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• W2079484449 abstract "Although the Toll-like receptors used by Mycobacterium tuberculosis membrane and secreted factors are known, the pathways activated by M. tuberculosis heat shock proteins are not. An efficient immune response against the intracellular pathogen M. tuberculosis is critically dependent on rapid detection of the invading pathogen by the innate immune system and coordinated activation of the adaptive immune response. Macrophage phagocytosis of M. tuberculosis is accompanied by activation of the transcription factor NF-κB and secretion of inflammatory mediators that play an important role in granuloma formation and immune protection during M. tuberculosis infection. The interaction between M. tuberculosis and the various Toll-like receptors is complex, and it appears that distinct mycobacterial components may interact with different members of the Toll-like receptor family. Here we show that recombinant, purified, mycobacterial heat shock proteins 65 and 70 induce NF-κB activity in a dose-dependent manner in human endothelial cells. Furthermore, we show that whereas mycobacterial heat shock protein 65 signals exclusively through Toll-like receptor 4, heat shock protein 70 also signals through Toll-like receptor 2. Mycobacterial heat shock protein 65-induced NF-κB activation was MyD88-, TIRAP-, TRIF-, and TRAM-dependent and required the presence of MD-2. A better understanding of the recognition of mycobacterial heat shock proteins and their role in the host immune response to the pathogen may open the way to a better understanding of the immunological processes induced by this important human pathogen and the host-pathogen interactions and may help in the rational design of more effective vaccines or vaccine adjuvants. Although the Toll-like receptors used by Mycobacterium tuberculosis membrane and secreted factors are known, the pathways activated by M. tuberculosis heat shock proteins are not. An efficient immune response against the intracellular pathogen M. tuberculosis is critically dependent on rapid detection of the invading pathogen by the innate immune system and coordinated activation of the adaptive immune response. Macrophage phagocytosis of M. tuberculosis is accompanied by activation of the transcription factor NF-κB and secretion of inflammatory mediators that play an important role in granuloma formation and immune protection during M. tuberculosis infection. The interaction between M. tuberculosis and the various Toll-like receptors is complex, and it appears that distinct mycobacterial components may interact with different members of the Toll-like receptor family. Here we show that recombinant, purified, mycobacterial heat shock proteins 65 and 70 induce NF-κB activity in a dose-dependent manner in human endothelial cells. Furthermore, we show that whereas mycobacterial heat shock protein 65 signals exclusively through Toll-like receptor 4, heat shock protein 70 also signals through Toll-like receptor 2. Mycobacterial heat shock protein 65-induced NF-κB activation was MyD88-, TIRAP-, TRIF-, and TRAM-dependent and required the presence of MD-2. A better understanding of the recognition of mycobacterial heat shock proteins and their role in the host immune response to the pathogen may open the way to a better understanding of the immunological processes induced by this important human pathogen and the host-pathogen interactions and may help in the rational design of more effective vaccines or vaccine adjuvants. Mycobacterium tuberculosis continues to be a major global health problem, infecting nearly one-third of the world's population and killing at least 3 million people each year (1Flynn J.L. Chan J. Annu. Rev. Immunol. 2001; 19: 93-129Crossref PubMed Scopus (1719) Google Scholar, 2Frieden T.R. Sterling T.R. Munsiff S.S. Watt C.J. Dye C. Lancet. 2003; 362: 887-899Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). The immune response mounted to the infection is generally successful in containing, although not eliminating, the pathogen (1Flynn J.L. Chan J. Annu. Rev. Immunol. 2001; 19: 93-129Crossref PubMed Scopus (1719) Google Scholar). Traditionally, protective immunity to M. tuberculosis has been ascribed to T cell-mediated immunity, with CD4+ T cells playing a crucial role. This is primarily because the organism lives within cells, usually macrophages; thus T cell effector mechanisms, rather than antibody responses, are required to control or eliminate the bacteria (1Flynn J.L. Chan J. Annu. Rev. Immunol. 2001; 19: 93-129Crossref PubMed Scopus (1719) Google Scholar). The breakdown of immune responses designed to contain the infection can result in reactivation and replication of the bacilli, with necrosis and damage to lung tissue (1Flynn J.L. Chan J. Annu. Rev. Immunol. 2001; 19: 93-129Crossref PubMed Scopus (1719) Google Scholar). Recent immunological and genetic studies support the model that innate immunity plays an important role in host defenses against M. tuberculosis (3van Crevel R. Ottenhoff T.H. van der Meer J.W. Clin. Microbiol. Rev. 2002; 15: 294-309Crossref PubMed Scopus (462) Google Scholar). An efficient immune response against the intracellular pathogen M. tuberculosis is critically dependent on the rapid detection of the invading pathogen by the innate immune system and the coordinated activation of the adaptive immune response, but a comprehensive understanding of the molecular mechanisms mediating these responses has only recently been addressed (4Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1421) Google Scholar, 5Means T.K. Wang S. Lien E. Yoshimura A. Golenbock D.T. Fenton M.J. J. Immunol. 1999; 163: 3920-3927PubMed Google Scholar, 6Underhill D.M. Ozinsky A. Smith K.D. Aderem A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14459-14463Crossref PubMed Scopus (675) Google Scholar, 7Brightbill 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 (1400) Google Scholar, 8Noss E.H. Pai R.K. Sellati T.J. Radolf J.D. Belisle J. Golenbock D.T. Boom W.H. Harding C.V. J. Immunol. 2001; 167: 910-918Crossref PubMed Scopus (359) Google Scholar, 9Moreno C. Taverne J. Mehlert A. Bate C.A. Brealey R.J. Meager A. Rook G.A. Playfair J.H. Clin. Exp. Immunol. 1989; 76: 240-245PubMed Google Scholar, 10Abel B. Thieblemont N. Quesniaux V.J. Brown N. Mpagi J. Miyake K. Bihl F. Ryffel B. J. Immunol. 2002; 169: 3155-3162Crossref PubMed Scopus (305) Google Scholar, 11Thoma-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 (586) Google Scholar).Emerging evidence suggests that Toll-like receptors (TLRs), 1The abbreviations used are: TLR, Toll-like receptor; Δ, dominant negative; HMEC, human dermal endothelial cell; HSP, heat shock protein; IL, interleukin; LPS, lipopolysaccharide; Mtb, mycobacterial; MyD88, myeloid differentiation factor 88; sCD14, soluble CD14; STF, soluble tuberculosis factor; TIR, Toll/IL-1 receptor; TIRAP, TIR domain-containing adapter protein; TRIF, TIR domain-containing adapter inducing interferon-β; TNF, tumor necrosis factor; TRAM, TRIF-related adapter molecule.1The abbreviations used are: TLR, Toll-like receptor; Δ, dominant negative; HMEC, human dermal endothelial cell; HSP, heat shock protein; IL, interleukin; LPS, lipopolysaccharide; Mtb, mycobacterial; MyD88, myeloid differentiation factor 88; sCD14, soluble CD14; STF, soluble tuberculosis factor; TIR, Toll/IL-1 receptor; TIRAP, TIR domain-containing adapter protein; TRIF, TIR domain-containing adapter inducing interferon-β; TNF, tumor necrosis factor; TRAM, TRIF-related adapter molecule. which are critical pattern recognition molecules that alert the host to the presence of microbial pathogens, contribute to innate immunity by detecting M. tuberculosis-associated molecular patterns and mediating the secretion of various cytokines and antibacterial effector molecules (12Barton G.M. Medzhitov R. Science. 2003; 300: 1524-1525Crossref PubMed Scopus (1044) Google Scholar). In addition, TLRs influence the adaptive immune response by up-regulating co-stimulatory molecules to support the development of a Th1 response (12Barton G.M. Medzhitov R. Science. 2003; 300: 1524-1525Crossref PubMed Scopus (1044) Google Scholar). TLRs comprise a family of at least eleven cell-surface pattern recognition molecules that alert the host to the presence of microbial pathogens (12Barton G.M. Medzhitov R. Science. 2003; 300: 1524-1525Crossref PubMed Scopus (1044) Google Scholar). TLR4 initiates signaling cascades in response to lipopolysaccharide (LPS) (13Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2759) Google Scholar), a major component of the outer membrane of Gram-negative bacteria, as well as to Taxol (14Kawasaki K. Nogawa H. Nishijima M. J. Immunol. 2003; 170: 413-420Crossref PubMed Scopus (72) Google Scholar) and endogenous (15Ohashi K. Burkart V. Flohe S. Kolb H. J. Immunol. 2000; 164: 558-561Crossref PubMed Scopus (1357) Google Scholar) or chlamydial heat shock protein 60 (HSP60) (16Bulut Y. Faure E. Thomas L. Karahashi H. Michelsen K.S. Equils O. Morrison S.G. Morrison R.P. Arditi M. J. Immunol. 2002; 168: 1435-1440Crossref PubMed Scopus (320) Google Scholar, 17Sasu S. LaVerda D. Qureshi N. Golenbock D.T. Beasley D. Circ. Res. 2001; 89: 244-250Crossref PubMed Scopus (246) Google Scholar, 18Costa C.P. Kirschning C.J. Busch D. Durr S. Jennen L. Heinzmann U. Prebeck S. Wagner H. Miethke T. Eur. J. Immunol. 2002; 32: 2460-2470Crossref PubMed Scopus (83) Google Scholar), whereas TLR2 recognizes various fungal, Gram-positive (4Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1421) Google Scholar, 13Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2759) Google Scholar), and mycobacterial cell wall components (6Underhill D.M. Ozinsky A. Smith K.D. Aderem A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14459-14463Crossref PubMed Scopus (675) Google Scholar, 19Means 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) such as peptidoglycan, lipoteichoic acid, soluble tuberculosis factor (STF), and lipoarabinomannan.It appears that distinct mycobacterial components may interact with different members of the TLR family (19Means 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), thus increasing the likelihood that a pathogen will be recognized by several mechanisms. Indeed, recent studies have demonstrated that the M. tuberculosis 19-kDa lipoprotein, a potent inducer of T cell responses, activates murine and human macrophages to secrete TNF and nitric oxide (7Brightbill 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 (1400) Google Scholar) and inhibits major histocompatibility complex II antigen processing via interaction with TLR2 (8Noss E.H. Pai R.K. Sellati T.J. Radolf J.D. Belisle J. Golenbock D.T. Boom W.H. Harding C.V. J. Immunol. 2001; 167: 910-918Crossref PubMed Scopus (359) Google Scholar). Lipoarabinomannan, a glycolipid that is abundant in the mycobacterial cell wall, is a key molecule in eliciting cytokine secretion by macrophages (9Moreno C. Taverne J. Mehlert A. Bate C.A. Brealey R.J. Meager A. Rook G.A. Playfair J.H. Clin. Exp. Immunol. 1989; 76: 240-245PubMed Google Scholar). Uncapped lipoarabinomannan (AraLAM) induces cell activation via TLR2 (5Means T.K. Wang S. Lien E. Yoshimura A. Golenbock D.T. Fenton M.J. J. Immunol. 1999; 163: 3920-3927PubMed Google Scholar, 6Underhill D.M. Ozinsky A. Smith K.D. Aderem A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14459-14463Crossref PubMed Scopus (675) Google Scholar). STF contains mannosylated phosphatidylinositol that possesses TLR2 agonist activity (20Jones 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). Furthermore, we demonstrated previously a functional interaction between TLR2 and TLR6 in the cellular response to STF (21Bulut Y. Faure E. Thomas L. Equils O. Arditi M. J. Immunol. 2001; 167: 987-994Crossref PubMed Scopus (342) Google Scholar). TLR2 activation leads to the killing of intracellular M. tuberculosis in both mouse and human macrophages. Whereas in mouse macrophages this pathway is nitric oxide-dependent, in human monocytes and alveolar macrophages it is nitric oxide-independent (11Thoma-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 (586) Google Scholar).In contrast to the findings in some earlier publications, more recent reports found that mycobacterial mannosylated phosphatidylinositol induces both TLR2- and TLR4-dependent signaling (10Abel B. Thieblemont N. Quesniaux V.J. Brown N. Mpagi J. Miyake K. Bihl F. Ryffel B. J. Immunol. 2002; 169: 3155-3162Crossref PubMed Scopus (305) Google Scholar). Means et al. reported that both soluble and cell-associated mycobacterial factors can activate TLR-dependent signaling in a CD14-independent manner (5Means T.K. Wang S. Lien E. Yoshimura A. Golenbock D.T. Fenton M.J. J. Immunol. 1999; 163: 3920-3927PubMed Google Scholar). They observed that a soluble, heat-stable, and protease-resistant mycobacterial factor mediated TLR2-dependent activation, whereas a heat-labile, cell-associated mycobacterial factor activated cells in a TLR4-dependent manner (5Means T.K. Wang S. Lien E. Yoshimura A. Golenbock D.T. Fenton M.J. J. Immunol. 1999; 163: 3920-3927PubMed Google Scholar, 19Means 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). Therefore, whereas purified mycobacterial antigens such as 19-kDa lipoprotein, lipoarabinomannan, and STF preferentially interact with TLR2, infection with whole bacilli evokes a more complex activation pattern involving at least TLR2 and TLR4 and leads to differential activation of antibacterial effector pathways (5Means T.K. Wang S. Lien E. Yoshimura A. Golenbock D.T. Fenton M.J. J. Immunol. 1999; 163: 3920-3927PubMed Google Scholar, 6Underhill D.M. Ozinsky A. Smith K.D. Aderem A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14459-14463Crossref PubMed Scopus (675) Google Scholar, 7Brightbill 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 (1400) Google Scholar, 8Noss E.H. Pai R.K. Sellati T.J. Radolf J.D. Belisle J. Golenbock D.T. Boom W.H. Harding C.V. J. Immunol. 2001; 167: 910-918Crossref PubMed Scopus (359) Google Scholar, 19Means 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, 22Stenger S. Modlin R.L. Curr. Opin. Immunol. 2002; 14: 452-457Crossref PubMed Scopus (109) Google Scholar).Several experimental animal models suggest that perhaps both TLR2 and TLR4 play a role in innate responses to M. tuberculosis infection in vivo (23Sugawara I. Yamada H. Li C. Mizuno S. Takeuchi O. Akira S. Microbiol. Immunol. 2003; 47: 327-336Crossref PubMed Scopus (148) Google Scholar, 24Sugawara I. Yamada H. Mizuno S. Takeda K. Akira S. Microbiol. Immunol. 2003; 47: 841-847Crossref PubMed Scopus (73) Google Scholar, 25Branger J. Leemans J.C. Florquin S. Weijer S. Speelman P. Van Der Poll T. Int. Immunol. 2004; 16: 509-516Crossref PubMed Scopus (93) Google Scholar). A recent study suggested that TLR2 and TLR4 are redundant to control M. tuberculosis infection and that only at extremely high infectious doses was survival reduced in TLR2-deficient mice (26Reiling N. Holscher C. Fehrenbach A. Kroger S. Kirschning C.J. Goyert S. Ehlers S. J. Immunol. 2002; 169: 3480-3484Crossref PubMed Scopus (376) Google Scholar). Another study demonstrated that both standard and high doses of aerosol infection with live M. tuberculosis resulted in significantly increased bacterial loads, defective granulomatous response, and chronic pneumonia in TLR2-deficient mice (27Drennan 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 (274) Google Scholar). Whereas studies with C3H/HeJ mice, a strain harboring a mutation in the signaling domain of TLR4 that renders mice unresponsive to LPS, showed that TLR4 may not play a significant role in immunity to M. tuberculosis (28Kamath A.B. Alt J. Debbabi H. Behar S.M. Infect. Immun. 2003; 71: 4112-4118Crossref PubMed Scopus (69) Google Scholar, 29Shim T.S. Turner O.C. Orme I.M. Tuberculosis (Edinb.). 2003; 83: 367-371Crossref PubMed Scopus (54) Google Scholar), other studies have shown that C3H/HeJ mice have a reduced capacity to eliminate mycobacteria from the lungs, with spreading of the infection to the spleen and liver, whereas the wild-type mice controlled the infection (10Abel B. Thieblemont N. Quesniaux V.J. Brown N. Mpagi J. Miyake K. Bihl F. Ryffel B. J. Immunol. 2002; 169: 3155-3162Crossref PubMed Scopus (305) Google Scholar). These authors suggested that TLR4 signaling appears to be required to control the local growth and dissemination of M. tuberculosis infection from lungs (10Abel B. Thieblemont N. Quesniaux V.J. Brown N. Mpagi J. Miyake K. Bihl F. Ryffel B. J. Immunol. 2002; 169: 3155-3162Crossref PubMed Scopus (305) Google Scholar, 23Sugawara I. Yamada H. Li C. Mizuno S. Takeuchi O. Akira S. Microbiol. Immunol. 2003; 47: 327-336Crossref PubMed Scopus (148) Google Scholar, 24Sugawara I. Yamada H. Mizuno S. Takeda K. Akira S. Microbiol. Immunol. 2003; 47: 841-847Crossref PubMed Scopus (73) Google Scholar). In summary, previous studies suggest that the interaction between M. tuberculosis and the various TLRs is complex, and it appears that distinct mycobacterial components may interact with different members of the TLR family.Mycobacterial (Mtb) HSPs may also participate in cytokine expression resulting from infection by M. tuberculosis (30Young D.B. Garbe T.R. Infect. Immun. 1991; 59: 3086-3093Crossref PubMed Google Scholar). HSPs stabilize cellular proteins in response to diverse sources of stress or injury (31Pockley A.G. Lancet. 2003; 362: 469-476Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, 32Young D.B. Kaufmann S.H. Hermans P.W. Thole J.E. Mol. Microbiol. 1992; 6: 133-145Crossref PubMed Scopus (195) Google Scholar). HSPs also have a number of immunological effects, including the induction of pro-inflammatory cytokines (15Ohashi K. Burkart V. Flohe S. Kolb H. J. Immunol. 2000; 164: 558-561Crossref PubMed Scopus (1357) Google Scholar, 16Bulut Y. Faure E. Thomas L. Karahashi H. Michelsen K.S. Equils O. Morrison S.G. Morrison R.P. Arditi M. J. Immunol. 2002; 168: 1435-1440Crossref PubMed Scopus (320) Google Scholar, 31Pockley A.G. Lancet. 2003; 362: 469-476Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, 32Young D.B. Kaufmann S.H. Hermans P.W. Thole J.E. Mol. Microbiol. 1992; 6: 133-145Crossref PubMed Scopus (195) Google Scholar, 33Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1244) Google Scholar, 34Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-15112Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). We have shown that chlamydial HSP60 signals through TLR4 and MD-2 (16Bulut Y. Faure E. Thomas L. Karahashi H. Michelsen K.S. Equils O. Morrison S.G. Morrison R.P. Arditi M. J. Immunol. 2002; 168: 1435-1440Crossref PubMed Scopus (320) Google Scholar), and other studies suggest that HSP60 and HSP70 may signal through pathways dependent upon both TLR2 and TLR4 (33Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1244) Google Scholar, 34Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-15112Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). It is not known whether Mtb HSPs are recognized by and signal through TLRs. Given the importance of Mtb HSPs 65 and 70 in the pathogenesis of the M. tuberculosis infection, we sought to clarify the role of TLRs in the innate immune response to these ligands. Here we report that recombinant purified M. tuberculosis HSP65 signals exclusively through TLR4, whereas M. tuberculosis HSP70 signals through both TLR4 and TLR2 to activate the innate immune system in a myeloid differentiation factor 88 (MyD88)-, Toll/interleukin-1 receptor (TIR) domain-containing adapter protein (TIRAP)-, TIR domain-containing adapter-inducing interferon-β (TRIF)-, and TRIF-related adapter molecule (TRAM)-dependent manner.EXPERIMENTAL PROCEDURESCell Cultures—Immortalized human dermal endothelial cells (HMECs) (a generous gift from Dr. Francisco J. Candal, Center for Disease Control and Prevention, Atlanta, GA) were cultured as described earlier (35Faure E. Equils O. Sieling P.A. Thomas L. Zhang F.X. Kirschning C.J. Polentarutti N. Muzio M. Arditi M. J. Biol. Chem. 2000; 275: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). Tissue culture reagents were from Invitrogen. HEK 293 cells were purchased from American Type Culture Collection (Manassas, VA) and cultured as recommended.Recombinant Purified M. tuberculosis HSP65 and HSP70 —Purified recombinant M. tuberculosis HSP65 and HSP70 were produced using a bacterial expression system. For Mtb HSP70, protein was obtained from heat-induced (42 °C) cells of Escherichia coli strain M1592 with plasmid pKAM2101. Cells were lysed by sonication. After centrifugation, the recombinant 70-kDa protein was purified by anion exchange chromatography (Q-Sepharose), followed by ATP affinity chromatography and again by anion exchange chromatography. The purified protein was dialyzed against 10 mm ammonium bicarbonate and lyophilized (32Young D.B. Kaufmann S.H. Hermans P.W. Thole J.E. Mol. Microbiol. 1992; 6: 133-145Crossref PubMed Scopus (195) Google Scholar, 36Scanga C.A. Bafica A. Feng C.G. Cheever A.W. Hieny S. Sher A. Infect. Immun. 2004; 72: 2400-2404Crossref PubMed Scopus (161) Google Scholar). For Mtb HSP65, the protein was obtained from the heat-induced (42 °C) E. coli K12 strain M1546, which carries the plasmid pRIB1300. Cells were lysed by sonication, and the supernatant was subjected to Q-Sepharose anion exchange chromatography. The fractions containing HSP65 were further purified on a Mono Q column. The buffer was exchanged to 10 mm ammonium bicarbonate by dialysis (32Young D.B. Kaufmann S.H. Hermans P.W. Thole J.E. Mol. Microbiol. 1992; 6: 133-145Crossref PubMed Scopus (195) Google Scholar). All reagents were verified to be LPS-free by the Pyrotell Limulus amebocyte lysate assay (<0.03 endotoxin units/ml; Associates of Cape Cod, East Falmouth, MA). Purified, protein-free E. coli K235 LPS was obtained from Dr. Stephanie N. Vogel (University of Maryland, Bethesda, MD). Mtb HSP65, Mtb HSP70, and LPS were treated with proteinase K at 56 °C for 30 min and subsequently at 95 °C for 5 min to inactivate proteinase K (Roche Applied Science). In separate experiments, Mtb HSP65, Mtb HSP70, and LPS were boiled at 100 °C for 30 min before use.cDNA Constructs and Transient Transfections—ELAM-NF-κB luciferase, pCMV-β-galactosidase, dominant negative (Δ) MyD88 (ΔMyD88), ΔTIRAP, and ΔTLR4 vectors were used as described previously (21Bulut Y. Faure E. Thomas L. Equils O. Arditi M. J. Immunol. 2001; 167: 987-994Crossref PubMed Scopus (342) Google Scholar, 35Faure E. Equils O. Sieling P.A. Thomas L. Zhang F.X. Kirschning C.J. Polentarutti N. Muzio M. Arditi M. J. Biol. Chem. 2000; 275: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 37Zhang F.X. Kirschning C.J. Mancinelli R. Xu X.P. Jin Y. Faure E. Mantovani A. Rothe M. Muzio M. Arditi M. J. Biol. Chem. 1999; 274: 7611-7614Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). A FLAG-tagged human MD-2 cDNA construct was obtained from Dr. Kensuke Miyake (University of Tokyo). Transient transfection of cultured cells was conducted using FuGENE 6 transfection reagent (Roche Applied Science) as described earlier (21Bulut Y. Faure E. Thomas L. Equils O. Arditi M. J. Immunol. 2001; 167: 987-994Crossref PubMed Scopus (342) Google Scholar, 35Faure E. Equils O. Sieling P.A. Thomas L. Zhang F.X. Kirschning C.J. Polentarutti N. Muzio M. Arditi M. J. Biol. Chem. 2000; 275: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). HMECs were also stimulated in the presence or absence of serum with or without recombinant human soluble CD14 (sCD14) (0.15 μg/ml; R&D Systems, Minneapolis, MN). The plasmid DNA for pCMV-β galactosidase (0.1 μg), ELAM-NF-κB luciferase (0.5 μg), pCMV empty vector (0.5 μg), wild-type TLR2 (0.5 μg), ΔTLR4 (0.5 μg), ΔTIRAP (0.1 μg), and ΔMyD88 (0.5 μg) were co-transfected as described earlier (21Bulut Y. Faure E. Thomas L. Equils O. Arditi M. J. Immunol. 2001; 167: 987-994Crossref PubMed Scopus (342) Google Scholar, 35Faure E. Equils O. Sieling P.A. Thomas L. Zhang F.X. Kirschning C.J. Polentarutti N. Muzio M. Arditi M. J. Biol. Chem. 2000; 275: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 38Faure E. Thomas L. Xu H. Medvedev A. Equils O. Arditi M. J. Immunol. 2001; 166: 2018-2024Crossref PubMed Scopus (401) Google Scholar). The total amount of DNA transfected was kept constant with a pCMV empty vector. After overnight transfection, cells were stimulated for 5 h with LPS (20 ng/ml), Mtb HSP65 (0.3–10 μg/ml), or Mtb HSP70 (0.3–10 μg/ml). Luciferase and β-galactosidase activity were measured as reported previously (35Faure E. Equils O. Sieling P.A. Thomas L. Zhang F.X. Kirschning C.J. Polentarutti N. Muzio M. Arditi M. J. Biol. Chem. 2000; 275: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 37Zhang F.X. Kirschning C.J. Mancinelli R. Xu X.P. Jin Y. Faure E. Mantovani A. Rothe M. Muzio M. Arditi M. J. Biol. Chem. 1999; 274: 7611-7614Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 38Faure E. Thomas L. Xu H. Medvedev A. Equils O. Arditi M. J. Immunol. 2001; 166: 2018-2024Crossref PubMed Scopus (401) Google Scholar).Generation of Bone Marrow-derived Macrophages from TLR2-, TLR4-, TRAM-, and TRIF-deficient Mice—TLR2-deficent mice, TRAM-deficient mice, and TLR4-deficient mice were kindly provided by Dr. Shizuo Akira (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) (39Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1011) Google Scholar). TrifLps2 mice were kindly provided by Dr. Bruce Beutler (The Scripps Research Institute, La Jolla, CA). Bone marrow cells were flushed from femurs and tibias of mice by complete medium (Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 2 mm glutamine, 100 μg/ml penicillin, and streptomycin) and washed three times with complete medium. Cells were cultured for 3 days in complete medium supplemented with murine macrophage colony-stimulating factor (M-CSF) (BIOSOURCE). On day 3 adherent cells were fed with fresh M-CSF-containing medium. On day 6 adherent cells were fed with complete Dulbecco's modified Eagle's medium and used in experiments on day 8. Briefly, macrophages were stimulated with Mtb HPS65, Mtb HSP70, LPS, or lipopeptide (2 μg/ml) (palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-Ala-Gly-OH; Bachem, Torrance, CA.) for 24 h. Supernatants were harvested and assessed for cytokine release using murine TNF-α or IL-6 enzyme-linked immunosorbent assay kits according to the manufacturer's instructions (BD Biosciences).Statistical Analysis—Data are shown as mean ± S.D. of one representative experiment from at least three independent experiments. Transfections have been performed in triplicate. Transfection data are expressed as the percentage luciferase activity induced by LPS or Mtb HSP65/HSP70 (indicated as 100%). Significance of differences in mean values was determined by Student's t test. p values of < 0.05 were considered statistically significant.RESULTSM. tuberculosis HSP65 and HSP70 Induce NF-κB Activation in HMECs in a Dose-dependent Manner—HMECs express TLR4 but not functional TLR2 (35Faure E. Equils O. Sieling P.A. Thomas L. Zhang F.X. Kirschning C.J. Polentarutti N. Muzio M. Arditi M. J. Biol. Chem. 2000; 275: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). 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