector encoding the IFN-γ orT-bet promoter, together with an Arid5a expression plasmid or an empty(control) plasmid. Luciferase activity was measured according to the protocoldescribed previously (11). Details are given in SI Materials and Methods.

mRNA Decay Experiments (Tet-off System). mRNA decay experiment of T-betmRNA was performed using quantitative RT-PCR (qRT-PCR) Tet-off system inHEK293 Tet-off cells by transfectingwith pTREtight-T-bet-CDS + 3′UTR (which has aT-bet coding and noncoding 3′UTR sequence), together with an Arid5a expressionplasmid or a control (empty) plasmid. Details are given in SIMaterials andMethods.

RIP Assay. HEK293T cells were transfected with an expression vector encod-ing the T-bet (CDS + 3′UTR), together with a Flag-tagged Arid5a expressionvector or an empty vector (control). Twelve hours after transfection, the cellswere collected, and an RNA immunoprecipitation (RIP) assay was performedusing the RiboCluster Profiler RIP assay kit (MBL) according to the manu-facturer’s instructions, using a Flag monoclonal antibody (Sigma) and controlmouse IgG. The eluted RNA was analyzed by RT-PCR.

qRT-PCR, RNA-EMSA, and Immunoblotting. qRT-PCR analysis, RNA-EMSA, andimmunoblotting were performed as previously described (11, 12, 17). Detailsare given in SI Materials and Methods.

ACKNOWLEDGMENTS.We thank Professor Hiroko Tsutsui (Hyogo College ofMedicine, Japan) for providing the heat-killed P. acnes bacteria and ChugaiPharmaceutical Co., Ltd. (Tokyo) for making the recombinant Arid5a pro-tein. This work was funded by the Kishimoto Foundation and WPI Immunol-ogy Frontier Research Center.

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