Produção Nacional
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Stimulation of Toll-like Receptor 2 by Coxiella burnetii Is Required for Macrophage Production of Pro-inflammatory Cytokines and Resistance to Infection
2004; Elsevier BV; Volume: 279; Issue: 52 Linguagem: Inglês
10.1074/jbc.m410340200
ISSN1083-351X
AutoresDario S. Zamboni, Marco Antônio Campos, Ana Cláudia Torrecilhas, Kati Kiss, James E. Samuel, Douglas T. Golenbock, Fanny N. Lauw, Craig R. Roy, Igor C. Almeida, Ricardo T. Gazzinelli,
Tópico(s)Mosquito-borne diseases and control
ResumoInnate and adaptive immune responses are initiated upon recognition of microbial molecules by Toll-like receptors (TLRs). We have investigated the importance of these receptors in the induction of pro-inflammatory cytokines and macrophage resistance to infection with Coxiella burnetii, an obligate intracellular bacterium and the etiological agent of Q fever. By using a Chinese hamster ovary/CD14 cell line expressing either functional TLR2 or TLR4, we determined that C. burnetii phase II activates TLR2 but not TLR4. Macrophages deficient for TLR2, but not TLR4, produced less tumor necrosis factor-α and interleukin-12 upon C. burnetii infection. Furthermore, it was found that TLR2 activation interfered with C. burnetii intracellular replication, as macrophages from TLR2-deficient mice were highly permissive for C. burnetii growth compared with macrophages from wild type mice or TLR4-deficient mice. Although LPS modifications distinguish virulent C. burnetii phase I bacteria from avirulent phase II organisms, electrospray ionization-mass spectrometry analysis showed that the lipid A moieties isolated from these two phase variants are identical. Purified lipid A derived from either phase I or phase II LPS failed to activate TLR2 and TLR4. Indeed, the lipid A molecules were able to interfere with TLR4 signaling in response to purified Escherichia coli LPS. These studies indicate that TLR2 is an important host determinant that mediates recognition of C. burnetii and a response that limits growth of this intracellular pathogen. Innate and adaptive immune responses are initiated upon recognition of microbial molecules by Toll-like receptors (TLRs). We have investigated the importance of these receptors in the induction of pro-inflammatory cytokines and macrophage resistance to infection with Coxiella burnetii, an obligate intracellular bacterium and the etiological agent of Q fever. By using a Chinese hamster ovary/CD14 cell line expressing either functional TLR2 or TLR4, we determined that C. burnetii phase II activates TLR2 but not TLR4. Macrophages deficient for TLR2, but not TLR4, produced less tumor necrosis factor-α and interleukin-12 upon C. burnetii infection. Furthermore, it was found that TLR2 activation interfered with C. burnetii intracellular replication, as macrophages from TLR2-deficient mice were highly permissive for C. burnetii growth compared with macrophages from wild type mice or TLR4-deficient mice. Although LPS modifications distinguish virulent C. burnetii phase I bacteria from avirulent phase II organisms, electrospray ionization-mass spectrometry analysis showed that the lipid A moieties isolated from these two phase variants are identical. Purified lipid A derived from either phase I or phase II LPS failed to activate TLR2 and TLR4. Indeed, the lipid A molecules were able to interfere with TLR4 signaling in response to purified Escherichia coli LPS. These studies indicate that TLR2 is an important host determinant that mediates recognition of C. burnetii and a response that limits growth of this intracellular pathogen. Coxiella burnetii is a Gram-negative, obligate intracellular bacterium that survives inside large replication vacuoles (LRVs) 1The abbreviations used are: LRVs, large replication vacuoles; ESI-MS, electrospray ionization-mass spectrometry; LPS, lipopolysaccharide; TLR, Toll-like receptor; PMA, phorbol 12-myristate 13-acetate; PBMC, peripheral blood mononuclear cells; IL, interleukin; CHO, Chinese hamster ovary; TNF-α, tumor necrosis factor-α; DAPI, 4,6-diamidino-2-phenylindole; IFN-γ, interferon-γ. 1The abbreviations used are: LRVs, large replication vacuoles; ESI-MS, electrospray ionization-mass spectrometry; LPS, lipopolysaccharide; TLR, Toll-like receptor; PMA, phorbol 12-myristate 13-acetate; PBMC, peripheral blood mononuclear cells; IL, interleukin; CHO, Chinese hamster ovary; TNF-α, tumor necrosis factor-α; DAPI, 4,6-diamidino-2-phenylindole; IFN-γ, interferon-γ. that display phagolysosomal characteristics such as low pH, the presence of lysosomal hydrolases and glycoproteins, and RAB7 on their membranes (1Baca O.G. Li Y.P. Kumar H. Trends Microbiol. 1994; 2: 476-480Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 2Maurin M. Benoliel A.M. Bongrand P. Raoult D. Infect. Immun. 1992; 60: 5013-5016Crossref PubMed Google Scholar, 3Beron W. Gutierrez M. Rabinovitch M. Colombo M.I. Infect. Immun. 2002; 70: 5816-5821Crossref PubMed Scopus (186) Google Scholar, 4Heinzen R.A. Hackstadt T. Samuel J.E. Trends Microbiol. 1999; 7: 149-154Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Two phase variants of C. burnetii have been described, the highly virulent phase I and the avirulent phase II. Avirulent phase II C. burnetii can be obtained by multiple passages through chicken cells. Although avirulent for mammals, phase II organisms effectively infect and grow in cultured host cells (1Baca O.G. Li Y.P. Kumar H. Trends Microbiol. 1994; 2: 476-480Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 2Maurin M. Benoliel A.M. Bongrand P. Raoult D. Infect. Immun. 1992; 60: 5013-5016Crossref PubMed Google Scholar, 5Baca O.G. Paretsky D. Microbiol. Rev. 1983; 46: 127-149Crossref Google Scholar). C. burnetii phase II, Nine Mile strain clone 4, contains a 26-kb deletion in the genome, which codes for a group of lipopolysaccharide biosynthetic genes (6Hoover T.A. Culp D.W. Vodkin M.H. Williams J.C. Thompson H.A. Infect. Immun. 2002; 70: 6726-6733Crossref PubMed Scopus (93) Google Scholar), a feature that provides genetic support for the reported differences in the composition of LPS from C. burnetii phase I and II (7Amano K. Williams J.C. J. Bacteriol. 1984; 160: 994-1002Crossref PubMed Google Scholar, 8Schramek S. Mayer H. Infect. Immun. 1982; 38: 53-57Crossref PubMed Google Scholar). C. burnetii phase I is the etiological agent of Q fever, a disease of worldwide distribution (9Maurin M. Raoult D. Clin. Microbiol. Rev. 1999; 14: 518-553Crossref Google Scholar, 10Norlander L. Microbes Infect. 2000; 2: 417-424Crossref PubMed Scopus (88) Google Scholar, 11Williams J.C. Thompson H.A. The Biology of Coxiella burnetii. CRC Press, Inc., Boca Raton, FL1991: 21-71Google Scholar). Because of its ability to survive in the environment and high infectivity when aerosolized, C. burnetii is classified as a category B bioterrorism agent (12Seshadri R. Paulsen I.T. Eisen J.A. Read T.D. Nelson K.E. Nelson W.C. Ward N.L. Tettelin H. Davidsen T.M. Beanan M.J. Deboy R.T. Daugherty S.C. Brinkac L.M. Madupu R. Dodson R.J. Khouri H.M. Lee K.H. Carty H.A. Scanlan D. Heinzen R.A. Thompson H.A. Samuel J.E. Fraser C.M. Heidelberg J.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5455-5460Crossref PubMed Scopus (407) Google Scholar, 13Marrie T.J. Curr. Opin. Infect. Dis. 2004; 17: 137-142Crossref PubMed Scopus (32) Google Scholar). The disease manifests either as an acute or chronic illness with symptoms such as debilitating headache and cyclic fever (9Maurin M. Raoult D. Clin. Microbiol. Rev. 1999; 14: 518-553Crossref Google Scholar). Acute Q fever is usually self-limiting in immunocompetent hosts, whereas the chronic form of the disease develops in individuals defective in cell-mediated immunity (9Maurin M. Raoult D. Clin. Microbiol. Rev. 1999; 14: 518-553Crossref Google Scholar, 13Marrie T.J. Curr. Opin. Infect. Dis. 2004; 17: 137-142Crossref PubMed Scopus (32) Google Scholar). These findings support the fundamental role of an effective innate immune recognition to the host resistance against C. burnetii. The innate immune system has evolved sophisticated mechanisms to sense invading microbes, to discriminate between different pathogens, and to initiate the production and secretion of inflammatory molecules that contribute to the development of an acquired immune response and host resistance to infection. Toll-like receptors (TLRs) constitute a family of pattern recognition molecules that can respond to molecular structures conserved in many microbial products. These transmembrane receptors contain ectodomains that have leucine-rich repeats that are involved in pattern recognition and intracellular signaling domains that initiate cellular responses to microbial products (14Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Crossref PubMed Scopus (5994) Google Scholar, 15Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4655) Google Scholar). To date, 11 functional TLRs have been described (TLR1–11). Many TLRs have the ability to respond to bacterial products (15Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4655) Google Scholar, 16Akira S. Curr. Opin. Immunol. 2003; 15: 5-11Crossref PubMed Scopus (469) Google Scholar, 17Zhang D. Zhang G. Hayden M.S. Greenblatt M.B. Bussey C. Flavell R.A. Ghosh S. Science. 2004; 303: 1522-1526Crossref PubMed Scopus (867) Google Scholar). TLR2 appears to be the most promiscuous, responding to multiple bacterial products, including lipoproteins, lipopeptides, lipoteichoic acid, and peptidoglycans (14Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Crossref PubMed Scopus (5994) Google Scholar, 15Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4655) Google Scholar, 18Aliprantis A.O. Yang R.B. Mark M.R. Suggett S. Devaux B. Radolf J.D. Klimpel G.R. Godowski P. Zychlinsky A. Science. 1999; 285: 736-739Crossref PubMed Scopus (1261) Google Scholar). In contrast, TLR4 is specifically activated by the lipid A moiety of LPS from Gram-negative bacteria (19Beutler B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (635) Google Scholar). However, the ability of TLR4 to respond to LPS is not universal. LPS molecules from bacteria such as Porphyromonas gingivalis, Leptospira interrogans, Legionella pneumophila, and Rhizobium species fail to activate TLR4; however, there is evidence to suggest that these bacteria activate host cells through a TLR2-dependent mechanism (20Darveau R.P. Pham T.T. Lemley K. Reife R.A. Bainbridge B.W. Coats S.R. Howald W.N. Way S.S. Hajjar A.M. Infect. Immun. 2004; 72: 5041-5051Crossref PubMed Scopus (390) Google Scholar, 21Girard R. Pedron T. Uematsu S. Balloy V. Chignard M. Akira S. Chaby R. J. Cell Sci. 2003; 116: 293-302Crossref PubMed Scopus (129) Google Scholar, 22Hirschfeld M. Weis J.J. Toshchakov V. Salkowski C.A. Cody M.J. Ward D.C. Qureshi N. Michalek S.M. Vogel S.N. Infect. Immun. 2001; 69: 1477-1482Crossref PubMed Scopus (553) Google Scholar, 23Werts C. Tapping R.I. Mathison J.C. Chuang T.H. Kravchenko V. Saint Girons I. Haake D.A. Godowski P.J. Hayashi F. Ozinsky A. Underhill D.M. Kirschning C.J. Wagner H. Aderem A. Tobias P.S. Ulevitch R.J. Nat. Immunol. 2001; 2: 346-352Crossref PubMed Scopus (565) Google Scholar). It is not clear if LPS from C. burnetii can activate TLR-dependent signaling pathways. Recently, Honstettre and colleagues (24Honstettre A. Ghigo E. Moynault A. Capo C. Toman R. Akira S. Takeuchi O. Lepidi H. Raoult D. Mege J.L. J. Immunol. 2004; 172: 3695-3703Crossref PubMed Scopus (103) Google Scholar) showed that C. burnetii are internalized by macrophages from TLR4–/– mice less efficiently and that infected TLR4-deficient mice have a defect in granuloma formation and cytokine production. However, TLR4–/– mice and macrophages were as effective at controlling infections by C. burnetii as wild type mice (24Honstettre A. Ghigo E. Moynault A. Capo C. Toman R. Akira S. Takeuchi O. Lepidi H. Raoult D. Mege J.L. J. Immunol. 2004; 172: 3695-3703Crossref PubMed Scopus (103) Google Scholar). In addition, compared with LPS from enteric bacteria, C. burnetii LPS has been shown to be a weak endotoxin (25Williams J.C. Waag D.M. Williams J.C. Thompson H.A. The Biology of Coxiella burnetii. CRC Press, Inc., Boca Raton, FL1991: 175-222Google Scholar, 26Toman R. Garidel P. Andra J. Slaba K. Hussein A. Koch M.H. Brandenburg K. BMC Biochem. 2004; 5: 1-28Crossref PubMed Scopus (27) Google Scholar). To determine mechanisms that underlie host recognition of C. burnetii, TLR2- and TLR4-dependent responses to C. burnetii were investigated. By using CHO/CD14 reporter cell lines stably transfected with either TLR2 or TLR4, it was determined that highly purified C. burnetii lipid A is a weak agonist of both TLR2 and TLR4. Purified lipid A from C. burnetii was found to be an antagonist of TLR4, inhibiting responses to purified Escherichia coli LPS. Live C. burnetii triggered TLR2 activation, a feature required for induction of inflammatory cytokines and macrophage resistance to C. burnetii infection. Finally, by mass spectrometry we show that highly purified lipid A from phase I and II LPS display the same ionic species and fragmentation profiles, supporting the idea they have very similar if not identical structures. These data suggest an important role for TLR2 in the host response to C. burnetii and a potentially immunosuppressive activity for TLR4 associated with C. burnetii lipid A. Bacterial Preparation to Cellular Studies—Infective inocula of C. burnetii phase II Nine Mile strain clone 4 (RSA439) were prepared as described (27Zamboni D.S. Mortara R.A. Rabinovitch M. J. Microbiol. Methods. 2001; 43: 223-232Crossref PubMed Scopus (28) Google Scholar) from confluent Vero cells infected with C. burnetii for 7 days. Prior to infection, suspensions containing about 109 infective bacteria per ml were mildly sonicated at 35 kHz for 15 min at room temperature. Except for cytokine determination, macrophages were infected with ∼100 infective organisms per cell. After 24 h, infected cultures were vigorously washed with Hanks' saline solution, and the appropriate fresh medium was added. LPS and Lipid A Extraction—C. burnetii Nine Mile strain phase I clone 7 (RSA493) was grown in Spf embryonated chicken eggs purified as described (28Samuel J.E. Frazier M.E. Mallavia L.P. Infect. Immun. 1985; 49: 775-779Crossref PubMed Google Scholar), whereas Nine Mile strain phase II (RSA439) was cultured and purified from persistently infected Vero cells as described (27Zamboni D.S. Mortara R.A. Rabinovitch M. J. Microbiol. Methods. 2001; 43: 223-232Crossref PubMed Scopus (28) Google Scholar). LPS was preliminarily purified with hot phenol (29Westphal O. Jann K. Methods Carbohydr. Chem. 1965; 5: 83-91Google Scholar) or, alternatively, by a procedure that involves extensive delipidation followed by butanolic water extraction, previously used for extraction of glycolipoproteins and glycolipids from protozoan parasites (30Almeida I.C. Camargo M.M. Procopio D.O. Silva L.S. Mehlert A. Travassos L.R. Gazzinelli R.T. Ferguson M.A. EMBO J. 2000; 19: 1476-1485Crossref PubMed Scopus (200) Google Scholar). This latter procedure resulted in an LPS less contaminated with other bacterial components. Briefly, inactivated bacteria were lyophilized and sequentially extracted (three times each) with 10 volumes of chloroform/methanol (2:1, 1:1, and 1:2, v/v, respectively) and chloroform/methanol/water (10:20:8, v/v/v). Delipidated pellet was then re-extracted (three times) with 10 volumes of 9% 1-butanol, for 4 h at room temperature, under constant shaking. Butanol extracts were grouped and dried using a rotatory evaporator (Büchi, Switzerland), dissolved in endotoxin-free deionized water, and filtered through a 0.2-μm polytrifluoroethylene filter disk. Final purity of the LPS was determined by SDS-PAGE on a 12% gel, subsequently silver-stained as described previously (22Hirschfeld M. Weis J.J. Toshchakov V. Salkowski C.A. Cody M.J. Ward D.C. Qureshi N. Michalek S.M. Vogel S.N. Infect. Immun. 2001; 69: 1477-1482Crossref PubMed Scopus (553) Google Scholar). The lipid A moiety was isolated from LPS of phases I and II after mild acid hydrolysis with 0.25 n HCl (prepared from sequencing grade 6 n HCl) for 1 h at 100 °C, followed by neutralization with 0.25 n NaOH and Folch's partition (31Folch J. Lees M. Stanley G.H.S. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Lipid A was recovered in the lower phase, dried under N2, and redissolved in chloroform/methanol (1:1, v/v), and quantified by estimating the inorganic phosphate content (32Rouser G. Fkeischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2853) Google Scholar). CHO Cell Lines and Flow Cytometry Analysis—The CHO reporter cell lines (CHO/CD14, expressing functional TLR4; 7.19/CD14/TLR-2, expressing TLR2; and the 7.19 clone, expressing neither TLR2 nor functional MD2) were generated as described (33Lien E. Sellati T.J. Yoshimura A. Flo T.H. Rawadi G. Finberg R.W. Carroll J.D. Espevik T. Ingalls R.R. Radolf J.D. Golenbock D.T. J. Biol. Chem. 1999; 274: 33419-33425Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar) and maintained as adherent monolayers in Ham's F-12/Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, at 37 °C, 5% CO2, and antibiotics as described (33Lien E. Sellati T.J. Yoshimura A. Flo T.H. Rawadi G. Finberg R.W. Carroll J.D. Espevik T. Ingalls R.R. Radolf J.D. Golenbock D.T. J. Biol. Chem. 1999; 274: 33419-33425Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar). These cell lines expressing TLRs contain the CD25 gene under the control of E-selectin promoter, which contains an NF-κB-binding site. Thus, CD25 expression is dependent upon NF-κB activation (33Lien E. Sellati T.J. Yoshimura A. Flo T.H. Rawadi G. Finberg R.W. Carroll J.D. Espevik T. Ingalls R.R. Radolf J.D. Golenbock D.T. J. Biol. Chem. 1999; 274: 33419-33425Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar, 34Delude R.L. Yoshimura A. Ingalls R.R. Golenbock D.T. J. Immunol. 1998; 161: 3001-3009PubMed Google Scholar). Cells were plated (1 × 105 cells/well in 24-well tissue culture dishes), cultured for 24 h, and stimulated with live C. burnetii phase II at a ratio of 10, 100, or 1000 bacteria per cell; 10, 100, 1000, or 10000 ng/ml of purified LPS; or 82 nmol (in regard to inorganic phosphate content) of purified lipid A (about 200 ng/ml). Controls included UV-killed E. coli (HB101) and Staphylococcus aureus (ATCC 12692), and LPS and lipid A extracted from E. coli and Bordetella pertussis. After 18 h stimulation, cells were stained with (R)-phycoerythrin-labeled anti-CD25 (mouse monoclonal antibody to human CD25, (R)-phycoerythrin conjugate; CALTAG Laboratories, Burlingame, CA) 1:200 and examined by flow cytometry (BD Biosciences) as described previously (35Campos M.A. Almeida I.C. Takeuchi O. Akira S. Paganini E. Procopio D.O. Travassos L.R. Smith J.A. Golenbock D.T. Gazzinelli R.T. J. Immunol. 2001; 167: 416-423Crossref PubMed Scopus (450) Google Scholar). Mice and Primary Macrophages Culture—TLR4 mutant (C3H/HeJ) and control (C3H/HePas) mice were purchased from the University of São Paulo (Brazil), and TLR2-null mice were provided by Shizuo Akira (Osaka University, Japan) and backcrossed 8 times to C57BL/6 to ensure similar genetic backgrounds. C57BL/6, used as control mice, were obtained from CEDEME/UNIFESP (São Paulo, Brazil). Bone marrow-derived macrophages were generated as described (36Zamboni D.S. Rabinovitch M. Infect. Immun. 2003; 71: 1225-1233Crossref PubMed Scopus (91) Google Scholar) from 6- to 8-week-old mice. Differentiated macrophages were counted and seeded (2 × 105) in either 96-well tissue culture plates (for cytokine determination) or 24-well tissue culture plates containing a glass coverslip (for infection studies). Cultures were kept at 36 °C in a 5% CO2 in RPMI supplemented with 10% of fetal bovine serum and 5% of conditioned medium derived from cultures of L929 cells. Human PBMC were isolated from heparinized blood by Ficoll-Paque gradient centrifugation as described (37Golenbock D.T. Hampton R.Y. Qureshi N. Takayama K. Raetz C.R. J. Biol. Chem. 1991; 266: 19490-19498Abstract Full Text PDF PubMed Google Scholar). Cells were resuspended in complete RPMI supplemented with fetal bovine serum and plated (1 × 105 cells/well) in 96-well plates. Production of IL-12/TNF-α by Infected Macrophages—PBMC were cultured and stimulated in 96-well tissue culture plates with C. burnetii phase I lipid A (0.1, 1, or 10 μg/ml), doubly extracted LPS from E. coli strain O111:B4 (0.1, 1, or 10 ng/ml), PMA (30 ng/ml), and/or E5564 molecule (1 μg/ml). Supernatants were harvested for TNF-α determination 18 h after stimulation. Murine macrophages were infected in 96-well tissue culture plates at a multiplicity of 10, 100, or 500 bacteria per cell (in a final volume of 200 μl/well) in the presence or absence of 50 units/ml of IFN-γ (R & D Systems, Minneapolis, MN). Aliquots of the supernatant were collected 24 and 48 h after infection, respectively, for the determination of the presence of TNF-α and IL-12 (p40). Cytokines were measured by a commercially available enzyme-linked immunosorbent assay kit (Duoset; R & D Systems). Determination of C. burnetii Viability and Percentage of Cells with LRVs—The viability of C. burnetii was determined as described previously (36Zamboni D.S. Rabinovitch M. Infect. Immun. 2003; 71: 1225-1233Crossref PubMed Scopus (91) Google Scholar). Briefly, infected macrophages were submitted to hypotonic lysis with H2O, a step that did not reduce the bacterial infectivity. Lysates were sonicated, diluted, and then used to infect monolayers of γ-irradiated (1000 rads) Vero cells to block cell multiplication. Irradiated Vero cells were cultured as described (36Zamboni D.S. Rabinovitch M. Infect. Immun. 2003; 71: 1225-1233Crossref PubMed Scopus (91) Google Scholar) and fixed/stained by DAPI 4 days after infection. An epifluorescence microscope equipped with a ×40 objective was used to score the percentage of infected cells. Dilutions chosen for counting contained in the range of 10–50% infected cells. The percentage of cells with LRVs was determined as described (36Zamboni D.S. Rabinovitch M. Infect. Immun. 2003; 71: 1225-1233Crossref PubMed Scopus (91) Google Scholar), using a ×40 objective in an inverted microscope to score the presence or absence of large C. burnetii vacuoles. Approximately 500 cells in each of triplicate coverslips were scored for either LRV formation or viability determination. Confocal Microscopy, Image Acquisition, and Determination of C. burnetii Load in LRVs—Images of fixed and DAPI-stained cells (stained for 15 min with 3.5 μm DAPI) were acquired in a Bio-Rad 1024UV confocal system as described (27Zamboni D.S. Mortara R.A. Rabinovitch M. J. Microbiol. Methods. 2001; 43: 223-232Crossref PubMed Scopus (28) Google Scholar). MetaMorph (Universal Imaging Corp.) version 3.5 was used for image processing. Polygons were drawn onto digitized images of infected cells, and the relative fluorescence intensity of DAPI-stained bacteria within the circumscribed areas was determined. Under the measurement conditions used, it was shown that the fluorescence intensity within each polygon is proportional to the bacterial load in the region measured (27Zamboni D.S. Mortara R.A. Rabinovitch M. J. Microbiol. Methods. 2001; 43: 223-232Crossref PubMed Scopus (28) Google Scholar). Between 60 and 90 vacuoles were measured in each of triplicate coverslips. Electrospray Ionization-Mass Spectrometry—Lipid A species were analyzed using an electrospray ionization-ion trap-mass spectrometer (ESI-MS) (LCQ-Duo, ThermoFinnigan, San Jose, CA). Samples were diluted in chloroform/methanol (1:1, v/v), containing 10 mm ammonium acetate (CM/AA), and introduced into the electrospray source through a fused silica capillary (50-μm internal diameter), using a microinfusion pump (Harvard Apparatus) at a flow rate of 5–10 μl/min, or through a 20-μl loop of the ESI-MS instrument with the assistance of a solvent delivery system (Omnifit, UK) containing CM/AA and pressurized with N2 (9–10 pounds/square inch). Spectra were collected in negative ion mode, using an ion source voltage of 4.2 kV and capillary voltage and temperature of 35–45 V and 200 °C, respectively. Full scans were acquired at a rate of 3 scans/s, over the mass range of 200–2000 m/z. Fragmentation analysis (ESI-MS/MS) was carried out using a relative collision energy of 20–40% (1–2 eV). Authentic lipid A preparations from E. coli and Salmonella minnesota (Sigma) were used to calibrate instrument parameters in both ESI-MS and ESI-MS/MS modes. C. burnetii Phase II Bacteria Activate TLR2 but Not TLR4 — CHO/CD14 reporter cell lines expressing either human TLR2 or TLR4 were stimulated for 18 h with live C. burnetii phase II at a ratio of 10, 100, and 1000 bacteria per cell. Controls included UV-killed S. aureus and E. coli. The former is known to activate only TLR2, whereas the latter contains components that activate both TLR2 (such as lipopeptides and peptidoglycan) and TLR4 (lipopolysaccharide) (38Lee H.K. Lee J. Tobias P.S. J. Immunol. 2002; 168: 4012-4017Crossref PubMed Scopus (112) Google Scholar, 39Muroi M. Ohnishi T. Azumi-Mayuzumi S. Tanamoto K. Infect. Immun. 2003; 71: 3221-3226Crossref PubMed Scopus (21) Google Scholar). NF-κB activation was assessed by measuring the expression of surface CD25 by flow cytometry (33Lien E. Sellati T.J. Yoshimura A. Flo T.H. Rawadi G. Finberg R.W. Carroll J.D. Espevik T. Ingalls R.R. Radolf J.D. Golenbock D.T. J. Biol. Chem. 1999; 274: 33419-33425Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar, 34Delude R.L. Yoshimura A. Ingalls R.R. Golenbock D.T. J. Immunol. 1998; 161: 3001-3009PubMed Google Scholar). No induction of CD25 expression was observed when cells expressing TLR4 were exposed to C. burnetii phase II. In contrast, induction of CD25 expression by TLR2-expressing cells was observed. These data indicate that live C. burnetii can activate TLR2 but not TLR4 (Fig. 1). TLR2 Is Important for the Production of Pro-inflammatory Cytokines by C. burnetii-infected Murine Macrophages—The experiments with CHO reporter cell lines suggested that C. burnetii stimulates TLR2 but not TLR4. To determine whether TLR2 is important for host recognition, macrophages from mice deficient for TLR2 (TLR2–/–) or TLR4 (C3H/HeJ) were infected with phase II C. burnetii, and the detection of bacteria by host cells was assessed by cytokine production. Macrophages from C3H/HePas and C3H/HeJ both produced high levels of TNF-α and IL-12p40 after C. burnetii infection (Fig. 2). In contrast, macrophages from TLR2–/– mice were severely defective in cytokine production when compared with macrophages from wild type mice (Fig. 2). Additionally, treating macrophages with IFN-γ before infection enhanced TNF-α and IL-12 production by wild type and TLR4-deficient macrophages but not by TLR2-deficient macrophages (Fig. 2, B and D). These findings indicate that TLR2 is required for the signaling pathway leading to production of pro-inflammatory cytokines by macrophages exposed to phase II C. burnetii and further suggest that TLR4 is not responding to C. burnetii. TLR2 Responses Limit Growth of C. burnetii in Murine Macrophages—To determine the contribution of TLR2 or TLR4 on host cell defense, macrophages from C3H/HeJ, C3H/HePas, TLR2–/–, and wild type (C57BL/6) mice were infected with C. burnetii phase II and monitored for 8 days after infection. Bacterial replication was determined by measuring focus-forming units over time. Fig. 3A shows that C. burnetii multiplication in TLR4-deficient macrophages was similar to that observed in control macrophages that produce functional TLR4. By contrast, TLR2-deficient macrophages were found to be highly susceptible to C. burnetii multiplication. We have demonstrated previously that murine macrophages have the ability to control the development of the LRVs in which C. burnetii multiplies (36Zamboni D.S. Rabinovitch M. Infect. Immun. 2003; 71: 1225-1233Crossref PubMed Scopus (91) Google Scholar, 40Zamboni D.S. Freymuller E. Mortara R.A. Rabinovitch M. Microbes Infect. 2002; 4: 591-598Crossref PubMed Scopus (26) Google Scholar). Bacterial multiplication correlates with LRV formation (36Zamboni D.S. Rabinovitch M. Infect. Immun. 2003; 71: 1225-1233Crossref PubMed Scopus (91) Google Scholar, 41Zamboni D.S. Rabinovitch M. Infect. Immun. 2004; 72: 2075-2080Crossref PubMed Scopus (19) Google Scholar, 42Zamboni D.S. Infect. Immun. 2004; 72: 2395-2399Crossref PubMed Scopus (20) Google Scholar). Thus, TLR2-deficient macrophages were examined to determine whether they contained a greater number of LRVs compared with control macrophages. Fig. 3B shows that macrophages from TLR2–/– mice had more LRVs than control macrophages infected in parallel. After 2 days of infection, more than 50% of the TLR2–/– macrophages contained LRVs, while less than 25% of the macrophages from C57BL/6 or C3H/HeJ or C3H/HePas displayed LRVs at this time. To investigate whether LRVs found in TLR2-deficient macrophages contained more C. burnetii cells, intravacuolar bacterial loads were measured using quantitative fluorescence in digital images of DAPI-stained cells (27Zamboni D.S. Mortara R.A. Rabinovitch M. J. Microbiol. Methods. 2001; 43: 223-232Crossref PubMed Scopus (28) Google Scholar). Fig. 3C shows that DAPI fluorescence of LRVs in macrophages from C3H/HePas mice did not differ from that found in TLR4-deficient macrophages. However, the DAPI intensities for LRVs found in macrophages deficient in TLR2 were significantly higher than those of LRVs from wild type macrophages (Fig. 3C). Fig. 3D shows representative fields of macrophage cultures infected for 4 days with C. burnetii phase II. Several macrophages from TLR2–/– mice display LRVs, whereas only a few macrophages from C57BL/6 mice developed these organelles. The images shown in Fig. 3D also highlight the higher bacterial load found in vacuoles formed in macrophages deficient in TLR2, as compared with wild type macrophages. Overall, these results show that wild type macrophages were more effective than TLR2-defi
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