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- W2123117008 abstract "Mycobacterium tuberculosis lipomannans (LMs) modulate the host innate immune response. The total fraction of Mycobacterium bovis BCG LM was shown both to induce macrophage activation and pro-inflammatory cytokines through Toll-like receptor 2 (TLR2) and to inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages through a TLR2-independent pathway. The pro-inflammatory activity was attributed to tri- and tetra-acylated forms of BCG LM but not the mono- and di-acylated ones. Here, we further characterize the negative activities of M. bovis BCG LM on primary murine macrophage activation. We show that di-acylated LMs exhibit a potent inhibitory effect on cytokine and NO secretion by LPS-activated macrophages. The inhibitory activity of mycobacterial mannose-capped lipoarabino-mannans on human phagocytes was previously attributed to their binding to the C-type lectins mannose receptor or specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). However, we found that di-acylated LM inhibition of LPS-induced tumor necrosis factor secretion by murine macrophages was independent of TLR2, mannose receptor, or the murine ortholog SIGNR1. We further determined that tri-acyl-LM, an agonist of TLR2/TLR1, promoted interleukin-12 p40 and NO secretion through the adaptor proteins MyD88 and TIRAP, whereas the fraction containing tetra-acylated LM activated macrophages in a MyD88-dependent fashion, mostly through TLR4. TLR4-dependent pro-inflammatory activity was also seen with M. tuberculosis LM, composed mostly of tri-acylated LM, suggesting that acylation degree per se might not be sufficient to determine TLR2 versus TLR4 usage. Therefore, LM acylation pattern determines the anti-inflammatory versus pro-inflammatory effects of LM through different pattern recognition receptors or signaling pathways and may represent an additional mean of regulating the host innate immunity by mycobacteria. Mycobacterium tuberculosis lipomannans (LMs) modulate the host innate immune response. The total fraction of Mycobacterium bovis BCG LM was shown both to induce macrophage activation and pro-inflammatory cytokines through Toll-like receptor 2 (TLR2) and to inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages through a TLR2-independent pathway. The pro-inflammatory activity was attributed to tri- and tetra-acylated forms of BCG LM but not the mono- and di-acylated ones. Here, we further characterize the negative activities of M. bovis BCG LM on primary murine macrophage activation. We show that di-acylated LMs exhibit a potent inhibitory effect on cytokine and NO secretion by LPS-activated macrophages. The inhibitory activity of mycobacterial mannose-capped lipoarabino-mannans on human phagocytes was previously attributed to their binding to the C-type lectins mannose receptor or specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). However, we found that di-acylated LM inhibition of LPS-induced tumor necrosis factor secretion by murine macrophages was independent of TLR2, mannose receptor, or the murine ortholog SIGNR1. We further determined that tri-acyl-LM, an agonist of TLR2/TLR1, promoted interleukin-12 p40 and NO secretion through the adaptor proteins MyD88 and TIRAP, whereas the fraction containing tetra-acylated LM activated macrophages in a MyD88-dependent fashion, mostly through TLR4. TLR4-dependent pro-inflammatory activity was also seen with M. tuberculosis LM, composed mostly of tri-acylated LM, suggesting that acylation degree per se might not be sufficient to determine TLR2 versus TLR4 usage. Therefore, LM acylation pattern determines the anti-inflammatory versus pro-inflammatory effects of LM through different pattern recognition receptors or signaling pathways and may represent an additional mean of regulating the host innate immunity by mycobacteria. Control of Mycobacterium tuberculosis infection involves both phagocytes and T cell-mediated innate and adaptive immune responses (for review, see Refs. 1Flynn J.L. Chan J. Annu. Rev. Immunol. 2001; 19: 93-129Crossref PubMed Scopus (1734) Google Scholar, 2Cooper A.M. Kipnis A. Turner J. Magram J. Ferrante J. Orme I.M. J. Immunol. 2002; 168: 1322-1327Crossref PubMed Scopus (265) Google Scholar, 3Ehlers S. Holscher C. Scheu S. Tertilt C. Hehlgans T. Suwinski J. Endres R. Pfeffer K. J. Immunol. 2003; 170: 5210-5218Crossref PubMed Scopus (121) Google Scholar), and interactions between the bacillus and host phagocytes, macrophages and dendritic cells, are central to both immunity to M. tuberculosis and tuberculosis pathogenesis. In the lungs alveolar macrophages are primary host cells for M. tuberculosis, which has evolved mechanisms to persist and multiply within these cells. Dendritic cells are critical to carry mycobacterial antigens from the infection site to the draining lymph nodes and establish an efficacious T cell-mediated immune response. In addition, macrophages and dendritic cells participate in modulation of the innate immune response by secreting cytokines after recognition of microbial motives through various pattern recognition receptors. Cytokines such as TNF 2The abbreviations used are: LAMlipoarabinomannanDC-SIGNdendritic cell-specific intracellular adhesion molecule-3 grabbing nonintegrinLMlipomannanManLAMmannose-capped LAMMyD88myeloid differentiation protein 88TLRToll-like receptorPIMphosphatidyl-myo-inositol mannosideLPSlipopolysaccharideILinterleukinMALDI-MSmatrix-assisted laser desorption ionization-mass spectroscopy. are an integral part of the pathological process, with induction of cachexia and necrosis, but TNF is also an essential mediator for granuloma formation and containment of M. tuberculosis infection. Similarly IL-12, a cytokine that polarizes T lymphocytes toward a protective interferon (IFN)-γ-secreting type 1 profile, IFNγ, but also IL-1, IL-18, IL-23, lymphotoxin α (LTα), LTβ, and nitric oxide are required for host defense as demonstrated both in murine experimental tuberculosis models (1Flynn J.L. Chan J. Annu. Rev. Immunol. 2001; 19: 93-129Crossref PubMed Scopus (1734) Google Scholar, 2Cooper A.M. Kipnis A. Turner J. Magram J. Ferrante J. Orme I.M. J. Immunol. 2002; 168: 1322-1327Crossref PubMed Scopus (265) Google Scholar, 3Ehlers S. Holscher C. Scheu S. Tertilt C. Hehlgans T. Suwinski J. Endres R. Pfeffer K. J. Immunol. 2003; 170: 5210-5218Crossref PubMed Scopus (121) Google Scholar, 77Fremond C.M. Togbe D. Doz E. Rose S. Vasseur V. Maillet I. Jacobs M. Ryffel B. Quesniaux V.F. J. Immunol. 2007; 179: 1178-1189Crossref PubMed Scopus (269) Google Scholar) and in some clinical situations (4Jouanguy E. Lamhamedi-Cherradi S. Altare F. Fondaneche M.C. Tuerlinckx D. Blanche S. Emile J.F. Gaillard J.L. Schreiber R. Levin M. Fischer A. Hivroz C. Casanova J.L. J. Clin. Investig. 1997; 100: 2658-2664Crossref PubMed Scopus (327) Google Scholar, 5Altare F. Durandy A. Lammas D. Emile J.F. Lamhamedi S. Le Deist F. Drysdale P. Jouanguy E. Doffinger R. Bernaudin F. Jeppsson O. Gollob J.A. Meinl E. Segal A.W. 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Med. 2001; 345: 1098-1104Crossref PubMed Scopus (3269) Google Scholar, 10Mohan A.K. Cote T.R. Siegel J.N. Braun M.M. Curr. Opin. Rheumatol. 2003; 15: 179-184Crossref PubMed Scopus (67) Google Scholar). Phagocytes also produce immuno-modulatory cytokines such as IL-10 and transforming growth factor-β that dampen the immune response and inflammation. Tuberculosis protection versus pathogenesis, thus, likely relies on a fine equilibrium between pro- and anti-inflammatory cytokines. How M. tuberculosis interferes with these inflammatory and immuno-modulatory networks is still not fully understood. A better comprehension of the molecular mechanisms by which the tubercle modulates such immune responses should help in the design of new strategies to prevent or treat tuberculosis primary infection or reactivation. lipoarabinomannan dendritic cell-specific intracellular adhesion molecule-3 grabbing nonintegrin lipomannan mannose-capped LAM myeloid differentiation protein 88 Toll-like receptor phosphatidyl-myo-inositol mannoside lipopolysaccharide interleukin matrix-assisted laser desorption ionization-mass spectroscopy. Macrophages and dendritic cells recognize mycobacterial structural motives through various pattern recognition receptors, including Toll-like receptors (TLRs) (11Brightbill H.D. Libraty D.H. Krutzik S.R. Yang R.B. Belisle J.T. Bleharski J.R. Maitland M. Norgard M.V. Plevy S.E. Smale S.T. Brennan P.J. Bloom B.R. Godowski P.J. Modlin R.L. Science. 1999; 285: 732-736Crossref PubMed Scopus (1408) Google Scholar, 12Heldwein K.A. Fenton M.J. 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Neyrolles O. J. Biol. Chem. 2003; 278: 5513-5516Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 17Pitarque S. Herrmann J.L. Duteyrat J.L. Jackson M. Stewart G.R. Lecointe F. Payre B. Schwartz O. Young D.B. Marchal G. Lagrange P.H. Puzo G. Gicquel B. Nigou J. Neyrolles O. Biochem. J. 2005; 392: 615-624Crossref PubMed Scopus (89) Google Scholar, 18Torrelles J.B. Azad A.K. Schlesinger L.S. J. Immunol. 2006; 177: 1805-1816Crossref PubMed Scopus (150) Google Scholar). DC-SIGN is also expressed by alveolar macrophages in tuberculosis patients (19Tailleux L. Pham-Thi N. Bergeron-Lafaurie A. Herrmann J.L. Charles P. Schwartz O. Scheinmann P. Lagrange P.H. de Blic J. Tazi A. Gicquel B. Neyrolles O. PloS. Med. 2005; 2: e381Crossref PubMed Scopus (139) Google Scholar). Interactions between M. tuberculosis and DC-SIGN on human dendritic cells lead to secretion of the anti-inflammatory cytokine IL-10 and partial deactivation of the cells (20Geijtenbeek T.B. Van Vliet S.J. Koppel E.A. Sanchez-Hernandez M. Vandenbroucke-Grauls C.M. Appelmelk B. Van Kooyk Y. J. Exp. Med. 2003; 197: 7-17Crossref PubMed Scopus (898) Google Scholar). Mycobacterial lipoglycans such as ManLAM and LM may contribute to modulate the regulation of macrophage and dendritic cell activation and, thus, control the inflammatory response. Mannose receptor and more recently DC-SIGN have been proposed to mediate the inhibition by ManLAM of LPS-induced IL-12 production in dendritic cells (20Geijtenbeek T.B. Van Vliet S.J. Koppel E.A. Sanchez-Hernandez M. Vandenbroucke-Grauls C.M. Appelmelk B. Van Kooyk Y. J. Exp. Med. 2003; 197: 7-17Crossref PubMed Scopus (898) Google Scholar, 21Nigou J. Zelle-Rieser C. Gilleron M. Thurnher M. Puzo G. J. Immunol. 2001; 166: 7477-7485Crossref PubMed Scopus (353) Google Scholar). We showed previously that mycobacterial LMs have a dual potential for pro-inflammatory and anti-inflammatory effects. The stimulatory effect of LM on TNF and IL-12 production was mediated by TLR2 and MyD88, whereas their inhibitory effect on LPS-induced TNF, IL-12, and NO production was TLR-independent (22Quesniaux V.J. Nicolle D.M. Torres D. Kremer L. Guerardel Y. Nigou J. Puzo G. Erard F. Ryffel B. J. Immunol. 2004; 172: 4425-4434Crossref PubMed Scopus (211) Google Scholar). These different studies led to the interesting hypothesis that tuberculosis protection versus pathogenesis may rely, at least in part, on the balance between TLRs versus C-type lectin signaling induced by mycobacterial motives in phagocytes (23Neyrolles O. Gicquel B. Quintana-Murci L. Trends Microbiol. 2006; 14: 383-387Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 24Kaufmann S.H. Schaible U.E. J. Exp. Med. 2003; 197: 1-5Crossref PubMed Scopus (63) Google Scholar). To address this point, we analyzed the molecular basis of mycobacterial LM stimulatory and inhibitory properties using a combination of purified LM acyl forms with genetically engineered macrophages lacking specific TLRs, TLR adaptors, or C-type lectins. TLR2 seems crucially involved in the innate response to mycobacteria since TLR2-dependent cell activation by mycobacterial cell wall lipoglycans such as phosphoinositol-capped LAM but also LM, PIM2, and PIM6 or the 19-kDa mycobacterial lipoprotein have been described (22Quesniaux V.J. Nicolle D.M. Torres D. Kremer L. Guerardel Y. Nigou J. Puzo G. Erard F. Ryffel B. J. Immunol. 2004; 172: 4425-4434Crossref PubMed Scopus (211) Google Scholar, 25Means T.K. Lien E. Yoshimura A. Wang S. Golenbock D.T. Fenton M.J. J. Immunol. 1999; 163: 6748-6755Crossref PubMed Google Scholar, 26Jones B.W. Means T.K. Heldwein K.A. Keen M.A. Hill P.J. Belisle J.T. Fenton M.J. J. Leukocyte Biol. 2001; 69: 1036-1044PubMed Google Scholar, 27Gilleron M. Quesniaux V.F. Puzo G. J. Biol. Chem. 2003; 278: 29880-29889Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 28Thoma-Uszynski S. Stenger S. Takeuchi O. Ochoa M.T. Engele M. Sieling P.A. Barnes P.F. Rollinghoff M. Bolcskei P.L. Wagner M. Akira S. Norgard M.V. Belisle J.T. Godowski P.J. Bloom B.R. Modlin R.L. Science. 2001; 291: 1544-1547Crossref PubMed Scopus (594) Google Scholar). TLR4 can also mediate cellular activation to soluble cell-associated mycobacterial factors distinct from LAM (29Means T.K. Wang S. Lien E. Yoshimura A. Golenbock D.T. Fenton M.J. J. Immunol. 1999; 163: 3920-3927Crossref PubMed Google Scholar), and M. tuberculosis-induced TNF production by murine macrophages is blocked by a TLR4 antagonist (30Means T.K. Jones B.W. Schromm A.B. Shurtleff B.A. Smith J.A. Keane J. Golenbock D.T. Vogel S.N. Fenton M.J. J. Immunol. 2001; 166: 4074-4082Crossref PubMed Scopus (246) Google Scholar). Mice deficient for TLR4 or TLR2 are defective in their long-term control of the M. tuberculosis infection (31Abel B. Thieblemont N. Quesniaux V.J. Brown N. Mpagi J. Miyake K. Bihl F. Ryffel B. J. Immunol. 2002; 169: 3155-3162Crossref PubMed Scopus (307) Google Scholar, 32Drennan M.B. Nicolle D. Quesniaux V.J. Jacobs M. Allie N. Mpagi J. Fremond C. Wagner H. Kirschning C. Ryffel B. Am. J. Pathol. 2004; 164: 49-57Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). In addition, other pattern recognition receptors such as the C-type lectins mannose receptor, human pulmonary surfactant protein A, or DC-SIGN have been implicated in binding and/or as key molecules participating in anti-inflammatory transduction signals from Man-LAM in dendritic cells (16Maeda N. Nigou J. Herrmann J.L. Jackson M. Amara A. Lagrange P.H. Puzo G. Gicquel B. Neyrolles O. J. Biol. Chem. 2003; 278: 5513-5516Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 20Geijtenbeek T.B. Van Vliet S.J. Koppel E.A. Sanchez-Hernandez M. Vandenbroucke-Grauls C.M. Appelmelk B. Van Kooyk Y. J. Exp. Med. 2003; 197: 7-17Crossref PubMed Scopus (898) Google Scholar, 21Nigou J. Zelle-Rieser C. Gilleron M. Thurnher M. Puzo G. J. Immunol. 2001; 166: 7477-7485Crossref PubMed Scopus (353) Google Scholar, 33Sidobre S. Nigou J. Puzo G. Riviere M. J. Biol. Chem. 2000; 275: 2415-2422Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 34Sidobre S. Puzo G. Riviere M. Biochem. J. 2002; 365: 89-97Crossref PubMed Scopus (33) Google Scholar). LAMs are lipoglycans ubiquitously found in the envelope of mycobacteria. They may have different immunomodulatory activities, depending upon their structure. Phosphoinositol-capped LAM from fast-growing and avirulent species, such as Mycobacterium smegmatis (35Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. J. Biol. Chem. 1995; 270: 12380-12389Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 36Gilleron M. Himoudi N. Adam O. Constant P. Venisse A. Riviere M. Puzo G. J. Biol. Chem. 1997; 272: 117-124Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) are pro-inflammatory molecules stimulating the production of TNF and IL-12, whereas LAM capped by mannosyl residues (ManLAM) from the slow-growing mycobacteria M. tuberculosis and Mycobacterium bovis BCG (37Chatterjee D. Roberts A.D. Lowell K. Brennan P.J. Orme I.M. Infect. Immun. 1992; 60: 1249-1253Crossref PubMed Google Scholar, 38Prinzis S. Chatterjee D. Brennan P.J. J. Gen. Microbiol. 1993; 139: 2649-2658Crossref PubMed Scopus (116) Google Scholar, 39Venisse A. Berjeaud J.M. Chaurand P. Gilleron M. Puzo G. J. Biol. Chem. 1993; 268: 12401-12411Abstract Full Text PDF PubMed Google Scholar) are anti-inflammatory molecules, inhibiting the production of IL-12 and TNF and increasing IL-10 production by dendritic cells or monocytic cell lines (20Geijtenbeek T.B. Van Vliet S.J. Koppel E.A. Sanchez-Hernandez M. Vandenbroucke-Grauls C.M. Appelmelk B. Van Kooyk Y. J. Exp. Med. 2003; 197: 7-17Crossref PubMed Scopus (898) Google Scholar, 21Nigou J. Zelle-Rieser C. Gilleron M. Thurnher M. Puzo G. J. Immunol. 2001; 166: 7477-7485Crossref PubMed Scopus (353) Google Scholar, 40Knutson K.L. Hmama Z. Herrera-Velit P. Rochford R. Reiner N.E. J. Biol. Chem. 1998; 273: 645-652Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Although phosphoinositol-capped LAM activates macrophages in a TLR2-dependent manner by activating the NF-κB signaling pathway (25Means T.K. Lien E. Yoshimura A. Wang S. Golenbock D.T. Fenton M.J. J. Immunol. 1999; 163: 6748-6755Crossref PubMed Google Scholar), the anti-inflammatory effects of Man-LAM have been attributed to their binding to the mannose receptor (21Nigou J. Zelle-Rieser C. Gilleron M. Thurnher M. Puzo G. J. Immunol. 2001; 166: 7477-7485Crossref PubMed Scopus (353) Google Scholar) or to DC-SIGN (16Maeda N. Nigou J. Herrmann J.L. Jackson M. Amara A. Lagrange P.H. Puzo G. Gicquel B. Neyrolles O. J. Biol. Chem. 2003; 278: 5513-5516Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 20Geijtenbeek T.B. Van Vliet S.J. Koppel E.A. Sanchez-Hernandez M. Vandenbroucke-Grauls C.M. Appelmelk B. Van Kooyk Y. J. Exp. Med. 2003; 197: 7-17Crossref PubMed Scopus (898) Google Scholar). Within ManLAM, the critical motifs for recognition by DC-SIGN have been shown to be the mannose caps as well as the fatty acids involved in a supramolecular organization of the molecule associated with increased avidity for their receptors (16Maeda N. Nigou J. Herrmann J.L. Jackson M. Amara A. Lagrange P.H. Puzo G. Gicquel B. Neyrolles O. J. Biol. Chem. 2003; 278: 5513-5516Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 41Riviere M. Moisand A. Lopez A. Puzo G. J. Mol. Biol. 2004; 344: 907-918Crossref PubMed Scopus (35) Google Scholar). LMs, the biosynthetic precursors of LAM, are composed of a carbohydrate backbone made of a α-d-mannan core and a mannosyl-phosphatidylinositol anchor at the reducing end of the mannan core but lack the d-arabinan domain and capping motifs found in LAM (42Besra G.S. Morehouse C.B. Rittner C.M. Waechter C.J. Brennan P.J. J. Biol. Chem. 1997; 272: 18460-18466Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 43Nigou J. Gilleron M. Puzo G. Biochimie (Paris). 2003; 85: 153-166Crossref PubMed Scopus (219) Google Scholar). LM are pro-inflammatory, but we described recently that LM from different mycobacterial origins, including M. bovis BCG, M. tuberculosis, Mycobacterium chelonae, and Mycobacterium kansasii, also present strong anti-inflammatory properties (22Quesniaux V.J. Nicolle D.M. Torres D. Kremer L. Guerardel Y. Nigou J. Puzo G. Erard F. Ryffel B. J. Immunol. 2004; 172: 4425-4434Crossref PubMed Scopus (211) Google Scholar). In particular, LM from M. bovis BCG was shown to induce macrophage activation and pro-inflammatory cytokines through TLR2 and the adaptor protein MyD88 and to inhibit pro-inflammatory cytokines production by LPS-activated macrophages through a TLR2- and MyD88-independent pathway. Recently, purification and structural characterization of four LM acyl forms from M. bovis BCG was reported using MALDI-MS and two-dimensional 1H,31P NMR analyses (44Gilleron M. Nigou J. Nicolle D. Quesniaux V. Puzo G. Chem. Biol. 2006; 13: 39-47Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Tri- and tetra-acylated fractions were strongly pro-inflammatory, but not the mono- and di-acylated LM, and the tri-acylated LM form was identified as the main LM TLR2 agonist using TLR2/TLR1 heterodimers for signaling. Here, we separate the negative and positive activities of M. bovis BCG LM. The purified mono-, di-, tri-, and tetra-acylated forms of M. bovis BCG LM were compared for anti-inflammatory or pro-inflammatory activities. Using primary macrophages derived from various TLR-, C-type lectins- and signal adaptor-deficient mice, we demonstrate a potent inhibitory effect of M. bovis BCG di-acylated LM on LPS-induced macrophage activation that turned out to be independent of TLR2 but also of mannose receptor and SIGNR1. Although mono-acylated LM was essentially inactive, the profound pro-inflammatory activity of M. bovis BCG tetra-acylated LM fraction was largely TLR4-dependent. The M. bovis BCG tri-acylated LM fraction exhibited both a strong TLR2/TLR1-dependent TNF-promoting activity mediated by MyD88 and TIRAP and some inhibition of LPS-induced TNF secretion that was independent of TLR2. This study demonstrates that mycobacterial LM acylation pattern determines the anti-inflammatory versus pro-inflammatory modulin effect of M. bovis BCG LM fractions. Purification of LM Acyl Forms—LMs from M. bovis BCG was prepared as previously described (45Nigou J. Gilleron M. Cahuzac B. Bounery J.D. Herold M. Thurnher M. Puzo G. J. Biol. Chem. 1997; 272: 23094-23103Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 46Gilleron M. Nigou J. Cahuzac B. Puzo G. J. Mol. Biol. 1999; 285: 2147-2160Crossref PubMed Scopus (66) Google Scholar), and M. bovis BCG LM mono-, di-, tri-, and tetra-acyl forms were further fractionated using hydrophobic interaction chromatography (44Gilleron M. Nigou J. Nicolle D. Quesniaux V. Puzo G. Chem. Biol. 2006; 13: 39-47Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The purity of the different acyl forms was assessed by 31P NMR and MALDI-MS and was estimated to be >95% with no other molecular species detected. LM from M. tuberculosis H37Rv purified according to established procedures (45Nigou J. Gilleron M. Cahuzac B. Bounery J.D. Herold M. Thurnher M. Puzo G. J. Biol. Chem. 1997; 272: 23094-23103Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 46Gilleron M. Nigou J. Cahuzac B. Puzo G. J. Mol. Biol. 1999; 285: 2147-2160Crossref PubMed Scopus (66) Google Scholar) was determined here by 31P NMR and MALDI-MS analysis to contain 88% tri-acylated and 12% di-acylated forms, and no other molecular contaminant was detectable (46Gilleron M. Nigou J. Cahuzac B. Puzo G. J. Mol. Biol. 1999; 285: 2147-2160Crossref PubMed Scopus (66) Google Scholar, 47Nigou J. Gilleron M. Puzo G. Biochem. J. 1999; 337: 453-460Crossref PubMed Scopus (48) Google Scholar). The endotoxin content of the LM preparations was quantified by limulus amebocyte lysate kinetic turbidimetric assays (Cambrex, Verviers, Belgium). A second source of LM from M. tuberculosis H37Rv was kindly provided by L. Kremer and was from J. Belisle (Colorado State University, Fort Collins, CO; endotoxin content of 17.6 pg per 10 μg). Mice—6–12-week old mice deficient for TLR4 and/or TLR2, obtained by intercross from TLR4-deficient mice (from S. Akira (48Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar)) and TLR2-deficient mice (from C. Kirsching (49Michelsen K.S. Aicher A. Mohaupt M. Hartung T. Dimmeler S. Kirschning C.J. Schumann R.R. J. Biol. Chem. 2001; 276: 25680-25686Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar)), for MyD88 (50Kawai T. Adachi O. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar), TIRAP (51Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Crossref PubMed Scopus (685) Google Scholar), mannose receptor (52Lee S.J. Evers S. Roeder D. Parlow A.F. Risteli J. Risteli L. Lee Y.C. Feizi T. Langen H. Nussenzweig M.C. 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After resuspension in cold phosphate-buffered saline, washing, and re-culturing for 3 days in fresh medium, the cell preparation contained a homogenous population of macrophages (verified periodically by Giemsa staining and CD11b expression). The bone marrow-derived macrophages were plated in 96-well microculture plates at a density of 105 cells/well in Dulbecco's minimal essential medium supplemented with 2 mml-glutamine and 2 × 10–5m β-mercaptoethanol and stimulated with 100 ng/ml LPS (Escherichia coli, serotype O111:B4, Sigma), 0.5 μg/ml synthetic bacterial lipopeptide Pam3CSK4 ([S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH] trihydrochloride, EMC Microcollections, Tuebingen, Germany), 0.125 μm CpG ODN1826 (tccatgacgttcctgacgtt), and LM or LAM (at the concentrations indicated). The macrophages were activated with interferon-γ (500 units/ml) to study IL-12 expression. After 6–24 h of stimulation, the supernatants were harvested and analyzed immediately or stored at –20 °C until further use. The absence of cytotoxicity of the stimuli was controlled using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide incorporation. SIGNR1-expressing RAW Cells—Murine macrophage RAW 264.7 cells expressing SIGN-R1 were prepared as described using SIGNR1 cDNA subcloned into the retroviral vector pFB (neo) (Stratagene, La Jolla, CA), and RAW-FB cells were used as controls (55Taylor P.R. Brown G.D. Herre J. Williams D.L. Willment J.A. Gordon S. J. Immunol. 2004; 172: 1157-1162Crossref PubMed Scopus (155) Google Scholar), both kind gifts from Prof. S. Gordon (University of Oxford, Oxford, UK). Human HEK Cell Lines Expressing TLR4:HEK-Blue™-4 Cells—HEK-BLue™-4 cells (InvivoGen, Toulouse, France) consisting in the human cell line HEK293 stably transfected with human TLR4, the co-receptors MD2 and CD14 genes, and a NF-κB-inducible reporter system (secreted alkaline phosphatase) were used. Cells were plated at 10,000 cells per well, and the different LM acyl forms were added at 10–100 ng/ml using two LPSs as controls (E. coli, serotypes O111:B4 and 055:B5) in the absence or in presence polymixin at 10 μg/ml, a concentration sufficient to neutralize 1000 ng/ml of LPS. Alkaline phosphatase activity was measured after 18–40 h by reading absorbance at 630 nm. Cytokine Enzyme-linked Immunosorbent Assay—Supernatants were harvested and assayed for cytokine content using commercially available enzyme-linked immunosorbent assay reagents for TNF and IL-12p40 (Duoset R&D Systems, Abingdon, UK). Nitrite Measurements—Nitrite concentrations in cell supernatants were determined using the Griess reaction (3% phosphoric acid, 1% p-aminobenzene sulfonamide, 1% n-1-napthylethylenediamide) as previously described (56Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10828) Google Scholar). Separation of the Inhibitory and the Stimulatory Activities of M. bovis BCG LM on Cytokine and NO Production after Purification of the Different Acyl Forms—ManLAM is a complex lipoglycan considered as" @default.
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- W2123117008 title "Acylation Determines the Toll-like receptor (TLR)-dependent Positive Versus TLR2-, Mannose Receptor-, and SIGNR1-independent Negative Regulation of Pro-inflammatory Cytokines by Mycobacterial Lipomannan" @default.
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