FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2143719444> ?p ?o ?g. }

• W2143719444 endingPage "34" @default.
• W2143719444 startingPage "24" @default.
• W2143719444 abstract "Bryostatin-1 (Bryo-1), a natural macrocyclic lactone, is clinically used as an anti-cancer agent. In this study, we demonstrate for the first time that Bryo-1 acts as a Toll-like receptor 4 (TLR4) ligand. Interestingly, activation of bone marrow-derived dendritic cells (in vitro with Bryo-1) led to a TLR4-dependent biphasic activation of nuclear factor-κB (NF-κB) and the unique induction of cytokines (IL-5, IL-6, and IL-10) and chemokines, including RANTES (regulated on activation normal T cell expressed and secreted) and macrophage inflammatory protein 1α (MIP1-α). In addition, EMSA demonstrated that Bryo-1-mediated induction of RANTES was regulated by NF-κB and the interferon regulatory factors (IRF)-1, IRF-3, and IRF-7 to the RANTES independently of myeloid differentiation primary response gene-88 (MyD88). Bryo-1 was able to induce the transcriptional activation of IRF-3 through the TLR4/MD2-dependent pathway. In vivo administration of Bryo-1 triggered a TLR-4-dependent T helper cell 2 (Th2) cytokine response and expanded a subset of myeloid dendritic cells that expressed a CD11chighCD8α− CD11b+CD4+ phenotype. This study demonstrates that Bryo-1 can act as a TLR4 ligand and activate innate immunity. Moreover, the ability of Bryo-1 to trigger RANTES and MIP1-α suggests that Bryo-1 could potentially be used to prevent HIV-1 infection. Finally, induction of a Th2 response by Bryo-1 may help treat inflammatory diseases mediated by Th1 cells. Together, our studies have a major impact on the clinical use of Bryo-1 as an anti-cancer and immunopotentiating agent. Bryostatin-1 (Bryo-1), a natural macrocyclic lactone, is clinically used as an anti-cancer agent. In this study, we demonstrate for the first time that Bryo-1 acts as a Toll-like receptor 4 (TLR4) ligand. Interestingly, activation of bone marrow-derived dendritic cells (in vitro with Bryo-1) led to a TLR4-dependent biphasic activation of nuclear factor-κB (NF-κB) and the unique induction of cytokines (IL-5, IL-6, and IL-10) and chemokines, including RANTES (regulated on activation normal T cell expressed and secreted) and macrophage inflammatory protein 1α (MIP1-α). In addition, EMSA demonstrated that Bryo-1-mediated induction of RANTES was regulated by NF-κB and the interferon regulatory factors (IRF)-1, IRF-3, and IRF-7 to the RANTES independently of myeloid differentiation primary response gene-88 (MyD88). Bryo-1 was able to induce the transcriptional activation of IRF-3 through the TLR4/MD2-dependent pathway. In vivo administration of Bryo-1 triggered a TLR-4-dependent T helper cell 2 (Th2) cytokine response and expanded a subset of myeloid dendritic cells that expressed a CD11chighCD8α− CD11b+CD4+ phenotype. This study demonstrates that Bryo-1 can act as a TLR4 ligand and activate innate immunity. Moreover, the ability of Bryo-1 to trigger RANTES and MIP1-α suggests that Bryo-1 could potentially be used to prevent HIV-1 infection. Finally, induction of a Th2 response by Bryo-1 may help treat inflammatory diseases mediated by Th1 cells. Together, our studies have a major impact on the clinical use of Bryo-1 as an anti-cancer and immunopotentiating agent. Activation of the innate immune response is essential to control the initial stage of pathogen invasion and for the establishment of adaptive immunity (1Montoya C.J. Jie H.B. Al-Harthi L. Mulder C. Patiño P.J. Rugeles M.T. Krieg A.M. Landay A.L. Wilson S.B. J. Immunol. 2006; 177: 1028-1039Crossref PubMed Scopus (68) Google Scholar). Dendritic cells (DCs) 3The abbreviations used are: DCdendritic cellBMDCbone marrow dendritic cellPBpolymyxin B sulfateTLRToll-like receptorRANTESregulated on activation normal T cell expressed and secretedIRFinterferon regulatory factorTIRToll-IL-1 resistancePEphycoerythrinIRFinterferon regulatory factorALLN-acetyl-Leu-Leu-Nle-CHOKCkeratinocyte chemoattractantNBDNEMO binding domain. play a critical role in the balance between immunity and immunological tolerance (2Banchereau J. Briere F. Caux C. Davoust J. Lebecque S. Liu Y.J. Pulendran B. Palucka K. Annu. Rev. Immunol. 2000; 18: 767-811Crossref PubMed Scopus (5619) Google Scholar) due to their uniqueness as cells highly specialized in the uptake, transport, processing, and presentation of antigens (3Zenke M. Hieronymus T. Trends Immunol. 2006; 27: 140-145Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The discrimination process leading to cell immunity or tolerance is highly regulated by pattern recognition receptors, such as the Toll-like receptor (TLR) family, which recognize pathogen-associated molecular patterns (1Montoya C.J. Jie H.B. Al-Harthi L. Mulder C. Patiño P.J. Rugeles M.T. Krieg A.M. Landay A.L. Wilson S.B. J. Immunol. 2006; 177: 1028-1039Crossref PubMed Scopus (68) Google Scholar, 4Medzhitov R. Janeway Jr., C. Trends Microbiol. 2000; 8: 452-456Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). Immature DCs express many members of the TLR family (5Kaisho T. Akira S. Trends Immunol. 2001; 22: 78-83Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Recognition of pathogen-associated molecular pattern by DCs results in the activation of TLR family members, which in turn activate the NF-κB and MAPK signaling pathways and induce DC maturation (6O'Neill L.A. Trends Immunol. 2002; 23: 296-300Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 7Yan X. Xiu F. An H. Wang X. Wang J. Cao X. Life Sci. 2007; 80: 307-313Crossref PubMed Scopus (29) Google Scholar). Upon maturation, DCs acquire the ability to produce a wide range of cytokines/chemokines and promote the differentiation of various T helper cell phenotypes such as Th1, Th2, Th17, and Tregs, thereby controlling the activation of adaptive immune responses (6O'Neill L.A. Trends Immunol. 2002; 23: 296-300Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 8Wang Z.Y. Yang D. Chen Q. Leifer C.A. Segal D.M. Su S.B. Caspi R.R. Howard Z.O. Oppenheim J.J. Exp. Hematol. 2006; 34: 1115-1124Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). dendritic cell bone marrow dendritic cell polymyxin B sulfate Toll-like receptor regulated on activation normal T cell expressed and secreted interferon regulatory factor Toll-IL-1 resistance phycoerythrin interferon regulatory factor N-acetyl-Leu-Leu-Nle-CHO keratinocyte chemoattractant NEMO binding domain. The TLR family is the essential recognition and signaling component of the host defense (9Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2068) Google Scholar, 10Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4416) Google Scholar). DCs express TLR4, which mediates the recognition of Gram-negative bacterial LPS and several other pathogen-associated molecular patterns (11Kato H. Sato S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. Akira S. Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1104) Google Scholar). Toll signaling to NF-κB occurs from the conserved Toll-IL-1 resistance (TIR) domain of TLR, which triggers the recruitment of the TIR domain-containing adaptor protein MyD88 (9Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2068) Google Scholar). The recruitment of MyD88 to the activated TLR results in NF-κB activation via the MyD88 adaptor-like protein (Mal/TIRAP) (12Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Crossref PubMed Scopus (996) Google Scholar) and the IκB kinase complex (13Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4073) Google Scholar). Although most of the TLRs seem to be exclusively dependent on the expression of MyD88 for all their functions, TLR3 and TLR4 are unique in that they are capable of activating both MyD88-dependent and MyD88-independent responses (9Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2068) Google Scholar, 14Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nat. Immunol. 2003; 4: 161-167Crossref PubMed Scopus (1009) Google Scholar), through the TIR domain-containing adaptor-inducing IFN-β (TRIF) (15Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1018) Google Scholar). It has been shown that stimulation of DC maturation and the induction of type 1 interferon (IFN-β) is mediated by the MyD88-independent signaling pathway (14Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nat. Immunol. 2003; 4: 161-167Crossref PubMed Scopus (1009) Google Scholar, 15Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1018) Google Scholar). However, whereas all TLRs activate NF-κB, not all TLRs induce IFN-β (8Wang Z.Y. Yang D. Chen Q. Leifer C.A. Segal D.M. Su S.B. Caspi R.R. Howard Z.O. Oppenheim J.J. Exp. Hematol. 2006; 34: 1115-1124Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Therefore, TIR domain-containing adaptors such as MyD88, TRIF, and Mal/TIRAP play crucial roles in TLR-signaling pathways, because they provide specificity to the response generated by signaling through each TLR (16Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6669) Google Scholar). Bryostatin-1 (Bryo-1) is a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina (17Ramsdell J.S. Pettit G.R. Tashjian Jr., A.H. J. Biol. Chem. 1986; 261: 17073-17080Abstract Full Text PDF PubMed Google Scholar). The potent anti-proliferative effects and anti-neoplastic properties of Bryo-1 against various tumor cells have led to its use as a chemotherapeutic agent. Recently, Bryo-1 has received much attention because of its immunomodulatory properties, both in vitro and in vivo. Bryo-1 has been shown to be a potent activator of human macrophages (18Bosco M.C. Rottschafer S. Taylor L.S. Ortaldo J.R. Longo D.L. Espinoza-Delgado I. Blood. 1997; 89: 3402-3411Crossref PubMed Google Scholar) and also to induce the production of immunomodulatory cytokines-IL-1, IL-6, IL-8, and TNF-α (18Bosco M.C. Rottschafer S. Taylor L.S. Ortaldo J.R. Longo D.L. Espinoza-Delgado I. Blood. 1997; 89: 3402-3411Crossref PubMed Google Scholar). It is also known that Bryo-1 induces the proliferation and activation of B and T cells (19Scheid C. Prendiville J. Jayson G. Crowther D. Fox B. Pettit G.R. Stern P.L. Cancer Immunol. Immunother. 1994; 39: 223-230Crossref PubMed Scopus (45) Google Scholar, 20Hess A.D. Vogelsang G.B. Silanskis M. Friedman K.A. Beschorner W.E. Santos G.W. Transplant. Proc. 1988; 20: 487-492PubMed Google Scholar). Studies from our laboratory have shown that Bryo-1 enhances the maturation and antigen-presenting abilities of DCs in vitro (21Do Y. Mainali E. Nagarkatti P.S. Nagarkatti M. Cell. Immunol. 2004; 231: 8-13Crossref PubMed Scopus (5) Google Scholar). We have demonstrated that Bryo-1 alone or in combination with calcium ionophore could activate cord blood monocyte-derived DCs to express higher levels of MHC class II antigens, as well as the co-stimulatory molecules CD1a, CD80, CD83, and CD86. Furthermore, Bryo-1 and calcium ionophore-activated DCs were capable of inducing the proliferation of cord blood-derived alloreactive T cells and the production of IFN-γ (21Do Y. Mainali E. Nagarkatti P.S. Nagarkatti M. Cell. Immunol. 2004; 231: 8-13Crossref PubMed Scopus (5) Google Scholar). However, the molecular mechanism(s) by which Bryo-1 exerts its biological properties on DCs is not clearly understood. In this study, we investigated the involvement of TLR4 in Bryo-1-mediated effects in vitro and in vivo. In this study, we have identified some unique properties of Bryo-1, which demonstrate that it can act as a TLR-4 ligand, thereby activating the innate immunity. Our findings are important and have major translational impact because Bryo-1 is currently being tested in humans as an anti-cancer agent. Furthermore, the ability of Bryo-1 to induce CC-chemokines, RANTES and MIP1-α, may have a major application as an inhibitor of HIV infection. Moreover, the inability of Bryo-1 to activate inflammatory cytokines such as IL-12 in DCs and its property to promote a Th2 response may find its use to treat inflammatory diseases by facilitating Th1 to Th2 switch. Adult (6–8 weeks of age) female C57BL/6 (H-2b) (referred to as wild type (WT) and C57/10ScNJ (H-2b) (referred to as TLR4−/− or TLR4 KO) mice were purchased from NCI, National Institutes of Health, and The Jackson Laboratory, respectively. MyD88−/− mice were kindly provided by Dr. Wei Chao, Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, Boston. Mice were housed in polyethylene cages and given standard animal feed and water ad libitum. Mice were housed in rooms maintaining a temperature of 23 ± 1 °C and on a 12-h light/dark cycle. HEK293 cell lines stably expressing pcDNA3, TLR3, TLR4, or both TLR4 and MD2 were gifts from D. Golenbock (University of Massachusetts Medical School, Worcester). All cell lines were maintained in DMEM-supplemented media as described previously (11Kato H. Sato S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. Akira S. Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1104) Google Scholar). Bryostatin-1 (Biomol), LPS (Sigma), and polymyxin B sulfate (Invitrogen) were purchased and used in various in vivo and in vitro assays. The Gal4-IRF-3 and Gal4-luciferase reporter gene were a gift from T. Fujita (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). NF-κB luciferase construct ELAM was from D. Golenbock. IFNβ-RE-luciferase reporter gene was a gift from S. Kwok (Albert Einstein Medical Center, Philadelphia, PA). LPS derived from Escherichia coli strain 011:B4 and bryostatin-1 were purchased from Sigma and Biomol, respectively. Poly(IC) was obtained from Amersham Biosciences. ALL, MG132 (Calbiochem), and TAT-NBD (IKKγ NEMO binding domain) peptides were obtained from Alexis Biochemicals. Murine DCs were obtained from bone marrow cells by culturing with murine recombinant granulocyte macrophage colony-stimulating factor (GM-CSF; 5 ng/ml; Pharmingen) for 6 days, as described previously (22Do Y. Hegde V.L. Nagarkatti P.S. Nagarkatti M. Cancer Res. 2004; 64: 6756-6765Crossref PubMed Scopus (26) Google Scholar). Twenty four hours after Bryo-1 (75 μg/kg body weight, i.p.) injection, WT and TLR4−/−mice were sacrificed and spleens removed. The RBCs were lysed, and the cell numbers were adjusted to 1 × 106 cells/ml in RPMI 1640 medium supplemented with 10% FCS. The cells were labeled for various DC activation markers and analyzed for the different DC populations (myeloid, lymphoid, and plasmacytoid). Phenotypic analysis of DCs was carried out by double or triple staining with phycoerythrin (PE)-conjugated, allophycocyanin-conjugated, or fluorescein isothiocyanate (FITC)-conjugated mAbs following incubation with Fc-block (anti-CD16/CD32 mAb; Pharmingen) to avoid nonspecific binding. The following mAbs were used: FITC-anti-CD40, PE-anti-CD80, PE-anti-CD86, allophycocyanin-anti-CD11c, FITC-anti-CD11b, FITC-anti-B220, FITC-anti-CD4, and PE-anti-CD8α (Pharmingen). Cells were analyzed by flow cytometry (EPICS FC500; Coulter Electronics, Miami, FL). Various cytokines and chemokines were assayed in the serum and supernatants of BMDCs from WT (TLR4+/+) and TLR4−/− mice, treated with vehicle, LPS, or Bryo-1. DCs from WT and TLR4−/− mice were treated with Byro-1 (10 ng/ml) for 24 h in vitro, after which the cells were spun down and the supernatants collected. Supernatants from LPS (100 ng/ml) and vehicle-treated BMDCs were used as positive and negative controls. For serum samples, blood was drawn at 24 h after Bryo-1 (75 μg/kg body weight), and serum was separated. Vehicle treated and untreated mice were used as controls. The serum was assayed using the multiplex bead-based assays (Bio-Plex assay, Bio-Rad), designed to quantitate multiple cytokine analysis, as described previously (23Singh N.P. Hegde V.L. Hofseth L.J. Nagarkatti M. Nagarkatti P. Mol. Pharmacol. 2007; 72: 1508-1521Crossref PubMed Scopus (149) Google Scholar). We looked at a panel of 18 cytokines and chemokines, including IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12(p40), IL-12(p70), IL-17, G-CSF, GM-CSF, TNF-α, IFN-γ, KC, MIP1-α, and RANTES. The supernatants from the cultures were harvested and analyzed using ELISA kit for the production of RANTES (PeproTech Inc., NJ) and IFN-β (PBL Biomedical Laboratories, NJ). BMDCs from WT and TLR4 KO mice were obtained as described above and subsequently treated with either LPS (100 ng/ml) or Bryo-1 (10 ng/ml). At the indicated times, nuclear extracts were prepared as described previously (24Fiorentino L. Stehlik C. Oliveira V. Ariza M.E. Godzik A. Reed J.C. J. Biol. Chem. 2002; 277: 35333-35340Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and electrophoretic mobility shift analysis was performed probing for the DNA binding activity of NF-κB. Briefly, 5 μg of nuclear extract was incubated with 1 μg of poly(dI-dC)-poly(dI-dC) (Amersham Biosciences) for 10 min at room temperature. To this mixture, 2 × 104 cpm of a 32P-labeled oligonucleotide (NF-κB, 5′-ATGTGAGGGGACTTTCCCAGGC-3′, Santa Cruz Biotechnology; RANTES-ISRE-IRF-1-RE, 5′-ACACAAAATGGAAAACTGAAATCACCCTTGG-3′; and IFNβ-PRD III-I, 5′-GAAAACTGAAAGGGAGAAGTGAAAGTG-3′, Integrated DNA Technologies) was added (binding site is underlined) in a buffer consisting of 4 mm Tris-HCl, pH 7.9, 12 mm Hepes-KOH, 60 mm KCl, 12% glycerol, 0.5 mm EDTA, and 1 mm dithiothreitol and incubated for an additional 20 min. Complexes were resolved on a nondenaturing 5% polyacylamide gel and subsequently exposed on Hyperfilm (Amersham Biosciences). For supershifts, antibodies raised against specific subunits of NF-κB, p65, p50 (nuclear localization signal), c-Rel, and RelB, or the IRF family members, IRF-1, IRF-3 (H-246), and IRF7 (FL-425) (Santa Cruz Biotechnology), were preincubated for 10 min at room temperature before the addition of 1 mm phenylmethylsulfonyl fluoride and 1 μg of poly(dI-dC)-poly(dI-dC). HEK293 cells (1 × 106) were seeded into 6-well plates and transiently transfected 24 h later using Lipofectamine transfection reagent (Invitrogen), following the recommendations of the manufacturer. For NF-κB and IFNβ reporter assays, cells were transfected with 0.5 μg of either ELAM or IFN-β, respectively, and 0.1 μg of CMV-β-Gal reporter vectors. For IRF-3 reporter assay, HEK293 cells (4 × 104) were seeded into 96-well plates and transfected with 40 ng/well of the Gal4-luciferase reporter gene. At 36 h following transfection, cells were treated with Bryo-1 (50 ng/ml) and LPS (100 ng/ml) for 6 h or left untreated. Cell lysates were prepared, and reporter gene activities were measured using the Dual-Luciferase reporter system (Applied Biosystems). Data were normalized for transfection efficiency and expressed as the mean relative stimulation ± S.D. To detect expression of RANTES in DCs from Myd88−/− mice, we performed intracellular staining using Cytofix/CytopermTM fixation/permeabilization kit following the recommendations of the manufacturer (BD Biosciences). Western blotting was performed using antibodies against total IRF3 and phosphorylated IRF3 (P-IRF3 Ser-396; Santa Cruz Biotechnology) at a dilution of 1:1000 and β-actin at dilution of 1:5000. HRP-conjugated secondary Ab was used at 1:4000 dilution (Cell Signaling). In brief, lysates from vehicle- or Bryo-1-treated BMDCs were prepared, and protein concentration was measured using standard Bradford assay (Bio-Rad). The proteins fractionated in SDS-PAGE were transferred onto PVDF membranes using a dry blot apparatus (Bio-Rad). The membrane was first incubated in blocking buffer for 1 h at room temperature, followed by incubation in primary antibody at 4 °C overnight. The membrane was incubated for 1 h in HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA) in blocking buffer after washing three times (10–15 min) with washing buffer (PBS + 0.2% Tween 20). The membranes were incubated in developing solution (ECL Western blotting detection reagents, GE Healthcare), and signal was detected using ChemiDoc System (Bio-Rad). Densitometric analyses of the Western blots were performed using ChemiDoc software (Bio-Rad). The statistical comparisons between different study groups were carried out using Student's t test and GraphPad software and differences of p < 0.05 were considered to be significant. Each experiment was repeated at least three times. Earlier studies from our laboratory have shown that Bryo-1 is capable of inducing maturation of DCs (21Do Y. Mainali E. Nagarkatti P.S. Nagarkatti M. Cell. Immunol. 2004; 231: 8-13Crossref PubMed Scopus (5) Google Scholar). To determine the cytokine/chemokine profile induced by Bryo-1, immature BMDC from WT and TLR4−/− mice were treated with Bryo-1 or vehicle. Next, supernatants were evaluated for the presence of cytokines and chemokines by ELISA, as described under “Experimental Procedures.” Specifically, we studied cytokines and chemokines that are induced following activation of DCs through TLRs, including IL-1α, IFN-β, IFN-γ, IL-12, TNF-α, IL-6, MIP1-α, KC, and RANTES. We observed that activation of BMDCs with LPS from WT mice led to significant induction of IL-12 and IL-1α as well as low levels of IFN-β (Fig. 1), and furthermore, these cytokines were dramatically reduced in LPS-activated BMDCs from TLR4 KO mice. Interestingly, Bryo-1 activated BMDCs produced little or no IL-12 and IL-1α and low levels of IFN-β. Moreover, of all the cytokines and chemokines screened, Bryo-1 activated BMDCs from wild-type mice produced only MIP1-α and RANTES (also known as CCL5) (Fig. 1). It was noted that Bryo-1-induced production of IFN-β, MIP1-α, and RANTES in DCs was significantly higher in WT BMDCs when compared with BMDCs from TLR4−/− mice, suggesting that the induction of IFN-β, MIP1-α, and RANTES by Bryo-1 was also regulated, at least in part, through TLR4 activation. Because all TLR signaling pathways culminate in the activation of NF-κB transcription factor, which in turn regulates the induction of cytokines, we investigated whether Bryo-1-induced activation of DCs involved modulation of NF-κB binding activity. To explore this possibility, nuclear extracts from Bryo-1 (10 ng/ml) or LPS (100 ng/ml)-stimulated BMDCs were prepared, and the NF-κB binding activity was examined by EMSA analysis, using a specific 32P-labeled NF-κB oligonucleotide. As shown in Fig. 2A, Bryo-1 induced NF-κB activation at 2 and 4 h following treatment. We used LPS-treated BMDCs as a positive control. To further evaluate the composition of the NF-κB complexes induced by Bryo-1, supershift analysis using antibodies against the NF-κB members p50, p65, c-Rel, and RelB was performed. As shown in Fig. 2B, the majority of the NF-κB complexes was composed of a p50 homodimer after a 2-h treatment with Bryo-1 and of p50/c-Rel and p50/p65 heterodimers following a longer (4 h) incubation time with Bryo-1. To determine the binding specificity of these complexes, competition analysis using a mutated NF-κB oligonucleotide was performed. As shown in Fig. 2, C and D, binding of the nuclear complexes to the DNA probe was specific because Bryo-1-mediated stimulation of DNA-protein complexes was not blocked by excess of unlabeled mutated NF-κB oligonucleotide (9th lane). Competition assays using 100-fold molar excess of unlabeled DNA probe as specific competitor further demonstrated the specificity of the band corresponding to NF-κB (Fig. 2E, cold probe lane). To determine whether TLR4 was implicated in Bryo-1-mediated activation of NF-κB, we examined the ability of BMDC nuclear extracts from C57BL/6 and TLR4−/− mice to bind to the NF-κB probe in response to Bryo-1. Interestingly, we found that the NF-κB binding activity of nuclear extracts from TLR4−/− BMDCs was significantly reduced relative to that of wild-type cells suggesting that TLR4 was in fact involved in Bryo-1 signaling in BMDCs (Fig. 3). We next addressed the molecular mechanism(s) by which Bryo-1 triggers the production of RANTES by DCs. To determine whether the increased RANTES production induced by Bryo-1 is mediated by IRFs, the binding of nuclear extracts from Bryo-1 or vehicle-treated BMDCs to a 32P-labeled oligonucleotide containing the IRF-binding motif in the RANTES gene promoter was examined by EMSA analysis, as described previously (24Fiorentino L. Stehlik C. Oliveira V. Ariza M.E. Godzik A. Reed J.C. J. Biol. Chem. 2002; 277: 35333-35340Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). As in LPS-stimulated BMDCs, binding of nuclear extracts to the IRF motif of the RANTES promoter was observed in response to Bryo-1 treatment of BMDCs (Fig. 4A). Furthermore, supershift analysis of the IRF complexes, using antibodies against the IRF-1, IRF-3, and IRF-7 proteins, revealed IRF-1 as the protein bound to the RANTES promoter in both Bryo-1- and LPS-stimulated BMDCs (Fig. 4A). Interestingly, IRF-3 (Fig. 4A, 8th and 12th lanes) and IRF-7 (9th and 13th lanes) also caused a gel shift when compared with the binding activity induced by Bryo-1 in the absence of antibody (Fig. 4A, 6th and 10th lanes) at both time points (2 and 4 h) suggesting that IRF-1 and possibly IRF-3 and IRF-7 may be the modulators of Bryo-1-mediated RANTES gene expression. The promoter of the RANTES gene carries IRF and NF-κB cis-acting elements both of which may be critical for RANTES gene expression (25Hoshino K. Kaisho T. Iwabe T. Takeuchi O. Akira S. Int. Immunol. 2002; 14: 1225-1231Crossref PubMed Scopus (237) Google Scholar). To further examine the specificity of Bryo-1-mediated increase in NF-κB binding activity, a series of proteosome inhibitors were used. Briefly, DCs were preincubated for 2 h with the NF-κB inhibitors ALL (10 μm), MG132 (1 μm), TAT-NBD peptide (200 μm), or left untreated followed by Bryo-1 treatment for 4 h. DCs were harvested for nuclear extract preparation, and the supernatant was collected for cytokine analysis by ELISA, as described under “Experimental Procedures.” The nuclear extracts were examined for their ability to bind to a 32P-labeled oligonucleotide containing the NF-κB consensus sequence. In the presence of nuclear extracts from vehicle-treated BMDCs, constitutive NF-κB complex formation was observed (Fig. 5, 2nd lane). Bryo-1 treatment, however, further increased protein complex binding to the NF-κB site (Fig. 5, 4th lane). Preincubation of BMDCs with various NF-κB inhibitors resulted in a decreased DNA binding activity of nuclear extracts relative to that of Bryo-1 alone. Furthermore, the NF-κB inhibitors ALL and NBD peptide were more effective at reducing protein-DNA complex formation than MG132 under our experimental conditions. In addition, the supernatants were also collected and analyzed for the presence of RANTES by ELISA (Fig. 5B). It was noted that Bryo-1-mediated induction of the NF-κB binding activity was considerably decreased in the presence of the proteosome inhibitors, ALL, TAT-NBD peptide, and to a lesser extent MG132. In addition, preincubation of BMDCs with the NF-κB inhibitors resulted in a decrease in the amount of RANTES chemokine released into the medium (Fig. 5B), suggesting that NF-κB was involved in the production of RANTES. Altogether, these data indicated that NF-κB and IRF-1 may be involved in the induction of RANTES in BMDCs in response to Bryo-1 stimulation. TLR4 activates both the MyD88- and the TRIF-dependent pathways. To investigate further the role of MyD88, immature BMDCs from WT and MyD88-deficient mice were cultured with Bryo-1 and analyzed for intracellular RANTES induction. The data indicated that BMDCs from MyD88 KO mice produced significant levels of RANTES comparable with the WT mice thereby suggesting that Bryo-1-induced RANTES production was independent of MyD88 (Fig. 6). These data were also consistent with the above observation that RANTES induction by Bryo-1 involved IRFs whose activation is independent of MyD88. There is increasing evidence that supports a key role for DCs in the production of type I interferons and the regulation of innate and adaptive immune responses (26Honda K. Yanai H. Takaoka A. Taniguchi T. Int. Immunol. 2005; 17: 1367-1378Crossref PubMed Scopus (284) Google Scholar, 27Tailor P. Tamura T. Ozato K. Cell Res. 2006; 16: 134-140Crossref PubMed Scopus (126) Google Scholar). We noted that activation of DCs with Bryo-1 failed to induce a majority of the cytokines characteristic of DCs except low levels of IFN-β. To further corroborate the role played by TLR-4 in the induction of IFN-β by Bryo-1, EMSA analysis of nuclear extracts from Bryo-1 (10 ng/ml) or LPS (100 ng/ml) stimulated WT or TLR4−/− BMDCs using a specific 32P-labeled DNA oligonucleotide containing the positive regulatory domains (PRDI-III) of the IFN-β gene promoter was performed. The data demonstrated that although Bryo-1 induced the binding of nuclear complexes to the IFN-β promoter DNA probe in the WT cells, Bryo-1-mediated stimulation of DNA-protein complex formation was significantly reduced and delayed in TLR4−/− BMDC (Fig. 7). These data suggested that TLR-4 was involved in the induction of IFN-β by Bryo-1, consistent with the observation that TLR4-deficient BMDC" @default.
• W2143719444 created "2016-06-24" @default.
• W2143719444 creator A5024920015 @default.
• W2143719444 creator A5036897140 @default.
• W2143719444 creator A5077284359 @default.
• W2143719444 creator A5077984302 @default.
• W2143719444 creator A5091773676 @default.
• W2143719444 date "2011-01-01" @default.
• W2143719444 modified "2023-09-30" @default.
• W2143719444 title "Bryostatin-1, a Naturally Occurring Antineoplastic Agent, Acts as a Toll-like Receptor 4 (TLR-4) Ligand and Induces Unique Cytokines and Chemokines in Dendritic Cells" @default.
• W2143719444 cites W145541864 @default.
• W2143719444 cites W1483162601 @default.
• W2143719444 cites W1485705546 @default.
• W2143719444 cites W1506899062 @default.
• W2143719444 cites W1547225947 @default.
• W2143719444 cites W1550510823 @default.
• W2143719444 cites W1966772446 @default.
• W2143719444 cites W1972922050 @default.
• W2143719444 cites W1983733845 @default.
• W2143719444 cites W1984811742 @default.
• W2143719444 cites W1990698093 @default.
• W2143719444 cites W1994718301 @default.
• W2143719444 cites W1996339149 @default.
• W2143719444 cites W1997719573 @default.
• W2143719444 cites W1998296865 @default.
• W2143719444 cites W2000175632 @default.
• W2143719444 cites W2004465286 @default.
• W2143719444 cites W2012054319 @default.
• W2143719444 cites W2013510722 @default.
• W2143719444 cites W2013601771 @default.
• W2143719444 cites W2018325317 @default.
• W2143719444 cites W2024445068 @default.
• W2143719444 cites W2031760220 @default.
• W2143719444 cites W2034038466 @default.
• W2143719444 cites W2036229725 @default.
• W2143719444 cites W2037368492 @default.
• W2143719444 cites W2040777447 @default.
• W2143719444 cites W2049651007 @default.
• W2143719444 cites W2049874360 @default.
• W2143719444 cites W2050133819 @default.
• W2143719444 cites W2062372651 @default.
• W2143719444 cites W2069873423 @default.
• W2143719444 cites W2077300798 @default.
• W2143719444 cites W2087717265 @default.
• W2143719444 cites W2089959931 @default.
• W2143719444 cites W2097747132 @default.
• W2143719444 cites W2107524856 @default.
• W2143719444 cites W2111220263 @default.
• W2143719444 cites W2113553995 @default.
• W2143719444 cites W2113948841 @default.
• W2143719444 cites W2121049876 @default.
• W2143719444 cites W2121284065 @default.
• W2143719444 cites W2130421043 @default.
• W2143719444 cites W2138677670 @default.
• W2143719444 cites W2140879800 @default.
• W2143719444 cites W2144787399 @default.
• W2143719444 cites W2147921405 @default.
• W2143719444 cites W2148387062 @default.
• W2143719444 doi "https://doi.org/10.1074/jbc.m110.135921" @default.
• W2143719444 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3012980" @default.
• W2143719444 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21036898" @default.
• W2143719444 hasPublicationYear "2011" @default.
• W2143719444 type Work @default.
• W2143719444 sameAs 2143719444 @default.
• W2143719444 citedByCount "31" @default.
• W2143719444 countsByYear W21437194442012 @default.
• W2143719444 countsByYear W21437194442013 @default.
• W2143719444 countsByYear W21437194442014 @default.
• W2143719444 countsByYear W21437194442015 @default.
• W2143719444 countsByYear W21437194442016 @default.
• W2143719444 countsByYear W21437194442017 @default.
• W2143719444 countsByYear W21437194442018 @default.
• W2143719444 countsByYear W21437194442019 @default.
• W2143719444 countsByYear W21437194442020 @default.
• W2143719444 countsByYear W21437194442021 @default.
• W2143719444 countsByYear W21437194442022 @default.
• W2143719444 crossrefType "journal-article" @default.
• W2143719444 hasAuthorship W2143719444A5024920015 @default.
• W2143719444 hasAuthorship W2143719444A5036897140 @default.
• W2143719444 hasAuthorship W2143719444A5077284359 @default.
• W2143719444 hasAuthorship W2143719444A5077984302 @default.
• W2143719444 hasAuthorship W2143719444A5091773676 @default.
• W2143719444 hasBestOaLocation W21437194441 @default.
• W2143719444 hasConcept C116569031 @default.
• W2143719444 hasConcept C13373296 @default.
• W2143719444 hasConcept C136449434 @default.
• W2143719444 hasConcept C170493617 @default.
• W2143719444 hasConcept C185592680 @default.
• W2143719444 hasConcept C203014093 @default.
• W2143719444 hasConcept C2776709828 @default.
• W2143719444 hasConcept C2778025104 @default.
• W2143719444 hasConcept C55493867 @default.
• W2143719444 hasConcept C86803240 @default.
• W2143719444 hasConcept C95444343 @default.
• W2143719444 hasConceptScore W2143719444C116569031 @default.
• W2143719444 hasConceptScore W2143719444C13373296 @default.
• W2143719444 hasConceptScore W2143719444C136449434 @default.
• W2143719444 hasConceptScore W2143719444C170493617 @default.

• next