Exosomes Released from Infected Macrophages Contain Mycobacterium avium Glycopeptidolipids and Are Proinflammatory
2007; Elsevier BV; Volume: 282; Issue: 35 Linguagem: Inglês
10.1074/jbc.m702277200
ISSN1083-351X
AutoresSanchita Bhatnagar, Jeffrey S. Schorey,
Tópico(s)Mycobacterium research and diagnosis
ResumoMycobacterium avium is a major opportunistic pathogen in HIV-positive individuals and is responsible for increased morbidity and mortality in AIDS patients. M. avium express glycopeptidolipids (GPLs) as a major cell wall constituent, and recent studies suggest that GPLs play an important role in M. avium pathogenesis. In the present study we show that M. avium-infected macrophages release GPLs, which are trafficked from the phagosome through the endocytic network to multivesicular bodies. Prior studies have shown that multivesicular bodies can fuse with the plasma membrane releasing small 50 to 100 nm vesicles known as exosomes. We found that M. avium-infected macrophages release exosomes containing GPLs leading to the transfer of GPLs from infected to uninfected macrophages. Interestingly, exosomes isolated from M. avium-infected but not from uninfected macrophages can stimulate a proinflammatory response in resting macrophages. This proinflammatory response is dependent on Toll like receptor (TLR) 2, TLR4, and MyD88 suggesting that released exosomes contain M. avium-expressed TLR ligands. Our studies are the first to demonstrate that exosomes isolated from mycobacteria-infected macrophages can induce a proinflammatory response, and we hypothesize that exosomes play an important role in immune surveillance during intracellular bacteria infections. Mycobacterium avium is a major opportunistic pathogen in HIV-positive individuals and is responsible for increased morbidity and mortality in AIDS patients. M. avium express glycopeptidolipids (GPLs) as a major cell wall constituent, and recent studies suggest that GPLs play an important role in M. avium pathogenesis. In the present study we show that M. avium-infected macrophages release GPLs, which are trafficked from the phagosome through the endocytic network to multivesicular bodies. Prior studies have shown that multivesicular bodies can fuse with the plasma membrane releasing small 50 to 100 nm vesicles known as exosomes. We found that M. avium-infected macrophages release exosomes containing GPLs leading to the transfer of GPLs from infected to uninfected macrophages. Interestingly, exosomes isolated from M. avium-infected but not from uninfected macrophages can stimulate a proinflammatory response in resting macrophages. This proinflammatory response is dependent on Toll like receptor (TLR) 2, TLR4, and MyD88 suggesting that released exosomes contain M. avium-expressed TLR ligands. Our studies are the first to demonstrate that exosomes isolated from mycobacteria-infected macrophages can induce a proinflammatory response, and we hypothesize that exosomes play an important role in immune surveillance during intracellular bacteria infections. Mycobacteria have a long history as infectious organisms and are the etiologic agents of numerous human diseases. One such disease is caused by the Mycobacterium avium complex (MAC), 2The abbreviations used are:MACM. avium complexGPLglycopeptidolipidBCGBacillus Calmette-GuerinTNF-αtumor necrosis factor-alphaILinterleukinTLRToll-like receptorMyD88myeloid differentiation factor 88MVBmultivesicular bodyDCdendritic cellsMHCmajor histocompatibility complexNKnatural killer cellLAMlipoarabinomannanPIMphosphatidylinositol dimannosidesLAMPlysosomal-associated membrane proteinBMMbone marrow-derived macrophagesRANTESregulated upon activation normal T-cell expressed and secretediNOSinducible nitric-oxide synthasePMAphorbol 12-myristate 13-acetateMAPKmitogen-activated protein kinaseLPSlipopolysaccharideN-Rh-PEN-rhodaminephosphatidylethanolamineBALbronchoalveolar lavageILVintraluminal vesicleICAMintracellular adhesion moleculehspheat shock proteinCTLcytotoxic T-lymphocytePBSphosphate buffered salineFITCfluorescein isothiocyanateELISAenzyme-linked immunosorbent assay. which consists of three mycobacterial species; M. avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum. MAC is one of the most common opportunistic pathogens found in AIDS patients particularly in individuals with low CD4+ T cells (∼50-100 cells/mm3) and is associated with increased morbidity and mortality in these patients (1Horsburgh Jr., C.R. J. Infect. Dis. 1999; 179 (Suppl 3): S461-S465Crossref PubMed Scopus (85) Google Scholar). M. avium appears to account for the majority of infections in AIDS patients. This may reflect the ubiquitous nature of M. avium in the environment, where it can be found both in water and soil. Moreover, pulmonary MAC infections appear to have increased in the United States over the past half century in non-AIDS patients (2Rusin P.A. Rose J.B. Haas C.N. Gerba C.P. Rev. Environ. Contam. Toxicol. 1997; 152: 57-83Crossref PubMed Scopus (192) Google Scholar). At present it is unclear if this reflects an increased incidence of infection or increased ability to recognize MAC-infected individuals. M. avium complex glycopeptidolipid Bacillus Calmette-Guerin tumor necrosis factor-alpha interleukin Toll-like receptor myeloid differentiation factor 88 multivesicular body dendritic cells major histocompatibility complex natural killer cell lipoarabinomannan phosphatidylinositol dimannosides lysosomal-associated membrane protein bone marrow-derived macrophages regulated upon activation normal T-cell expressed and secreted inducible nitric-oxide synthase phorbol 12-myristate 13-acetate mitogen-activated protein kinase lipopolysaccharide N-rhodaminephosphatidylethanolamine bronchoalveolar lavage intraluminal vesicle intracellular adhesion molecule heat shock protein cytotoxic T-lymphocyte phosphate buffered saline fluorescein isothiocyanate enzyme-linked immunosorbent assay. M. avium is an intracellular pathogen that resides within the phagosome of a host macrophage. Upon phagocytosis, M. avium, like other species in the genus Mycobacterium, inhibit phagosome maturation and thereby limit their exposure to hydrolytic enzymes present within a mature phagolysosome (3Frehel C. de Chastellier C. Lang T. Rastogi N. Infect. Immun. 1986; 52: 252-262Crossref PubMed Google Scholar). This is an essential component of mycobacterial pathogenesis. Pathogenic mycobacteria also modulate macrophage signaling responses and thereby limit the ability of macrophages to produced or respond to immune modulators. The capacity of mycobacteria to alter these macrophage functions requires the expression of specific mycobacterial surface components. For example, mannosylated lipoarabinomannan (LAM) expressed by M. tuberculosis has been shown to both inhibit phagosome maturation and to block interferon-γ-mediated activation of macrophages (4Fratti R.A. Vergne I. Chua J. Skidmore J. Deretic V. Electrophoresis. 2000; 21: 3378-3385Crossref PubMed Scopus (39) Google Scholar, 5Fratti R.A. Chua J. Vergne I. Deretic V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5437-5442Crossref PubMed Scopus (408) Google Scholar). Although M. avium express a single mannosylated form of mannosylated LAM, the major surface molecule, which accounts for 10% of the total extractable lipid from the cell wall, is the glycopeptidolipids (GPLs) (6Rulong S. Aguas A.P. da Silva P.P. Silva M.T. Infect. Immun. 1991; 59: 3895-3902Crossref PubMed Google Scholar). The high expression levels and its presence on the cell surface make the M. avium GPLs an excellent candidate for altering host microbicidal function. Moreover, our data indicates that GPLs are a major M. avium virulence factor as the absence/alteration of GPL significantly attenuates the virulence of M. avium (7Bhatnagar S. Schorey J.S. Cell Microbiol. 2006; 8: 85-96Crossref PubMed Scopus (22) Google Scholar, 8Krzywinska E. Bhatnagar S. Sweet L. Chatterjee D. Schorey J.S. Mol. Microbiol. 2005; 56: 1262-1273Crossref PubMed Scopus (29) Google Scholar). However, it is not clear how GPL can modulate the immune response and therefore accentuate the survival of M. avium in the host. The GPLs consist of a tripeptide amino alcohol core modified with an amide-linked fatty acid, a methylated rhamnose and a 6-deoxytalose (9Chatterjee D. Khoo K.H. Cell. Mol. Life Sci. 2001; 58: 2018-2042Crossref PubMed Scopus (122) Google Scholar). The M. avium GPLs can be further modified in the length and composition of sugars attached to the 6-deoxytalose residue. The GPLs are non-covalently attached to the cell wall of mycobacteria. Interestingly, studies by Vergne et al. (10Vergne I. Prats M. Tocanne J.F. Laneelle G. FEBS Lett. 1995; 375: 254-258Crossref PubMed Scopus (20) Google Scholar) showed that GPLs can interact with host membranes and suggests that GPLs could promote mycobacteria survival by interfering with the membrane-mediated functions of the host cells. There is also evidence showing that GPLs accumulate inside infected cells (11Horgen L Barrow E.L. Barrow W.W. Rastogi N. Microb. Pathog. 2000; 29: 9-16Crossref PubMed Scopus (31) Google Scholar, 12Tereletsky M.J. Barrow W.W. Infect. Immun. 1983; 41: 1312-1321Crossref PubMed Google Scholar, 13Draper P. Rees R.J. Nature. 1970; 228: 860-861Crossref PubMed Scopus (76) Google Scholar). Moreover, due to the extensive movement of membrane within the endocytic pathway, the mycobacterial GPL could be trafficked to other cellular compartments and modulate cellular functions. In the present study, we hypothesized that the mycobacterial GPL are released from the M. avium surface and trafficked throughout the cell as has been shown for mannosylated LAM and other mycobacterial lipids (14Beatty W.L. Rhoades E.R. Ullrich H.J. Chatterjee D. Heuser J.E. Russell D.G. Traffic. 2000; 1: 235-247Crossref PubMed Scopus (303) Google Scholar). However, our studies indicate that GPLs are more restrictive in their distribution; with the majority of the GPL trafficking to a distinct compartment within the endocytic pathway designated the multivesicular body or MVB. Prior studies have shown that fusion of MVBs with the plasma membrane results in the release of small 50-100 nm vesicles called exosomes (15Raposo G. Nijman H.W. Stoorvogel W. Liejendekker R. Harding C.V. Melief C.J. Geuze H.J. J. Exp. Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2630) Google Scholar). We found that exosomes released from M. avium-infected macrophages contain GPLs and that these exosomes can interact with uninfected macrophage leading to retention of GPLs in these "bystander" cells. Interestingly, exosomes purified from the culture supernatant of M. avium but not from uninfected cells induced a proinflammatory response in exosome-treated macrophages. To our knowledge, our studies are first to show that the exosomes carrying bacterial components could induce a proinflammatory response and suggests a novel mechanism by which stimulator molecules present on intracellular pathogens can be released from infected cells to promote an immune response. Mφ Isolation and Culture—BALB/c and C57BL/6 mice were purchased from Harlan (Mutant Mouse Regional Resource Center, Indianapolis IN). TLR2-/- (C57BL/6 background) and TLR4-/- (BALB/c background) mice were purchased from Jackson Laboratory (Bar Harbor, ME). MyD88-/- mice (C57BL/6 background) were generously provided by Dr. Soon-Cheol Hong, Indiana University Medical School. Bone marrow-derived macrophages (BMMs), used in all experiments, was isolated from 6- to 8-week-old BALB/c mice as previously described (16Roach S.K. Schorey J.S. Infect. Immun. 2002; 70: 3040-3052Crossref PubMed Scopus (111) Google Scholar). The macrophages were used 7-14 days after isolation or frozen after 7 days of culture in freezing media (50% Dulbecco's modified Eagle's medium, 40% fetal bovine serum, and 10% endotoxin-tested Me2SO (Sigma). Thawed or fresh macrophages were cultured on non-tissue culture plates for 3-7 days and then re-plated at 3 × 105 cells per 35-mm tissue culture plate. The cells were allowed to adhere for 24 h prior to treatments with mycobacteria or inhibitors. The plates were then incubated at 37 °C in 5% CO2 for various times. The infection was done for 4 h, and then the media on the cells was replaced with fresh media. All tissue culture reagents were found to be negative for the endotoxin contamination by the E-Toxate assay (Sigma). The mouse macrophage cell line J774 was maintained at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Invitrogen), 25 mm HEPES, 100 units/ml penicillin, and 100 μg/ml streptomycin (BioWhittaker, Walkersville, MD). Bacteria Culture—To generate the M. avium 2151 variant stocks, the bacteria were grown on Middlebrooks 7H11 plates for 20-30 days. The different morphotypes of M. avium 2151 were then resuspended in freezing media (Middlebrooks 7H9, 10% glycerol, glucose, oleic acids, Tween 20, and NaCl (Ref. 16Roach S.K. Schorey J.S. Infect. Immun. 2002; 70: 3040-3052Crossref PubMed Scopus (111) Google Scholar). All the stocks were quantitated by the serial dilutions. M. avium 101 and M. avium A5 were also prepared as described for M. avium 2151. Infection assays evaluated by fluorescence microscopy was performed on each stock of mycobacteria to determine the infection ratio required to obtain ∼80% of the macrophages infected. The amount of mycobacteria required to obtain this infection rate varied between species and batch preps of mycobacteria. However, the results were consistent across the different mycobacteria-to-macrophage ratios when the infection levels were kept constant. Complement Opsonization—Appropriate concentration of mycobacteria were suspended in macrophage culture media containing 10% horse serum as a source of complement components and incubated for 2 h at 37 °C (16Roach S.K. Schorey J.S. Infect. Immun. 2002; 70: 3040-3052Crossref PubMed Scopus (111) Google Scholar). Antibodies and Immunofluorescence Staining—The immunofluorescence staining and confocal microscopy conducted as previously described (17Boshans R.L. Szanto S. van Aelst L. D'Souza-Schorey C. Mol. Cell. Biol. 2000; 20: 3685-3694Crossref PubMed Scopus (157) Google Scholar). Briefly, infected cells were fixed in 2% paraformaldehyde (Sigma) in PBS. Fixed cells were permeabilized with 0.02% Triton X-100 (Sigma) and washed with PBS, 1% bovine serum albumin, and 0.02% gelatin (Sigma). The anti-mouse monoclonal antibody against serovar-specific 1, 2, and 4 glycopeptidolipids, the anti-rat monoclonal antibody against LAMP1 (1D4B, The Developmental Studies Hybridoma Bank, University of Iowa), the anti-rat monoclonal antibody against LAMP2 (ABL2, The Developmental Studies Hybridoma Bank), the anti-glucose-regulated protein (Grp-78, QED Bioscience Inc., San Diego, CA), the anti rab-11 (Zymed Laboratories Inc.), and the anti-MHCII (Lab Vision). The bisalkylaminoanthraquinone fluorphore Draq5 (Ex: 646 nm; Em: 681 nm, Axxora), which stains DNA, was used to visualize the nucleus. TLC Immunostaining—The TLC immunostaining of GPLs on TLC was performed according to the method of Watarai et al. (18Mise K. Akifusa S. Watarai S. Ansai T. Nishihara T. Takehara T. Infect. Immun. 2005; 73: 4846-4852Crossref PubMed Scopus (47) Google Scholar) with slight modifications. Briefly, alkali-stable GPLs were separated by TLC using Silica Gel-60 plates (EM Science) with chloroform/methanol/water (30:8:1, v/v) as the developing solvent. The GPLs were visualized using a-naphthol/sulfuric acid as the spray reagent. The dried plate was soaked for 1 min in a 0.02% solution of polyisobutylmethacrylate (Glycotech, MD) dissolved in acetone, allowed to air dry, and then blocked by incubation in PBS containing 10% heat-inactivated horse serum at 37 °C for 2 h. The plate was then rinsed with PBS containing 0.1% Tween 20 (washing buffer) and incubated with anti-GPL monoclonal antibody in PBS for 2 h at room temperature. Following this, the plate was washed thrice with washing buffer and probed with horseradish peroxidase-conjugated anti-mouse immunoglobulin G antiserum (Amersham Biosciences) at room temperature for 2 h. The plate was washed, and the bound Abs was detected using SuperSignal West Femto enhanced chemiluminescence reagents (Pierce). Isolation of Exosomes and Apoptotic Vesicles—The fetal calf serum used in the cell culture media for exosome isolation was centrifuged at 100,000 × g, 15 h to remove any contaminating exosomes from the media. J774 cells were infected with M. avium 2151 SmT for 4 h and washed extensively to remove extracellular bacteria. After 72 h, the culture medium was collected and centrifuged twice at 300 × g, 10 min to remove whole cells, followed by centrifugation at 1200 × g for 10 min to remove any bacilli. The supernatant from the previous spins was centrifuged at 10,000 × g for 30 min. This was followed with ultracentrifugation at 100,000 × g for 1 h. The resulting pellet following 100,000 × g centrifugation was further purified by sucrose gradient. Apoptotic vesicles were purified by consecutive centrifugations as described previously (19Schaible U.E. Winau F. Sieling P.A. Fischer K. Collins H.L. Hagens K. Modlin R.L. Brinkmann V. Kaufmann S.H. Nat. Med. 2003; 9: 1039-1046Crossref PubMed Scopus (435) Google Scholar, 20Winau F. Weber S. Sad S. de Diego J. Hoops S.L. Breiden B. Sandhoff K. Brinkmann V. Kaufmann S.H. Schaible U.E. Immunity. 2006; 24: 105-117Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Briefly, apoptosis was induced by fetal calf serum deprivation (21Katoh N. Kraft S. Wessendorf J.H. Bieber T. J. Clin. Invest. 2000; 105: 183-190Crossref PubMed Scopus (89) Google Scholar, 22Roth K.A. Motoyama N. Loh D.Y. J. Neurosci. 1996; 16: 1753-1758Crossref PubMed Google Scholar), and culture supernatant was collected 72 h post-treatment. The culture supernatant was centrifuged at 800 × g (15 min), 1800 × g (15 min), and 25,000 × g (20 min), and the remaining supernatant was spun at 100,000 × g (1 h) to pellet small apoptotic vesicles. Apoptotic vesicles were coated onto latex beads as described below and labeled with antibodies for analysis by flow cytometry or fluorescence microscopy. Sucrose Density Gradient Centrifugation—For further purification of exosomes, the 100,000 × g pellet was resuspended in 1 ml of 2.5 m sucrose, 20 mm Hepes/NaOH, pH 7.2. A linear sucrose gradient (2-0.25 m sucrose, 20 mm Hepes/NaOH, pH 7.2) was layered on top of the exosomes suspension in a tube, and the sample was centrifuged at 100,000 × g for 15 h. Gradient fractions (7 × 1 ml) were collected from the top of the tube, diluted with 3 ml of PBS, and ultracentrifuged at 100,000 × g for 1 h. Caspase 3 Inhibitor Treatment and Staining of Apoptotic Cells—Cells were stained for annexin V as per manufacturer's instructions (Annexin V-FITC apoptosis Detection kit II, Calbiochem, WI). Briefly, cells were washed with binding buffer and stained with annexin V (1:40) for 10 min at room temperature. Cells were washed with binding buffer and stained with propidium iodide (1 μg/ml) and analyzed by confocal microscopy. For caspase inhibition, the caspase-3-specific inhibitor Ac-DEVD-CHO (Calbiochem) was added to macrophages at a final concentration of 50 μm 1 h before infection and maintained for the duration of assay (23Huesmann G.R. Clayton D.F. Neuron. 2006; 52: 1061-1072Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 24Mohr S. McCormick T.S. Lapetina E.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5045-5050Crossref PubMed Scopus (92) Google Scholar, 25Ulett G.C. Maclean K.H. Nekkalapu S. Cleveland J.L. Adderson E.E. J. Immunol. 2005; 175: 2555-2562Crossref PubMed Scopus (32) Google Scholar). Electron Microscopy—Exosome pellets were resuspended and fixed in phosphate buffer containing 2% glutaraldehyde and then loaded on Formvar/carbon-coated electron microscopy grids. The samples were contrasted in uranyl acetate and viewed with Hitachi H-600 transmission electron microscope. Labeling with N-Rh-PE—The fluorescent phospholipids analog N-Rh-PE was inserted into the plasma membrane as previously described (26Savina A. Furlan M. Vidal M. Colombo M.I. J. Biol. Chem. 2003; 278: 20083-20090Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). Briefly, an appropriate amount of lipid was resuspended in ethanol. The ethanol solution was injected with a Hamilton syringe into serum-free Dulbecco's modified Eagle's medium while vigorously vortexing. This mixture was then added to the cells already infected with SmT M. avium 2151, and they were incubated for 60 min at 4 °C. Subsequently, the medium was removed, and the cells were extensively washed with PBS. Labeled cells were fixed after 3 h of incubation at 37 °C and immediately analyzed by confocal microscopy. Analysis of Uninfected Bystander Cells—BMMs were infected with FITC-labeled SmT M. avium 2151, and, after 4 h of infection, an equal number of uninfected BMMs labeled with the cell tracker dye 7-amino-4-chloromethylcoumarin (10 μm for 60 min) were added. Cells were examined by fluorescence microscopy for the presence of GPL in uninfected bystander cells. Coupling of Exosomes or Apoptotic Vesicles to Latex Beads—The purified exosomes or apoptotic vesicles (30 μg) were incubated with 4-μm diameter aldehyde/sulfate latex beads (Interfacial Dynamics) for 15 min at room temperature. This was followed by the dilution with PBS, and binding reaction was continued for another 2 h. The reaction was stopped by addition of 100 mm glycine. Vesicle-coated beads were then washed three times in PBS and stained with specific antibodies. Western Blot Analysis—For Western blots, equal concentration of protein from cell lysates or exosomes, as quantitated by the Micro BCA Protein Assay, were loaded on 10% SDS-PAGE gels, electrophoresed, and transferred onto polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were probed for iNOS (BD Transduction Laboratories) or total p38 (Cell Signaling) as described (16Roach S.K. Schorey J.S. Infect. Immun. 2002; 70: 3040-3052Crossref PubMed Scopus (111) Google Scholar). Blots containing 10 μg of exosome material were probed using antibodies against the M. tuberculosis LAM (1:500 dilution) or host hsp70 (R&D, 1/1000 dilution). Flow Cytometric Analysis of Exosomes and Apoptotic Vesicles—For flow cytometric analysis, beads coated with exosomes were labeled with the following FITC monoclonal antibodies: Grp78 (QED Biosciences Inc., San Diego, CA), LAMP1 (1D4B, The Developmental Hybridoma Bank), and LAMP2 (ABL2, the Developmental Studies Hybridoma Bank), MHCII (MACS, Auburn, CA), CD81 (Biolegend), and CD86 (Biolegend). The exosome-coated beads were also incubated with anti-GPL antibody and probed with FITC-tagged anti-mouse secondary antibody (Biolegend). Apoptotic vesicles were labeled with Annexin V (Calbiochem) or FcγRII/III (eBioscience). Exosomes or apoptotic vesicle-coated beads were incubated for 1 h with each primary antibody (1:100 dilution), followed when necessary by a 30-min incubation with FITC-conjugated secondary antibody (Biolegend) at 1/100 dilution, washed, and analyzed on Cytomics FC500 MPL Flow Cytometer (Beckman Coulter, FL). Macrophage Incubation with Exosomes or Apoptotic Vesicles—Thawed or fresh macrophages were cultured on non-tissue culture plates for 3-7 days and then re-plated at 1 × 105 cells per well in 24-well tissue culture plate. The cells were allowed to adhere for 24 h prior to the addition of exosomes or apoptotic vesicles. The BMMs in culture media minus antibiotics were incubated with isolated vesicles for 24 h. The culture supernatant was harvested at indicated times for subsequent cytokine/chemokine analysis. ELISA—The levels of TNF-α and RANTES secreted into the culture medium by exosome-treated macrophages were measured using the ELISA kits from BD Pharmingen and eBioscience, respectively. Culture media collected from the macrophages were analyzed for cytokines according to manufacturer's instructions, and the cytokine concentrations were determined against standard curves. Statistical Analysis—Data obtained from independent experiments were analyzed by a one-tailed Student t test. Differences were considered significant for p < 0.05. Our prior studies established that GPLs are M. avium virulence factors (7Bhatnagar S. Schorey J.S. Cell Microbiol. 2006; 8: 85-96Crossref PubMed Scopus (22) Google Scholar, 8Krzywinska E. Bhatnagar S. Sweet L. Chatterjee D. Schorey J.S. Mol. Microbiol. 2005; 56: 1262-1273Crossref PubMed Scopus (29) Google Scholar). Moreover, GPLs are shown to accumulate inside the cell and have a long half-life inside a phagosome (11Horgen L Barrow E.L. Barrow W.W. Rastogi N. Microb. Pathog. 2000; 29: 9-16Crossref PubMed Scopus (31) Google Scholar). To determine if GPLs can enter the endocytic pathway, we followed the trafficking of GPL inside infected cells using anti-GPL monoclonal antibodies. The specificity of the antibody was confirmed by TLC immunostaining comparing extracted lipids from SmT M. avium 2151, which express serotype 2 GPLs to an Rg 2151 isolate, which lacks GPL expression (28Barrow W.W. Brennan P.J. J. Bacteriol. 1982; 150: 381-384Crossref PubMed Google Scholar). As shown in Fig. 1A, we observed a single band only in lanes containing lipid extract from SmT M. avium 2151. This serotype-2 anti-GPL antibody was used to characterize the distribution of GPLs in SmT M. avium 2151-infected macrophages at 4, 24, 48, 72, and 96 h post infection. The fluorescence staining indicated that the GPLs were not only present on the mycobacterial surface (shown in merged images as yellow) but also separate from the mycobacteria (Fig. 1B). No cellular staining was observed with the anti-GPL monoclonal antibodies when the macrophages were infected with the GPL-deficient Rg M. avium 2151 (data not shown). The number of cells staining positive for GPLs was quantitated over time and indicated a gradual increase in the percentage of total GPL-positive macrophages between 4 and 96 h post-infection (Fig. 1, C and D). Interestingly, this increase appeared to be due to enhanced staining of uninfected macrophages with the anti-GPL monoclonal antibodies suggesting transfer of GPLs from infected to uninfected cells (Fig. 1D). To further confirm this cell-to-cell transfer of GPLs, BMMs were infected with FITC-labeled SmT M. avium 2151. The infected cells were washed with PBS to remove any free mycobacteria prior to the addition of equal number of uninfected macrophages labeled with the fluorescent marker aminochloromethylcoumarin. As expected, by 24 and 48 h we observed a significant number of the uninfected, aminochloromethylcoumarin-stained cells to be positive for GPLs demonstrating the transfer of GPLs from infected to uninfected cells (Fig. 1E). Analysis of SmT M. avium 2151-infected bone marrow-derived macrophages by fluorescence microscopy revealed significant release and trafficking of glycopeptidolipids. As noted above, mycobacteria are maintained within phagosomes that do not mature to phagolysosomes in non-activated macrophages. However, it is less clear whether released mycobacterial components can enter the late endosomal/lysosomal pathway or whether they can accumulate in specific compartments within infected cells. There are recent reports indicating that some mycobacterial lipids, including phosphoinositolmannoside (PIM) can enter distinct compartments in the endocytic pathway (14Beatty W.L. Rhoades E.R. Ullrich H.J. Chatterjee D. Heuser J.E. Russell D.G. Traffic. 2000; 1: 235-247Crossref PubMed Scopus (303) Google Scholar). Other mycobacterial lipids such as LAM show a more diffuse staining pattern in M. tuberculosis-infected macrophages (14Beatty W.L. Rhoades E.R. Ullrich H.J. Chatterjee D. Heuser J.E. Russell D.G. Traffic. 2000; 1: 235-247Crossref PubMed Scopus (303) Google Scholar). Previous studies looking at the trafficking of hydrazidelabeled mycobacterial lipids also reported these distinct membrane organelles carrying mycobacterial lipids, which were subsequently identified as MVBs (29Beatty W.L. Ullrich H.J. Russell D.G. Eur. J. Cell Biol. 2001; 80: 31-40Crossref PubMed Scopus (105) Google Scholar). To elucidate the route taken by GPL after release from mycobacterial phagosome and to characterize the GPL-positive compartment, we studied the colocalization of GPL with different endocytic markers, including LAMP1 (late endosomes), LAMP2 (late endosomes), and MHC class II (MIIC compartment). Studies with these markers showed significant colocalization with GPL (Fig. 2A and data not shown), indicating that GPLs traffic to one of the late compartments within the BMMs endocytic pathway. Moreover, the GPL-positive compartments were found negative for the Golgi marker Grp78 (Fig. 2A) and transferrin (data not shown). Based on these results and previous published studies, we hypothesized that the GPLs are trafficked predominately to the MVB. To further test our hypothesis, we looked for whether N-rhodamine-phosphatidylethanolamine (N-Rh-PE) colocalized with the GPL. N-Rh-PE when added to cells is efficiently internalized via endocytosis and has been shown to traffic specifically to MVBs in reticulocytes (30Vidal M. Mangeat P. Hoekstra D. J. Cell Sci. 1997; 110: 1867-1877Crossref PubMed Google Scholar) and RAW 264.7 cells (31Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). N-Rh-PE added to the M. avium 2151-infected cells showed marked colocalization with GPL (Fig. 2B). Together the data indicate that GPLs are released from M. avium-containing phagosomes, enter the endocytic pathway and localize, at least in part, to MVBs within infected cells. MVBs can be trafficked toward the plasma membrane where they can fuse and release the intraluminal vesicles (ILVs) known as exosomes into the extracellular environment. However, this phenomenon of the transporting, docking, and fusion of MVBs with the plasma membrane is not well understood. Recent studies by Colombo and colleagues demonstrate the recruitment of Rab11 to MVBs and its role in the docking and fus
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