Triptolide Inhibits IL-12/IL-23 Expression in APCs via CCAAT/Enhancer-Binding Protein α

This information is current as Yan Zhang and Xiaojing Ma of September 29, 2021. J Immunol 2010; 184:3866-3877; Prepublished online 1 March 2010; doi: 10.4049/jimmunol.0903417 http://www.jimmunol.org/content/184/7/3866 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Triptolide Inhibits IL-12/IL-23 Expression in APCs via CCAAT/Enhancer-Binding Protein a

Yan Zhang* and Xiaojing Ma*,†

Triptolide is a biologically active component purified from Chinese herbal plant Hook F.It is widely used in East Asia for treatment of systemic lupus erythematosus, rheumatoid arthritis, nephritis, Bechect’s disease, psoriasis, atopic dermatitis, and asthma. However, its immunological mechanisms are poorly understood. IL-12 and IL-23 are closely related heterodimeric cytokines that share the common subunit p40. They are produced by APCs and are key factors in the generation and effector functions of Th1 and Th17 cells, respectively. They have been strongly implicated in the pathogenesis of several autoimmune disorders. In this study, we investigated the molecular mechanism whereby triptolide inhibits the expression of the p40 gene in APCs. We demonstrate that triptolide does so at the transcriptional level in part through targeting CCAAT/enhancer-binding protein-a (C/EBPa), which

directly interacts with the p40 promoter and inhibits its transcription in inflammatory macrophages. Triptolide can activate the Downloaded from transcription of C/EBPa, and phosphorylation of Ser21 and Thr222/226 critical for C/EBPa inhibition of p40. Further, activation of C/EBPa by triptolide is dependent on upstream kinases ERK1/2 and Akt-GSK3b. This study provides mechanistic insights into the immunomodulatory capacity of triptolide and has strong implications for its therapeutic applications in autoimmune diseases. The Journal of Immunology, 2010, 184: 3866–3877.

riptolide, a diterpene triepoxide, is a biologicallyactive com- (12). It has also been shown that triptolide inhibits DC-mediated http://www.jimmunol.org/ ponent purified from the Chinese herbal plant Tripterygium chemoattraction of neutrophils and T cells through inhibiting Stat3 T wilfordii Hook F (TWHF). The therapeutic use of TWHF in phosphorylation and NF-kB activation (13). Zhu et al. showed that China as a natural medicine can be dated back several centuries ago triptolide prevented the differentiation of immature monocyte- (1). Currently, it is used for treatment of systemic lupus eryth- derived DC (MoDC) by inhibiting CD1a, CD40, CD80, CD86, and ematosus, rheumatoid arthritis, nephritis, Bechect’s disease, psoria- HLA-DR expression, and by reducing the capacity of MoDC to sis, atopic dermatitis, asthma, and very recently, in the prevention of stimulate lymphocyte proliferation in allogeneic MLR. However, transplant rejection (2–8). However, until recently, the mechanisms of expression of surface CD14 and phagocytic capacity of MoDC was the antineoplastic and anti-inflammatory effects of triptolide have not enhanced by triptolide (14). Therefore, the suppression of DC dif- been given sufficient attention. The immunosuppressive effect of ferentiation, maturation, and function of immature DCs by triptolide by guest on September 29, 2021 triptolide on T cells has been somewhat characterized. It inhibits T may explain some of its immunosuppressive properties. cell activation and cytokine gene transcription in T cells (9), and IL-12 is a heterodimeric cytokine composed of the p40 and p35 suppresses the expression of genes for transcription factors, signal chains. It is a pivotally important cytokine produced by APCs in host transduction pathway regulators, DNA binding protein, and MAPK in defense against intracellular pathogens and malignancies (15). IL-12 Jurkat cells (10). In addition, triptolide was reported to inhibit lym- and its close relative IL-23, which shares the p40 chain with IL-12 phocyte activation and T cell expression of IL-2 at the level of tran- plus its own unique chain p19, have been strongly implicated in the scription by inhibiting NF-kB transcriptional activation (11). pathogenesis of several types of autoimmune disorders, such as However, little is known about the effect of triptolide on accessory systemic lupus erythematosus, rheumatoid arthritis, multiple scle- cells, particularly the professional APCs, such as dendritic cells (DCs) rosis, inflammatory bowel disease, psoriasis, and atopic asthma (10, and macrophages. It has been suggested that DCs are a primary target 16–18). It has been previously demonstrated by several groups that of the immunosuppressive activity of triptolide. At high concen- triptolide could inhibit IL-12 expression (8, 19, 20), in DCs (19) and trations ($20 ng/ml) triptolide induces apoptosis of DCs through human monocytic cell line (THP-1) (20). sequential p38 MAP kinase phosphorylation and caspase 3 activation A recent study demonstrated that in (NZB 3 NZW)F1 mice, which develop spontaneous lupus nephritis (LN) by 28 wk, proteinuria and BUN levels were significantly reduced after treatment with either *Department of Microbiology and Immunology, Weill Medical College and †Program in triptolide or tripdiolide as compared with those treated with vehicle. Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sciences, Cor- There was no hypoalbuminemia or apparent evidence of LN in mice nell University, New York, NY 10065 treated with either of the two diterpenoids. At 44 wk of age, the survival Received for publication October 20, 2009. Accepted for publication January 22, 2010. rate in mice treated with vehicle was markedly lower than that in mice Address correspondence and reprint requests to Prof. Xiaojing Ma, 1300 York Ave- treated with either triptolide or tripdiolide. The mean level of anti- nue, Weill Medical College of Cornell University, New York, NY 10065. E-mail dsDNA Ab in mice treated with tripdiolide was lower than that in the address: [email protected] vehicle-treated mice on completion of the treatment course. Pro- Abbreviations used in this paper: C/EBPa, CCAAT/enhancer-binding protein-a; duction of TNF, IL-6, and MCP-1 by spleen cells was also decreased ChIP, chromatin immunoprecipitation; DC, dendritic cell; KO, knockout; LN, lupus nephritis; MoDC, monocyte-derived DC; MPM, mouse peritoneal macrophage; poly after diterpenoid therapy (2). These results support the conclusions that (I:C), polyinosinic-polycytidylic acid; PTP, protein tyrosine phosphatase; qPCR, real- the therapeutic value of extracts of TWHF in LN can be accounted for time quantitative PCR; ROS, reactive oxygen species; TWHF, Tripterygium wilfordii by the triptolide and tripdiolide contents, and that cooperation between Hook F; WT, wild type. components is not required for therapeutic benefit, and that either di- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 terpenoid could be effective therapy for LN. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903417 The Journal of Immunology 3867

Given the use of triptolide as an effective alternative therapeutic agent Quantitative real-time PCR forautoimmuneandinflammatorydiseasesinwhichAPC-derivedIL-12 Total RNAs were isolated using the RNeasy mini kit (Qiagen, Hilden, and IL-23 play strong roles, we investigated the molecular mechanism Germany) and reverse-transcribed into cDNA. Real-time PCR was per- whereby triptolide regulates expression of the IL-12/IL-23p40 gene in formedwithanABI7400SystemusingtheSYBRGREENPCRkit.Weselect inflammatory macrophages and MoDC. GAPDH as the endogenous control for the real-time PCR relative quantifi- cation analysis. The following primers were used for PCR amplification: 1) mouse IL-12p40,upper:59-GGAAGCACGGCAGCAGAATAAAT-39, lower: Materials and Methods 59-AACTTGAGGGAGAAGTAGGAATGG-39;2)mouse IL-12p35, upper: 59-CCCTTGCCCTCCTAAACCAC-39,lower:59-TAGTAGCCAGGCAAC- Cells TCTCG-39;3)immature mouse IL-12p40, upper: 59-GACACGCCTGAA- Human PBMCs were obtained from whole blood from disease-free vol- GAAGATGA-39,lower:59-TTGTGGAGCAGCAGATGTGA-39;4)imma- unteers by density gradient centrifugation with Ficoll (Invitrogen, Carlsbad, ture mouse IL-12p35,upper:59-TAGCCGCTCCTCACTCCTCT-39, lower: a CA). CD14+ monocytes were purified from fresh PBMCs with anti-CD14 59-TGACTGAAGCCTGCGATGAC-39;5)mouse C/EBP , upper: 59-AGCC- magnetic beads (Miltenyi Biotec, Auburn, CA), as recommended by the AAGAAGTCGGTGGACAAGAA-39,lower:59-GCGGTCATTGTCACTG- b manufacturer. Isolated monocytes were allowed to adhere to plastic by GTCAACTC-39;6)mouse C/EBP , upper: 59-GAAGTGGCCAACTTCTA- plating 106 cells per/ml in RPMI 1640 medium for 2 h. Adherent mono- CTACGAG-39,lower:59-AGAGGTCGGAGAGGAAGTCGTGGT-39;and7) d cytes were washed with RPMI 1640 medium and cultured for 6 d at 106 mouse C/EBP ,upper:59-GCCATGTACGACGACGAGAG-39,lower:59-GT- cells/ml in DC culture medium supplemented with 50 ng/ml of rGM-CSF TGAAGAGGTCGGCGAAGA-39. PCR cycling conditions were as follows: (R&D Systems, Minneapolis, MN) and 1000 U/ml of rIL-4 (R&D Sys- initial incubation step of 2 min at 50˚C, reverse transcription of 60 min at 60˚C tems). The murine macrophage-like cell line, RAW264.7, was obtained and 94˚C for 2 min, followed by 40 cycles of 15 s at 95˚C for denaturation and from American Type Culture Collection (Manassas, VA) and maintained in 2 min at 62˚C for annealing and extension. All experiments were performed RPMI 1640 containing 10% FCS, 2 mM L-glutamine, and penicillin/ three times independently. Downloaded from streptomycin. Peritoneal macrophages were isolated from C57BL/6J mice. Briefly, mice were injected i.p. with 2 ml 4% thioglycollate. After 3 d, Transfections peritoneal exudate cells were isolated by washing the peritoneal cavity Transient transfections were performed by electroporation as previously with ice-cold HBSS. Cells were incubated for 2 h, and adherent cells were described (22). Transfection efficiency was routinely monitored by b- used as peritoneal macrophages. galactosidase assay by cotransfection with 3 mg pCMV–b-galactosidase , Mice plasmid. Variability between samples was typically 10%. Lysates were

used for both luciferase and b-galactosidase assays. All experiments were http://www.jimmunol.org/ CCAAT/enhancer-binding protein-b deficient (C/EBPb2/2) mice were performed three times independently. obtained for this study by breeding heterozygous female mice with a tar- geted deletion in the gene of C/EBPb (C/EBPb+/2) with heterozygous EMSA b2/2 male mice. The generation of the C/EBP mice and their genetic Nuclear extraction, EMSA, and supershifts were performed as described a background was described previously by Screpanti et al. (21). C/EBP previously (22). Briefly, 5 mg nuclear protein were incubated for 20 min at a conditional knockout (KO) mice are homozygous for C/EBP floxed allele room temperature with 0.1 pmol IRDye-700 labeled–double-stranded oligo- and hemizygous for the Mx1-cre transgene. Mx1-Cre transgene expression nucleotide synthesized by IDT (Coralville, IA) in the presence of 2 mgpoly(dI: can be induced by administration of polyinosinic-polycytidylic acid [poly dC), and complexeswere resolvedon nondenaturing4.5% polyacrylamidegels (I:C)], leading to deletion of the floxed gene. For induction of Cre ex- that were scanned by Odyssey-infrared imaging system.Oligo used for EMSA: pression, 4–7-wk-old mice were injected i.p. with 250 mg poly(I:C) (Sigma-

59-TGTTTTCAATGTTGCAACAAGTCAGT-39. All experiments were per- by guest on September 29, 2021 Aldrich, St. Louis, MO) every other day for three doses. All mice were formed three times independently. housed in microisolator cages, were monitored daily for evidence of disease, and were sacrificed when moribund. Chromatin-immunoprecipitation assays Reagents and plasmids Chromatin-immunoprecipitation (ChIP) assay was performed as previously Recombinant human and murine IFN-g were purchased from Genzyme (Bos- described (24). The primers for C/EBPa and C/EBPb in the mouse IL-12p40 ton, MA). Recombinant human M-CSF was purchased from PeproTech (Rocky promoters: upper primer 59-GTCTCCAAGCACCTTGGCCATGAT-39;lower Hill, NJ). LPS from Escherichia coli 0127:B8 was from Sigma-Aldrich. Trip- primer 59-GCTGCTGTTGCTGGTACTGGAACT-39. All experiments were tolide (PG490) was from Biomol (Plymouth Meeting, PA). MAPK inhibitors performed three times independently. SB 202190, PD 98059, and JNK inhibitor II were from Calbiochem (Madison, SiRNA WI). Poly(I:C) was from InvivoGen (San Diego, CA). LiCl was from Sigma- Aldrich (Milwaukee, WI). Abs for C/EBPa,C/EBPb,C/EBPd,andMKP-1 To generate C/EBPa siRNA expression construct siRNA corresponding to were from Santa Cruz Biotechnologies (Santa Cruz, CA). Abs for phosphor-C/ the coding sequence of mouse C/EBPa gene (59-GCAAGAGCCGAGA- EBPa (Ser21), phosphor-C/EBPa (Thr222/226), PTEN, PP2A C subunit, TAAAGC-39) was cloned into the siRNA expression vector pSUPER.neo phosphor-ERK1/2, ERK1/2, phosphor-p38, p38, phosphor-JNK, JNK, phos- (OligoEngine, Seattle, WA) according to the manufacturer’s instructions. phor-MEK1/2, MEK1/2, phosphor-Akt, Akt, phosphor-GSK3b, and GSK3b Briefly, equimolar amounts of complementary sense and antisense strands were from Cell Signaling (Danvers, MA). were separately mixed, annealed, and slowly cooled to 10˚C in a 50 ml All deletions and mutations of human IL-12p40 promoter-luciferase reaction buffer (100 mM NaCl and 50 mM HEPES, pH 7.4). The annealed constructs have been described previously (22, 23). The wild-type (WT) C/ oligonucleotides were inserted into the BglII/HindIII sites of pSUPER.neo EBPa expression vector and mutant C/EBPa expression vectors (S21A, vector. RAW264.7 cells stably depleted of C/EBPa by C/EBPa siRNA was T222A/T226A, S192A, and S248A) were generously provided by Dr. established by transfecting the C/EBPa siRNA expression construct into MacDougald (University of Michigan Medical School, Ann Arbor, MI). The RAW264.7 cells by electroporation. After 24 h, the cells were cultured in dominant negative mutant of C/EBPa (3heptad-F) was generously provided 500 mg/ml G418, and expression of C/EBPa was analyzed in the selected by Dr. C. Vinson (National Cancer Institute, National Institutes of Health, cells. All experiments were performed three times independently. Bethesda, MD). The murine C/EBPa promoter construct was generated by PCR (sense primer: 59-TACGAGCTCCTATCGCTCTGGCCTGGAGAC- 39; antisense primer: 59-GTCAAGCTTCTCTGGAGGTGACTGCTCATC- Results 39) from mouse genome. The fragment was inserted into pGL3-basic vector. Suppression of p40 gene transcription in macrophages and DCs The construct was confirmed by sequence verified. All plasmids were puri- fied using the Qiagen Endotoxin-free kit (Qiagen, Valencia, CA). by triptolide Cytokine assays We first investigated whether triptolide could regulate IL-12/IL-23 production in LPS stimulated mouse peritoneal macrophage (MPM) Cytokine secretion was measured by ELISA, using appropriately diluted and human MoDCs. Fig. 1A and 1C show that triptolide suppressed culture supernatants. Human IL-12p40 and p70, and mouse IL-12p40, p70 were measured by the respective ELISA kits from BD Pharmingen (San Diego, IL-12p40 release from both MPM and MoDCs in a dose-dependent CA), mouse IL-23 was measured by an ELISA kit from Biolegend (San Diego, manner. No cytotoxic effect of triptolide was observed at these con- CA), in triplicates. All experiments were performed three times independently. centrations, measured by crystal violet staining (data not shown). 3868 TRIPTOLIDE REGULATES IL-12 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Effects of triptolide on IL-12 gene expression in LPS-treated MPM and MoDC (A–D)MPM(A, B), and MoDCs (C, D) were incubated for 15 h with different concentrations of triptolide and subsequently cultured for an additional 6 h in the presence (filled bars) or absence (open bars) of 1 mg/ml LPS. Mouse IL- 12p40 (A), mouse IL-12p70 (B), human IL-12p40 (C), human IL-12p70 (D), and mouse IL-23 (E) levels in the supernatants were measured by ELISA. Experiment shown is representative of three to four independent experiments with mean 6 SE. pp , 0.05. F–H, Total RNA was isolated from 1 3 106 MPM incubated with different concentrations of triptolide for 15 h with 1 mg/ml LPS (filled bars) or without (open bar) for an additional 3 h. Real-time PCR was performed to measure p40 (F), p35 (H) mRNA expression, and nascent transcripts for p40 (G). GAPDH mRNAwas analyzed as an internal control. The experiment shown is representative of two independent experiments.

Because the p35 and p40 subunits of IL-12 may be regulated in- Localization of the triptolide-responsive element in the human dependently, we determined whether triptolide has inhibitory effect p40 promoter on the production of the heterodimeric IL-12 or p70. Whereas p70 To further understand the transcriptional mechanism of the in- levels were markedlyreduced by triptolide inLPS-stimulated MoDCs hibition of triptolide on p40 expression we took an approach in D B (Fig. 1 ), the reduction was less pronounced in MPM (Fig. 1 ). which we sought to identify the promoter element(s) through which Compared with IL-12, IL-23 production by MPM was inhibited triptolide mediates its inhibitory effects on IL-12p40 transcription, significantly by triptolide at all doses (Fig. 1E). using a series of 59 deletion mutants of the p40 promoter-luciferase To further determine the molecular level at which triptolide exerts reporter constructs (Fig. 2A) transfected into the mouse macro- itsinhibitoryeffectonIL-12p40andp70 expression, weanalyzedthe steady-state mRNA and de novo synthesized transcripts of p40 and phage cell line RAW264.7 cells treated or not with triptolide. As p35 in triptolide-treated MPM by real-time quantitative PCR shown in Fig. 2B and 2C,59 deletion of the 3300-bp p40 promoter 2 (qPCR). Triptolide dose-dependently suppressed LPS-induced to 122 reduced the overall promoter activities but did not affect mRNA (Fig. 1F) and new transcripts (Fig. 1G) of the p40 gene, and the triptolide-mediated inhibition of the IFN-g/LPS-induced tran- p35 mRNA levels (Fig. 1H). These data indicate that triptolide scription. Deletion of the promoter to 2105, which eliminated the suppresses IL-12p40 at the transcriptional level in both MPM and NF-kB site, resulted in a significant loss of triptolide-induced in- MoDCs. In addition, the inhibitory effect of triptolide is likely in- hibition. This is consistent with previous finding that NF-kBis dependent of IL-10 because no change was observed in IL-10 pro- a target of triptolide (9). However, further deletion of the promoter duction in the presence or absence of triptolide (data not shown). We to 275 disrupting the functional composite C/EBP/AP-1 site (23) decided also to focus the study on MPM for simplicity. drastically reduced triptolide-mediated inhibition of the IL-12p40 The Journal of Immunology 3869 Downloaded from http://www.jimmunol.org/

FIGURE 2. Localization of the triptolide-responsive element in the human IL-12p40 promoter. A, Putative and verified transcription factor binding sites in the 2292/+108 p40 promoter region. The core binding sequences are in bold and uppercase, and positions are relative to the transcription initiation site (TIS), which is defined as 21. B, A series of 59deletion mutants of the full-length human IL-12p40 promoter-luciferase construct, which spans 3.3 kb upstream and 108-bp downstream of the TIS, were transfected transiently into RAW264.7. Cells were incubated for 15 h with (filled bars) or without 10 ng/ml triptolide (open bars), then stimulated with IFN-g (15 h), followed by LPS (7 h). Cell lysates were assayed for luciferase activity. Data are summaries of four separate ex- periments. C, Rate of triptolide-induced inhibition of IL-12p40 promoter activities shown in B, expressed as 1-(triptolide-treated/nontreated). Results represent by guest on September 29, 2021 three independent experiments showing mean 6 SE. D, Mutant promoter luciferase constructs of IL-12p40 promoter in the context of 2292/+108 were transfected into RAW264.7. Luciferase activity was measured from cell lysates after stimulation of RAW264.7 cells with IFN-g and LPS in the absence or presence of triptolide (15 h). E, Rate of triptolide-induced inhibition of IL-12p40 promoter activities shown in D. Results represent three independent ex- periments showing mean 6 SE. pp , 0.05. promoter activity, suggesting that this sequence element, in addi- We first performed EMSAs to examine physical DNA-protein tion to NF-kB, is a critical target in triptolide’s inhibitory activity interactions in MPM using the composite C/EBP/AP-1 element of on p40 expression. the p40 promoter as probe. Fig. 3A shows that two discernible Because of the reported role of the C/EBP/AP-1 site in the reg- binding activities were present constitutively (complexes I and II, ulation of the mouse IL-12p40 transcription (25), we sought to de- lane 3). LPS treatment resulted in a loss of complex II and in- termine whether it was important for triptolide-mediated inhibition tensification of complex I plus the appearance of three additional of the human IL-12p40 gene transcription. Base substitutions were fast-moving complexes (complex III–V, lane 1). Triptolide alone introduced into these two elements by site-directed mutagenesis in did not impact on the constitutive activities (lane 4). Triptolide the context of the 2292/2108 IL-12p40-luc construct, which re- treatment in the presence of LPS restored these activities to pre- tained full responsiveness to triptolide. Mutation of the C/EBP half LPS state (lane 2). Supershift experiment using Abs against the of the composite site resulted in a substantial reduction of inhibition three isoforms of C/EBP (Fig. 3B) revealed that complex II was of triptolide on the human IL-12p40 promoter activity, whereas the primarily composed of C/EBPa (lanes 2 and 6), whereas complex AP-1 site mutation did not (Fig. 2D,2E). Taken together, we con- III contained C/EBPb (lane 3), and complex I consisted of C/ clude that triptolide inhibitory effects on IL-12p40 promoter acti- EBPd (lane 8). Together, these data indicate that triptolide treat- vation are most likely mediated through the C/EBP element. ment of LPS-activated MPM likely leads to restoration of the binding activities to the pre-LPS state, that is, by altering the ratios Regulation of C/EBP binding to p40 promoter in vitro and in vivo of C/EBPa to C/EBPb and d. Next, we sought to further elucidate the molecular basis of the in- To demonstrate that triptolide also affects C/EBP binding activities volvementofthevariousmembersoftheC/EBPfamilyoftranscription in vivo, we performed ChIP assays. After LPS stimulation of MPM, factors in the inhibition of IL-12 p40 transcription by triptolide. Six there was a significant increase of C/EBPb binding, which was re- different members of C/EBP family (a, b, g, d, ε,andz)havebeen duced slightly in the presence of triptolide (Fig. 3C, upper), whereas isolated and characterized to date all sharing a strong homology in the the same treatment resulted in enhanced C/EBPa binding (Fig. 3C, C-terminal domain (26). The activity and/or expression level of three lower). These data further corroborate the results from the in vitro C/EBP members (a, b,andd) are regulated by a number of in- binding studies. flammatory agents, including LPS and a range of cytokines (27–29), To further establish whether the triptolide-induced change of which led us to focus mainly on these three members. C/EBP binding activities was caused by changes in C/EBP 3870 TRIPTOLIDE REGULATES IL-12 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Regulation of C/EBPa and b binding to IL-12p40 promoter by triptolide in vitro and in vivo. A, EMSA was performed to examine the DNA- protein interaction at the composite C/EBP/AP-1 site in the IL-12p40 promoter in vitro using nuclear extracts prepared from MPM after incubation withor without triptolide, and stimulation with LPS (3 h). The five discernible complexes are labeled I-V. B, Supershift EMSA was performed to identify the components of the C/EBP complexes. The nuclear extracts used in this procedure were from LPS-stimulated MPM treated with or without triptolide. C, Triptolide regulates C/EBP binding to the p40 promoter in vivo. After incubation with or without triptolide, and stimulation with LPS (3 h), MPM were crosslinked by treatment and lysed. Cell lysates were subjected to immunoprecipitation with control IgG or anti-C/EBPb (upper) or anti-C/ EBPa (lower). Input DNA and DNA recovered from the immunoprecipitation were amplified by real-time PCR with primers spanning the C/EBP/AP-1- binding site in the IL-12p40 promoter. D–F, Western blotting was performed to detect the endogenous C/EBPa (D), C/EBPb (E), and C/EBPd (F) ex- pression levels in 3 h LPS-stimulated MPM treated with or without triptolide, in the nucleus and cytoplasm. To aid in the appropriate identification of the various isoforms of endogenous C/EBP recombinant C/EBPs were loaded onto the same blots. Expression of a-tubulin was analyzed as a nuclear loading control whereas GAPDH expression was used as a loading control for whole cell lysate. pp , 0.05. expression, we analyzed C/EBPa and -b at the protein level in the the level of the most abundant C/EBPb isoform, LAP (Fig. 3E), nucleus and cytoplasm of MPM by Western blot. As revealed in whereas it did not impact on C/EBPd level (Fig. 3F). Fig. 3D, the 42 kDa and 30 kDa isoforms of C/EBPa, generated by Collectively, these data demonstrate that triptolide causes a shift alternative translational initiation, were present in the nucleus and in the binding activities of C/EBPa and b isoforms in inflammatory cytoplasm constitutively. LPS treatment caused a strong reduction macrophages by altering their respective nuclear levels, namely, of the level of this protein in both compartments, whereas trip- increasing C/EBPa, whereas decreasing C/EBPb. tolide caused a strong reversal of the reduction, particularly in the a nucleus. LPS stimulation induced nuclear levels of the three dif- C/EBP as a major physiological mediator in triptolide’s ferentially translated isoforms of C/EBPb, the 38 kDa full-length inhibition of IL-12p40 transcription protein, the functional 33 kDa LAP, and the dominant negative 20 To further demonstrate the important role of C/EBPa in the in- kDa LIP (30). Triptolide treatment led to an ∼50% reduction in hibition of IL12p40 transcription by triptolide we analyzed the The Journal of Immunology 3871 effect of triptolide on IL-12p40 production in C/EBPa KO mice. Regulation of transcription and phosphorylation of C/EBPa by C/EBPa conditional KO mice are homozygous for C/EBPa floxed triptolide allele and hemizygous for the Mx1-Cre transgene, whose ex- The above data suggested that triptolide may inhibit IL12p40 pression can be induced by administration of poly(I:C), leading transcription by increasing the protein level of C/EBPa in the to deletion of the floxed gene. Mx1-Cre expression was induced nucleus of MPM. To further understand the mechanism of this by i.p. injection of poly(I:C) for 1, 2, and 3 wk, respectively, effect, we analyzed the mRNA expression and transcription of C/ and IL-12p40 level in serum was measured by ELISA. Fig. 4A EBPa. Fig. 5A shows that LPS stimulation of MPM inhibited C/ shows that C/EBPa expression was strongly reduced 2 wk after EBPa mRNA level, whereas triptolide treatment increased the poly(I:C) injection in KO mice. There was an accompanying, level by 4-fold 1 h after LPS stimulation. significant reversal of the inhibition of p40 production by tripto- Next, we sought to determine whether C/EBPa was regulated by lide (Fig. 4B). Notably, IL-12p40 levels in both WT and KO mice triptolide at the level of transcription by using a mouse C/EBPa were decreased 2 wk after poly(I:C) injection. This may have been promoter-luciferase reporter construct, which spans 809-bp upstream caused by the toxicity of poly(I:C). Studies by others have found and 187-bp downstream of the transcription initiation site. The that 5 mg/kg poly(I:C) injection for up to 28 d can induce some proximal promoter region of the mouse C/EBPa gene has been clinical changes, including anemia, elevations of transaminases, characterized and DNA-protein interactions within the first 350-bp lactic dehydrogenase, and alkaline phosphatase, decreased clot- are essential for high levels of ubiquitous activity of the promoter (32). ting rate, and increased prothrombin time (31). In addition, we Studies have shown that the C/EBPa gene is autoactivated in a spe- found that loss of C/EBPb, compared with WT mice, did not in- cies-specific manner. Its promoter can be autoactivated in transfected fluence triptolide-induced inhibition of IL-12p40 production cells by expression plasmids that specify for C/EBPa or C/EBPb, Downloaded from (data not shown). which act via a C/EBP recognition sequence (32, 33). C/EBPa pro- To circumvent the toxicity problems caused by in vivo adminis- moter contains two potential binding sites for C/EBP: one between trationofpoly(I:C)weusedanexvivomethodforthedeletionoftheC/ nucleotides 2252 and 2239, the other betweennucleotides 2203 and EBPa gene. MPMs were isolated from WT and C/EBPa conditional 2176 (33). We transfected the C/EBPa luciferase construct into KO mice, and treated with different concentrations of poly(I:C) RAW264.7, followed by triptolide treatment and IFN-g/LPS stimu- in vitro for 24 h, followed by triptolide and LPS treatment. RT-PCR lation. As shown in Fig. 5B, in resting RAW264.7 cells, triptolide did http://www.jimmunol.org/ analysis of C/EBPa mRNA expression confirmed the significant not affect C/EBPa promoter activity. However, IFN-g/LPS stimula- deletion of the gene at this dose of poly(I:C) administration in KO tion resulted in some inhibition of the promoter activity, which was cells (Fig. 4C). As shown in Fig. 4D, the poly(I:C) treatment had no strongly reversed by triptolide. These data suggest that triptolide significant effect on IL-12p40 secretion in both WT and KO MPMs. could forcefully antagonize the negative signaling of LPS that leads to Although inhibition of p40 production by triptolide was not affected transcriptional inhibition of C/EBPa. Taken together, these results by the poly(I:C) treatment in WT cells, it was strongly reversed in C/ demonstrate that C/EBPa, not C/EBPb, plays a major role in tripto- EBPa KO MPM in a dose-dependent manner, reaching a highly lide-induced inhibition of IL-12p40 transcription. Triptolide can significant level at 10 mg/ml poly(I:C). regulate C/EBPa’s transcription in inflammatory macrophages. by guest on September 29, 2021

FIGURE 4. Effects of triptolide in C/EBPa knockout mice. A–C, Four- to 6-week-old WTand Mx1-cre/C/EBPa KO mice were injected with 250 mg poly(I:C) every other day for three doses in the first week. After 1–3 wk, in the treatment group, a dose of 1 mg/kg (body weight) of triptolide was administrated to mice by i. p. injection overnight, followed by stimulation of LPS (15 mg/kg) for 4 h. Proteins from splenocytes were analyzed for C/EBPa expression by Western blotting (A). Simultaneously, IL-12p40 levels in the serum were measured by ELISA (B). Each group had three mice. Results represent mean 6 SE of the three mice. C,MPM from WT and Mx1-cre/C/EBPa KO mice were treated with different concentrations of poly(I:C) for 24 h, incubated with triptolide (filled bars) or without (open bars), followed by stimulation of LPS for 6 h. Total RNAwas prepared, and analyzed for mRNA expression of C/EBPa and GAPDH by RT-PCR. Simultaneously, IL-12p40 levels in the supernatants were measured by ELISA (D). Results represent three independent experiments showing mean 6 SE. pp , 0.05. 3872 TRIPTOLIDE REGULATES IL-12 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Regulation of transcription and phosphorylation of C/EBPa by triptolide. A, MPM were incubated for 15 h with (dotted line) or without (solid line) 10 ng/ml triptolide and subsequently cultured for different times in the presence of 1 mg/ml LPS or absence (gray line and gray dotted line). B,A mouse C/EBPa promoter-luciferase construct, which spans 809-bp upstream and 187-bp downstream of the transcription initiation site, was transfected transiently into RAW264.7 cells. Cells were incubated for 15 h with (filled bars) or without 10 ng/ml triptolide (open bars), then stimulated with or without IFN-g (15 h), followed by LPS (7 h). Cell lysates were assayed for luciferase activity. Data are summaries of four separate experiments. C, Western blotting was performed to detect Ser21- and Thr222/226-phosphorylation of C/EBPa in MPM after incubation with or without triptolide, and stimulation with LPS (1 h). D, Stably expressed siRNA against C/EBPa efficiently inhibited expression of the endogenous C/EBPa at both mRNA (left) and protein (right) levels in RAW264.7 cells. E, Mouse C/EBPa promoter-luciferase construct was cotransfected with WT or various phosphorylation mutants of C/EBPa expression vector into RAW264.7 cells depleted of endogenous C/EBPa by stable siRNA expression. Cell lysates were assayed for luciferase activity 36 h after transfection. F, IL-12p40 luciferase reporter was cotransfected with WT or various phosphorylation mutants of C/EBPa expression vector into RAW264.7 cells depleted of endogenous C/EBPa. Luciferase activity was measured from cell lysates after stimulation of RAW264.7 cells with IFN-g and LPS in the presence or absence of triptolide (15 h). G, Western blot analysis of recombinant C/EBPa and phosphorylation mutants expressed in transfected RAW264.7 cells that had been depleted of the endogenous C/EBPa via siRNA, then stimulated with IFN-g/LPS in the presence of triptolide. Expression of GAPDH was analyzed as a loading control. All transfection results represent three independent experiments showing mean 6 SE. pp , 0.05. The Journal of Immunology 3873

Posttranslational modifications of C/EBPa, such as phosphorylation suggesting that C/EBPa’s ability to autoregulate is only dependent at different serine/threonine residues, have been found to be an im- on Thr222/Thr226. In contrast, for its ability to inhibit IL-12p40, portant mechanism of regulating its transcriptional activity (34, 35). We Ser21, and Thr222/Thr226 seemed to be important because their examined the potential effect of triptolide on this type of modification mutations individually and collectively reduced triptolide-induced of C/EBPa using Abs specific for phosphorylated C/EBPa at Ser21 inhibition of the p40 promoter activity (Fig. 5F). Western blotting and Thr222/Thr226. As shown in Fig. 5C, LPS induced phosphoryla- analysis of phosphorylation of C/EBPa protein expression at tion of C/EBPa at Ser21, and triptolide further enhanced this effect. At Ser21 and Thr222/Thr226 confirmed the effect of site-mutant Thr222/Thr226, LPS alone reduced the phosphorylation, whereas constructs of the gene (Fig. 5G). triptolide restored it to the control levels. The phosphorylation patterns In conclusion, triptolide induced inhibition of IL-12p40 tran- of C/EBPa were different from that of its expression (lower panels), scription is mediated by C/EBPa via both elevating the transcription suggesting that triptolide regulates both the expression and phosphor- factor’s expression and its serine/threonine phosphorylation. ylation of C/EBPa, both impacting the latter’s transcriptional activity. Next, we wanted to investigate the functional effects of the Regulation of both MAPK and PI3K-Akt-GSK pathways by phosphorylation of C/EBPa using different phosphorylation mu- triptolide tants of C/EBPa. To minimize the interference of the endogenous Previous studies by others have shown that in adipocytes and hep- C/EBPa in this cell line, we stably transfected a C/EBPa-specific atocytes C/EBPa is phosphorylated at Thr222/Thr226 by GSK-3b siRNA expression vector, which resulted in reduction of its target (36, 37), and in hematopoietic and hepatic cell lines Ser21 is phos- both at the mRNA and protein levels (Fig. 5D). We then tested the phorylated by ERK1/2 (38, 39). To explore the mechanism of trip- ability of the exogenously introduced C/EBPa to regulate the tolide-induced phosphorylation of C/EBPa, we examined the effect of Downloaded from cotransfected C/EBPa promoter, that is, its autoregulation as re- triptolide on these two C/EBPa-modifying kinases by assessing their ported by others (32, 33). Fig. 5E shows that WT C/EBPa in- respective phosphorylation/activation using phosphor-specific Abs. creased its own promoter activity ∼2-fold, which was consistent Fig. 6A shows that in MPM, ERK1/2 was activated rapidly inresponse with the previously published results. All other phosphorylation to LPS, with peak activity attained at ∼10minaftertheadditionof mutants, except Thr222A/Thr226A, acted similarly as the WT, LPS, followed by declines at 30 and 60 min. In contrast, ERk1/2 http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Regulation of MAPK and PI3K-AKT-GSK pathways by triptolide. A and B, Western blot analyses were performed to detect levels of phosphor-ERK1/2 (A) and phosphor-GSK-3b (B) in whole cell lysate prepared from MPM after incubation with or without triptolide, and stimulation with LPS. C, MPM were incubated for 15 h in the presence (filled bars) or absence (open bars) of triptolide, followed by 2 h treatment with p38 inhibitor (SB202190), ERK inhibitor (PD98059), and JNK inhibitor II, and subsequently cultured for an additional 6 h with LPS. Mouse IL-12p40 level in the supernatants was measured by ELISA. D, A similar protocol as that of C was used, except that cells were treated with the GSK-3b inhibitor LiCl or PD98059, or both. E, Same protocol was used as that of D. C/EBPa levels in whole cell lysate were determined by Western blotting. F, Same protocol as D was used. ChIP was performed to examine C/EBPa binding to the IL-12p40 promoter region in vivo. G–I, Western blot analyses were performed to detect phosphor-MEK1/2 (G), phosphor-AKT (H), and expression of MKP-1 and PTEN (I) in whole cell lysate. Results in C, D, and F represent three independent experiments showing mean 6 SE. pp , 0.05. 3874 TRIPTOLIDE REGULATES IL-12

phosphorylation stimulated by LPS was sustained at higher levels by degradation of the PtdIns(3,4,5)P3 generated by PI3K. PTEN in- triptolide during this period. Phosphorylation of GSK-3b on the in- hibits downstream functions mediated by the PI3K pathway, re- activating residue, Ser9, increased transiently after stimulation of sulting in the activation of Akt/protein kinase B, cell survival, and LPS, whereas triptolide strongly suppressed this phosphorylation cell proliferation (45, 46). In this study, we demonstrated that LPS (Fig. 6B). Because phosphorylation of Ser9 is inhibitory for GSK-3b alone did not induce significant PTEN expression in MPM, which activity, these results suggest that triptolide may increase the activities was in accord with previous studies (47). On triptolide treatment, of both ERK1/2 and GSK-3b in LPS-activated MPM. however, PTEN expression was elevated at 30 min after stimu- To further demonstrate the role of ERK1/2 in triptolide-induced in- lation with LPS (Fig. 6I, middle). hibition of IL-12p40, we treated MPM with small molecule inhibitors of p38 (SB 202190), ERK1/2 (PD 98059), and JNK (inhibitor II) 2 h prior to Discussion LPS stimulation with or without triptolide, and analyzed IL12p40 pro- In this study, we investigated the detailed molecular mechanism, duction. Fig. 6C shows that only PD 98059 rescued to a significant whereby triptolide inhibits the transcription of the p40 gene encoding degree the triptolide-induced inhibition of IL-12p40 in a dose-dependent the shared subunit of IL-12 and IL-23 in APCs. It led to the identifi- manner without cytotoxicity. These data indicate that ERK1/2, not p38 cation of C/EBPa as the major and direct mediator of triptolide’s in- and JNK, plays a role in mediating the IL-12–inhibiting activity of hibitory effect on p40 transcription. C/EBP-related proteins comprise triptolide. By a similar approach using a selective GSK-3b inhibitor, a family of basic region-leucine zipper transcription factors. These LiCl (40), we investigated the role of GSK-3b in triptolide’s inhibitory proteins dimerize through a leucine zipper and bind to a consensus function. As shown in Fig. 6D, LiCl treatment of MPM slightly resisted DNA motif through an adjacent basic region (48). The activity and triptolide’s inhibitory effect. However, when LiCl was combined with expression of three C/EBP members (a, b and d) are regulated by Downloaded from PD 98059, it strongly diminished triptolide’s effect on p40 production, a number of inflammatory signals, including LPS and a range of cy- by .50%. These data suggest that triptolide inhibits IL-12p40 tran- tokines (26–29, 49, 50). C/EBPb-deficient mice display impaired scription through ERK1/2- and GSK-3b–dependent pathways sepa- expression of TNF-a, serum amyloid A and P proteins, a1-acid gp, rately and interactively. and complement C3 component (28, 51, 52). However, induction of To further assess the role of ERK1/2 and GSK-3b in mediating the a number of cytokines, such as IL-12, IL-10, IFN-g, MIP-1a,andGM- 2/2 effects of triptolide on C/EBPa’s function, we treated MPM with CSF, in C/EBPb mice was comparable to that observed in WT http://www.jimmunol.org/ chemical inhibitors of the kinases, PD 98059 and LiCl, respectively, mice, with the exception of G-CSF (51). This may be due to functional and examined C/EBPa expression and in vivo binding of C/EBPa to compensation by other members that are expressed at normal levels, the p40 locus by ChIP analysis (Fig. 6E,6F). Inhibitors of GSK-3b for example, C/EBPd (48). These data are consistent with our results and ERK1/2 individually resulted in reduction of C/EBPa expres- (Fig 4) in that IL-12p40 expression was not impaired in C/EBPb2/2 sion and C/EBPa’s binding activity, whereas the combination of the MPM, nor was triptolide-induced inhibition of IL-12p40. two inhibitors caused a synergistically greater reduction. Most of the previously published work on C/EBPa focused on its Next,weevaluatedtheimpactoftriptolideontheupstreamkinases relationship with cellular proliferation and differentiation. C/EBPa is of ERK1/2 and GSK-3b: MEK1/2 and Akt1/2, respectively, by expressed at high levels in terminally differentiated cells and down- analyzing their phosphorylation. In LPS-stimulated MPM, phos- regulated during proliferation (53). C/EBPa is a strong inhibitor of by guest on September 29, 2021 phorylation of MEK1/2 was rapidly induced in 10 min, followed by cell proliferation when overexpressed in cultured cells (53, 54). In declines at 30 and 60 min. This pattern was very similar in triptolide- treated cells (Fig. 6G). On the other hand, phosphorylation of Akt was more sustained in LPS-stimulated MPM, but reduced overall by triptolide treatment (Fig. 6H). The lack of a triptolide-induced effect on the phosphorylation of MEK1/2 prompted us to examine the MAPK phosphatases (MKPs). MKPs are dual-specificity phosphatases that can dephosphorylate both phosphotyrosine and phosphothreonine residues on MAPKs (41). MKP-1, the archetypal member of this family, is widely studied and selectively inactivates all three MAPK families by de- phosphorylating them at catalytic tyrosine and threonine residues (42). We examined the effect of triptolide on LPS-induced MKP-1 expression by Western blotting. Fig. 6I (upper) shows that MKP-1 was strongly induced in MPM by LPS at ∼1 h, and triptolide treatment strongly decreased the expression. This is consistent with a previous study showing that triptolide blocked MKP-1 induction by LPS in macrophages (43). Triptolide had little effect on the ex- pression of PP2A (lower), which belongs to the same family of dual- specificity phosphatases as MKP-1. Because triptolide reduced phosphorylation of GSK-3b and Akt, and affected the phosphoinositol 3-kinase (PI3K)-Akt-GSK-3b pathway (Fig. 6B,6H), we sought to determine whether triptolide has an effect on phosphatase and tensin homolog deleted on chromosome ten (PTEN), one of the enzymes in the protein ty- FIGURE 7. Model for regulation of IL-12p40 production by triptolide. rosine phosphatases (PTPs) superfamily that negatively regulates Triptolide augments TLR4-mediated activation of ERK1/2 and GSK-3b by cell survival mediated by the PI3K-Akt pathway. PTEN acts as interfering with the expression of phosphatases MKP-1 and PTEN. ERK1/2 a phospholipid phosphatase dephosphorylating the PtdIns(3,4,5)P3 and GSK-3b enhance the phosphorylation of C/EBPa, and its expression and PtdIns(3,4)P2, resulting in PtdIns(4,5)P2 and PtdIns(4)P, re- because of autoregulation. C/EBPa functions as an inhibitor of p40 tran- spectively (44). PTEN modulates the PI3K pathway by catalyzing scription via a yet to be elucidated mechanism. The Journal of Immunology 3875 addition, C/EBPa-deficient mice have defective myeloid cell de- both. The additive effects of simultaneous chemical inhibition of both velopment, increased hematopoietic stem cell repopulating activity, ERK1/2 and GSK-3b (Fig. 6F) may be attributed to the independent and a significantly increased myeloblast population in the bone effects of targeting Ser21 and Thr222/226 of C/EBPa,respectively. marrow compartment. These phenotypes are probably caused by Control over kinase signaling networks is exerted at the level of a specific maturational block of myeloid precursors toward mature both phosphorylation and dephosphorylation, catalyzed by protein granulocyte and macrophage (55). The activity and/or mRNA and kinases and phosphatases, respectively (66). The phosphatases protein levels of various C/EBP genes are differentially modulated include tyrosine-specific phosphatases (PTPs), dual specificity in response to inflammatory stimuli and to recombinant cytokines. C/ (threonine/tyrosine), MKPs, and protein serine/threonine phos- EBPa is downregulated under these conditions. The mechanisms on phatases (PSPs). Our study showed that triptolide, in its inhibition the downregulation and the relationship between C/EBPa and ex- of p40 transcription, decreased LPS-induced MKP-1 and in- pression of cytokines have not been elucidated. Our study shows that creased PTEN (a member of PTPs), whereas having little effect on deletion of C/EBPa per se does not impact LPS-induced level of IL- PP2A (a member of PSPs) (Fig. 6K). The mechanism of the dif- 12p40 (Fig. 4B) or those of TNF-a and IL-10 (data not shown); ferential regulation of MKP-1 and PTEN expression by triptolide however, it strongly impeded triptolide-induced inhibition of p40 ex- is of great interest for further investigation. One possible mecha- pression, demonstrating the critical role of C/EBPa in this mechanism. nism is through induction of reactive oxygen species (ROS) in Furthermore, our data indicate that triptolide augments/increases macrophages. Phagocytic cells, such as macrophages, contain phosphorylation of LPS-induced C/EBPa at Ser21 and Thr222/ a multiprotein NADPH oxidase complex that produces a burst of Thr226 through ERK1/2 and GSK-3b, which potentiates C/EBPa’s ROS as part of the innate immune response to infection in re- transcriptional inhibition of p40 (Figs. 5C,6E). The MAPK cascade sponse to a wide variety of physiological stimuli, including LPS Downloaded from is very important, for many processes in immune responses (56). On (66, 67). There are reports that ROS might be involved in MKP-1 TLR4 engagement by LPS, MAPKs are activated by dual phos- induction in human epithelial and endothelial cells (68–70), and in phorylation at the tripepetide motif Thr-Xaa-Tyr, which leads to the decreased levels of PTEN in alveolar macrophages and increased production of inflammatory cytokines, such as IL-12, IL-1, and activity of AKT (71). Many studies of the relationship between TNF-a, etc. MAPK inactivation is carried out by a family of MAPK ROS and PTEN focused on inactivation of PTEN by ROS through

phosphatases (42, 57, 58). MKP-1 selectively inactivates all three posttranscriptional regulation (66, 72). Triptolide possesses http://www.jimmunol.org/ MAPK families by dephosphorylating them at catalytic tyrosine and a 9,11-epoxy-14b-hydroxy system, which may be involved in threonine residues (58). Activation of ERK1/2 has been shown to selective alkylation by nucleophilic groups, such as thiols, present play an important role in Th1/Th2 polarization and in regulating in key target enzymes (73); oxidation and reduction of thiol pro- cytokine production from APCs. ERK1-deficient mice display teins is thought to be the major mechanism by which reactive a bias toward Th1 type immune response. Consistent with this ob- oxidants integrate into cellular signal transduction pathway (74). servation, DCs from ERK1-deficient mice show enhanced IL-12p70 Onthebasisoftheseconsiderations,weproposeanewmodelonhow and reduced IL-10 secretion in response to TLR stimulation (59). triptolide inhibits IL-12/IL-23p40 in macrophages and DCs (Fig. 7). This suggests that ERK1/2 is an inhibitor of IL-12. In contrast, mice Our study has uncovered several novel targets of triptolide in MPM: deficient of the p38-activating kinase, MKK3, exhibit a selective C/EBPa, ERK1/2, GSK-3b, MKP-1, and PTEN, which may prove to by guest on September 29, 2021 defect in LPS-induced IL-12 production in macrophages (60). We have strong implications in guiding the therapeutic applications of found that triptolide-induced inhibition of p40 transcription was triptolide and its derivatives in the treatment of inflammatory auto- preceded by sustained phosphorylation of ERK1/2 (Fig. 6A), and immune diseases and cancer. that blocking the activity of ERK1/2 but not those of p38 and JNK can substantially rescued p40 production inhibited by triptolide Disclosures (Fig. 6C,6D). The authors have no financial conflicts of interest. Numerous studies have implicated the PI3K-AKT pathway in TLR signaling. GSK-3 can act as an intermediary in this pathway.GSK-3 is References constitutively active in resting cells but can be inactivated through 1. Kupchan, S. M., W. A. Court, R. G. Dailey, Jr., C. J. Gilmore, and R. F. Bryan. phosphorylation by AKT or other protein kinases (61, 62). PTEN is 1972. Triptolide and tripdiolide, novel antileukemic diterpenoid triepoxides from Tripterygium wilfordii. J. Am. Chem. Soc. 94: 7194–7195. a lipid phosphatase and dephosphorylates PIP3 to negatively regulate 2. Tao, X. L., Y. Sun, Y. Dong, Y. L. Xiao, D. W. Hu, Y. P. Shi, Q. L. Zhu, H. Dai, PI3K signaling (63). However, there are conflicting reports con- and N. Z. Zhang. 1989. A prospective, controlled, double-blind, cross-over study of tripterygium wilfodii hook F in treatment of rheumatoid arthritis. Chin. Med. cerning the physiological role of PI3K in the signaling pathway; the J. (Engl.) 102: 327–332. inconsistent results are partly due to the specificity of two inhibitors 3. Qin, W. Z., G. D. Zhu, S. M. Yang, K. Y. Han, and J. Wang. 1983. Clinical ob- (wortmannin and LY294002) in these investigations. Recently, some servations on Tripterygium wilfordii in treatment of 26 cases of discoid lupus er- ythematosus. J. Tradit. Chin. Med. 3: 131–132. research groups used genetic methods to knock out the regulatory 4. Jiang, X. 1994. Clinical observations on the use of the Chinese herb Triptery- subunit (p85) of PI3K, and found that TLR4-mediated induction of gium wilfordii Hook for the treatment of nephrotic syndrome. Pediatr. Nephrol. IL-12, IFN-g and TNF-a were increased in DCs (64, 65); and 8: 343–344. 5. Xu, W. Y., J. R. Zheng, and X. Y. Lu. 1985. Tripterygium in dermatologic therapy. knockdown of GSK-3b by siNA results in increased IL-10 production Int. J. Dermatol. 24: 152–157. and suppressed IL-12 synthesis (65). These results are in disagree- 6. Mao, H., X. R. Chen, Q. Yi, S. Y. Li, Z. L. Wang, and F. Y. Li. 2008. Myco- phenolate mofetil and triptolide alleviating airway inflammation in asthmatic ment with our data. We showed that triptolide strongly inhibited LPS- model mice partly by inhibiting bone marrow eosinophilopoiesis. Int. 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